Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Lanolin alcohol is negative in the Ames test, the mouse lymphoma assay and in the chromosome aberration test with and without metabolic activation ( Bowles &Thompson 2010, Brown 2010, Bowles 2010).

Lanolin fatty acids is negative in the Ames test, the mouse lymphoma assay and in the chromosome aberration test with and without metabolic activation ( Bowles &Thompson 2010, Brown 2010, Bowles 2010)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 25 February 2010 and 26 March 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon for Salmonella
Tryptophan operon for Escherichia
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Deletion through the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Requires tryptophan and can be reverse mutated by base substitution to tryptophan independence. This strain also has a deletion in an excision repair gene (uvrA-).
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthaflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation tests (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide at 50 mg/ml but was fully soluble in acetone in solubility checks performed in-house. Acetone was, therefore, selected as the vehicle. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix for 2 µg plate for WP2uvrA, 3 µg/plate for TA100 and 5µg/plate for TA1535
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix for 80 µg/plate for TA 1537
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix for 0.2 µg/plate for TA98
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Actone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix for 5 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With S9 mix for 1 µg/plate for TA 100, 2 µg/plate for TA1535 and TA1537, and 10 µg/plate for WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicate plating

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
-Lawn deficiency and colony reduction

OTHER EXAMINATIONS:
- Stability and sterility
Evaluation criteria:
Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
- but tested up to maximum recommended dose level of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
- but tested up to maximum recommended dose level of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated in report
- Effects of osmolality: none stated in report
- Evaporation from medium: none stated in report
- Water solubility: insoluble in water, so acetone used as solvent
- Precipitation: A greasy, particulate precipitate was noted at and above 1500 and 500 µg/plate in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.
- Other confounding effects: none stated in report

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-) at any of the doses tested. (Up to 5000 µg/plate). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.


ADDITIONAL INFORMATION ON CYTOTOXICITY: not applicable

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A greasy, particulate precipitate was noted at and above 1500 and 500 µg/plate in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A greasy, particulate precipitate was noted at and above 1500 and 500 µg/plate in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 08 March 2010 and 07 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
mammalian cell line, other: human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM) supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum.

- Properly maintained: yes

the average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthaflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml

Chromosome aberration test
Group Final concentration of Lanolin Alcohols (µg/ml)
4(20)-hour without S9 0*, 39.06, 78.13, 156.25, 312.5*, 625*, 1250*, MMC 0.4*
4(20)-hour with S9 0*, 39.06, 78.13, 156.25, 312.5*, 625*, 1250*, CP 5*
24-hour without S9 0*, 39.06, 78.13, 156.25*, 312.5*, 468.75*, 625*, MMC 0.2*
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: not stated in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9, mitomycin C (MMC) was used at 0.4 and 0.2 µg/ml for cultures in the 4 and 24-hour exposures respectively. It was dissolved in Minimal Essential Medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9, cyclophosphamide (CP) was used at 5 µg/ml for cultures in the 4-hour exposure. It was dissolved in dimethyl sulphoxide.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours with and without S9, 24 hours continuous exposure without S9
- Expression time (cells in growth medium): 20 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): DEMI-C
STAIN (for cytogenetic assays):Giemsa

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30-50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: In addition, cells with 69 chromosomes or more will be scored as polyploid cells and the incidence of polyploid cells (%) will be reported.
- Determination of endoreplication: not applicable
- Other: none
Evaluation criteria:
In all circumstances where increases in the frequency of cells with aberrations are seen, statistical comparisons will be made with the vehicle. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
mammalian cell line, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant changes in pH
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Evaporation from medium: not detailed in report
- Water solubility: not applicable, as dissolved in acetone
- Precipitation:
Precipitate observations were taken and a precipitate of test material was noted at and above 78.13 µg/ml in the 4(20)-hour exposure group in the presence of S9, at and above 156.25 µg/ml in the absence of S9 and at and above 312.5 µg/ml in the 24-hour continuous exposure group.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A precipitate of the test material was observed in the parallel blood-free cultures at the end of exposure, at and above 78.13 µg/ml, in the 4(20)-hour exposure groups and at and above 156.25 µg/ml (as cloudy precipitate) in the 24-hour continuous exposure group. The precipitate aggregated and became greasy and/ or oily in appearance at and above 2500 µg/ml in both of the 4(20)-hour exposure groups, and at and above 312.5 µg/ml in the 24-hour continuous exposure group. It was considered that test material exposure to the cells would be reduced at these dose levels.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/ml in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 625 µg/ml. This data shows that clear dose-related toxicity was observed in the 24-hour continuous exposure group only.
The selection of the maximum dose level for the main experiment was based upon precipitate for the 4(20)-hour exposure groups, and on toxicity for the 24 hour continuous exposure group.


COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the sensitivity of the assay and the efficacy of the metabolising system.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material did not induce a toxicological statistically significant increase in the frequency of cells with aberrations either in the absence or presence of metabolic activation. However, there was a small statistically significant increase in the frequency of cells with aberrations in the 4(20)-hour exposure group in the absence of S9 at the mid dose level assessed, 625 µg/ml. This increase was only observed in the A culture, where the mitotic index indicated that the test material induced a higher level of toxicity over the other duplicate culture. The response was predominantly due to break-type aberrations, and was only marginally above the historical control range and increases were not observed at the maximum dose level assessed indicating a lack of a dose-related response. Therefore, the increase was considered to be of no toxicological significance.

The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 1250 µg/ml in the 4(20)-hour in the presence and absence of metabolic activation (S9), and at 625 µg/ml in the 24 hour continuous exposure in the absence of S9.

The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in the 4(20)‑hour exposure groups, both with and without S9, and that 51% mitotic inhibition was achieved at 625 µg/ml in the 24 hour continuous exposure group in the absence of S9. It was therefore considered that the test material had been adequately tested.

Conclusions:
negative

The test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction. This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems in so far as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24 hours continuous exposure in the absence of metabolic activation.

Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the sensitivity of the assay and the efficacy of the metabolising system.

The test material was non-toxic in the 4 hours exposure in the presence and absence of metabolic activation (S9) with a 20-hour expression period. However, the test material was toxic in the 24 hours continuous exposure (in the absence of metabolic activation) at 625 µg/ml.

The test material did not induce any toxicologically statistically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included a dose level that induced approximately 50% mitotic inhibition or was the lowest precipitating dose level, depending on the exposure conditions.

Conclusion. The test material was, therefore, considered to be non-clastogenic to human lymphocytesin vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 08 March 2010 and 07 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009
Type of assay:
mammalian cell gene mutation assay
Target gene:
The L5178Y TK+/- locus of the L5178Y mouse lymphoma cell line
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air.

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: yes

- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ beta-naphthaflavone
Test concentrations with justification for top dose:
The dose range used in the preliminary toxicity test was 9.77 to 2500 µg/ml for all three of the exposure groups.

Main experiment - 4 hours with and without S9: 0, 39.06, 78.13, 156.25, 312.5, 625 and 937.5 µg/ml
Main experiment - 24 hours without S9: 0, 39.06, 78.13, 156.25, 208.33, 260.41 and 312.5 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: not stated in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (acetone) treatment groups were used as the vehicle control
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Used at 400 µg/ml and 150 µg/ml for the 4 and 24 hours exposures respectively, in absence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (acetone) treatment groups were used as the vehicle control
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used at 2 µg/ml in presence of S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (with S9) and 24 hours (without S9)
- Expression time (cells in growth medium): 2-day expression period

SELECTION AGENT (mutation assays): TFT = Trifluorothymidine Deoxyriboside

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: The cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG), induced mutant frequency (IMF), gloval evaluation factor (GEF)

OTHER EXAMINATIONS:
- None
Evaluation criteria:
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Dose levels that have survival values less than 10% are usually excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
39.06 µg/ml and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test material was dosed into media
- Effects of osmolality: osmolality did not increase by more than 50 mOsm.
- Evaporation from medium: not applicable to this test substance
- Water solubility: not soluble in water
- Precipitation: Increased levels of precipitation reduced exposure to the cells.
- Other confounding effects: none stated

RANGE-FINDING/SCREENING STUDIES: In all three of the exposure groups there was a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. Precipitate of the test material was observed at and above 19.53 µg/ml, which was seen to aggregate at and above 625 µg/ml and effectively reduce exposure to the cells. This is supported by the fact that maximum toxicity in both of the 4-hour exposure groups was achieved at 625 µg/ml. In the subsequent mutagenicity test the maximum dose was limited by the estimated maximum test material exposure and induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional

Preliminary toxicity test

The dose range of the test material used in the preliminary toxicity test was 9.77 to 2500 µg/ml. The results for the Relative Suspension Growth (%) were as follows:

Dose

(µg/ml)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

9.77

103

118

110

19.53

98

92

145

39.06

84

112

132

78.13

93

79

104

156.25

65

74

73

312.5

50

62

4

625

38

50

0

1250

88

91

0

2500

91

105

10

4 -Hour Exposure With and Without Metabolic Activation

There was evidence of toxicity following exposure to the test material in both the presence and absence of metabolic activation, as indicated by the %and RTG values. There was no evidence of a reduction in viability (%V), therefore indicating that no residual toxicity occurred, in both the absence and presence of metabolic activation. Optimum levels of toxicity were not achieved in the absence of metabolic activation with a maximum of 55% (RTG) being observed at 625 µg/ml, and 21% toxicity at the higher dose of 937.5 µg/ml. This confirmed the observation in the preliminary toxicity test that maximum exposure was achieved at 625 µg/ml. In the presence of metabolic activation optimum levels of toxicity were observed at 312.5 µg/ml, whilst less toxicity was observed at higher dose levels again confirming that increased levels of precipitate were reducing exposure to the cells. It was considered that the test material had been adequately tested. Acceptable levels of toxicity were seen with both positive control substances.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell in both the presence and absence of metabolic activation. In both of the 4-hour exposure groups, some of the test material dose levels had mutant frequencies greater than the vehicle control value. However, there were no clear dose-related responses and all the values were within the acceptable range for vehicle controls and, therefore, they were considered to be of no toxicological significance.

24-Hour Exposure Without Metabolic Activation

As was seen previously, there was evidence of a marked reduction in %and RTG values in cultures dosed with the test material . There was also evidence of modest reductions in viability (%V), therefore indicating that modest residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved at 208.33 µg/ml.  The positive control induced acceptable levels of toxicity.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test material.

The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10-6viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell. As was seen previously, any increases in mutant frequency over the controls were small, were within the acceptable range for vehicle controls and were therefore considered to be of no toxicological significance (Table 9). A precipitate of test material was observed at and above 78.13 µg/ml.

Conclusions:
negative

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Executive summary:

Introduction. This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008..

Methods. One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six levels, in duplicate, together with vehicle (acetone) and positive controls. The exposure groups used were as follows: 4‑hour exposures both with and without metabolic activation, and 24–hour exposure without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 39.06 to 937.5µg/ml for the 4 -hour exposure groups in the presence and absence of metabolic activation, and 39.06 to 312.5µg/ml for the 24 hour exposure in the absence of metabolic activation.

Results. The maximum dose level used in the mutagenicity test was limited by the estimated maximum exposure to the test material and test material induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

Conclusion. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 25 February 2010 and 26 March 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009. Date of signature: 26th November 2009
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon for Salmonella
Tryptophan operon for Escherichia
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicate plating

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction

OTHER EXAMINATIONS:
- None
Evaluation criteria:
Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
, but tested up to the meximum recommended dose level of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
, but tested up to the meximum recommended dose level of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated in report
- Effects of osmolality: none stated in report
- Evaporation from medium: none stated in report
- Water solubility:insoluble in water, so acetone used as solvent
- Precipitation: A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: none described

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.


ADDITIONAL INFORMATION ON CYTOTOXICITY: not applicable

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction. Th method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 09 March 2010 and 28 May 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
mammalian cell line, other: human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM)
- Properly maintained: yes

The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test:
0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml

Chromosome aberration test:
Group Final concentration of Lanolin Fatty Acids (µg/ml)
4(20)-hour without S9 0*, 78.13, 156.25, 312.5, 625*, 1250*, 2500*, MMC 0.4*
4(20)-hour with S9 0*, 78.13, 156.25, 312.5*, 625*, 1250*, 2500, CP 5*
24-hour without S9 0*, 78.13*, 156.25*, 312.5*, 468.8, 625, 1250, MMC 0.2*

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: not stated in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9, mitomycin C (MMC) was used at 0.4 and 0.2 µg/ml for cultures in the 4 and 24-hour exposures respectively. It was dissolved in Minimal Essential Medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9, cyclophosphamide (CP) (Acros Organics, Batch No. A0164185) was used at 5 µg/ml. It was dissolved in dimethyl sulphoxide.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours with and without S9, 24 hours continuous exposure without S9
- Expression time (cells in growth medium): 20 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Demi-C
STAIN (for cytogenetic assays): Giemsca

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30-50% of cells with aberrations, slide evaluation was terminated at 50 cells.

DETERMINATION OF CYTOTOXICITY
- Mitotic index and cell count

OTHER EXAMINATIONS:
- Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.
- Determination of endoreplication: not applicable
- Other: none

OTHER:
Evaluation criteria:
In all circumstances where increases in the frequency of cells with aberrations are seen, statistical comparisons will be made with the vehicle. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
mammalian cell line, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant changes in pH
- Effects of osmolality: osmalality did not increase by more than 50 mOsm
- Evaporation from medium: not detailed within report
- Water solubility: not applicable, as dissolved in acetone
- Precipitation: Precipitate observations were taken from the whole blood cultures and a precipitate of test material was noted at and above 312.5 µg/ml. This is a marginally higher dose level to the one where the onset of precipitate was observed in the media cultures in the preliminary toxicity test. Precipitate persisted on the slides at and above 1250 µg/ml in the presence of metabolic activation.

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
A precipitate of the test material was observed in the parallel blood-free cultures at the end of exposure, at and above 78.13 µg/ml (in the absence of S9) and from 156.25 µg/ml (in the presence of S9) in the 4(20)-hour pulse exposure groups. Precipitate was also observed at and above 156.25 µg/ml in the 24-hour continuous exposure group. The precipitate was observed to aggregate at 5000 µg/ml in the 4(20)-hour exposure groups, and this was considered to effectively reduce the exposure to the cells particularly in these groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/ml in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9) and up to 625 mg/ml in the continuous exposure group. The test material induced evidence of toxicity in the 24-hour continuous exposure group only.

The selection of the maximum dose level for the main experiment was based on the toxicity observed in the 24 hour exposure group and not on the onset of precipitate. Therefore the maximum dose level for the 4(20)-hour exposure groups was 2500 µg/ml, the 5000 µg/ml dose was dropped as it was considered maximum exposure was achieved at the lower dose level due to the aggregation of the precipitate. A maximum dose level of 1250 µg/ml was selected for the 24-hour continuous exposure group.


COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Chromosome aberration test

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test material (2500 µg/ml) in the 4(20)‑hour exposure group in the presence of metabolic activation (S9). Precipitate on the slide, though, was a limiting factor and, therefore, the maximum dose level was limited to 1250 µg/ml. In the 4(20)-hour exposure group in the absence of S9, there were metaphases suitable for scoring at 2500 µg/ml and up to 312.5 µg/ml in the 24 hour continuous exposure group.

Mitotic index data confirm the qualitative observations in that a modest dose-related inhibition of mitotic index was observed in both of the 4(20)-hour exposure groups, and a marked dose-related reduction of mitotic index was observed in the 24-hour continuous exposure group.

The maximum dose level selected for metaphase analysis was 2500 µg/ml in the 4(20)‑hour group in the absence of S9 with 30% mitotic inhibition, and 312.5 µg/ml in the 24-hour continuous exposure group where 67% mitotic inhibition was observed. In the presence of metabolic activation the maximum dose level selected was 1250 µg/ml with 38% mitotic inhibition and precipitate present on the slides.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

In the OECD 473 Guideline, it is recommended that a repeat of the 4(20)-hour exposure with metabolic activation is performed if a negative response is seen in the first experiment, unless there is scientific justification for its omission. This study was run in conjunction with a Mouse Lymphoma Assay (MLA) onLanolinFatty Acidsusing L5178Y cells (Harlan Laboratories Ltd Project Number 2724/0033) which has the capability of detecting clastogenic activity. The quoted study was performed to meet the requirements of the OECD 476 Guideline. It was, therefore, considered that referencing a study such as the Mouse Lymphoma Assay (MLA) using L5178Y cells, gave adequate scientific justification for the omission of the repeat of the with metabolic activation exposure group.

Conclusions:
The test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction. This report describes the results of anin vitrostudy for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24 hours continuous exposure in the absence of metabolic activation.

Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was non-toxic in the 4 hours exposure in the presence and absence of metabolic activation (S9) with a 20-hour expression period. However, the test material was toxic in the 24 hours continuous exposure (in the absence of metabolic activation) at and above 468.8 µg/ml. The dose range selected was based upon both precipitate and toxicity.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

Conclusion. The test material was considered to be non-clastogenic to human lymphocytesin vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 08 March 2010 and 28 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at approximately 37°C with 5% CO2 in humidified air.

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthoflavone
Test concentrations with justification for top dose:
The dose range used in the preliminary toxicity test was 9.77 to 2500 µg/ml for all three of the exposure groups.

Main experiment - 4 hours without S9: 0, 18.75, 37.5, 75, 150, 300, 400, 500 and 600 µg/ml
Main experiment - 4 hours with S9: 0, 75, 150, 200, 250, 300, 350 and 400 µg/ml

Main experiment - 24 hours without S9: 0, 20, 40, 80, 160, 200, 240, 280 and 320 µg/ml

Vehicle and positive controls were used in parallel with the test material. Solvent (acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for the 4-hour and 24-hour exposures, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: not stated in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Solvent (acetone) treatment groups were used as the vehicle controls.)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Used at 400 and 150 µg/ml for the 4-hour and 24-hour esposures in absence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Solvent (acetone) treatment groups were used as the vehicle controls.)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used at 2 µg/ml in presence of metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 2-day expression period

SELECTION AGENT (mutation assays): TFT = Trifluorothymidine Deoxyriboside

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: The cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG), induced mutant frequency (IMF), gloval evaluation factor (GEF)

OTHER EXAMINATIONS:
- None
Evaluation criteria:
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Dose levels that have survival values less than 10% are usually excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:
Small significant increases designated by the UKEMS statistical package reviewed using the above criteria.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked change in pH when the test material was dosed into media
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Evaporation from medium: not applicable to this test substance
- Water solubility: not soluble in water
- Other confounding effects: due to formulation practicalities the maximum achievable dose level of 2500 µg/ml was investigated in the preliminary toxicity test.

RANGE-FINDING/SCREENING STUDIES: In all three of the exposure groups there was a marked dose-related reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. Precipitate of the test material was observed at and above 156.25 µg/ml. In the subsequent mutagenicity test the maximum dose was limited by test material-induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The dose range of the test material used in the preliminary toxicity test was 9.77 to 2500 µg/ml. The results for the Relative Suspension Growth (%) were as follows:

Dose

(µg/ml)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

9.77

93

102

102

19.53

106

102

98

39.06

98

103

82

78.13

86

104

82

156.25

94

92

57

312.5

40

69

7

625

0

28

0

1250

0

5

0

2500

0

0

0

4-Hour Exposure With and Without Metabolic Activation

There was evidence of toxicity following exposure to the test material in both the presence and absence of metabolic activation, as indicated by the %and RTG values. There was no evidence of a reduction in viability (%V), therefore indicating that no residual toxicity occurred, in both the absence and presence of metabolic activation. Near optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 400 µg/ml in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 400 µg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell in either the absence or presence of metabolic activation. A precipitate of test material was observed at and above 150 µg/ml in both the absence and presence of metabolic activation.

24-Hour Exposure Without Metabolic Activation

As was seen previously, there was evidence of a marked reduction in %and RTG values in cultures dosed with the test material . However there was no evidence of reductions in viability (%V), therefore indicating that residual toxicity had not occurred. Optimum levels of test material-induced toxicity was achieved. The excessive toxicity observed at and above 280 µg/ml resulted in these dose levels not being plated for viability or 5-TFT resistance. The positive control induced acceptable levels of toxicity.

The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10-6viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell. A precipitate of test material was observed at and above 160 µg/ml.

Conclusions:
The test material 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 under the conditions of the test.
Executive summary:

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of.

Methods. One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (Acetone) and positive controls. The exposure groups used were as follows: 4‑hour exposures both with and without metabolic activation, and 24–hour exposure without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 18.75 to 600µg/ml for the 4 -hour exposure group in the absence of metabolic activation, 75 to 400µg/ml for the 4 hour exposure group with metabolic activation, and 20 to 320µg/ml for the 24 hour exposure group without metabolic activation.

Results. The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

Conclusion. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The ester of lanolin alcohols and fatty acids is expected to be hydrolysed by esterases and is hydrolysed abiotically.

Therefore the genotoxicity of the hydrolysis products lanolin alcohols and lanolin fatty esters is considered representative for the genotoxicity of the substance.

Additional information

Justification for classification or non-classification

Based on the information available, the substance does not need to be classified for mutagenicity according to Regulation (EC) No 1272/2008 (CLP).