SCCS/1591/17
Final version
Version S
Scientific Committee on Consumer Safety
SCCS
OPINION ON
the safety of Butylphenyl methylpropional (p-BMHCA) in
cosmetic products
- Submission II -
The SCCS adopted this Opinion by written procedure on 10 May 2019
SCCS/1591/17
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Opinion on the safety Butylphenyl methylpropional (p- BMHCA) in cosmetic products - Submission II
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ACKNOWLEDGMENTS
SCCS members listed below are acknowledged for their valuable contribution to the
finalisation of this Opinion.
For the Preliminary Opinion
SCCS Members
Dr U. Bernauer
Dr L. Bodin
Dr L. Celleno
Prof. Q. Chaudhry
Prof. P.J. Coenraads (Chairperson)
Prof. M. Dusinska
Dr J. Ezendam
Dr E. Gaffet
Prof. C. L. Galli
Dr B. Granum
Prof. E. Panteri
Prof. V. Rogiers
Dr Ch. Rousselle
Dr M. Stępnik (Rapporteur)
Prof. T. Vanhaecke
Dr S. Wijnhoven
For the Final Opinion
SCCS Members
Dr U. Bernauer
Dr L. Bodin
Prof. Q. Chaudhry
Prof. P.J. Coenraads (Chairperson)
Prof. M. Dusinska
Dr J. Ezendam
Dr E. Gaffet
Prof. C. L. Galli
Dr B. Granum
Prof. E. Panteri
Prof. V. Rogiers
Dr Ch. Rousselle
Dr M. Stępnik (Rapporteur)
Prof. T. Vanhaecke
Dr S. Wijnhoven
This Opinion has been subject to a commenting period of a minimum eight weeks after its
initial publication (from 20 December 2017 until 19 February 2018). Comments received
during this time were considered by the SCCS.
For this Opinion, comments received resulted in the following main changes: sections 3.1.5,
3.1.8, 3.3.4.1 SCCS comment, 3.3.6.1, 3.3.6.2 overall SCCS discussion, 3.3.8.2 SCCS
comment, 3.3.8.3, 3.3.12 SCCS comment, 3.3.13, as well as the discussion part
accordingly, the conclusions and the references’ list.
All Declarations of Working Group members are available on the following webpage:
http://ec.europa.eu/health/scientific_committees/experts/declarations/sccs_en.htm
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1. ABSTRACT
The SCCS concludes the following:
1. Does the SCCS consider Butylphenyl methylpropional (p-BMHCA) safe for use as a
fragrance ingredient in cosmetic leave-on and rinse-off type products in a concentration
limit(s) according the ones set up by IFRA as reported above?
On individual product basis, Butylphenyl methylpropional (p-BMHCA) (CAS 80-54-6) with
alpha-tocopherol at 200 ppm, can be considered safe when used as fragrance ingredient in
different cosmetic leave-on and rinse-off type products. However, considering the first-tier
deterministic aggregate exposure, arising from the use of different product types together,
Butylphenyl methylpropional at the proposed concentrations cannot be considered as safe.
This Opinion is not applicable to the use of p-BMHCA in any sprayable products that could
lead to exposure of the consumer’s lung by inhalation.
2. Does the SCCS have any further scientific concerns with regard to the use of Butylphenyl
methylpropional (p-BMHCA) as a fragrance ingredient in cosmetic leave-on and/or rinse-off
type products?
Evaluation of this substance by other scientific bodies (e.g. under REACH) should also be
taken into consideration by the Applicant for potential future assessment of the substance.
Butylphenyl methylpropional is also used as a fragrance ingredient in some non-cosmetic
products such as household cleaners and detergents. As no specific exposure data were
made available to SCCS to assess exposure following these non-cosmetic uses, it was not
possible to include them in the aggregated exposure scenarios. Therefore, the actual total
exposure of the consumer may be higher than exposure from cosmetic products alone.
Keywords: SCCS, scientific opinion, Butylphenyl methylpropional (p-BMHCA) in cosmetic
products Submission II, Regulation 1223/2009, CAS 80-54-6, EC 201-289-8.
Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on the
safety of Butylphenyl methylpropional (p-BMHCA) in cosmetic products - Submission II,
preliminary version of 14 December 2017, final version of 10 May 2019, SCCS/1591/2017.
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About the Scientific Committees
Two independent non-food Scientific Committees provide the Commission with the scientific
advice it needs when preparing policy and proposals relating to consumer safety, public
health and the environment. The Committees also draw the Commission's attention to the
new or emerging problems that may pose an actual or potential threat.
They are: the Scientific Committee on Consumer Safety (SCCS) and the Scientific
Committee on Health, Environmental and Emerging Risks (SCHEER) and are made up of
scientists appointed in their personal capacity.
In addition, the Commission relies upon the work of the European Food Safety Authority
(EFSA), the European Medicines Agency (EMA), the European Centre for Disease prevention
and Control (ECDC) and the European Chemicals Agency (ECHA).
SCCS
The Committee shall provide Opinions on questions concerning health and safety risks
(notably chemical, biological, mechanical and other physical risks) of non-food consumer
products (for example cosmetic products and their ingredients, toys, textiles, clothing,
personal care and household products such as detergents, etc.) and services (for example:
tattooing, artificial sun tanning, etc.).
Scientific Committee members
Ulrike Bernauer, Laurent Bodin, Qasim Chaudhry, Pieter-Jan Coenraads, Maria Dusinska,
Janine Ezendam, Eric Gaffet, Corrado Lodovico Galli, Berit Granum, Eirini Panteri, Vera
Rogiers, Christophe Rousselle, Maciej Stępnik, Tamara Vanhaecke, Susan Wijnhoven
Contact
European Commission
Health and Food Safety
Directorate C: Public Health, country knowledge and crisis management
Unit C2 – Country knowledge and Scientific Committees
Office: HTC 03/073
L-2920 Luxembourg
©
European Union, 2019
ISSN ISBN
Doi ND
The opinions of the Scientific Committees present the views of the independent scientists
who are members of the committees. They do not necessarily reflect the views of the
European Commission. The opinions are published by the European Commission in their
original language only.
http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm
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TABLE OF CONTENTS
ACKNOWLEDGMENTS ........................................................................................... 2
1. ABSTRACT .................................................................................................. 3
2. MANDATE FROM THE EUROPEAN COMMISSION ................................................ 6
3. OPINION ..................................................................................................... 8
3.1 Chemical and Physical Specifications ....................................................... 8
3.1.1 Chemical identity .................................................................... 8
3.1.2 Physical form ......................................................................... 9
3.1.3 Molecular weight .................................................................... 9
3.1.4 Purity, composition and substance codes .................................... 9
3.1.5 Impurities / accompanying contaminants ................................. 10
3.1.6 Solubility ............................................................................. 11
3.1.7 Partition coefficient (Log P
ow
) .................................................. 11
3.1.8 Additional physical and chemical specifications .......................... 11
3.1.9 Homogeneity and Stability ..................................................... 11
3.2 Function and uses .............................................................................. 11
3.3 Toxicological evaluation ...................................................................... 13
3.3.1 Acute toxicity ....................................................................... 13
3.3.2 Irritation and corrosivity ........................................................ 14
3.3.3 Skin sensitisation .................................................................. 15
3.3.4 Dermal / percutaneous absorption........................................... 16
3.3.5 Repeated dose toxicity .......................................................... 20
3.3.6 Mutagenicity / Genotoxicity .................................................... 21
3.3.7 Carcinogenicity ..................................................................... 34
3.3.8 Reproductive toxicity ............................................................. 34
3.3.9 Toxicokinetics ...................................................................... 41
3.3.10 Photo-induced toxicity ........................................................... 43
3.3.11 Human data ......................................................................... 44
3.3.12 Special investigations ............................................................ 45
3.3.13 Safety evaluation (including calculation of the MoS) ................... 46
3.3.14 Discussion ........................................................................... 49
4. CONCLUSION ............................................................................................ 53
5. MINORITY OPINION .................................................................................... 53
6. REFERENCES ............................................................................................. 54
7. GLOSSARY OF TERMS ................................................................................. 68
8. LIST OF ABBREVIATIONS ............................................................................ 68
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2. MANDATE FROM THE EUROPEAN COMMISSION
Background
The substance 2-(4-tert-Butylbenzyl)propionaldehyde (BMHCA, Lysmeral) CAS No. 80-54-6
with INCI name Butylphenyl methylpropional is a fragrance ingredient used in many
compounds for cosmetic products as well as in non-cosmetic products.
Butylphenyl methylpropional (BMHCA) is currently regulated for labelling purposes in Annex
III entry 83 of the Cosmetics Regulation No 1223/2009 when present in a concentration
above 10 ppm for leave-on products and above 100 ppm for rinse-off products.
Following a proposal for a harmonised classification as Toxic for Reproduction 2 substance
under Regulation (EC) No 1272/2008, a dossier on the safety assessment of BMHCA was
submitted to the Commission by the International Fragrance Association (IFRA) in April
2013 (Submission I).
The SCCS issued the opinion in 2015 (SCCS/1540/14 Revision of 16 March 2016) on the
safety of Butylphenyl methylpropional (BMHCA) in cosmetic products concluding that:
"The SCCS is of the opinion that BMHCA is not safe for use as fragrance ingredient in
cosmetic leave-on and rinse-off type products, neither at concentration limits according to
the ones set up by IFRA in 2013 (MoS = 3.6) nor at concentration limits as set up by IFRA
in the revised proposal that has been submitted in 2015 belatedly (MoS = 53). In addition,
no firm conclusion could be drawn on mutagenicity.
BMHCA poses a risk of inducing skin sensitisation in humans."
In March 2017 IFRA submitted to the Commission services a new safety dossier on p-
BMHCA (p-Lysmeral) Submission II to address the concerns expressed by the SCCS. The
dossier clearly aims to defend the use of para-isomer distinguishing between para- and
meta-Lysmeral, since the SCCS addressed critics on the impurities present in BMHCA,
amongst which meta-Lysmeral is a critical one.
This dossier also includes a revised proposal for maximum use levels of p-BMHCA in the
finished cosmetic product types as follows:
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Terms of reference
1. Does the SCCS consider Butylphenyl methylpropional (p-BMHCA) safe for use as a
fragrance ingredient in cosmetic leave-on and rinse-off type products in a concentration
limit(s) according the ones set up by IFRA as reported above?
2. Does the SCCS have any further scientific concerns with regard to the use of
Butylphenyl methylpropional (p-BMHCA) as a fragrance ingredient in cosmetic leave-on
and/or rinse-off type products?
Product types
Finished product
concentration (%)
Hydroalcoholic-based fragrances (e.g. Eau de Toilette,
perfume, Aftershave, Cologne)*
1.42
Deodorants
0.09
Make up products (e.g. eye make-up, make-up remover,
liquid foundation, mascara, eyeliner)
0.04
Face cream
0.05
Hand cream
0.05
Body lotion
0.06
Hair styling
0.04
Bath cleansing products (e.g. soaps, shower gel, rinse-off
conditioner, shampoo)
0.1
*Maximum finished product concentration for hydroalcoholics on shaved skin is 0.6%
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3. OPINION
3.1 Chemical and Physical Specifications
3.1.1 Chemical identity
3.1.1.1 Primary name and/or INCI name
Chemical name: 2-(4-tert-Butylbenzyl)propionaldehyde
INCI name: Butylphenyl methylpropional
Ref.: BASF SE, 2014, 2015 SMII: 3, 4
3.1.1.2 Synonyms
IUPAC name: 3-(4-tert-Butylphenyl)-2-methylpropanal
EC name: 2-(4-tert-Butylbenzyl)propionaldehyde
Benzenepropanal, 4-(1,1-dimethylethyl)-alpha-methyl-Butylphenyl methylpropional
para-tert-Bucinal; 2-(4-tert-Butylbenzyl) propionaldehyde;
para-t-Butyl-α-methyl-hydrocinnamaldehyde
α-Methyl-β-(p-t-butylphenyl)propionaldehyde
3.1.1.3 Trade names and abbreviations
Lilestralis
Lilial
®
Lysmeral
®
Extra
BMHCA
Other names such as:
Lilyal
NSC 22275
pt-bucinal
Source: European Chemicals Agency, http://echa.europa.eu
Ref.: BASF SE, 2014, 2015, 2017 SMII: 3, 4, 5
3.1.1.4 CAS / EC number
CAS: 80-54-6, containing two enantiomers, namely (2S)-3-(4-tert-butylphenyl)-2-methyl-
propanal (75166-30-2) and (2R)-3-(4-tert-butylphenyl)-2-methylpropanal (CAS 75166-31-
3)
EINECS: 201-289-8
Ref.: BASF SE, 2017, SMII: 5
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3.1.1.5 Structural formula
Lysmeral®Extra is always a racemic mixture covering two enantiomers, namely (2S)-3-(4-
tert-butylphenyl)-2-methyl-propanal and (2R)-3-(4-tert-butylphenyl)-2-methylpropanal.
The synthesis and isolation of the pure enantiomers is difficult due to the fact, that Lysmeral
is an α-chiral aldehyde (asymmetric secondary carbon atom that is a close neighbour to the
carbonyl group). The pure enantiomers would easily racemize after isolation via keto-enol
tautomerism.
Ref.: Dossier BASF-IFRA, 2017
3.1.1.6 Empirical formula
Formula: C
14
H
20
O
3.1.2 Physical form
Physical state at 20°C (1013 hPa): liquid, colourless to pale yellow; odour: mildly floral,
reminiscent of cyclamen and lily of the valley.
3.1.3 Molecular weight
Molecular weight: 204.31 g/mol
3.1.4 Purity, composition and substance codes
The degree of para-Lysmeral (CAS 80-54-6) in BASF’s quality Lysmeral®Extra is specified
to be > 99.0% (Reference: BASF 2010 SMII: 2). Analyses of the purity are constantly
performed during production (Reference: BASF 2013, SMII: 33) and additionally before the
conduct of toxicological studies (References: BASF 2014, 2015, 2013 SMII: 3, 4, 33).
Main constituent in Lysmeral®Extra, as outlined in the Certificates of Analysis:
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SCCS comment
The applicant used GC-FID method with two different GC columns (DB-1 and DB-1701) for
the peak purity evaluation of the BATCH AP13-105. Peak purity was calculated based on %
of area measurements to be 99.4%. Certificates of Analysis have been provided for the rest
of the batches.
3.1.5 Impurities / accompanying contaminants
According to the applicant several known and unknown impurities are constantly analysed
during the manufacturing process and documented in the Certificates of Analysis
(Reference: BASF 2013 SMII: 33). Among the known impurities, special attention is given
to the meta isomer 3-(m-tert-butylphenyl)-2-methylpropionaldehyde (CAS 62518-65-4),
which was self-classified by BASF as CMR Repr. 1B; H360D (May damage the unborn child)
in 2011 and which has since then been subject to rigorous concentration restriction (< 0.1%
in Lysmeral®Extra) (References: BASF 2010, 2011, 2013, 1998 SMII: 2, 33, 34; C. Supp to
SMII: 5). TBA (4-tert-butylbenzoic acid) is a metabolite systemically formed in vivo or in
hepatocytes in vitro. The direct autoxidation product of para-Lysmeral is Lysmerylic acid (2-
(4-tert-butylbenzyl)propionic acid). However, since alpha-tocopherol (CAS 59-02-9) is
added as a stabilizer directly after the production process (References: BASF 2017 SMII: 5,
36), only low concentrations of the corresponding acid are found in Lysmeral®Extra.
Ref.: BASF 2013 SMII: 33
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SCCS comment
The applicant proceeded with chemical characterisation of the impurities using a GC-EI/MS
method (BASF-Study No13L00139, SMII 32). The applicant has self-classified meta-
Lysmeral as a CMR 1B (Repr 1B) substance and therefore subjected it to rigorous
concentration restriction (< 0.1%) and surveillance. According to the analytical data
provided, the meta-Lysmeral content is < 0.1%.
3.1.6 Solubility
Water solubility: 33 mg/L at 20°C (”Flask method”, OECD Guideline#105)
3.1.7 Partition coefficient (Log P
ow
)
Log P
ow
= 4.2 (24°C, HPLC, 7, OECD Guideline#117)
3.1.8 Additional physical and chemical specifications
Melting point: <–20°C (1013 hPa)
Boiling point: 279.5°C (1013 hPa)
Flash point: 79°C (EU method A.9)
Vapour pressure: 0.0025 hPa at 20°C
Density: 0.94 at 25°C
Viscosity: dynamic 12.3 mPa.s at 20
o
C (OECD TG 114)
pKa: Substance without any ionic structure
Refractive index: 1.503 – 1.507 at 20°C
UV_Vis spectrum:
max
≈ 263 nm
3.1.9 Homogeneity and Stability
General Comments to physicochemical characterisation
Further information (ECHA data dossiers): In aqueous solution and in the presence of air at
pH 7 and 25°C, Lilial
®
(p-BMHCA) undergoes significant oxidation (about 30% during a
period of 168 h). Thus, it can be assumed that p-BMHCA has a rather short life in the
environment (around two weeks) and that its oxidation product, lilic acid (lysmerylic acid),
is the major component to be considered in an environmental risk assessment. Given its
rapid oxidation at ambient air conditions, it is furthermore reasonable to assume that p-
BMHCA is unlikely to preserve its high purity of ≥99.5% (w/w) when being applied in
toxicological studies.
Lysmeral®Extra is prevented from auto-oxidating to the corresponding acid by alpha-
tocopherol, which is present in the final product at 200 ppm (References: BASF 2016, 2017
SMII: 5, 6, 35). The shelf life of Lysmeral®Extra is 730 days at 25°C (References: BASF
1998 SMII: 34). Moreover, the stability of Lysmeral®Extra as a test item is analytically
monitored during the conduct of toxicological studies to make sure that only high purity
substances are used throughout the experiment.
3.2 Function and uses
According to CLH Report, BASF SE, 30.9.2013:
“Lysmeral (2-(4-tert-butylbenzyl)propionaldehyde) is used as a fragrance in a wide number
of industries. It has an intensive, radiant, floral odour with a typical lily-of-the-valley note.
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As a component of fragrance mixtures, the main uses include cosmetic/personal care
products and washing/cleaning products. Lysmeral may also be included as a fragrance
substance in hair care products, biocidal products, coatings and paints, fillers/plasters,
ink/toners, polishes/wax blends and scented articles (clothes, eraser, toys, paper articles).”
According to IRSC/IFRA Dossier, 28.3.2013:
“BMHCA (2-(4-tert-butylbenzyl)propionaldehyde) is a fragrance ingredient used in many
compounds for dermal application in decorative cosmetics, fine fragrances, shampoos, toilet
soaps and other toiletries, as well as in non-cosmetic products such as household cleaners
and detergents. BMHCA is not used in flavour applications.”
According to BASF/IFRA Dossier, 24.2.2017:
“2-(4-tert-Butylbenzyl)propionaldehyde (BMHCA, Lysmeral) CAS No. 80-54-6 is a fragrance
ingredient used in many compounds for cosmetic products as well as in non-cosmetic
products such as household cleaners and detergents.
The proposed maximum use levels of BMHCA in the finished cosmetic product types are as
follows:
BMHCA is not used in flavour applications (Reference: BASF SE, 2016, SMII: 6) nor in
lipstick, toothpaste or mouthwash products (Reference: IFRA 2015b, SMII: 19).”
Product types
Finished product
concentration (%)
Hydroalcoholic-based fragrances (e.g. Eau de
Toilette, perfume, aftershave, cologne)*
1.42
Deodorants
0.09
Make up products (e.g. eye make-up, make-up
remover, liquid foundation, mascara, eyeliner)
0.04
Face cream
0.05
Hand cream
0.05
Body lotion
0.06
Hair styling
0.04
Bath products (e.g. soaps, shower gel, rinse-off
conditioner, shampoo)
0.1
* Maximum finished product concentration for hydroalcoholics on shaved skin is 0.6%
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3.3 Toxicological evaluation
3.3.1 Acute toxicity
3.3.1.1 Acute oral toxicity
From submission I
SCCS overall comment on acute oral toxicity
The acute oral toxicity (LD
50
) in rats was determined at 1390 mg/kg bw (95% confidence
limits: 1019 – 1867 mg/kg bw).
Additional data from Applicant’s submission II dossier
In a non-GLP, non-guideline study, the test substance was administered orally to each of 10
rats at dose levels of 1220; 2470; 5000; 10140 mg/kg bw. The animals were observed for
treatment-related effects for a 14-day observation period. There were no deaths at 1220
mg/kg bw. One rat died at 2470 mg/kg bw and seven died at 5000 mg/kg bw. The highest
dose was lethal for all animals. The acute oral toxicity (LD
50
) was 3700 mg/kg bw (95%
confidence limits: 2600 – 5400 mg/kg bw).
Ref.: MB Research Laboratories, 1977, SMI: 75, #1695
A further non-GLP, non-guideline screening study was conducted on groups of 2 rats
(1/sex). The animals were administered, by gavage, p-BMHCA in vegetable oil at dose
levels of 100, 500, 1000, 2000, and 5000 mg/kg bw. Observations were conducted for 13
days. One death occurred in the 2 highest dose groups. The study findings suggested that
the oral LD
50
of p-BMHCA in rats ranged between 1000 - 2000 mg/kg bw.
Ref.: Bush Boake Allen, 1980a, SMI: 18, #52291
3.3.1.2 Acute dermal toxicity
Additional data from Applicant’s submission II dossier
There was no additional data that would have impacted the SCCS's previous conclusion
(SCCS/1540/14).
3.3.1.3 Acute inhalation toxicity
Additional data from Applicant’s submission II dossier
No additional data
3.3.1.4 Acute intraperitoneal toxicity
Additional data from Applicant’s submission II dossier
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The acute toxicity after intraperitoneal injection was investigated in the range-finding part
of a guideline (OECD 474, ICH) mouse micronucleus study under GLP conditions. ICR Mice
in groups of 5/sex/dose received single intraperitoneal injections of p-BMHCA (lot
9000349505) in corn oil at 300, 500, 700 or 1000 mg/kg bw each. Animals were observed
for clinical signs after injection and daily thereafter for 3 days. Lethargy and piloerection
were observed at all doses. The mice exhibited prostration, irregular breathing and crusty
eyes in the two highest dose groups. Convulsions occurred at 1000 mg/kg bw, and all mice
at this dose died by the third day of the study. No deaths occurred in other dose groups.
Ref.: RIFM 2000b, SMI: 91, #35691
In a GLP-compliant non-guideline study, NMRI mice (5 per sex and dose) were treated by
intraperitoneal injections of p-BMHCA in carboxymethyl cellulose at 200 or 700 mg/kg bw.
The animals were observed daily for clinical signs for 14 days following administration. Body
weight determination was performed at regular intervals and gross pathology was
performed. Half of the animals died in the 700 mg/kg bw dose group (4/5 males and 1/5
females) and no mortality was observed in the 200 mg/kg dose group. Unspecific signs in
the form of dyspnea, apathy, staggering, spastic gait, rough fur coat and poor general
condition were observed in both dose groups and body weight loss, abnormal position,
twitching, tremor, tonic convulsions, skin erythema, and dehydration were found in the
high-dose animals only.
Ref.: BASF SE 1981, SMI: 3, #63831
SCCS conclusions on acute toxicity based on Submission I and II
Acute toxicity after all relevant routes of application of BHMCA was investigated in rats,
mice and rabbits (oral, dermal, intraperitoneal, inhalation). The acute oral LD
50
value in rats
was determined to be 1390 mg/kg bw and the acute dermal LD
50
value in rabbits >2000
mg/kg bw. Thus the acute toxicity of p-BMHCA can be considered moderate (oral route). An
inhalation toxicity test in rats led to no mortalities but signs of systemic toxicity after
exposure to a p-BMHCA saturated atmosphere continued to be observed for 7 hours.
However, the assessment of inhalation toxicity on the basis of this study is limited due to
the low volatility of p-BMHCA (vapour pressure: 0.0025 hPa at 20°C).
3.3.2 Irritation and corrosivity
From submission I
SCCS conclusion on irritation
Under the conditions tested, p-BMHCA as neat compound was revealed to be irritating to
the skin and eyes of rabbits. In addition, 2% p-BMHCA in propylene glycol led to mild skin
erythema; however, the scoring of the solvent was comparable. In general, the observed
effects occurred transiently and were reversible. In a special investigation, p-BMHCA also
displayed the potential of inducing respiratory irritation.
3.3.2.1 Skin irritation
Additional data from Applicant’s submission II dossier
In a non-GLP and non-guideline study, occlusive dermal application of neat p-BMHCA on 2-3
rabbits for 5 minutes, 2 or 24 hours resulted in desquamation in all animals at the end of
the observation period (8 days) for all exposure periods. Questionable to slight edema
(reversible for all exposure periods) and erythema (reversible for 5 min exposure period)
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were observed. Longer exposure periods led to persisting erythema at the end of the
observation period.
Ref.: BASF SE 1981, SMI: 3, #63831
3.3.2.2 Mucous membrane irritation / Eye irritation
Additional data from Applicant’s submission II dossier
In a non-GLP and non-guideline study, undiluted p-BMHCA was applied into one eye of 3
rabbits, each without washing out after application, and animals were observed daily for 72
hours. Slight conjunctival redness was found in all animals 24 hours after application and in
1 of 3 animals 48 hours after application, resulting in a mean score of 0.7 over all animals
and observation time points. No adverse findings, i.e. chemosis, iritis or corneal opacity,
were observed at any time point.
Ref.: BASF SE, 1981, SMI: 3, #63831
SCCS conclusion on irritation
The data on irritation potential of p-BMHCA provided in Submission II do not change the
SCCS's previous conclusion (SCCS/1540/14).
3.3.3 Skin sensitisation
From submission I
SCCS conclusion on skin sensitisation
BMHCA was comprehensively tested in experimental animals, mostly according to guideline
procedures and under GLP conditions. Several positive LLNA resulted in EC3 values
indicative for sensitisation. Depending on the solvent, the EC3 values ranged from 2.97%
(in EtOH) to 13.91% (in 25% EtOH/75% DEP), and up to 18.7% by application of p-BMHCA
in acetone/olive oil (4:1). Another LLNA with EtOH as vehicle showed SI>3 for all tested
doses of p-BMHCA (10, 25, 50, 100%). An EC3 value of about 2.9% p-BMHCA in the LLNA
has been substantiated by data from the International Fragrance Association directly
submitted to SCCS in 2009 (SCCS, 2012). By contrast, GPMTs performed were
contradictory and thus ambiguous. Finally, dermal reactions have been observed in a KAO
test in guinea pigs.
Based on the animal data obtained, the overall potency classification of p-BMHCA is a
"moderate sensitiser" (Basketter et al., 2001; SCCP, 2005 and 2012).
Local Lymph Node Assay (LLNA)
Additional data from Applicant’s submission II dossier
No additional data.
Guinea pig maximization test (GPMT)
Additional data from Applicant’s submission II dossier
A guinea pig maximization test was performed on 10 Hartley Dunkin guinea pigs. In the
intradermal induction phase, neat p-BMHCA was injected and for topical induction a filter
paper patch saturated with neat p-BMHCA was applied for 48 hours under occlusion. For
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topical challenge 2 weeks after the topical induction, a filter paper saturated with p-BMHCA
was applied for 24 hours under occlusion. The challenge concentrations included the neat
material and 50% in mineral oil. One concentration was applied to each flank. After patch
removal, only limited signs of irritation in individual tests and control group animals were
observed, but there was no indication of skin sensitisation.
Ref.: Bush Boake Allen, 1980b, SMI: 19, #52292
In a poorly reported study on an unspecified number of guinea pigs, strong sensitising
effects were reported when p-BMHCA at 10% in an unspecified vehicle was used for
induction and challenge.
Ref.: Ishihara et al., 1986, SMI: 67, #5601
Buehler test
Additional data from Applicant’s submission II dossier
No additional data.
SCCS comment
The data on the sensitisation potential of p-BMHCA provided in Submission II do not change
the SCCS's previous conclusion (SCCS/1540/14) that p-BMHCA is a moderate skin
sensitiser.
3.3.4 Dermal / percutaneous absorption
From submission I
SCCS conclusion on dermal/percutaneous absorption
Dermal absorption studies in vitro demonstrated species-specific effects. The bioavailable
portion was found to be much higher in rats (66.1 and 50.8%) when compared to mini pigs
(0.8% and 4.9%), depending on the solvent used (methylcarbitol or ethanol). In a second
study, applying two real cream formulations (that contained 0.6% p-BMHCA), rat skin again
allowed a much higher penetration (45.2% and 78.4%) than mini pig skin (23.6% and
25.7%). Nevertheless, the fraction of bioavailable p-BMHCA was found strongly increased in
the mini pig experiment when moving from dissolved p-BMHCA to real cream formulations
(4.9% vs. 25.7%).
Concurrently, administration of p-BMHCA onto the skin of experimental animals and humans
demonstrated the permeation and systemic availability of this compound. Percutaneous
absorption of p-BMHCA in humans was lower than it was in rats (1.4 vs. 19%).
Upon dermal application of [
14
C]-p-BMHCA (11.37 mg test substance in 70% ethanol on 10
cm² back skin) on 3 human volunteers for 6 hours, a mean of 1.4% (range 0.8 – 2.4%) of
the applied dose was excreted in urine within 24 hours, whereas radioactivity was below the
detection limit in urine samples of later time points and in all faeces and blood plasma
samples. The overall mean total recovery of topical application of [
14
C]-p-BMHCA was 71 ±
10%. In comparison to the in vitro observations, the absorption rate found in humans for
ethanolic solutions of p-BMHCA was comparable to what has been found in excised mini pig
skin. Given that the absorption of p-BMHCA in mini pig skin was much higher when this
compound was applied via real cream formulations, it is reasonable to conclude that p-
BMHCA might also better penetrate human skin when it is applied in cream formulations.
Since there is no further experimental data on this subject, the SCCS concludes that the
maximum fraction of p-BMHCA being absorbed by human skin might be in the range of 25%
rather than at 2.4%.
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In consideration of the comparability of pig skin with human skin, the dermal bioavailability
of ethanolic (dissolved) p-BMHCA to be used in the calculation of the systemic exposure
dose (SED) and margin of safety (MoS) will be set at 5% (worst case scenario based on 1%
p-BMHCA in EtOH applied at 120 µg substance/cm
2
onto 5 cm
2
excised mini pig skin; result:
total of 5.87 µg substance/cm
2
found in stripped skin and chamber fluid after 16 hrs of
exposure). On the other hand, the penetration rate of p-BMHCA applied onto the skin as an
ingredient in creamy formulations will be set at 25% (worst case scenario based on 36 µg
substance/cm
2
applied onto 5 cm
2
excised mini pig skin; mean out of two experiments: total
of 8.88 µg substance/cm
2
found in stripped skin and chamber fluid after 16 hrs of
exposure). The SCCS is aware of the issue that the exact identity of the cream formulations
applied in the latter study remains obscure.
The results obtained from the part of the study with 1% ethanolic p-BMHCA can further be
used to assess the SED for hydroalcoholic products to be applied on a defined surface area
of shaved or unshaved skin once daily (1 x 305 cm
2
/day). Here, an absorption of about 6 µg
substance/cm
2
can be assumed for unshaved skin (stratum corneum intact). For shaved
skin (stratum corneum compromised), however, the total absorption would be 11 µg
substance/cm
2
(with the addition of the portion of 4.66 µg/cm
2
that was found sticking in
the stratum corneum in the respective experiment; cf. above).
3.3.4.1. Dermal /percutaneous absorption in vitro
Additional data from Applicant’s submission II dossier
Guideline: OECD TG 428, OECD GD No. 28, SCCP/0970/06
Test system: Frozen dermatomed human skin (200 – 400 μm)
Number of donors: Per dose group min. 8 samples from 12 donors (< 65 years)
Membrane integrity: Visual inspection and electrical resistance barrier integrity test,
membranes with a resistance < 1 kΩ were excluded
Test substance: BMHCA (Lysmeral Extra)
Test item: [
14
C]-BMHCA in 4 test formulations:
1) 70 % ethanol in water
2) “silicone in water”
3) “water in oil”
4) “oil in water”
Batch: 00046877L0 (non-radiolabeled); 969-2005 (radiolabelled)
Purity: 99.5% (non-radiolabelled, GC); 97.7 % (radiolabelled)
Dose applied: Group 1: 1.9 % [[
14
C]-p-BMHCA in formulation 1, 95.0 μg p-
BMHCA/cm²
Group 2: 0.1 % [
14
C]-p-BMHCA in formulation 2, 5.0 μg p-
BMHCA/cm²
Group 3: 0.1 % [
14
C]-p-BMHCA in formulation 3, 5.0 μg p-
BMHCA/cm²
Group 4: 0.1 % [
14
C]-p-BMHCA in formulation 4, 5.0 μg p-
BMHCA/cm²
Exposed area: 1 cm
2
Exposure period: 24h
Sampling period: up to 72h post dose
Receptor fluid: Tap water; for prolonged observation time experiments: tap water
with 0.01 % sodium azide (NaN
3
)
Solubility in receptor
fluid: 0.033 g/L in water
Mass balance analysis: Provided
Tape stripping: Yes (20)
Method of Analysis: Liquid scintillation counting
GLP: In compliance
Study period: July - December 2016
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Human abdominal and breast skin samples were obtained from 12 different donors. The
skin was dermatomed (200 - 400 µm) and then the split-thickness membranes stored
frozen, at approximately -20° C until use. The dermatomed skin membranes were checked
for integrity visually and by the Transepithelial/Endothelial Electrical Resistance (TEER)
method prior to use. Only visually intact skin samples with a TEER (impedance value) above
1 kΩ were used. Each skin preparation was hydrated in physiological saline for about 10
minutes before mounting to the diffusion cells which were filled up with physiological saline
with a protease inhibitor. The prepared diffusion cells were covered with Fixomull
®
Stretch
and stored overnight in a refrigerator. The integrity of the skin preparations was also
visually checked immediately before starting the experiment. The receptor fluid was
pumped through the receptor chambers at 2.3 mL/h. The samples were maintained at a
constant temperature of 32 ± 1 °C.
Penetration of [
14
C]-p-BMHCA (Lysmeral Extra) through and into human skin was assessed
by a single topical application of target doses of 95.0 μg/cm² and 5.0 μg/cm² of test
substance formulated in different test-substance preparations, representative of in-market
cosmetic formulations: Group 1 consisted of a hydro-alcoholic preparation with 1.9 % of p-
BMHCA in 70 % ethanol in water; Group 2 of 0.1 % p-BMHCA in a “silicone in water”
formulation, Group 3 of 0.1 % p-BMHCA in a “water in oil” and Group 4 of 0.1 % p-BMHCA
in a “oil in water” formulation. Dermal absorption of p-BMHCA was assessed by a two-step
experimentation procedure: 24h post dosing and with prolonged observation time: 72h
sampling period for each formulation, for which 6-8 cells were used.
Absorption of p-BHMCA was evaluated by collecting receptor fluid every hour from 0 to 8h
post dose, then every 2 hours from 8 to 24h post dose. After the exposure time of 24h and
after the sampling period, skin membranes were washed with sodium-laurylethersulfate,
diluted 1:140 w/w in tap water, followed by tap water. The tape-stripping procedure was
performed on dried skin samples. Twenty tape strips were taken and pooled into three
samples (the first 2 tapes as sample 1, the subsequent 9 tapes as sample 2 and the last 9
samples as sample 3) for analysis. The remaining skin from the 24h experiments was
separated into dermis and epidermis by heat separation and subsequently analysed. The
remaining skin of the 72h prolonged observation experiments and the skin of the control
experiments were not separated into epidermis and dermis, but were extracted immediately
after the stripping procedure or application.
No rate limiting effects on the diffusion process by saturation of the aqueous receptor fluid
were present. The stability of the test item over the exposure period was assessed. The
concentration of test-substance preparations of > 80.2 % and mean radiochemical purities
of > 87.8 % radiolabelled [
14
C] were determined over the application period.
Results
In the 24h experiments, the mean total recoveries ranged between 80.44 and 97.32 %
(with individual values between 74.43 and 119.37 %) of the applied dose. Lysmeral is
volatile and major parts of the test substance evaporated during the exposure period and
were recovered in the charcoal filter.
Given the evaporation observed, the recovery range expressed as percentage was 80.44 ±
1.83% and 84.67 ± 13.80% for formulation 1, 83.08 ± 3.28% and 88.72 ± 2.97% for
formulation 2, 97.32 ± 3.91% and 91.01 ± 13.82% for formulation 3, 96.21 ± 2.98% and
87.88 ± 3.44% for formulation 4, after 24h and 72h post-exposure, respectively.
Under these test conditions, 5.31 ± 2.22% (4.85 ± 2.03 µg), 3.50 ± 1.31% (0.16 ± 0.06
µg), 4.83 ± 3.54% (0.23 ± 0.17 µg), and 4.77 ± 2.16% (0.23 ± 0.1 µg) of the applied
dose of
14
C Lysmeral were recovered as absorbed dose in the 24h absorption experiments
for the hydro-alcoholic solution, the “silicone in water”, the “water in oil”, and the “oil in
water” based formulations, respectively. When an additional 48 hours post-observation
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period was included after the 24h exposure, 5.29 ± 2.52% (5.07 ± 2.42 µg), 5.04 ± 2.60%
(0.21 ± 0.11 µg), 7.82 ± 5.42% (0.39 ± 0.27 µg), and 4.97 ± 2.26% (0.23 ± 0.10 µg) of
the applied dose of
14
C-Lysmeral were recovered as absorbed dose after 72h for the hydro-
alcoholic solution, the “silicone in water”, the “water in oil”, and the “oil in water” based
formulations, respectively.
In the 72h experiments, the residues of p-BMHCA in the skin preparations were
differentiated into an extractable portion and a non-extractable portion. The non-extractable
portion of p-BMHCA in living skin is assumed to be bound to the skin matrix and therefore
represents a non-absorbable fraction excluded from the final calculations:
Percentage of p-BMHCA in living skin not extractable = Mean percent of the applied dose in
skin residue (+ 1SD) * 100 / Mean percent of the applied dose in skin residue (+ 1SD) +
skin extract (+ 1SD)
- “Ethanol in water” = (0.32 + 0.11) * 100 / (0.32 + 0.11 + 1.31 + 0.33) = 21%
- “Silicone in water” = (0.25 + 0.10) * 100 / (0.25 + 0.10 + 0.71 + 0.24) = 27%
- “Water in oil” = (0.24 + 0.15) * 100 / (0.24 + 0.15 + 0.50 + 0.48) = 28%
- “Oil in water” = (0.18 + 0.06) * 100 / (0.18 + 0.06 + 0.28 + 0.12) = 38%
Conclusion
The dermal penetration data using the hydro-alcoholic vehicle showed that an additional
72h observation time did not result in any evident movement of p-BMHCA from different
skin compartments (i.e. the skin reservoir) to the receptor fluid. Therefore, the fraction
found in the epidermis was not included as bioavailable. For the other vehicles, the dose
associated to the remaining skin (dermis+ epidermis) was reduced by the non-extractable
portion determined in the living skin. The percentage of dermally absorbed p-BMHCA was
calculated as follows:
- “Ethanol in water” (24h): (Absorbed dose+1SD) + (Dermis+1SD) =
5.31+2.22+0.71+0.28 = 8.52%
- “Water in oil”: (Absorbed dose+1SD) + ((Epidermis+1SD) + (Dermis+1SD) * 72%) =
4.83+3.54+((0.74+0.31+0.73+0.35)*72%) = 9.90%
- “Oil in water” (24h): (Absorbed dose+1SD) + (Epidermis+1D) + (Dermis+1SD) * 62%)
= 4.77+2.16+((0.69+0.31+0.78+0.17)*62%) = 8.14%
Ref.: BASF SE, 2016a, SMII, 7
SCCS comment
According to SCCS/1358/10, recovery of the test substance should be between 85 - 115%.
The overall recovery of p-BMHCA tested in formulations 1 (“ethanol in water”) and 2
(“silicone in water”) was not within this acceptance range, even under the semi-occlusive
conditions used.
According to SCCS/1602/18, in the case of substances with very low dermal absorption and
limited permeation (e.g. colourants or UV-filters with high molecular weight and low
solubility), the epidermis may be excluded when it is demonstrated that no movement of
the chemicals from the skin reservoir to the receptor fluid occurs. BMHCA does not fulfil
these criteria. Therefore, all p-BMHCA present in the living epidermis has to be taken into
account for the dermal absorption.
Based on the SCCS requirements, the mean + 1 SD will be taken for MoS calculation for:
- “Water in oil” (24h): (Absorbed dose+1SD) + (Epidermis+1SD) + (Dermis+1SD) =
(4.83+3.54) + (0.74+0.31) + (0.73+0.35) = 10.5%
- “Oil in water” (24h): (Absorbed dose+1SD) + (Epidermis+1SD) + (Dermis+1SD) =
(4.77+2.16) + (0.69+0.31) + (0.78+0.17) = 8.9%
Based on significant deviations from the SCCS requirements, the mean + 2 SD will be taken
for MoS calculation for:
- “Ethanol in water” (24h) = (Absorbed dose+2SD) + (Epidermis+2SD) +
(Dermis+2SD) = (5.31+2*2.22) + (1.50+2*0.49) + (0.71+2*0.28) = 13.5%
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- “Silicone in water” (24h) = (Absorbed dose+2SD) + (Epidermis+2SD) +
(Dermis+2SD) = (3.50+2*1.31) + (0.96+2*0.18) + (0.64+2*0.23) = 8.5%.
3.3.4.2. Dermal /percutaneous absorption in vivo
Additional data from Applicant’s submission II dossier
No additional data.
3.3.5 Repeated dose toxicity
From submission I
SCCS conclusion on subacute and subchronic dose toxicity
The toxicity of p-BMHCA after repeated application was investigated in several species.
Decreases in body weights and food consumption and/or clinical signs of toxicity were
observed after subacute oral administration of p-BMHCA at doses of ≥50 mg/kg bw/day
(rats) and ≥200 mg/kg bw/day (dogs). In oral studies, rats were found to be more sensitive
than dogs to this compound irrespective of the length of treatment. Clinical chemistry and
histopathological examinations repeatedly revealed adverse effects on the liver and male
reproductive system. Decreases in plasma cholinesterase activity levels in both sexes of rats
were observed after oral exposure to ≥25 mg/kg bw/day for 90 days. In addition, effects on
adrenal glands in females were also observed at the same dose levels. From this most
meaningful oral study, with respect to the doses administered, a NOAEL of 5 mg/kg bw/day
can be derived for systemic effects.
On the other hand, dermal administration in rats for 5 days led to adverse effects (including
testicular toxicity) only at excessive dose levels (2000 mg/kg bw/day). No 90-day studies
on dermal or inhalative administration were available.
3.3.5.1 Repeated dose short-term oral / dermal / inhalation toxicity
Additional data from Applicant’s submission II dossier
The results of screening studies provided (BASF SE, 2011b, SMI: 15, #59014 and Givaudan,
2009, SMI: 60, #57411) confirmed the known potential of p-BMHCA (orally for 5-14 days,
at 50-250 mg/kg bw/d) to affect the reproductive organs in rats.
3.3.5.2 Sub-chronic (90 days) toxicity (oral, dermal, inhalation)
Additional data from Applicant’s submission II dossier
No additional data.
3.3.5.3 Chronic (> 12 months) toxicity
Additional data from Applicant’s submission II dossier
No additional data.
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3.3.6 Mutagenicity / Genotoxicity
From submission I
The applicant’s overall conclusion on mutagenicity/genotoxicity
Based on the data provided, the applicant came to the following conclusion on the overall
mutagenicity/ genotoxicity: No genotoxic/mutagenic potential was found in bacterial gene
mutation assays with S. typhimurium or E. coli strains in the presence or absence of
metabolic activation. BMHCA also did not induce gene mutations at the Hprt locus in
Chinese hamster V79 cells. Structural and numerical chromosomal aberrations were found
in the absence of S9, while no aberration occurred in its presence in CHO cells.
Intraperitoneal treatment of mice with p-BMHCA did not induce increases in the incidence of
chromosomal aberrations in bone marrow cells. Hence occasionally emerging clastogenicity
in vitro remained unconfirmed in vivo. Based on the data available, p-BMHCA can be
considered not mutagenic/genotoxic.
SCCS comment and conclusion
SCCS disagrees with the applicant’s conclusion. Neither in vitro gene mutation nor in vitro
chromosomal damage can be excluded based on the data provided. Similarly, due to the
lack of sufficient and detailed information, it is also not possible to draw a conclusion from
the in vivo micronucleus report provided.
3.3.6.1 Mutagenicity / Genotoxicity in vitro
Additional data from Applicant’s submission II dossier
Guideline: OECD 471
Test system: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537,
Escherichia coli strain WP2uvrA
Replicates: Three experiments, triplicate plates
Test substance: p-BMHCA (Lilestralis Pure: 32229) with 200 ppm of alpha-tocopherol
Batch: A100423A (purity: > 99%)
Concentrations: Experiment I – Plate incorporation test:
±S9 mix: S. typhimurium strains: 0, 1.5, 5, 15, 50, 150, 500, 1500
μg/plate; E. coli strain: 0, 50, 150, 500, 1500, 5000 μg/plate
Experiment II – Pre-incubation test:
-S9 mix: S. strains TA100 and TA1537: 0, 0.15, 0.5, 1.5, 5, 15, 50,
150 μg/plate
-S9 mix: S. strain TA1535 and E. coli strain: 0, 0.05, 0.15, 0.5, 1.5,
5, 15, 50 μg/plate
-S9 mix: S. strain TA98: 0, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate
+S9 mix: all S. strains: 0, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate; E.
coli strain: 0, 5, 15, 50, 150, 500, 1500, 5000 μg/plate
Experiment III (confirmatory test) – Plate incorporation test:
+S9 mix: S. strain TA1535: 0, 50, 100, 150, 200, 300 μg/plate
Vehicles: DMSO
Positive Controls: -S9 mix: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate for
WP2uvrA, 3 μg/plate for TA100, 5 μg/plate for TA1535; 9-
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Aminoacridine (9AA): 80 μg/plate for TA1537; 4-Nitroquinoline-1-
oxide (4NQO): 0.2 μg/plate for TA98
+S9 mix: 2-Aminoanthracene (2AA): 1 μg/plate for TA100; 2
μg/plate for TA1535 and TA1537, 10 μg/plate for WP2 uvrA;
Benzo(a)pyrene (BP): 5 μg/plate for TA98
Negative controls: Vehicle control
GLP: In compliance
Study period: 06 Jan 2011 – 30 Jun 2011
Material and methods
Para-BMHCA with 200 ppm of alpha-tocopherol was tested for mutagenicity in the reverse
mutation assay with and without metabolic activation in S. typhimurium strains TA1535,
TA1537, TA98, TA100 and E. coli strain WP2 uvrA using both the Ames plate incorporation
and pre-incubation methods at up to seven dose levels, in triplicate, both with and without
the addition of a rat liver homogenate metabolising system (induced with Phenobarbitone/β
Naphthoflavone, 10% liver S9 in standard co-factors). The dose range for the first
experiment was determined in a preliminary toxicity assay and ranged between 1.5 and
5000 µg/plate, depending on bacterial strain type. The experiment was repeated (pre-
incubation method) using fresh cultures of the bacterial strains and fresh test item dilutions.
The test item dose range was slightly expanded, based on the results of Experiment 1, and
ranged between 0.05 and 5000 μg/plate, depending on bacterial strain type and presence
or absence of S9-mix. Additional dose levels and an expanded dose range were selected in
both experiments. This was done in order to achieve both four non-toxic dose levels and the
toxic limit of the test item. In addition, a third experiment was performed to confirm
whether a two-fold increase in TA1535 revertant colony frequency, noted in Experiment 1,
was real or spurious. The experiment was carried out using bacterial strain TA1535
(presence of S9-mix only) and employed a narrowed test item dose range of 50, 100, 150,
200 and 300 μg/plate.
Results
Equivocal findings were observed in this study for the Salmonella strain TA 1535 in the plate
incorporation test with and without metabolic activation. Increased numbers of revertant
colonies were observed for TA 1535 in the first experiment (plate incorporation method) but
not in the follow-up pre-incubation test. The increase observed consisted of an isolated
statistically significant increase in colony frequency at non-bacteriotoxic concentrations,
noted in one single concentration (150 μg/plate) in the presence of S9. This finding was not
reproducible in a confirmatory plate incorporation test. At higher test item concentrations, a
concentration dependent increase of colony numbers associated with a sparse bacterial
background lawn was noted for TA 1535 in experiment 1 and 3. The authors suggest that
this increase in colony number might have resulted from residual histidine levels that were
available to a small number of surviving His- bacteria in the presence of bacteriotoxic p-
BMHCA concentrations (although likely, this has not been confirmed experimentally). These
histidine levels would allow the surviving His- bacteria to undergo several additional cell
divisions: resulting colonies do therefore not represent revertant (mutant) colonies.
SCCS comment
The SCCS disagrees with the applicant's conclusion and considers the results obtained as
positive. In Experiment I, p-BMHCA was shown to be positive in S. typhimuruim TA1535,
both ±S9-mix (almost 10 fold increase in revertants, starting from 150 µg/plate –S9-mix
and 500 µg/plate +S9-mix). In Experiment II –S9-mix p-BMHCA was not tested at the same
concentrations, but only up to 50 µg/plate. In Experiment III, p-BMHCA was tested only
with S9-mix at up to 300 µg/plate.
Ref.: Innospec Ltd., 2011a, SMII: 16
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Guideline: OECD 471
Test system: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537,
Escherichia coli strain WP2uvrA
Replicates: Two experiments, triplicate plates
Test substance: p-BMHCA
Batch: not stated (source: Sigma–Aldrich, St. Louis, MO, USA, purity:
> 90%)
Concentrations: ±S9 mix:
Preliminary test:
All strains: 0.05, 0.25, 0.5, 2.5, 5.0, 25 μM/plate
(corresponding to 10, 51, 102, 510, 1022, 5108 μg/plate based
on the molecular weight of 204.31 g/mol)
Main test:
S. typhimurium and E. coli strains: 0.01; 0.02; 0.05; 0.07; 0.1;
0.2; 1.0; 2.0; 10.0 μM/plate (corresponding to 2; 4; 10; 14;
20; 40; 200; 400; 2000 μg/plate based on the molecular weight
of 204.31 g/mol)
Vehicles: DMSO
Positive Controls: -S9 mix:
- sodium azide (SA): 1 μg/plate for TA1535 and TA100
- 9-aminoacridine (9AA): 50 μg/plate for TA1537
- 2-nitrofluorene (2NF): 2 μg/plate for TA98
- methyl methanesulfonate (MMS): 500 μg/plate for WP2 uvrA
+S9 mix:
- 2-aminoanthracene (2AA): 1 μg/plate for TA98 and TA100; 10
μg/plate for TA1535, TA1537, WP2 uvrA
- benzo[a]pyrene (BaP): 50 μg/plate for TA98, TA100, WP2
uvrA, 100 μg/plate for TA1535; 50, 100, 500 μg/plate for
TA1537
Negative controls: Vehicle control
GLP: No
Published: Yes, date of publication: 2014
Material and methods
Para-BMHCA (alpha-tocopherol content unknown) was tested for mutagenicity in the
reverse mutation assay on bacteria with and without metabolic activation (liver
postmitochondrial supernatant of rats treated with phenobarbital/β-naphthoflavone)
according to the pre-incubation test method. In a pre-test, the Salmonella typhimurium
strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA were exposed to
the test substance (dissolved in DMSO) at concentrations ranging from 0.05 – 25 μM/plate
(10-5108 µg/plate) to check solubility and cytotoxicity. For the main test, concentrations
ranging from 0.01-10.0 μM/plate were tested.
Results
In the preliminary test, p-BMHCA was cytotoxic in strains TA1535 and TA1537 in the
absence of S9 at a concentration of 0.25 μM/plate (51 µg/plate). In the presence of S9,
cytotoxicity occurred at 0.25 µM/plate (51 µg/plate) in TA1537 and 0.5 μM/plate (102
µg/plate) in TA1535 and at 5 µM/plate (510 µg/plate) in TA98 and WP2uvrA.
In the main mutagenicity assay, p-BMHCA did not increase the number of revertant colonies
in any of the bacterial strains tested at non-cytotoxic concentrations, either with or without
the metabolic activator S9.
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Conclusion
Para-BMHCA was considered to be non-mutagenic in this bacterial gene mutation test, with
or without S9-mix metabolic activation, when tested up to cytotoxic concentrations.
Ref.: Di Sotto et al., 2014 a, b, SMII: 11, 12
SCCS comment
The study has several limitations: it was not conducted under GLP conditions; positive
controls used did not clearly demonstrate positive response. The positive control substance,
i.e. BaP for TA1535, did not induce mutant frequency up to the concentration of 500
μM/plate. Para-BMHCA was tested in low concentrations. No data on historical controls are
provided. Overall, the results have limited value and no firm conclusion can be drawn from
this study.
Additional study on the Ames test provided in December 2018
Guideline: OECD TG 471
Test system: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537
Escherichia coli strain WP2 uvrA
Replicates: Three experiments, triplicate plates
Test substance: p-BMHCA (Lysmeral extra) with 200 ppm of alpha-tocopherol
Batch: 00046877L0 (purity: 99.4 area-%)
Concentrations: Experiment I – Plate incorporation test:
S. typ., E. coli strains: 0, 33, 100, 333, 1000, 2500, 5000 µg/plate
(with and without S9 mix).
Experiment II – Plate incorporation test:
- TA1535: 0, 1, 3.3, 10, 33, 100, 150, 333 µg/plate (without S9 mix)
and 0, 3.3, 10, 33, 100, 333, 1000 µg/plate (with S9 mix)
- TA100: 0, 1, 3.3, 10, 33, 100, 333 µg/plate (without S9 mix) and 0,
3.3, 10, 33, 100, 333, 1000 µg/plate (with S9 mix)
- TA98: 0, 1, 3.3, 10, 33, 100, 333 µg/plate (without S9 mix)
- TA1537: 0, 0.33, 1, 3.3, 10, 33, 100 µg/plate (without S9 mix) and
0, 1, 3.3, 10, 33, 100, 333 µg/plate (with S9 mix)
Experiment III – Pre-incubation test:
- TA1535: 0, 1, 3.3, 10, 33, 100, 150, 333 µg/plate (without S9 mix)
and 0, 1, 3.3, 10, 33, 100, 333, µg/plate (with S9 mix)
- TA100: 0, 1, 3.3, 10, 33, 100, 333 µg/plate (without and with S9
mix)
- TA98: 0, 1, 3.3, 10, 33, 100, 333 µg/plate (without S9 mix) and 0,
3.3, 10, 33, 100, 333, 1000 µg/plate (with S9 mix)
- TA1537: 0, 0.33, 1, 3.3, 10, 33, 100 µg/plate (without S9 mix) and
0, 1, 3.3, 10, 33, 100, 333 µg/plate (with S9 mix)
- WP2 uvrA: 10, 33, 100, 333, 1000, 2500 µg/plate (without S9 mix)
and 0, 3.3, 10, 33, 100, 333, 1000 µg/plate (with S9 mix)
Vehicles: Acetone
Positive Controls: Without S9 mix:
- MNNG: 5 µg/plate for TA1535, TA100
- NOPD: 10 µg/plate for TA98
- AAC: 100 µg/plate for TA1537
- 4NQO: 5 µg/plate for WP2 uvrA
With S9 mix:
- 2AA: 2.5 µg/plate for TA98, TA100, TA1535, TA1537
60 µg/plate for WP2 uvrA
Negative controls: Vehicle controls, sterility controls
GLP: In compliance
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Study period: 13 Jul 2018 – 19 Oct 2018
Material and methods
Para-BMHCA was tested for mutagenicity in the bacterial reverse mutation assay in S.
typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2 uvrA. Both, the
Ames plate incorporation and pre-incubation methods were used at up to seven dose levels,
in triplicate, both with and without the addition of a rat liver homogenate metabolising
system (induced with phenobarbital/β-naphthoflavone, 10% liver S9 in standard co-
factors). The dose range for the first experiment (plate incorporation method) was selected
including a maximum test dose of 5000 µg p-BMHCA/plate. The plate incorporation
experiment was repeated, and the test item dose range was adjusted according to the
bacteriotoxicity observed in Experiment 1 (i.e. 0.33-1000 μg/plate dependent on tester
strain and testing condition used). A similar concentration range (up to 2500 μg/plate) was
tested in a third experiment according to the preincubation method.
Bacteriotoxicity was determined by a decrease in the number of revertants and/or clearing
or diminution of the background lawn (i.e. reduced his- or trp- background growth).
In case of inconsistent and untypical colonies, a representative number of colonies were
randomly picked, suspended in 100 μL 0.9% NaCl per colony and spread on minimal
glucose agar plate. At least 5 colonies identified to be true revertants from the vehicle or
the respective positive controls were also picked as an additional control. After incubation at
37°C for 4 days the agar plates were assessed by unaided eye for bacterial growth.
To determine the spontaneous mutation rate and to assess the mutability of the bacteria
and the activity of the S9 mix, the negative/vehicle (acetone) control and positive controls
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD) 9-
aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO) and 2-aminoanthracene (2-AA)
have been incubated in parallel at adequate concentrations.
Results
A bacteriotoxic effect was observed in the plate incorporation test from about 33 μg/plate
onward and in the preincubation assay from about 10 μg/plate onward, depending on the
strain and test conditions. No test substance precipitation was found in any of the test
conditions chosen.
The number of revertant colonies in the negative and positive controls, were within the
range of the historical control data for each tester strain and fulfilled the acceptance criteria
of the testing laboratory.
No relevant increase in the number of his+ or trp+ revertants was observed in the tester
strains TA98, TA100, TA1537 and E.coli WP2 uvrA in the plate incorporation and pre-
incubation method after incubation with p-BMHCA with and without metabolic activation.
For the tester strain TA1535 no relevant increase in colony numbers was found in the pre-
incubation test with and without metabolic activation and in the plate incorporation test in
the presence of a metabolic system. An increase in colony numbers was observed in one out
of the three assessed plates in the plate incorporation test at concentrations of 333
(Exp.1&2) and 2500 μg/plate (Exp.1) each without metabolic activation. At these test
concentrations, p-BMHCA was significantly bacteriotoxic as shown by a reduced background
growth and the absence of any colony formation in some plates. Further, the morphology of
these colonies from the plates in question completely differed from those of true revertants.
The assessment for histidine independent growth potential of representative colonies from
these plates showed, that all picked colonies did not grow in minimal glucose agar plate.
Therefore, none of the inconsistent colonies picked were his+ revertants.
Conclusion
According to the results of the present study, p-BMHCA did not lead to a relevant increase
in the number of revertant colonies with and without a metabolizing system in three
independent experiments for any of the tester strains. The sporadic increases in the number
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of colonies observed in the strain TA1535 in the absence of S9 mix is not considered as
biologically relevant. These increases were not reproducible within the triplicate plates, were
strongly associated with bacteriotoxicity and no dose trend was observed. Furthermore, the
morphology of the colonies was different from true revertants and 54 representative
colonies (including 30 colonies with an untypical/inconsistent morphology) were therefore
checked for growth in absence of histidine, a hallmark of mutant colonies. This assessment
for histidine independent growth showed that none of these colonies grew in the absence of
histidine, confirming that they are not mutant colonies. The result of growth is presumably
triggered by the strong test substance toxicity observed at the respective test
concentrations. Accordingly, p-BMHCA was determined to be not mutagenic in the bacterial
reverse mutation test in the absence and the presence of metabolic activation under the
experimental conditions chosen.
Ref.: BASF SE 2018a, 40M0369/01M027 (C. Sup I: 1)
SCCS comment
In the experiments very often four (sometimes 3) analysable concentrations (i.e. not
inducing a reduction of background growth) of p-BMHCA were used.
The results indicate that p-BMHCA with alpha-tocopherol at 200 ppm is not mutagenic in the
bacterial reverse mutation test in the absence and the presence of metabolic activation.
Guideline: OECD 476
Test system: L5178Y mouse lymphoma cell line (Tk+/-)
Replicates: Two independent experiments, each two parallel cultures
Test substance: p-BMHCA (Lilestralis pure: 32229) with 200 ppm of alpha-
tocopherol
Batch: A100423A (purity: > 99%)
Concentrations: Preliminary test:
±S9 mix (4 h exposure) and -S9 mix (24 h exposure): 7.97,
15.94, 31.88, 63.75, 127.5, 255, 510, 1020, 2040 μg/mL
Main test:
Experiment I:
-S9 mix (4 h exposure): 4, 8, 16, 20, 24, 28, 32, 36 μg/mL
+S9 mix (4 h exposure): 8, 16, 32, 40, 48, 56, 64, 72 μg/mL
Experiment II:
-S9 mix (24 h exposure): 1.25, 2.5, 5, 10, 15, 20, 25, 30
μg/mL
+S9 mix (4 h exposure): 20, 30, 40, 50, 55, 60, 65, 70 μg/mL
Vehicle controls: DMSO
Positive Controls: -S9 mix: ethyl methanesulfonate (EMS), 150 μg/mL
+S9 mix: Cyclophosphamide (CP), 2 μg/mL
GLP: Yes
Study period: 25 Jun 2010 – 22 Jun 2011
Material and methods
The in vitro mammalian cell gene mutation assay was conducted to investigate the potential
of p-BMHCA (with 200 ppm of alpha-tocopherol) dissolved in DMSO to induce gene
mutations at the TK +/- locus of the L5178Y mouse lymphoma cell line. Prior to the main
study, a preliminary toxicity test was performed on cell cultures using a 4-hour exposure
time both with and without metabolic activation (S9, liver post mitochondrial supernatant of
rats treated with phenobarbital/β-naphthoflavone) and using a 24-hour exposure without
S9-mix. The dose range used was 7.97 to 2040 μg/mL for all three exposure groups.
The following main study was performed in two independent experiments, using two parallel
cultures each. In the first experiment of the main study, p-BMHCA treatments were
performed in duplicate (A + B) both with and without metabolic activation (S9-mix) at eight
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dose levels of the test item (4 - 36 μg/mL in the absence of S9-mix, and 8 - 72 μg/mL in
the presence of metabolic activation), vehicle and positive controls. The treatment vessels
were incubated at 37°C for 4 hours with continuous shaking. In the second experiment of
the main study, the dose range of the test item was 1.25 - 30 μg/mL in the absence and 20
– 70 μg/mL in the presence of S9-mix. The treatment vessels were incubated at 37°C with
continuous shaking for 24 hours in the absence of metabolic activation and 4 hours in the
presence of S9-mix.
Results
In the preliminary test, toxicity in the form of marked reductions in %Relative Survival
Growth (%RSG) was observed in all three of the exposure groups starting at 31.88 μg/mL
(15% RSG, -S9). At the end of the exposure periods, precipitation of test item was
observed at and above 127.5 μg/mL in the 4h exposure groups, and at and above 255
μg/mL in the 24h exposure group and increased in intensity as the concentration increased.
In both experiments of the main test performed, a marked test item-induced toxicity in both
the absence and presence of S9-mix, as indicated by the %RSG and Relative Total Growth
(RTG) values was observed (Exp. I: 4 h, -S9: at/above 32 μg/mL (31% RSG); 4 h, +S9:
at/above 64 μg/mL (40% RSG), Exp. II: 24 h, -S9: at/above 15 μg/mL (44% RSG), 4 h,
+S9: at/above 50 μg/mL (18 % RSG)). The test item did not induce any statistically
significant or dose-related increases in the mutant frequency at any of the concentrations,
neither in the absence nor presence of metabolic activation, including the concentration in
the absence of metabolic activation exceeding the upper limit of acceptable toxicity (10% -
20% RSG).
The vehicle control mutant frequency values were within the acceptable range and the
positive controls produced marked increases in the mutant frequency demonstrating the
sensitivity of the assay and the efficacy of the S9-mix. Precipitation of the test item was not
observed at any of the concentrations tested in both main study experiments.
Conclusion
Under the conditions of the study, p-BMHCA did not induce any toxicologically significant
increases in the mutant frequency at the Tk +/- locus in L5178Y cells and is therefore
considered to be non-mutagenic in mammalian cells.
Ref.: Innospec Ltd., 2011b, SMII: 17
Guideline: Comparable to OECD 487
Test system: Human peripheral blood lymphocytes (two healthy non-smoker
males, less than 40 years old, supplied by AVIS (Italian
Association of Voluntary Blood donors))
Replicates: Each treatment on cells of 2 donors, each in 2 separate cultures
(i.e. 4 cultures/treatment)
Test substance: p-BMHCA
Batch: Not stated (source: Sigma–Aldrich, St. Louis, MO, USA, purity:
> 90%)
Concentrations: 5, 10, 25, 35, 50, 100, 250, 500 μM
Vehicles: p-BMHCA: dissolved in ethanol (50 % v/v), diluted in RPMI
1640 medium to avoid precipitation
Positive controls: ethyl methanesulfonate (EMS): 120 μM
Colcemid (COL): 0.02 μM
Negative controls: DMSO
GLP: No
Published: Yes, date of publication: 2014
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Material and methods
Para-BMHCA (alpha-tocopherol content unknown) was tested for its clastogenic and
aneugenic potential in vitro on peripheral blood lymphocytes of two healthy non-smoker
males (less than 40 years old). Prior to the main test, the cytotoxicity of p-BMHCA on the
peripheral blood lymphocytes was evaluated by scoring at least 1000 cells per treatment for
the presence of one, two, three or more nuclei and determining the nuclear division index
(NDI). The cells that did not undergo mitosis were not included in the count. Genotoxicity
was assayed in the main tests starting from the highest concentration to concentrations at
which neither necrosis nor cytotoxic or cytostatic effects were observed. The cultured
lymphocytes, supplemented with Cytochalasin-B (6.25 μM final concentration), were treated
for 24h at 37°C with test material at concentrations of 5, 10, 25, 35, 50, 100, 250 and 500
μM in the absence of an exogenous source of metabolic activation. Each treatment was
carried out on the cells obtained from two donors and in two separate cultures (i.e. four
cultures were set up for each treatment group). For each treatment, at least 1000
lymphocytes were scored to determine the NDI value, and at least 2000 binucleated cells
(BNCs) were examined for the presence of micronuclei. A positive response was defined as
a statistically significant increase of MN frequencies in the treated cultures respect to the
vehicle.
Results
The preliminary cytotoxicity test showed that at the concentration of 100 μM, p-BMHCA
reduced the cell proliferation, inducing a less than 70% value of NDI and early signs of
cytotoxicity. At 250 and 500 μM, the NDI was not applicable due to the advanced necrosis.
Para-BMHCA, when tested on the human lymphocyte cultures at non-cytotoxic
concentrations of 5 - 50 μM for 24 hours, did not increase the mean micronuclei frequency
in binucleated cells in comparison with the vehicle. The positive controls, EMS and COL
increased the micronuclei frequency significantly, showing that the lymphocytes were
suitable for detecting both clastogenic and aneuploidic damage.
Conclusion
It was shown that p-BMHCA revealed no potential to induce clastogenic or aneuploidic
damage under the chosen testing conditions.
Ref.: Di Sotto et al., 2014 a, b, SMII: 11, 12
SCCS comment
The study was not performed under GLP. It was performed without metabolic activation.
Only information from public literature is available. Limited information is provided on
treatment of cells, cytotoxicity and how the study was done. Also, no data on historical
controls are provided. Results have limited value.
Additional study on micronucleus test provided in December 2018
Guideline: OECD TG 487
Test system: Human peripheral blood lymphocytes (two healthy non-smoker
females; age of 33 and 35 years; not receiving medication)
Replicates: Each treatment on cells of 1 donor in 2 separate cultures (i.e. 2
cultures/treatment)
Test substance: p-BMHCA (Lysmeral Extra) with 200 ppm of alpha-tocopherol
Batch: 00046877L0 (purity: > 99%)
Concentrations: Experiment 1A (4h exposure, without S9 mix): 0.9, 1.7, 3.5,
6.9*, 13.9*, 27.8*, 55.6, 111, 222, 667, 2000 μg/mL
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Experiment 1B (4h exposure, without S9 mix): 2.5, 5, 7.5, 10,
15, 20*, 30*, 40*, 60, 80, 100 μg/mL
Experiment 2 (20h exposure, without S9 mix): 1.9, 3.4, 5.9,
10.4, 18.1*, 31.7*, 55.6*, 111 μg/mL
Experiment 2 (4h exposure, with S9 mix): 31.7*, 55.6*, 111*,
222, 444, 667, 1000 μg/mL
*Cytogenetic evaluation performed
Positive Controls: Without S9 mix:
MCC: 1.0 μg/mL (4h treatment)
Demecolcine: 75 ng/mL (20h treatment)
With S9 mix:
CPA: 17.5 μg/mL (4h treatment)
Negative controls: Solvent control (culture medium with 0.5 % Acetone)
GLP: In compliance
Study period: 22 March 2018 – 05 Nov 2018
Material and methods
Para-BMHCA (with 200 ppm of alpha-tocopherol) was tested for its clastogenic and
aneugenic potential in vitro on peripheral blood lymphocytes of two healthy non-smoker
females (age of 33 and 35 years). The cytogenetic experiments were performed by applying
p-BMHCA for 4 hours (followed by a recovery period for 16 hours; pulse exposure) or 20
hours (continuous exposure). Both protocols included a subsequent exposure with
cytochalasin B (4 μg/mL) for 20 hours until cell preparation. Phenobarbital/β-
naphthoflavone induced rat liver S9 was used as the metabolic activation system.
The cytotoxic potential on the peripheral blood lymphocytes was characterized up to 2000
μg/mL p-BMHCA via reduction in the cytokinesis-block proliferation index CBPI in
comparison with the controls by counting 500 cells per culture in duplicate (Experiment 1A;
pulse exposure; with and without S9 mix). The cultures without S9 mix fulfilled the
requirements and were used for cytogenetic evaluation. The cultures with S9 mix were
repeated with an adjusted p-BMHCA concentration range in Experiment 2 due to increases
in micronucleated cells above the historical control data in the solvent control. Furthermore,
Experiment 2 was repeated due to lack of evaluable concentrations (with S9 mix, pulse
exposure) and solvent controls with micronucleated cell values above the historical controls
(without S9 mix, continuous exposure). Experiment 1B was performed with adjusted p-
BMHCA concentrations due to lack of evaluable concentrations in a cytotoxic or phase
separation range in Experiment 1A (pulse treatment, without S9 mix).
The cytogenicity of p-BMHCA was assessed by counting micronuclei in 1000 (controls) or
2000-6000 (p-BMHCA) binucleate cells per culture. Each treatment was carried out on the
cells obtained from one donor in two separate cultures. Dose selection for the cytogenicity
assessment was based on phase separation and cytotoxicity of p-BMHCA. Culture medium
with 0.5% acetone was used as negative control and 1.0 μg/mL mitomycin C (MCC) (pulse
treatment, without S9 mix), 75 ng/mL demecolcine (continuous treatment, without S9 mix)
and 17.5 μg/mL cyclophosphamide (CPA) (pulse treatment, with S9 mix) was used as
positive controls.
Results
Precipitation in the form of phase separation of p-BMHCA in the culture medium was
observed at 111 μg/mL and above at the end of treatment (Experiment 1A and Experiment
2). In Experiment 1B, phase separation was observed already at 40.0 μg/mL and above at
the end of treatment. The cytotoxicity of p-BMHCA has been assessed up to the first dose
showing precipitation, i.e. phase separation. In Experiment 1A (pulse exposure without S9
mix), no cytotoxicity was observed up to 27.8 μg/mL (i.e. the highest dose evaluated for
cytogenicity). However, the next higher tested dose (55.6 μg/mL) showed excessive
cytotoxic effects resulting in non-evaluable cells for cytogenicity. In Experiment 1B (pulse
exposure without S9 mix) and Experiment 2 (pulse exposure with S9 mix), no evident
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cytotoxicity was observed up to the first concentration showing phase separation. In the
experiment with continuous treatment (Experiment 2, without S9 mix), clear cytotoxicity
was observed at 55.6 μg/mL (i.e. the highest dose evaluated for cytogenicity) and excessive
cytotoxicity was observed at 111 μg/mL (i.e. the first dose showing phase separation).
No relevant increases in the numbers of micronucleated cells were observed after treatment
with p-BMHCA in any of the testing conditions. In Experiment 2 (continuous treatment,
without S9 mix) a statistical trend test identified a dose dependent increase of
micronucleated cells (0.97%, 0.97% and 1.05% at 18.1 μg/mL, 31.7 μg/mL and 55.6
μg/mL, respectively). Since none of the individual dose groups were statistically significantly
increased compared to the control and were all within the range of the historical control
data (0.00 – 1.11% micronucleated cells) and very close to the corresponding solvent
control (0.88%), the dose-dependency can be regarded as biologically irrelevant. The
positive controls demecolcine, MMC and CPA showed distinct increases in micronucleated
cells, met all acceptance criteria of the testing laboratory and confirm the suitability of the
chosen testing conditions.
Conclusion
It was shown, that p-BMHCA did not induce micronuclei under the chosen testing conditions.
Therefore, p-BMHCA revealed no potential to induce clastogenic or aneuploidic damage as
determined by the in vitro micronucleus test in human lymphocytes.
Ref.: BASF SE 2018b, 31M0369/01X101 (C. Sup I: 2)
SCCS comment
The results indicate that p-BMHCA with alpha-tocopherol at 200 ppm does not induce a
positive effect in the in vitro micronucleus test in human lymphocytes.
The SCCS notes that in Experiment IA and Experiment II, phase separation of the test item
in the culture medium was observed at 111 μg/mL and above at the end of treatment, while
in Experiment IB phase separation was observed at 40.0 μg/mL and above at the end of
treatment.
Guideline/method: Alkaline Comet assay according to published literature
(Aviello et al., 2010, J. Cell. Mol. Med. 14, 2006–2014)
Test system: Human colonic epithelial cells (HCEC, obtained from
Fondazione Callerio Onlus, Trieste, Italy)
Replicates: Three experiments
Test substance: p-BMHCA
Batch: not stated (source: Sigma–Aldrich, St. Louis, MO, USA,
purity: > 90%)
Concentrations: 100 μM
Vehicles: DMSO
Positive control: H
2
O
2
, 75 μM
Negative controls: Vehicle control
GLP: No
Published: Yes, date of publication: 2014
Material and methods
Para-BMHCA (alpha-tocopherol content unknown) was tested for its potential to induce DNA
damage in an indicator test in the form of the alkaline Comet assay in Human colonic
epithelial cells (HCEC, obtained from Fondazione Callerio Onlus, Trieste, Italy). Prior to the
main test, the cytotoxicity on HCEC cells was evaluated by the neutral red uptake assay.
The cells were seeded in 96-well plates and allowed to adhere for 48 h. Thereafter, they
were incubated with serial dilutions of the test substance in the range between 1 – 300 μM
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for 24 h and subsequently with the neutral red dye solution for 3 h, and the absorbance was
read at 532 nm.
In the main test, DNA damage was evaluated by the alkaline comet assay. HCEC were
seeded in 6 well-plates. After 48 h, the cells were incubated with 100 μM for 24 h and
subsequently, cells were trypsinised. Aliquots of cell suspension were centrifuged and
pellets were collected, mixed with 0.85% low melting point agarose and laid on pre-coated
glass slides. The slides were then suspended at 4°C for 1 h for lysis and electrophoresed in
alkaline buffer at 26 V, and 300 mA for 20 min. After neutralization in Tris-HCl, the gels
were stained with ethidium bromide. Images were analyzed using a Leica microscope
equipped with image analysis Comet Assay™ software.
Results
In the preliminary test, p-BMHCA at concentrations ranging from 1 to 300 μM did not affect
HCEC cell viability after 24 h exposure. The vehicle DMSO (0.1% v/v) did not modify the
response, while DMSO at higher concentration (20% v/v) and used as positive control,
significantly reduced HCEC viability.
In the main test, p-BMHCA at the tested non-toxic concentration of 100 μM induced no DNA
damage in the form of an increase in DNA tail after electrophoresis compared to the vehicle
and following a 24 hour exposure. The positive control (H
2
O
2
) increased the DNA tail
significantly, indicating induction of single strand breaks. In summary, no evidence was
found for p-BMHCA to induce single-strand breaks.
Conclusion
Para-BMHCA was considered to induce no DNA damage in the form of single-strand breaks
under the conditions of this indicator test in human colonic epithelial cells.
Ref.: Di Sotto et al., 2014 a, b, SMII: 11, 12
SCCS comment
The comet assay experiment was not performed under GLP. So far there is no OECD TG for
the comet assay in vitro. Only 24h exposure was used though it would also be required to
use short (3-4 h) treatment as during 24h exposure DNA repair is taking place and thus
effects may not be detected. In a preliminary cytotoxicity test neither of the used
concentrations induced cytotoxicity. The concentrations should range from non-toxic up to
mildly toxic (around 80% viability). Testing only one concentration of p-BMHCA of 100 µM in
the comet assay is not justified and results of the test would have limited value.
Genotoxicity assessment of Lysmerylic acid (p-BMHCA related metabolite)
The acid form of p-BMHCA (Lysmerylic acid) was identified as the quantitatively main
metabolite in vitro (BASF SE, 2010b, SMI: 13, #63830) and no parent compound but the
acid was detectable in blood plasma directly after oral application of p-BMHCA to rats or
mice (BASF SE, 2006b, SMI: 7, #53648 (rat); BASF SE, 2006c, SMI: 8,#63832
(mouse)). Therefore, in December 2018 the Applicant provided supplementary genotoxicity
data for Lysmerylic acid.
In an Ames test (according to OECD TG 471 and GLP), Lysmerylic acid was tested in a
range of 33 μg - 5200 μg/plate (plate incorporation test) and 10 μg - 5200 μg/plate (pre-
incubation test) using S. typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and E. coli
WP2 uvrA with and without liver S9 mix from phenobarbital/β-naphthoflavone induced rats
(BASF SE 2018c; C. Sup I: 3). No precipitation of the test substance was found but a
bacteriotoxic effect was observed depending on the strain and test conditions from about
2600 μg/plate onward. Lysmerylic acid did not show any relevant increase in the number of
his+ or trp+ revertants (factor ≤ 2 for TA 100, TA 98 and WP2 uvrA and factor ≤ 3: TA
1535 and TA 1537) in any of the testing conditions chosen.
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In a micronucleus test in vitro in human lymphocytes (according to OECD TG 487 and GLP),
Lysmerylic acid was applied for 4 hours (followed by a recovery period for 16 hours; pulse
exposure) or 20 hours (continuous exposure) with subsequent exposure to Cytochalasin B
(4 µg/mL) for 20 hours (BASF SE 2018d; C. Sup I: 4). Phenobarbital/β-naphthoflavone
induced rat liver S9 was used as the metabolic activation system in a pulse exposure
experiment. Concentration ranges tested were 219-670 µg/mL (pulse exposure, with and
without S9 mix) and 364-714 μg/mL (continuous exposure, without S9 mix). No cytotoxicity
was observed up to the highest concentration evaluated for micronuclei, however,
precipitation was found (i.e. at 670 μg/mL after pulse exposure and 714 μg/mL after
continuous exposure). Neither a statistically significant nor a biologically relevant increase in
the number of micronucleated cells was observed after treatment with Lysmerylic acid in
any of the testing conditions chosen.
Accordingly, the main metabolite of p-BMHCA (Lysmerylic acid) was not mutagenic in
bacteria and not cytogenic/ aneugenic in mammalian cells under the chosen experimental
conditions of these studies.
3.3.6.2 Mutagenicity / Genotoxicity in vivo
Additional data from Applicant’s submission II dossier
No additional data.
Overall discussion and conclusion on mutagenicity/genotoxicity
From submission II
The applicant’s overall conclusion on mutagenicity/genotoxicity
The mutagenic/genotoxic potential of p-BMHCA was investigated in a wide range of
validated and scientifically robust studies in vitro and in vivo. The overall picture of several
bacterial reverse mutation assays performed over more than 3 decades is mostly
consistent. The majority of mutagenicity data in bacteria provide no evidence for a
mutagenic potential of p-BMHCA. However, equivocal findings were reported in one of the
submitted Ames tests for Salmonella strain TA1535 but this study is considered insufficient
in terms of procedure and reporting (Innospec Ltd., 2011a, SMII: 16). Moreover, this
observation in TA1535 was not confirmed in the respective pre-incubation test and no
corresponding increases of other strains (i.e. TA100) were observed. Further, this finding is
in contrast to the results of a GLP and guideline Ames plate incorporation test (Reference:
RIFM, 1999b, SMI: 89, #35168) and the Ames pre-incubation test in line with OECD TG 471
reported in literature (References: Di Sotto et al., 2014 a, b, SMII: 11, 12). Further,
sporadic but no relevant increases in the mean number of revertant colonies were reported
for the Salmonella strain TA1538 (without metabolic activation only) (Roche, 1984, SMI:
103). These findings were not reproducible in further trials and followed no concentration
response and the study is considered to have limited validity, since spontaneous revertant
frequencies were unusually low. The lack of biological relevance of this variation is
confirmed by the results in TA98. In this tester strain, investigating the same type of
mutagenic lesions, no effects/variations were observed.
Two different mutagenicity studies in mammalian cells investigating the same mutagenic
endpoint (gene mutation at both the HPRT- and the tk+/- locus) supported the absence of a
mutagenic potential of p-BMHCA (BASF SE, 2010a, SMI: 12; Innospec Ltd., 2011b, SMII:
17). Although methodological shortcomings exist, the highly sensitive indicator test for DNA
damage that was reported in the literature, namely the Comet assay in human colonic
epithelial cells, provided further evidence for the absence of p-BMHCA's DNA-damaging
potential (Di Sotto et al., 2014 a, b, SMII: 11, 12).
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Thus, the negative result generated in mammalian cells as well as the absence of an effect
in the Comet assay support the weight of evidence that p-BMHCA is non-genotoxic in vitro.
Para-BMHCA was found to induce structural and numerical chromosomal aberrations in the
absence of a metabolic system, while no induction occurred in the presence of metabolic
activation in Chinese hamster ovary cells and it is therefore considered clastogenic in CHO
cells (RIFM 2000a, SMI: 94,). However, these cells have previously been shown to generate
a high percentage of false-positive results compared with other cell types, e.g. primary
human cells, cell lines with functional p53 etc., and are consequently considered of
questionable value in the investigation of this endpoint (Fowler et al, 2012, SMII: 13). A
chromosomal damage potential of p-BMHCA was not observed in a non-GLP but a
scientifically reliable micronucleus test in human peripheral lymphocyte cultures that is
comparable to OECD 487 (Di Sotto et al., 2014 a, b, SMII: 11, 12). Thus, p-BMHCA does
not appear to have the potential to induce clastogenic or aneugenic damage in primary
human peripheral lymphocytes under the chosen testing conditions. Therefore, no
conclusive result with regards to chromosomal damage was observed in vitro.
The absence of a relevant potential of induction of chromosomal aberrations was confirmed
by an in vivo micronucleus assay where no relevant increase in the incidence of micronuclei
in bone marrow cells was observed following i.p. application of p-BMHCA to mice (RIFM
2000b, SMI: 95,). Systemic bioavailability was clearly demonstrated by the PCE/total
erythrocyte ratio in the top dose group at 24 hours sacrifice interval (-15% or -30% of
control [male; female]). This ratio evidenced cytotoxicity in the bone marrow as target
tissue of the test substance/metabolites after intraperitoneal administration.
Overall, p-BMHCA is unlikely to pose a genotoxic hazard to humans in a weight of evidence.
Some isolated equivocal findings in a few in vitro assays were not considered relevant due
to lack of reproducibility and insufficiencies in terms of procedure and reporting. In vivo,
there was no evidence of a genotoxic potential of p-BMHCA in a micronucleus assay
following i.p. application in mice.
In its preliminary opinion (December 2017), the SCCS, based on studies from
submission I and II on mutagenicity/genotoxicity of p-BMHCA, commented the
following:
In its previous Opinion (SCCS/1540/14) the SCCS concluded that neither in vitro gene
mutation nor in vitro chromosomal damage could be excluded based on the data provided in
submission I. Similarly, due to the lack of sufficient and detailed information, it was also
impossible to draw a firm conclusion from the in vivo micronucleus report provided.
Based on the analysis of additional reports provided in submission II, the SCCS considered
that the data did not allow to exclude potential genotoxic effects of p-BMHCA because:
1. In the tests on gene mutations in bacteria:
o p-BMHCA was confirmed to induce gene mutations in TA1535 strain (Ref.
Innospec Ltd., 2011a, SMII: 16)
o The study by Di Sotto et al. (2014a, b) using the Ames test was considered to
be of limited value as: the positive controls used did not clearly demonstrate
positive response, no information on historical controls was available and p-
BMHCA was tested in low concentrations,
2. In the tests on chromosomal aberrations in vitro:
o The study by Di Sotto et al. (2014a) using a micronucleus test on human
peripheral blood lymphocytes was considered to be of limited value as: p-
BMHCA was tested without metabolic activation, limited information was
provided on the treatment of cells, cytotoxicity and how study was done and
no information on historical controls was available,
3. In the comet assay in vitro:
o The study by Di Sotto et al. (2014a) using human colonic epithelial cells was
considered to be of limited value as: only 24h exposure was used though
shorter incubation times (3-4h treatment) should also have been used, at
least 3-5 concentrations ranging from non-toxic up to mildly toxic (around
80% viability) should be used, testing only one concentration of 100 µg/mL
was not justified.
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Based on analysis of data provided in submission I and additionally in submission II, the
SCCS maintains its previous opinion that no firm conclusion could be drawn on the
mutagenicity of p-BMHCA.
Overall discussion and conclusion on mutagenicity/genotoxicity of p-BMHCA based
on all available data including additional studies from the Supplement I to
Submission II
In response to the SCCS preliminary opinion the Applicant committed to conduct two
additional genotoxicity tests, i.e. an AMES test (according to OECD TG 471, GLP) and an in
vitro micronucleus test (according to OECD TG 487, GLP) using a representative and
market-relevant specification of p-BMHCA as a test substance.
The results of the new bacterial gene mutation test provided in December 2018 confirmed a
negative effects of p-BMHCA (BASF SE 2018a; C. Sup I: 1). Para-BMHCA did not lead to a
relevant increase in the number of revertant colonies in any of the tested strains. The
sporadic increases in the number of colonies observed in the strain TA1535 (also observed
in previous GLP study by Innospec Ltd., 2011a, SMII: 16) were not reproducible, were
strongly associated with bacteriotoxicity, lacked a clear dose trend and colonies were
confirmed to be non-mutant according to histidine independent growth experiments.
Based on the evaluation of all available gene mutation data (including the findings
from the gene mutation tests in mammalian cells), the SCCS is of the opinion that
a potential of p-BMHCA with 200 ppm alpha-tocopherol to induce gene mutations
can be excluded.
The results of the new in vitro micronucleus test in human lymphocytes (BASF SE 2018b; C.
Sup I: 2) provided in December 2018 confirmed that p-BMHCA with 200 ppm alpha-
tocopherol did not induce any relevant increase in the number of cells containing
micronuclei. Considering all available cytogenotoxicity data, the SCCS is of the
opinion that p-BMHCA with 200 ppm alpha-tocopherol does not induce clastogenic
or aneuploidic damage.
3.3.7 Carcinogenicity
From submission I
SCCS conclusion on carcinogenicity
No carcinogenicity data are available for p-BMHCA. Currently there is no evidence from
repeated dose studies that p-BMHCA is able to induce hyperplasia or neoplasia.
Additional data from Applicant’s submission II dossier
No additional data.
3.3.8 Reproductive toxicity
From submission I
SCCS conclusion on reproductive toxicity
Adverse effects of p-BMHCA on the male reproductive system have been consistently
observed in several repeated dose and reproduction toxicity studies. A NOAEL of 25 mg/kg
bw/day in male rats with regard to this endpoint is substantiated by studies applying the
compound for 5 days, 90 days or in the frame of a 1-generation study over 6 weeks prior to
mating. It is to be emphasised that reproductive toxicity already became occasionally visible
after a single application of 50 mg/kg bw/day. In all investigations available, testicular
toxicity in rats was accompanied by signs of systemic toxicity. By contrast, other species
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such as mice and dogs were less sensitive. In dogs, a NOAEL of 40 mg/kg bw/day has been
established based on the onset of testicular toxicity after treatment periods of 2 weeks and
3 months. So, from the animal data available, male rats revealed as most sensitive species
with regard to p-BMHCA-mediated testicular toxicity. On the other hand, in female rats
developmental toxicity was accompanied by systemic toxicity and was already found at
lower concentrations. Here, a NOAEL based on developmental toxicity is to be set at 5
mg/kg bw/day. This value is identical to the one defined for general systemic toxicity in rats
based on repeated dose (90-days) toxicity studies. Since the onset of developmental
toxicity was tightly accompanied by maternal toxicity, the malformations and tissue
variations observed likely resulted from general fetotoxicity rather than from specific
teratogenicity.
3.3.8.1 Two generation reproduction toxicity
Additional data from Applicant’s submission II dossier
No additional data.
3.3.8.2 Other data on fertility and reproduction toxicity
Additional data from Applicant’s submission II dossier
Guideline/method: OECD 443, Modified Extended one-generation reproduction toxicity
study
Species/strain: Rat/Wistar (strain Crl:WI(Han))
Group size: 35 male and 35 female rats per group for diet control, placebo
alginate control, low- and mid-dose groups
40 male and 40 female rats per group for high-dose group (F0
parental generation)
10 male and 10 female rats as positive control (Cohort 3 –
developmental immunotoxicity)
Test substance: p-BMHCA (Lysmeral encapsulated)
Batch: 1420-0552/201400167 (purity/content: 17.7 g/100 g, (3-(4-tert-
butylphenyl)-2-methylpropanoic acid): 0.2 g/100g, Reference: BASF
SE, 2015, SMII: 4))
Dose levels: Target: 0, 1, 3, 10 mg/kg bw/d
Encapsulated in the diet: 0, 75, 230, 750 ppm (corresponding to 0, 13, 41 and 133 ppm
active ingredient (a.i.))
Placebo alginate: 750 ppm consisting of 67.6 % Glycerin (Lot: GR335), 20.6 % Alginat
BR- L (Lot: G2600301) and 11.8 % Alginat BR-GM (Lot: G7708901).
The nucleus consists of 100 % sunflower oil, refined (Lot: 5603206)
Positive control: Cyclophospamide monohydrate (Batch: MKBS0021V, purity: 99.9%
and 6.9% water) used for Cohort 3 – (Immunotoxicity)
Route: Oral (diet (microcapsules of Lysmeral homogenously added to food))
Exposure period: F0 animals: approximately 2 weeks prior to breeding and continuing
through breeding (up to two weeks), and for a maximum of 6 post-
mating weeks (males) or gestation (three weeks) and lactation (three
weeks) for females. Selected F1 offspring (cohorts 1A, 1B, 2A, 2B, 3,
4A and 4B) were maintained on the test diet until sacrifice or one day
before.
Exposure frequency: daily
GLP: Yes
Study period: 21 April 2015 - 30 Jan 2017
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The study was performed to fulfil the requirements of a decision on a substance evaluation
pursuant to Article 46(1) of the REACh regulation, not for the purposes of the cosmetic
safety evaluation.
Material and methods
Para-BMHCA (Lysmeral encapsulated) was investigated in an extended one-generation
reproduction toxicity study to obtain general information on the possible effects on the
integrity and performance of the male and female reproductive systems, including gonadal
function, estrous cyclicity, mating behaviour, conception, gestation, parturition, lactation
and weaning, as well as on growth and development of the offspring. This study also
provided information on neonatal morbidity, mortality, target organs of the pups and
preliminary data on prenatal and postnatal developmental toxicity including possible effects
on the embryonic, fetal and pre-adult development of the nervous and immune systems as
well as alterations in endocrine function including thyroid perturbations.
The test substance was administered to groups of 35 male and 35 female healthy young
Wistar rats in the control, low- and mid-dose groups and to 40 male and 40 female healthy
young Wistar rats in the high dose groups (F0 parental generation) as a homogeneous
addition to the food in concentrations of 75, 230 and 750 ppm (corresponding to 13, 41 and
133 ppm of the active ingredient or to target dose levels of 1, 3 and 10 mg/kg bw/d due to
its content of 17.7%). The negative control group was fed a plain diet and an additional
placebo control group was dosed with Placebo Alginat (encapsulated) without p-BMHCA via
the diet in parallel.
F0 animals were treated at least for 13 days prior to mating to produce a litter
(F1generation). Mating pairs were from the same dose group. Pups of the F1 litter were
selected (F1 rearing animals) and assigned to 7 different cohorts, which continued in the
same fashion as their parents and which were subjected to specific post-weaning
examinations. Cohort 1B was selected to produce F2 pups. F1 Cohort 1B animals selected
for breeding were continued in the same dose group as their parents, and the breeding
programme was repeated to produce a F2 litter. The study was terminated with the terminal
sacrifice of the F2 weanlings and F1 Cohort 1B parental animals. Test diets containing p-
BMHCA (encapsulated) were offered continuously throughout the study.
- Cohort 1A (Reproductive PND90); Puberty: Yes; Approx. age at necropsy: 13 weeks
- Cohort 1B (Reproductive = F1 parental animals); Puberty: Yes; Approx. age at
necropsy: 19-25 weeks
- Cohort 2A (Neurotoxicity PND75-90); Puberty: Yes; Approx. age at necropsy: 11
weeks
- Cohort 2B (Neurotoxicity PND22); Puberty: No; Approx. age at necropsy: 3 weeks
- Cohort 3 (developmental immunotoxicity); Puberty: Yes; Approx. age at necropsy:
8-9 weeks
- Cohort 4A (Cholinesterase PND22); Puberty: No; Approx. age at necropsy: 3 weeks
- Cohort 4B (Cholinesterase adult); Puberty: Yes; Approx. age at necropsy: 11-12
weeks
The parents' and the pups' state of health was checked each day, and parental animals
were examined for their mating and reproductive performances. Food consumption of the
F0 and F1 parents and F1 rearing animals was determined regularly once weekly and
weekly during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation periods (days 1 - 4, 4 -
7, 7 - 14 and 14 - 21). In general, body weights of F0 and F1 parents and F1 rearing
animals were determined once weekly. However, during gestation and lactation F0/F1
females were weighed on gestation days (GD) 0, 7, 14 and 20 and on postnatal days (PND)
1, 4, 7, 14 and 21. A detailed clinical observation (DCO) was performed in all F0 parents
and F1 animals in cohorts 1A, 1B, 2A, 3 and 4B before initial test substance administration
(only F0 parents) and, as a rule, thereafter at weekly intervals. Estrous cycle data were
evaluated for F0 and cohort 1B (=F1 generation) females over a two weeks (F0 females) or
three weeks (F1 females) time period prior to mating until evidence of mating occurred. In
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all cohort 1A females, vaginal smears were collected after the vaginal opening until the first
cornified smear (estrous) was recorded. The estrous cycle was also evaluated in cohort 1A
females for 2 weeks around PND 75. Moreover, the estrous stage of each female was
determined on the day of scheduled sacrifice. An auditory startle response test was carried
out in all animals of cohort 2A on PND 24. A functional observational battery examination
(FOB) was performed in all animals of cohort 2A on PND 69. Motor activity was measured in
all animals of cohort 2A on PND 68. The F1 and F2 pups were sexed on the day of birth
(PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14
and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically
(including weight determinations of brain, spleen and thymus in one pup/sex/litter).
Anogenital distance (defined as the distance from the anus [centre of the anal opening] to
the base of the genital tubercle) measurements were conducted in a blind randomized
fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving
male pups were examined for the presence or absence of nipple/areola anlagen on PND 13.
If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined
one day prior to necropsy. Time of sexual maturation, i.e. day of vaginal opening (females)
or balanopreputial separation (males), of all F1 pups brought up beyond weaning was
recorded. Blood samples for clinical pathological investigations were withdrawn from 10
selected F0 and cohort 1A animals per sex and group. Further blood samples were taken
from 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22
pups per sex and group. Blood samples for acetyl cholinesterase investigations (AChE) were
withdrawn from 10 selected F0 animals per sex and group as well as from 10 surplus
(culled) PND 4 and 10 PND 22 (=cohort 4A) pups per sex and group and in all cohort 4B
animals.
Various sperm parameters (motility, sperm head count, morphology) were assessed in the
F0 and F1 generation males at scheduled sacrifice or after appropriate staining. All F0 and
F1 parental animals were assessed by gross pathology (including weight determinations of
several organs) and subjected to an extensive histopathological examination; special
attention being paid to the organs of the reproductive system. A quantitative assessment of
primordial and growing follicles in the ovaries was performed for all control and high-dose
F1 parental females.
All F1 rearing animals were assessed by different pathological, neuro- and histopathological
examinations.
Results
The stability of the test substance preparations over a period of 35 days at ambient
temperature and the homogeneous distribution of the test substance in the diet was
analytically verified. The mean recovery of p-BMHCA from the diet preparation in the first
analysis ranged between 60 and 80% of the expected values, the recovery rates in the
remaining 4 analyses were 63 - 95%, 92 - 102%, 81 - 107% and 86 - 97% of the expected
values. With regard to the very low concentration of p-BMHCA in the applied formulation as
well as in the diet preparations, and the high complexity of the extraction and analytical
method, these recovery rates were considered acceptable and demonstrated the
correctness of the diet preparations. The overall mean dose of p-BMHCA throughout all
study phases and across all cohorts was approx. 1.4 mg/kg mg/kg bw/d in the 75 ppm
group, approx. 4.5 mg/kg bw/d in the 230 ppm group and approx. 15.1 mg/kg bw/d in the
750 ppm group indicating that the targeted dose levels were achieved or exceeded.
There were no test substance-related mortalities or adverse clinical observations noted in
any of the groups. In particular, regularly conducted detailed clinical observations revealed
no effects at all.
The high-dose of the test substance led to some adverse systemic effects in the F0 parental
rats and F1 offspring. In the 10 mg/kg bw/d F0 females and F1 females of Cohort 1B, food
consumption was consistently reduced during lactation (F0 females: 5% and F1 cohort B1
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females: 13% below placebo-control). The food consumption of all animals in other cohorts
in all dose groups remained unchanged.
Organ weights: absolute and relative ovary weights were reduced significantly in a dose-
dependent manner in the F0 females. The weight decrease (absolute 97.067 mg; relative
0.045%) was below the historical control range values (absolute 109.542–130.320 mg;
relative: 0.046 – 0.056%). This change was judged by the authors to be “attributed to
physiological differences in the phases of the sexual cycle and not treatment-related”. This
reasoning is not clear, since the values were mean values compared to the mean values
from the control animals.
Table 1. F0 ovary weight change (%) relative to the placebo controls. **p <=0.01
Dose (mg/kg bw/d)
0
1
3
10
Absolute ovary
weight
100
99
94
88**
Relative ovary
weight
100
96
93
89**
In the high-dose F0 parental females, body weights were consistently reduced during
gestation and the first two weeks into lactation, which was caused by a reduced body
weight gain during different sections of premating and gestation. No such effects were
observed in the high-dose F0 parental males. Body weights of the high-dose Cohort 1A,
Cohort 1B, Cohort 2A and Cohort 4B males were below the concurrent control throughout
the in-life period after weaning (up to 11%). The difference gained statistical significance in
Cohorts 1B and 2A, but was consistently present in all these cohorts. High-dose F1 females
of Cohort 1B were similarly affected, and the decrease of body weight persisted throughout
gestation and lactation period for the F2 litters. The high-dose F1 females of Cohort 1B were
also affected by a reduction of body weight gain during pregnancy. Although all these
changes were not consistent and mild, a substance relationship is considered as likely.
In addition, there were some changes in blood and enzyme parameters in F0 and F1
females at 10 mg/kg bw/d such as prolonged prothrombin time (i.e. reduced synthesis of
coagulation factors), increased γ-glutamyl transferase (GGT) activity and reduced albumin
levels indicative of an altered metabolic activity of the liver cells. A prolonged prothrombin
time was also noted for the corresponding F0 and F1 males at this dose. In F0 females at 10
mg/kg bw/d higher red blood cell (RBC) counts, hemoglobin and hematocrit values were
detected. This effect was also present at 10 mg/kg bw/d in F1 males and females, both with
higher RBC and haemoglobin values.
Regarding pathology, the target organ was the liver. In the high-dose F0 females and
Cohort 1A and 1B, a significant increase in absolute and relative liver weights was observed.
When assessed histopathologically, these increases were associated with minimal to slight
centrilobular hypertrophy accompanied by minimal to slight apoptosis/single cell necrosis of
hepatocytes. Furthermore, periportal vacuolation and multinucleated hepatocytes were
noted in a few animals. All of these findings together were considered as treatment-related
and adverse. At 3 mg/kg bw/d a significant liver weight increase in F0, Cohort 1A and 1B
females was within the historical control range values and occurred without a
histopathological correlate, thus, it was clearly considered not adverse. There were no
indications from clinical examinations or from gross and histopathology that BMHCA
(encapsulated) adversely affected the fertility or reproductive performance of the F0 and F1
parental animals up to and including the high dose of 10 mg/kg bw/d.
Estrous cycle data, on the whole sperm quality of males, mating behaviour, conception,
gestation, parturition, lactation and weaning as well as sexual organ weights and gross and
histopathological findings of these organs (specifically the differential ovarian follicle count)
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were comparable between the rats of all groups including control and ranged within the
historical control data of the test facility.
The only notable findings were slightly higher incidences of abnormal sperm in the cauda
epididymidis in the high-dose F0 males (9.8+/-13.2% compared with 6.3+/-0.6% in the
control males, mean +/-SD respectively). However, this effect was not present in the
corresponding high-dose F1 males, had no influence on fertility and reproductive
performance of the affected males and had no testicular histopathological correlate. Thus,
the adversity and toxicological relevance of this finding is rather questionable.
For all liveborn male and female pups of the F0 and F1 parents, no test substance-induced
signs of developmental toxicity were noted at dose levels as high as 3 mg/kg bw/d.
Postnatal survival, pup body weight gain as well as post-weaning development of the
offspring of this test group until puberty remained unaffected by the test substance.
Furthermore, clinical and/or gross necropsy examinations of the F1 and F2 pups revealed no
adverse findings. Pup body weight development of the high-dose F1 and F2 offspring was
affected as these offspring weighed about 14-15% less than control after birth and did not
recover until weaning. Organ weight changes observed at this dose were considered to be
secondary to the changes in body weight.
There was no influence on postnatal pup survival.
Measurement of thyroid hormones revealed no effect caused by the test item, either in the
F0 parental animals or in the F1 offspring.
Anogenital distance of all test substance treated F1 pups were comparable to the concurrent
placebo-control values. Anogenital distance of the high-dose F2 male and female pups was
statistically significantly below the concurrent placebo-control values (about 4%,
respectively) and at the lower limit of historical control. In contrast, anogenital index of the
high-dose male and female F1 and F2 pups were statistically significantly above the
concurrent placebo-control values. Thus, the observed findings were solely a consequence
of the lower body weight and not considered as a specific treatment-related effect.
The incidence of present nipples/areolas revealed no test substance-related effect. No
treatment-related adverse effects were noted for the vaginal opening in all female F1
offspring or preputial separation in male F1 offspring, indicating no influence on sexual
maturation of the F1 progeny. An observed 1 day delay in preputial separation of the male
F1 offspring (10 mg/kg bw/d) was well within the historical control range of the test facility
and can be attributed to the general developmental delay. It is thus not considered to be a
direct test substance-related effect on male sexual maturation. No effect at all on the timing
of male puberty was noted in the lower-dose groups.
Lower peripheral acetylcholinesterase (AChE) activities in serum erythrocytes and
diaphragm tissue were found in male pups at PND 4 and in females at PND 76 of the high
dose group, while no changes were found in the animals of the opposite sex at these time
points. Although these results were not fully conclusive an inhibitory effect of the compound
on the peripheral AChE activity in pups and adolescent rats cannot be excluded. However,
no corresponding clinical signs of developmental neurotoxicity were evident in male and
female F1 offspring at any dose level. There were no compound related effects on motor
activity, auditory startle habituation, and in-the-field observation battery following exposure
to the test compound in these animals.
The only notable finding in neurobehavioral testing was lower maximum amplitudes in the
auditory startle response test of the high-dose F1 males of Cohort 2A. However, in
comparison to corresponding vehicle control data and high-dose F1 Cohort 2A female data
the placebo control values were rather unusually high. Moreover, no such findings were
noted in the high dose F1 females and no corresponding effects were recorded for startle
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response latency. Thus, this isolated observation was not considered as a treatment-related
effect.
Neuropathology examinations in the form of brain weight determination, brain length and
width measurements as well as brain morphometry and neuropathological examination by
light microscopy did not reveal any neurotoxicological treatment-related findings.
There was no evidence that the test substance produced any developmental
immunotoxicity. Neither T-cell dependent anti-SRBC IgM antibody response, nor absolute
and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, T-lymphocytes,
CD4-, CD8- T lymphocytes and natural killer (NK) cells) displayed any treatment-related
changes.
Conclusion
The extended one-generation reproduction toxicity study is predominantly designed to focus
on reproductive and developmental effects that may occur as a result of pre- and postnatal
exposure to a substance as well as an evaluation of systemic toxicity in pregnant and
lactating females and young and adult offspring. Under the conditions of this study, the
NOAEL for fertility and reproductive performance in the F0 and F1 parental rats was 10
mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity in the F1 and F2 progeny was 3 mg/kg bw/d
(equivalent to a mean overall oral dose of 4.5 mg/kg bw/d), based on reduced pup body
weights in the F1 and F2 offspring, which were observed at 10 mg/kg bw/d. As these weight
reductions were only observed in the presence of maternal toxicity, including lower weight
gain during pregnancy, they are not considered as an indication for specific developmental
toxicity.
The NOAEL for developmental neurotoxicity (DNT) for the F1 progeny is 10 mg/kg bw/d, the
highest dose tested. Although an inhibitory effect at this dose on the peripheral AChE
activity in pups and adolescent rats cannot be excluded, there were no corresponding
effects evident in the neurobehavioral or neuropathological examinations.
The NOAEL for developmental immunotoxicity (DIT) for the F1 progeny is 10 mg/kg bw/d,
the highest dose tested.
The NOAEL for general, systemic toxicity is 3 mg/kg bw/d (equivalent to a mean overall oral
dose of 4.5 mg/kg bw/d) for the F0 and F1 parental as well as adolescent animals, based on
evidence for distinct liver toxicity, as well as corresponding effects on food consumption,
body weights and clinical pathological parameters, which were observed at 10 mg/kg bw/d
predominantly in females.
Ref.: BASF SE, 2015, 2017b, SMII, 4, 8
SCCS comment
The SCCS agrees that NOAEL for fertility and reproductive as well as systemic toxicity of p-
BMHCA in this study is 10 mg/kg bw/d and the NOAEL for developmental toxicity is 3 mg/kg
bw/d. However, the SCCS does not agree with the developmental neurotoxicity NOAEL since
inhibition of AChE in different tissues at 10 mg/kg bw/d should be considered adverse.
Based on the overall assessment, the NOAEL value of 4.5 mg/kg bw/d could be applied for
MoS calculation.
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3.3.8.3 Developmental Toxicity
Additional data from Applicant’s submission II dossier
No additional data.
SCCS overall comment on reproductive toxicity based on studies from submission I
and II
In the previous Opinion (SCCS/1540/14) the SCCS concluded that based on the study in
which pregnant female rats were exposed to p-BMHCA at 5, 15 or 45 mg/kg bw/d (BASF
SE, 2004, RIFM# 52014), a NOAEL based on developmental toxicity could be set at 5 mg/kg
bw/day. This value was identical to the one defined for general systemic toxicity in rats
based on repeated dose (90-days) toxicity studies (Givaudan, 1990a, RIFM #12144,
Givaudan, 1990i, RIFM #12143).
However, based on the study provided with submission II, the SCCS considers that the
NOAEL for developmental toxicity should be set 4.5 mg/kg bw/d.
3.3.9 Toxicokinetics
From submission I
SCCS conclusion on toxicokinetics
Quantitative data on the toxicokinetics of p-BMHCA are available from rat, mouse, rabbit,
guinea pig, dog and rhesus monkey and human studies. Given its physicochemical
properties, p-BMHCA is likely to have high bioavailability via the oral route. After oral and
dermal administration to experimental animals and humans, there is clear evidence of
systemic absorption of p-BMHCA. However, in humans compared to rats, only limited
percutaneous absorption of p-BMHCA (in EtOH) could be observed in vivo (1.4% vs. 19%).
Species-specific differences in the metabolism of p-BMHCA have been identified both in vitro
and in vivo. Lysmerylic acid was the main hepatic metabolite in all species tested.
Quantitative evaluation of metabolic profiles for different species in an in vitro metabolism
study demonstrated much higher levels of p-tert-butylbenzoic acid (TBBA) formation by rat
hepatocytes when compared to other species. In particular, TBBA levels observed in human
hepatocytes were about 4-fold lower compared to rat hepatocytes at corresponding
concentrations. Comparative assessment of the urinary metabolites in different animal
species again uncovered differences in the urinary excretion of TBBA (and p-tert-butyl-
hippuric acid, TBHA), with rats being the species that predominantly forms TBBA. However,
the differences observed between rats and monkeys did not mirror the 4-fold difference in
TBBA formation as seen with rat and human liver microsomes in vitro.
3.3.9.1 Metabolism in vitro
Additional data from Applicant’s submission II dossier
No additional data.
3.3.9.2 Toxicokinetics in laboratory animals
Additional data from Applicant’s submission II dossier
No additional data.
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3.3.9.3 Toxicokinetics in humans
Additional data from Applicant’s submission II dossier
Guideline/Method: Explorative metabolism and excretion study after a single oral dose
according to an ethically approved protocol
Species: Human
Group size: 5 healthy volunteers (3 females, 2 males, age range: 23 – 32 years)
Test substances: p-BMHCA
Batch: no data (purity: no data)
Dose level: 5.26 mg/volunteer
Vehicle: Ethanol
Route: Oral
Exposure: Single
Application procedure: 52.6 mg p-BMHCA dissolved in ethanol using a 10 mL
volumetric flask. Each volunteer received a chocolate-coated edible
waffle cup containing 1 mL spiked ethanol (exact 5.26 mg p-BMHCA,
equivalent to 25.7 μM) and approximately 20 mL coffee, milk or
water, depending on the choice of the volunteers
Urine samples: immediately prior to exposure up to 48h after exposure
GLP: No data
Study period: No data
Material and methods
The metabolism and excretion kinetics of p-BMHCA was investigated in an explorative study
in human volunteers after application of a single oral dosage. The study was performed in
accordance with the ethical standards of the Declaration of Helsinki (1964) and was
approved by the Ethics Commission of the Ruhr University Bochum (Reg. No.:5105-14). The
primary intention of this investigation was to develop a human biomonitoring method (HBM)
including identification of suitable biomarkers of exposure in human urine and basic
toxicokinetics. In addition, urinary conversion factors (CF) were deduced from the
toxicokinetics results to allow the back-calculation of the exposure doses of p-BMHCA from
urinary metabolite levels of the 40 adult volunteers.
Five healthy subjects (3 females, 2 males) were orally dosed once with p-BMHCA. Each
volunteer received a chocolate-coated edible waffle cup containing 1 mL spiked ethanol
(exact 5.26 mg lysmeral, equivalent to 25.7 µmol) and approximately 20 mL coffee, milk or
water, depending on the choice of the volunteers. Urine was collected immediately before
and for 48h after administration and frozen (< - 20°C) until analysis. The p-BMHCA
metabolites lysmerol, lysmerylic acid, hydroxylated lysmerylic acid and 4-tert-butylbenzoic
acid (TBBA) were determined in all urine samples by a newly developed UPLC-MS/MS (ultra-
high-pressure liquid chromatography combined with tandem mass spectrometry) method.
The derived conversion factors (CFs) were applied to spot urines samples of 40 health
subjects (33 males, 7 females). The toxicokinetic variables for the urinary excretion of the
p-BMHCA metabolites were evaluated individually for each subject. Where appropriate,
means, standard deviations (SD) and medians were calculated. The amount of metabolites
excreted after 3, 6, 12 and 24h were obtained by linear interpolation.
Results
The peak amounts of the 4 metabolites were excreted between 3 and 6h after oral p-
BMHCA application. The primary metabolites lysmerol (2) and lysmerylic acid (3) appeared
slightly earlier in the urine than the secondary metabolites hydroxyl-lysmerylic acid (9) and
TBBA (4). After 12 and 24h more than 90 and 97%, respectively, of the p-BMHCA
metabolites were excreted in the urine. The authors regarded excretion of these metabolites
as complete by 48h after the oral intake.
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After 48h, the urinary excreted metabolites of p-BMHCA are dominated by TBBA (4)
representing about 14.3% of the administered dose, followed by lysmerol (2), yielding
1.82% of the dose after 48 h. Hydroxy-lysmerylic acid (9) and lysmerylic acid (3)
represented only 0.20% and 0.16% of the dose, respectively. In total, the 4 metabolites
represented about 16.5% of the dose. Average times for peak excretion (tmax) were 2.2 h
and 4.64 h for lysmerol (2) and TBBA (4) and 3.1 h for both lysmerylic acid (3) and
hydroxyl-lysmerylic acid (9). After 24 h, between 95% (TBBA) and 99% (lysmerol) were
excreted. The elimination half-lives (t½) were found to be lower for the primary metabolites
lysmerol (2) and lysmerylic acid (3) (1.19 h and 1.25 h, respectively) than for the
secondary metabolites hydroxyl-lysmerylic acid (9) and TBBA (4) (1.39 h and 1.40 h,
respectively).
Volunteers CF values were applied to 40 urine samples collected by subjects of the general
population. Creatinine standardised urinary p-BMHCA metabolite levels were used for back
calculation of the uptake dose. The CF derived with the molar sum of all four metabolites (2
+ 3 + 9 + 4) yielded a median uptake dose of 224 μg/d.
Conclusion
This explorative metabolism study confirmed that TBBA, lysmerol, lysmerylic acid and
hydroxyl-lysmerylic acid are major urinary p-BMHCA metabolites in humans. Therefore,
they can be considered as possible biomarkers for assessing exposure in human
biomonitoring studies. While TBBA is quantitatively the most dominant p-BMHCA metabolite
in urine, its specificity might be hampered by other sources of TBBA apart from p-BMHCA.
The three other metabolites, carrying the full p-BMHCA backbone, are considered as specific
to p-BMHCA representing about 2% of the oral dose.
Peak excretion for all metabolites occurred between 2 and 5h after oral application, with the
primary metabolites (lysmerol and lysmerylic acid) being excreted about 1h earlier than the
secondary metabolites (hydroxylated lysmerylic acid and TBBA). More than 90% of all
measured lysmeral metabolites were excreted after 12h, with the renal excretion being
virtually complete after 48h. After this time period, TBBA, lysmerol, lysmerylic acid and
hydroxyl-lysmerylic acid represent on average 14.3, 1.82, 0.20 and 0.16%, respectively, of
the dose administered. In total, the 4 metabolites determined represent about 16.5% of the
dose. Back-calculation of the exposure dose in 40 adult subjects from the general
population resulted in median daily doses of 140–220 μg/d p-BMHCA, depending on the
inclusion or exclusion of TBBA in the combined urinary conversion factors.
Ref.: Scherer et al., 2016, SMII: 22
3.3.10 Photo-induced toxicity
From submission I
SCCS conclusion on photo-induced toxicity
Based on the data and studies available, p-BMHCA is unlikely to exhibit photo-induced
toxicity (irritation or sensitisation) in guinea pigs.
3.3.10.1 Phototoxicity / photo-irritation and photosensitisation
Additional data from Applicant’s submission II dossier
No additional data.
3.3.10.2 Photomutagenicity / photoclastogenicity
Additional data from Applicant’s submission II dossier
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No additional data.
3.3.11 Human data
From submission I
SCCS conclusion on human data
There is no evidence that p-BMHCA exhibits photo-induced toxicity. However, undiluted p-
BMHCA is a proven skin irritant. In most HRIPT studies, p-BMHCA – when being dissolved in
a mixture of ethanol and diethyl phthalate – did not provoke skin sensitising reactions after
dermal application at concentrations of up to 25%. Conversely, p-BMHCA dissolved in
petrolatum already caused positive skin reactions in this assay at concentrations of 5%,
thus demonstrating the influence of the vehicle being used to administer the compound
onto skin. Additional data from clinical populations also point to sensitising properties of p-
BMHCA, albeit at only low frequencies. Reactions were only occasionally observed at
concentrations of <5%. Overall, mainly based on clinical studies, the SCCS considers p-
BMHCA as an “established contact allergen in humans”, an opinion it has held since 2012
(SCCS, 2012).
3.3.11.1. Irritation
Additional data from Applicant’s submission II dossier
No additional data.
3.3.11.2. Sensitisation
Additional data from Applicant’s submission II dossier
No additional data.
3.3.11.3. Other clinical data
Recently, the working group of Schnuch et al. 2015 analysed the frequency of sensitisation
to 26 fragrances in 5451 products including p-BMHCA to be labelled according to current EU
legislation.
Use volumes were provided by the International Fragrance Association (IFRA). Data on
sensitization frequency generated by the Information Network of Departments of
Dermatology (IVDK) network between 2007 and 2009 and specifically 2008 were used.
Results of patch testing on the 26 labelled fragrances (1870 patients (in 2008: n=823) were
analysed. The proportion of reactions to single constituents in breakdown testing in
fragrance mix positives from testing the standard series was extrapolated to the study
population (n = 1870) yielding the frequency of sensitisation to single constituents. The
relative frequency of sensitisation was calculated as the share of sensitisation to a single
allergen (%) relative to the total of sensitisation (=100%) to fragrances. Sensitisation
exposure quotient (SEQ) as an estimate of sensitisation risk associated with exposure to the
respective fragrance was calculated as the quotient of the relative frequency of sensitisation
divided by the relative frequency of use. The SEQ varied greatly, offering a ranking
regarding risk of sensitisation.
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Although p-BMHCA was highly used in terms of the relative volume sold (standardised
market share) of 19.42%, only 0.7% of the 1870 patients tested showed a positive allergic
reaction. The share of positive reactions to p-BMHCA was calculated to be 2.9% (confidence
interval (CI): 1.5 - 4.9) and the resulting sensitisation exposure quotient (SEQ) of 0.15
indicated a low risk of sensitisation.
Ref.: Schnuch et al., 2015 (SMII: 23)
SCCS comment
The study confirms that, while p-BMHCA is a sensitiser, however, the risk of sensitisation at
current use levels is low.
3.3.12 Special investigations
Additional data from Applicant’s submission II dossier
The possible estrogenic activity of p-BMHCA (source: Sigma, Poole, UK, purity: ≥ 95%) was
examined in an explorative screening assay in MCF7 human breast cancer cells in vitro. At
3.000.000-fold molar excess, p-BMHCA partially displaced [
3
H]-estradiol from recombinant
human estrogen receptors ERα and ERβ and from cytosolic estrogen receptor of MCF7 cells.
At concentrations in the range of 5×10
-5
to 5×10
-4
M it increased the expression of a stably
integrated estrogen-responsive reporter gene (ERE-CAT) and of the endogenous estrogen-
responsive pS2 gene in MCF7 cells. However, the increase was clearly below the positive
control 17β-estradiol (10
-8
M). Para-BMHCA led to an increase of the proliferation of the
estrogen-dependent MCF7 cells over 7 days. This effect was inhibited by the anti-estrogen
fulvestrant, suggesting an ER-mediated mechanism.
Although the extent of stimulation of proliferation over 7 days was lower with p-BMHCA
than with 10
-8
M 17β-estradiol, given a longer time period of 35 days the extent of
proliferation with 10
-4
M increased to the same magnitude as observed with 10
-8
M 17β-
estradiol over 14 days. Based on these observations the authors concluded that p-BMHCA is
able to induce estrogenic responses in the MCF7 human breast cancer cell line in vitro.
Ref.: Charles and Darbre, 2009, SMII: 10
However, in vitro receptor-binding alone does not inform whether specific exposures to that
substance may lead to adverse effects in vivo. For p-BMHCA, there is ample evidence from
a variety of in vivo studies of a lack of adverse effects on female reproductive organs or
fertility. Especially the most recent extended one-generation reproduction toxicity study led
to no effects on fertility or on reproductive or endocrine organs (see section 6.8.2).
Furthermore, the adverse testicular effects of p-BMHCA in sensitive species appear not to be
endocrine-related, but due to overt toxicity to seminiferous tissues including a clear
threshold.
Para-BMHCA (no data on source, batch or purity) and its main metabolite p-tert-
butylbenzoic acid (TBBA) (no data on source, batch or purity) were tested for agonist and
antagonist activities against human RARα, RARβ and RARγ receptors under GLP conditions.
The tested concentrations ranged between 0.0013 – 100 μM. All treatment concentrations
were performed in triplicate. DMSO was used as solvent and examined as negative control.
Agonists (9-cis-retinoic acid, all-trans-retinoic acid) and antagonists (BMS195614, CD2665)
were used as reference compounds. For all treatment groups, the DMSO concentration was
normalised to a final concentration of 0.1%. 100 µl of each treatment medium was
dispensed into triplicate assay wells pre-dispensed with the Reporter Cells. Assay plates
were incubated at 37°C for 24 h. In the agonist assays, p-BMHCA and TBBA exhibited no
agonist activity towards human RARα and RARβ receptors. Para-BMHCA showed very low-
level, non-dose-dependent agonist activity towards human RARγ receptor (about 2.5 fold
activation at 4.0 μM only), which is finally considered as biologically not relevant. In the
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antagonist assays, none of the test compounds showed antagonist activity towards human
RARα and RARβ and RARγ receptors.
Ref.: Indigo Biosciences, 2016, SMII: 15
SCCS comment
In in vitro experiments a potential p-BMHCA estrogenic activity has been noted but at a
higher concentration than observed for the reference. However, as only estrogenic activity
was investigated, the SCCS cannot exclude an endocrine mediated mode of action for p-
BMHCA.
3.3.13 Safety evaluation (including calculation of the MoS)
According to the mandate/ToR, there are the following different categories to be considered
in the exposure assessment (numbers for relative daily exposure [mg/kg bw/day] according
to the SCCS’s NoG 2018 and Tozer et al., 2004):
Product types
Finished product
concentration (%)
Relative daily exposure
[mg/kg bw/day]
Hydroalcoholic-based
fragrances (e.g. Eau de
Toilette, perfume,
Aftershave, Cologne)*
1.42
7.17
Deodorants
0.09
22.08
Make up products
eye make-up
make-up remover
liquid foundation
mascara
eyeliner
0.04
0.33
8.33
7.9
0.42
0.08
Face cream
0.05
24.14
Hand cream
0.05
32.7
Body lotion
0.06
123.2
Hair styling
0.04
5.74
Bath cleansing products
soap
shower gel
rinse-off conditioner
shampoo
0.1
3.33
2.79
0.67
1.51
* Maximum finished product concentration for hydroalcoholics on shaved skin is 0.6%
For calculation of MoS the percutaneous absorption study using human skin (BASF, 2016)
was used. Based on the SCCS requirements, the mean absorption + 1 SD was taken for:
- “Water in oil” (24h): (Absorbed dose+1SD) + (Epidermis+1SD) + (Dermis+1SD) =
(4.83+3.54) + (0.74+0.31) + (0.73+0.35) = 10.5%
- “Oil in water” (24h): (Absorbed dose+1SD) + (Epidermis+1SD) + (Dermis+1SD) =
(4.77+2.16) + (0.69+0.31) + (0.78+0.17) = 8.9%
Based on significant deviations from the SCCS requirements, the mean + 2 SD was taken
for:
- “Ethanol in water” (24h) = (Absorbed dose+2SD) + (Epidermis+2SD) +
(Dermis+2SD) = (5.31+2*2.22) + (1.50+2*0.49) + (0.71+2*0.28) = 13.5%
- “Silicone in water” (24h) = (Absorbed dose+2SD) + (Epidermis+2SD) +
(Dermis+2SD) = (3.50+2*1.31) + (0.96+2*0.18) + (0.64+2*0.23) = 8.5%
By considering the percentage of p-BMHCA suggested permissible in the product, the
application of these numbers would result in the following SED values:
SCCS/1591/17
Final version
Product types
Finished product
concentration
(p-BMHCA %)
Relative daily
exposure
[µg/kg bw/day]
Type of
product
p-BMHCA
fraction
absorbed
Systemic Exposure Dose
[µg/kg bw/day]
Hydroalcoholic-based
fragrances (e.g. Eau de
Toilette, perfume,
Aftershave, Cologne)
1.42
7170*
Hydroalcoholic
0.135
13.745
Deodorants
0.09
22080
Hydroalcoholic
0.135
2.683
Make up products
eye make-up
make-up remover
liquid foundation
mascara
eyeliner
0.04
330
8330
7900
420
80
Oil in water
0.089
0.089
0.089
0.089
0.089
0.012
0.297
0.281
0.015
0.003
Face cream
0.05
24140
Water in oil
0.105
1.267
Hand cream
0.05
32700
Water in oil
0.105
1.717
Body lotion
0.06
123200
Oil in water
0.089
6.579
Hair styling
0.04
5740
Oil in water
0.089
0.204
Bath cleansing products
soap
shower gel
rinse-off conditioner
shampoo
0.1
3330
2790
670
1510
Oil in water
0.089
0.089
0.089
0.089
0.296
0.248
0.060
0.134
Aggregated SED
28.15
* value of systemic exposure based on Tozer et al., 2004
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The use of a first-tier deterministic approach (adding all of the numbers derived above)
leads to the total SED level of 0.0281 mg/kg bw/day.
CALCULATION OF THE MARGIN OF SAFETY (aggregate exposure)
Total systemic exposure dose (SED) = 0.0281 mg/kg bw
No observed adverse effect level NOAEL
sys
= 4.5 mg/kg bw/d
(EOGRTS, oral, rat)Bioavailability 50% * NOAEL
sys
= 2.25 mg/kg bw/d
Margin of Safety (MOS) NOAEL
sys
/SED = 80
* Standard procedure according to the SCCS's Notes of Guidance for the testing of cosmetic
ingredients and their safety evaluation, 2018.
Individual MoS calculations for the respective product groups:
Product types
Systemic
Exposure Dose
[µg/kg bw/day]
Individual MoS
calculations for the
respective product
groups
Hydroalcoholic-based fragrances (e.g.
Eau de Toilette, perfume, Aftershave,
Cologne)
13.745
327
Deodorants
2.683
1677
Make up products
eye make-up
make-up remover
liquid foundation
mascara
eyeliner
0.012
0.297
0.281
0.015
0.003
7409
Face cream
1.267
3551
Hand cream
1.717
2621
Body lotion
6.579
684
Hair styling
0.204
22022
Bath cleansing products
soap
shower gel
rinse-off conditioner
shampoo
0.296
0.248
0.060
0.134
6092
Para-BMHCA is also used as a fragrance ingredient in some non-cosmetic products such as
household cleaners and detergents (see section 3.2). As no specific exposure data were
made available to SCCS to assess exposure following these non-cosmetic uses, it was not
possible to include them in the aggregated exposure scenarios. Therefore, the actual total
exposure of the consumer may be higher than exposure from cosmetic products alone.
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3.3.14 Discussion
Physicochemical properties
Based on the previous SCCS Opinion (SCCS/1540/14):
Para-BMHCA is a colourless to pale yellow liquid carrying a mildly floral odour, reminiscent
of cyclamen and lily of the valley. It is commercially available at a purity of ≥97.5% (w/w).
According to the applicant the degree of purity can be as high as ≥99.5% (w/w). Possible
impurities include 3-(3-tert-butylphenyl)-2-methylpropanal and lysmerylic (lilac) acid. The
latter compound results from air oxidation in aqueous solutions at pH7 and 25°C. However,
since alpha-tocopherol (CAS 59-02-9) is added as a stabilizer directly after the production
process, only low concentrations of the corresponding acid are found in Lysmeral®Extra,
i.e. a market-relevant ingredient.
General toxicity
Based on the previous SCCS Opinion (SCCS/1540/14) and submission II:
The acute toxicity after all relevant routes of application of BHMCA was investigated in rats
and rabbits. Based on the LD
50
values obtained, the acute (lethal) toxicity of p-BMHCA can
be considered moderate (>1300 mg/kg bw, oral route) or low (>2000 mg/kg bw, dermal
route). However, a single oral application of 50 mg p-BMHCA per kg body weight in male
rats already led to testicular atrophy in 2 out of 5 animals. An inhalation toxicity test in rats
led to no mortalities but to signs of systemic toxicity after exposure to a saturated
atmosphere.
The data on acute toxicity of p-BMHCA provided in Submission II do not change the
previous SCCS conclusion (SCCS/1540/14).
Repeated dose toxicity
Based on the previous SCCS Opinion (SCCS/1540/14):
The toxicity of p-BMHCA after repeated oral application was investigated in several species.
Decreases in body weights and food consumption and/or clinical signs of toxicity were
observed after subacute oral administration of p-BMHCA at doses of ≥50 mg/kg bw/day
(rats) and ≥200 mg/kg bw/day (dogs). Clinical chemistry and histopathological
examinations repeatedly revealed adverse effects on the liver and male reproductive system
(testicular toxicity). In a 90-day GLP study in rats BMHCA dose-dependently induced
systemic toxicity in both sexes at levels of ≥25 mg/kg bw/day and testicular toxicity in
males at ≥50 mg/kg bw/day. Thus oral NOAEL values of 5 mg/kg bw/day and 25 mg/kg
bw/day were derived for systemic effects and reproductive effects, respectively.
Reproductive toxicity
Based on the previous SCCS Opinion (SCCS/1540/14) and submission II:
Adverse effects of p-BMHCA on the male reproductive system have been consistently
observed in several repeated dose and reproduction toxicity studies. A NOAEL of 25 mg/kg
bw/day in male rats with regard to this endpoint is substantiated by studies applying the
compound for 5 days, 90 days or in the frame of a 1-generation study over 6 weeks prior to
mating. In all investigations available, testicular toxicity in rats was accompanied by signs of
systemic toxicity. By contrast, other species such as mice and dogs were less sensitive. In
dogs, a NOAEL of 40 mg/kg bw/day has been established based on the onset of testicular
toxicity after treatment periods of 2 weeks and 3 months. So, from the animal data
available, male rats revealed as the most sensitive species with regard to p-BMHCA-
mediated testicular toxicity. On the other hand, in female rats developmental toxicity was
accompanied by systemic toxicity and found already at lower concentrations. Here, a NOAEL
is to be set at 5 mg/kg bw/day. This value is identical to the one defined for general
systemic toxicity in rats based on repeated dose toxicity studies. The data available point to
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rats as most sensitive animal species tested. Toxicokinetic studies revealed that hepatic
metabolism of p-BMHCA in rats results in significantly higher levels of p-tert-butylbenzoic
acid (TBBA) when compared to other species. The SCCS is aware of older short-term studies
applying TBBA to rats via the oral route and suggesting that this metabolite may also exert
testicular toxicity (along with systemic toxicity). However, the doses applied in these studies
from the 1960s – 1980s were high and the quality of the studies generally low. The data
available therefore do not support the conclusion that this metabolite would be mainly
responsible for the testicular effects observed with p-BMHCA in rats.
In the extended one-generation reproduction toxicity study which results were provided in
submission II, the NOAEL for general, systemic toxicity of p-BMHCA applied in encapsulated
form at 1, 3, or 10 mg/kg bw/d, was established at 3 mg/kg bw/d for the F0 and F1
parental as well as adolescent animals, based on evidence for distinct liver toxicity. This
value was further supported by corresponding effects on food consumption, body weights
and clinical pathological parameters, which were observed at 10 mg/kg bw/d predominantly
in females. The NOAEL for fertility and reproductive toxicity of p-BMHCA in this study could
be established at 10 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 and F2
progeny was 3 mg/kg bw/d (equivalent to a mean overall oral dose of 4.5 mg/kg bw/d),
based on reduced pup body weights in the F1 and F2 offspring, which were observed at 10
mg/kg bw/d. This NOAEL for developmental toxicity is used for the calculation of the MoS.
As these weight reductions were only observed in the presence of maternal toxicity,
including lower weight gain during pregnancy, they are not considered as an indication for
specific developmental toxicity.
Irritation/sensitisation
Based on the previous SCCS Opinion (SCCS/1540/14):
Para-BMHCA as neat compound is irritating to the skin and eyes of rabbits. A solution of 2%
p-BMHCA in propylene glycol led to mild skin erythema. In general the observed effects
occurred transiently and were reversible. In a special investigation, p-BMHCA also displayed
the potential of inducing respiratory irritation at high concentrations (starting at about 70
μg/L in the atmosphere). In humans 10 and 20% p-BMHCA (dissolved in 75% ethanol/25%
diethyl phthalate) led to faint, minimal erythema in 1 and 2 out of 25 volunteers,
respectively.
Para-BMHCA is considered to be a moderate skin sensitiser based on several positive LLNA
studies. However, human patch-test data show that the risk of sensitisation in consumers at
current use levels is low.
Dermal absorption
Based on the previous SCCS Opinion (SCCS/1540/14) and submission II:
Administration of p-BMHCA onto the skin of both experimental animals and humans
demonstrated permeation and systemic availability of this compound. Further, in vitro
studies demonstrated solvent dependent and species specific effects. The bioavailable
portion was found much higher in rats (>50%) when compared to mini pigs (<5%).
Applying real cream formulations of 0.6% p-BMHCA, again rat skin allowed a much higher
absorption (>45%) than mini pig skin (about 25%). In the latter the fraction of bioavailable
p-BMHCA increased from 4.9% (EtOH solution) to 25% (cream formulation).
In vivo, percutaneous absorption of p-BMHCA in humans was lower when compared with
rats (1.4 vs. 19%). The range in 3 volunteers observed was 0.8 – 2.4% (excreted in urine
within 24 hours). So, the absorption found in humans for ethanolic solutions of p-BMHCA
was comparable to that was has been found in excised mini pig skin.
In the study on percutaneous study provided in submission II the SCCS identified significant
deviations from the SCCS requirements. According to SCCS 1358/10, recovery should be
between 85 - 115%. The overall recovery of p-BMHCA tested in formulations 1 (“ethanol in
water”) and 2 (“silicone in water”) was not within this acceptance range, even under the
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semi-occlusive conditions used. According to SCCS 1564/15, in the case of substances with
very low dermal absorption and limited permeation (e.g. colourants or UV-filters with high
molecular weight and low solubility), the epidermis may be excluded when it is
demonstrated that no movement of the chemicals from the skin reservoir to the receptor
fluid occurs. Para-BMHCA did not fulfil these criteria. Therefore, all p-BMHCA present in the
living epidermis had to be taken into account for the dermal absorption. Based on the SCCS
requirements, the mean + 1 SD was taken for MoS calculation for “Water in oil” (24h) =
10.5% and “Oil in water” (24h) =8.9%; the mean + 2 SD was taken for MoS calculation for
“Ethanol in water” (24h) = 13.5% and “Silicone in water” (24h) = 8.5%.
Mutagenicity
Based on the previous SCCS Opinion (SCCS/1540/14) and submission II:
In its previous Opinion (SCCS/1540/14) the SCCS concluded that neither in vitro gene
mutation nor in vitro chromosomal damage could be excluded based on the data provided in
submission I. Similarly, due to the lack of sufficient and detailed information, it was also
impossible to draw a firm conclusion from the in vivo micronucleus report provided.
Based on the analysis of additional reports provided in submission II the SCCS considered
that the data did not allow excluding potential genotoxic effects of p-BMHCA because:
- In the tests on gene mutations in bacteria:
o p-BMHCA was confirmed to induce gene mutations in TA1535 strain
o The study based on the Ames test was considered to be of limited value as:
positive controls used did not clearly demonstrate positive response, no
information on historical controls was available and p-BMHCA was tested in
low concentrations,
- In the tests on chromosomal aberrations in vitro:
o The study on micronucleus test on human peripheral blood lymphocytes was
considered to be of limited value as: p-BMHCA was tested without metabolic
activation, limited information was provided on treatment of cells, cytotoxicity
or on study methodology and no information on historical controls was
available,
- In the comet assay in vitro:
o The study on human colonic epithelial cells was considered to be of limited
value as: only 24h exposure was used though shorter incubation times (3-4h
treatment) should have also been used, at least 3-5 concentrations ranging
from non-toxic up to mild toxic (around 80% viability) should be used, testing
only one concentration of 100 µg/mL was not justified.
Based on analysis of data provided in submission I and additionally in submission II, the
SCCS maintained its previous opinion that no firm conclusion could be drawn on
mutagenicity of p-BMHCA.
Based on Supplement I to Submission II:
In response to the SCCS preliminary opinion (December 2017) the Applicant committed to
conduct two additional genotoxicity tests, i.e. an AMES test and an in vitro micronucleus
test using a representative and market-relevant specification of p-BMHCA as a test
substance.
The results of the new bacterial gene mutation test provided in December 2018 confirmed
negative effects of p-BMHCA.
Based on evaluation of all available gene mutation data (including the findings from the
gene mutation tests in mammalian cells), the SCCS is of the opinion that a potential of p-
BMHCA with 200 ppm alpha-tocopherol to induce gene mutations can be excluded.
The results of the new in vitro micronucleus test in human lymphocytes confirmed that p-
BMHCA with 200 ppm alpha-tocopherol did not induce any relevant increase in the number
of cells containing micronuclei. Based on evaluation of all available cytogenotoxicity data the
SCCS is of the opinion that p-BMHCA with 200 ppm alpha-tocopherol has no potential to
induce clastogenic or aneuploidic damage
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Carcinogenicity
Based on the previous SCCS Opinion (SCCS/1540/14):
No specific investigations available. There is no evidence from repeated dose studies in
animals that p-BMHCA is capable of inducing cancer.
Toxicokinetics
Based on the previous SCCS Opinion (SCCS/1540/14) and submission II:
Quantitative data on the toxicokinetics of p-BMHCA are available from rat, mouse, rabbit,
guinea pig, dog and rhesus monkey and humans. Given its physicochemical properties, p-
BMHCA is likely to have high bioavailability via the oral route. Similarly, data after dermal
administration clearly demonstrates that p-BMHCA becomes systemically available in
animals and humans.
Species specific differences in the metabolism of p-BMHCA have been identified in vitro as
well as in vivo. Still, lysmerylic acid (oxidation product) was the main hepatic metabolite in
all species tested. Quantitative evaluation of the metabolic profiles in different species in
vitro demonstrated much higher levels of p-t-butyl-benzoic acid (TBBA) formation by rat
hepatocytes when compared to other species. Older studies with rats also provided some
evidence of testicular toxicity induced by TBBA, suggesting that this metabolite might be
involved in the effects triggered upon application of its parent.
TBBA levels observed in human hepatocytes were about 4-fold lower compared to rat
hepatocytes at corresponding concentrations. Comparative assessment of the urinary
metabolites in different animal species again uncovered differences in the urinary excretion
of TBBA (and TBHA), with rats being the species that predominantly forms TBBA. However,
the differences observed between rats and monkeys did not mirror the 4-fold difference in
TBBA formation as seen with rat and human liver microsomes in vitro.
The data on metabolism of p-BMHCA provided in submission II confirmed that TBBA,
lysmerol, lysmerylic acid and hydroxyl-lysmerylic acid are major urinary p-BMHCA
metabolites in humans. Peak excretion for all metabolites occurred between 2 and 5 h after
oral application, with the primary metabolites (lysmerol and lysmerylic acid) being excreted
about 1 h earlier than the secondary metabolites (hydroxylated lysmerylic acid and TBBA).
After 48 h, TBBA, lysmerol, lysmerylic acid and hydroxyl-lysmerylic acid represent on
average 14.3, 1.82, 0.20 and 0.16%, respectively, of the dose administered.
As there are no oral bioavailability data available, a default value of 50% is used to
calculate systemic NOAEL.
Human data
/
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4. CONCLUSION
1. Does the SCCS consider Butylphenyl methylpropional (p-BMHCA) safe for use as a
fragrance ingredient in cosmetic leave-on and rinse-off type products in a concentration
limit(s) according the ones set up by IFRA as reported above?
On individual product basis, Butylphenyl methylpropional (p-BMHCA) (CAS 80-54-6) with
alpha-tocopherol at 200 ppm, can be considered safe when used as fragrance ingredient in
different cosmetic leave-on and rinse-off type products. However, considering the first-tier
deterministic aggregate exposure, arising from the use of different product types together,
Butylphenyl methylpropional at the proposed concentrations cannot be considered as safe.
This Opinion is not applicable to the use of p-BMHCA in any sprayable products that could
lead to exposure of the consumer’s lung by inhalation.
2. Does the SCCS have any further scientific concerns with regard to the use of Butylphenyl
methylpropional (p-BMHCA) as a fragrance ingredient in cosmetic leave-on and/or rinse-off
type products?
Evaluation of this substance by other scientific bodies (e.g. under REACH) should also be
taken into consideration by the Applicant for potential future assessment of the substance.
Butylphenyl methylpropional is also used as a fragrance ingredient in some non-cosmetic
products such as household cleaners and detergents. As no specific exposure data were
made available to SCCS to assess exposure following these non-cosmetic uses, it was not
possible to include them in the aggregated exposure scenarios. Therefore, the actual total
exposure of the consumer may be higher than exposure from cosmetic products alone.
5. MINORITY OPINION
/
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6. REFERENCES
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B: Submission II (SMII) References
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24. Scientific Committee on Consumer Safety (SCCS, 2016) The SCCS Notes of
Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation, 9th
Revision, adopted at 11th plenary meeting, 29-Sep 2015, revised 25-Apr-2016,
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adverse) Changes—Conclusions from the 3rd International ESTP Expert Workshop.
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methylpropional (BMHCA), SCCS/1540/14, adopted by written procedure on 12
August 2015, revision of 16 March 2016
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human and in vivo rat skin absorption studies. Human & Experimental Toxicology
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Proliferation: Differentiation between Adaptation and Toxicity. TOXICOLOGIC
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32. BASF SE (2016) Certificates of Analysis on Lysmeral®Extra. Collection of selected
examples from the years 2014-2016
33. BASF SE (2013) Final Report – Characterization of “Lysmeral Extra”, Study No.
13L00139 (confidential), Competence Center Analytics, BASF SE, Ludwigshafen,
Germany, unpublished, confidential data, 18 November 2013
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Document No. LJ17/009, BASF SE, Ludwigshafen, Germany, unpublished,
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data
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38. Dossier on the safety of p-BMHCA (2-(4-tert-butylbenzyl)propionaldehyde
(Lysmeral) CAS 80-54-6) in cosmetic products Submission II BASF-IFRA, 24
February, 2017
39. Scientific Committee on Consumer Safety (SCCS, 2018) The SCCS Notes of
Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation, 10th
Revision, adopted at plenary meeting on 24 – 25 October 2018.
C: Supplement I to Submission II (SMII) References
1. BASF SE (2018a) Lysmeral Extra - SALMONELLA TYPHIMURIUM / ESCHERICHIA
COLI REVERSE MUTATION ASSAY. Report No. 40M0369/01M027.
2. BASF SE (2018b) Lysmeral Extra - Micronucleus Test In Human Lymphocytes In
Vitro. Report No. 31M0369/01X101.
3. BASF (2018c) Lysmeralsäure - SALMONELLA TYPHIMURIUM / ESCHERICHIA COLI
REVERSE MUTATION ASSAY. Report No. 40M0491/14M347.
4. BASF (2018d) Lysmeralsäure - Micronucleus Test In Human Lymphocytes In Vitro.
Report No. 31M0491/14X669.
5. BASF SE (2011) Study Report – m-Lysmeral. Enhanced One-Generation
Reproduction Toxicity Study in Wistar Rats. Oral Administration (Gavage), Study
No. 77R0875/08070, BASF, Ludwigshafen, Germany, unpublished, confidential
data, 12 July 2011
7. GLOSSARY OF TERMS
See SCCS/1602/18, 10th Revision of the SCCS Notes of Guidance for the Testing of
Cosmetic Ingredients and their Safety Evaluation – from page 141
8. LIST OF ABBREVIATIONS
See SCCS/1602/18, 10th Revision of the SCCS Notes of Guidance for the Testing of
Cosmetic Ingredients and their Safety Evaluation – from page 141
