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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: Gene mutation (Bacterial reverse mutation, Ames test), OECD 471, GLP: S. typhimurium TA 1535, TA 1537, TA 98, TA 100: negative with and without metabolic activation

Genetic toxicity in vitro: Gene mutation (mouse lymphoma assay), OECD 490, GLP: mouse lymphoma L5178Y cells: negative with and without metabolic activation

Genetic toxicity in vitro: Chromosome aberration (Micronucleus test), OECD 487, GLP: human lymphocytes: negative with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented bacterial reverse mutation assay, conducted not in accordance with current guideline. However, it is in accordance with OECD guideline 471 as adopted 1983-05-26. There were only four strains tested, and spacing of test concentration was greater than 10exp(1/2). However, these deficiencies are considered to be minor as the fifth strain was added to the guideline because the former common four strains may not detect certain oxidising mutagens, cross-linking agents and hydrazines. Based on the chemical structure, this mode of action is not to be expected from the test item. There was no dose-dependent and biologically relevant increase of the mutant figures to the double of the negative controls. Actually, there was no increase in the mutant frequency in any of the tester strains at all, leading to the conclusion that any variation of the testing method could not lead to the observation of any mutagenic effect.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
as adopted 1983-05-26
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
B14, as set out in EEC Directive 84/449/EEC
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
his-
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: pKM 101 in TA100 and TA98
Metabolic activation:
with and without
Metabolic activation system:
induced S9
Test concentrations with justification for top dose:
0, 8, 40, 200, 1000, 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol (test item); DMSO (positive controls)
- Justification for choice of solvent/vehicle: The used solvent was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: nitrofuranthion; 4-nitro-1,2-phenylene diamine, 2-aminoanthracene
Remarks:
sodium azide 10 µg/plate (only TA1535), nitrofuranthion 0.2 µg/plate (only TA100), 4-nitro-1,2-phenylene diamine 10 µg/plate (only TA1537) / 0.5 µg/plate (only TA98), 2-aminoanthracene 3 µg/plate
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 30 sec
- Exposure duration: 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): his-negative agar

NUMBER OF REPLICATIONS: 4 plates per strain, dose, ± S9

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity of the substance was assessed in three ways. The first was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with background growth. (The same applies to the signs "c", "v", "p", "n" or "%", which may also be used in the tables.) Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 1000 µg/plate, the substance started to precipitate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses >200 µg/plate had a weak, strain-specific bacteriotoxic effect. Nevertheless, these doses could be used for assessment up to and including 5000 µg/plate.
Conclusions:
Interpretation of results: negative

Testing for mutagenicity in bacteria was performed according to OECD guideline 471 as adopted 1983-05-26. Although not being conducted to recent guidelines, the test was conducted scientifically reasonable with negligible deficiencies. Also, the testing was sufficiently documented, positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently relieable to assess the mutagenic potential of 2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) in bacteria. A dose-dependent and biologically relevant increase of the mutant figures to the double of the negative controls could not be observed with any of the four strains used. This applied to the examinations with and without the S-9 mix. In consequence, 2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) is considered to be non-mutagenic under the conditions of this test.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471), histidine-auxotrophic strains TA1535, TA100, TA1537 and TA98 of S. typhimurium were exposed to 2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) in ethanol at concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate in the presence and absence of mammalian metabolic activation (induced S9) via plate co-incubation.

2,2'-Methylene-bis-(6-cyclohexyl-4-methylphenol) was tested up to limit concentration 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

Doses up to and including 200 µg/plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes. Substance precipitation occurred at the dose 1000 µg/plate and above.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 (as adopted 1983-05-26) for in vitro mutagenicity (bacterial reverse gene mutation) data with minor deviations from the recent Guideline.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-09 - 2016-06-20 (experimental phase
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, adopted 28 July 2015, Guideline No. 490 "In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene“
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden
Type of assay:
other: mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
TK +/-
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Suitability of cells: The L5178Y cell line has been used successfully in in vitro experiments for many years. L5178Y cells are characterized by a high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiency of untreated cells of usually more than 50 % both necessary for the appropriate performance of the study. The cells have a stable karyotype with a near diploid (40 ± 2) chromosome number
A low metabolic activity of cells under in vitro conditions is a disadvantage of assays with cell cultures as many chemicals develop a mutagenic potential after metabolization by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).
. Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of Envigo allowing the repeated use of the same cell culture batch in experiments. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
- Methods for maintenance in cell culture if applicable: Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment. The cell cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
- Modal number of chromosomes: 40 ± 2
- Normal (negative control) cell cycle time: 10 - 12 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 15 % horse serum (HS) (3 % HS during 4 hour treatment), 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal agent.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as metabolic activation system.
Test concentrations with justification for top dose:
The highest concentration level should be 10 mM, but not higher than 2.0 mg/mL of the pure substance unless limited by the solubility or toxicity.
RSG (Relative Suspension Growth, pre-experiment) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest concentration of the main experiment should reduce the RSG or RTG value to approx. 10 - 20%, if possible.
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 15.6 µg/mL and 2000 µg/mL were used.
Relevant toxic effects indicated by a relative suspension growth (RSG) below 50% were observed at 15.6 µg/mL and above following 4h treatment without metabolic activation and at 31.3 µg/mL and above following 4h treatment with metabolic activation. After 24h treatment cytotoxicity as described above was noted at 62.5 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation occurred at 62.5 µg/mL and above following 4h treatment with and without metabolic activation and at 125.0 µg/mL and above after 24h treatment without metabolic activation.
The dose range of the main experiments was set according to the pre-experiment, spacing factor of 2.0. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.
Both, the pH value and the osmolarity was determined in the pre-experiment at the highest two concentrations of the test item and in the solvent control without metabolic activation. No relevant shift of the osmolarity or the pH value was observed (solvent control: 351 mOsm, pH 7.56 versus 291 mOsm and pH 7.58 at 2000 µg/mL or 325 mOsm and pH 7.53 at 1000 µg/mL).
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO, purity 99.99%
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Negative controls:
yes
Remarks:
solvent controls
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Methyl Methane Sulfonate (MMS)
Remarks:
The stability of both positive control substances in solution is proven by the mutagenic response in the expected range.
Details on test system and conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): In the mutation experiment, 1x10E7 (3x10E6 during 24 h exposure) cells/flask (80 cm² flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % during 24 h exposure)

DURATION
- Exposure duration: 4 or 24 h
- Expression time (cells in growth medium): expression and growth period of totally 48 h
- Selection time (if incubation with a selection agent): 10 - 15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 - 18 days

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: Cells from each experimental group were seeded into 2 microtiter plates.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Rationale for test conditions:
precipitation / cytotoxicity
Evaluation criteria:
Evaluation of Results
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.
Statistics:
Statistical Analysis
A linear regression will be performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance will be considered together.
experimental group p-value
experiment I, culture I without S9 mix 0.202
experiment I, culture II without S9 mix 0.551
experiment I, culture I with S9 mix 0.793
experiment I, culture II with S9 mix 0.992
experiment II, culture I without S9 mix 0.111
experiment II, culture II without S9 mix 0.122
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Remarks:
solvent controls
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES:
Relevant toxic effects indicated by a relative suspension growth (RSG) below 50% were observed at 15.6 µg/mL and above following 4h treatment without metabolic activation and at 31.3 µg/mL and above following 4h treatment with metabolic activation. After 24h treatment cytotoxicity as described above was noted at 62.5 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation occurred at 62.5 µg/mL and above following 4h treatment with and without metabolic activation and at 125.0 µg/mL and above after 24h treatment without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) (Number of mutant colonies per 10E6 cells)
- Positive historical control data: MMS, 4h, -S9: 407 ± 181; CPA, 4h, +S9: 488 ± 341; MMS, 24h, -S9: 486 ± 238
- Negative (solvent/vehicle) historical control data: Solvent control (medium, DMSO, water, ethanol, acetone, THF): 4h, -S9: 84 ± 30; 44, +S9: 85 ± 31; 24h, -S9: 81 ± 29
Conclusions:
The study was conducted under GLP according to OECD guideline 490 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of CAS 4066-02-8 to induce mutations in mammalian cells. No substantial and reproducible increase of the mutation frequency was noted in both experiments with and without metabolic activation.
In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed under GLP to investigate the potential of CAS 4066-02-8 to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The maximum test item concentration of the pre-experiment was 2000 µg/mL based on the OECD 490 guideline. The concentration range of the main experiments was limited by test item induced cytotoxicity.

The test substance was dissolved in DMSO.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal analyzable concentration of the test item.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Therefore, CAS 4066-02-8 is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay using the cell line L5178Y.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490 and EU method B.17.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 October 2015 - 22 March 2016 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well-documented GLP OECD 487 guideline study with slight, suitable deviations regarding the test design on the registered substance itself
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 487 “In vitro Mammalian Cell Micronucleus Test”, adopted 26 September 2014
Deviations:
yes
Remarks:
In-house non-GLP validation experiments were performed to get distinct significant responses using the specified positive controls. So the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Target gene:
n/a
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
0, 2.4, 4.2, 7.4 µg/ml (Exposure period 4 hrs without S9 mix)
0, 7.4, 13.0, 22.7 µg/ml (Exposure period 20 hrs without S9 mix)
0, 4.2, 7.4, 13.0 µg/ml (Exposure period 4 hrs with S9 mix)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Negative controls:
yes
Remarks:
solvent control
Solvent controls:
yes
Remarks:
DMSO 0.5%
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin
Remarks:
1.5 μg/mL MMC (-S9, pulse treatment); 100.0 ng/mL Demecolcin (-S9, continuous treatment); 17.5 μg/mL CPA (+S9)
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h
- Exposure duration: 4h or 20h
- Expression time (cells in growth medium): 16h + 20h with CytB (pulse exposure) or 20h with CytB (continuous exposure)
- Fixation time (start of exposure up to fixation or harvest of cells): 40h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 parallel cultures

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells.
Statistics:
Statistical significance was confirmed by using the Chi-squared test (α < 0.05) for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Remarks:
pulse treatment
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
continuous treatment, at 22.7 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on pH was observed.
- Effects of osmolality: No relevant influence on osmolarity was observed.
- Evaporation from medium: none noted
- Precipitation: In Experiment I, precipitation of the test item in the culture medium was observed at 7.4 μg/mL and above in the absence of S9 mix and at 13.0 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 69.6 μg/mL and above at the end of treatment.
Conclusions:
Interpretation of results: negative

The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 2,2'-methylenebis[6-cyclohexyl-p-cresol] to induce micronuclei in mammalian cells.
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, of 2,2'-methylenebis[6-cyclohexyl-p-cresol] is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating or the highest evaluable concentrations.
Executive summary:

In a mammalian cell cytogenetics assay, Micronucleus test (OECD 487), primary human lymphocyte cultures were exposed to 2,2'-methylenebis[6-cyclohexyl-p-cresol] in DMSO at concentrations of0, 2.4, 4.2, 7.4 µg/ml (Exposure period 4 hrs without S9 mix), or 0, 7.4, 13.0, 22.7 µg/ml (Exposure period 20 hrs without S9 mix), or 0, 4.2, 7.4, 13.0 µg/ml (Exposure period 4 hrs with S9 mix) (Phenobarbital/β-naphthoflavone induced rat liver S9).

2,2'-methylenebis[6-cyclohexyl-p-cresol] was tested up to precipitating concentrations. Positive controls induced the appropriate response. There was no evidence or a concentration related positive response of Micronuclei induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenetic mutagenicity data.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

As all available and required in vitro genotoxicity tests, i.e. testing for gene mutations in both bacteria and mammalian cells as well as chromosome mutations in mammalian cells, revealed negative results, no conclusion on a mode of action for genotoxic events can be drawn. No indication is given that the obtained results are not relevant for humans, as in vivo metabolism of the test item is sufficiently mimicked by addition of S9 mix, human lymphocytes were also tested and direct genotoxins act commonly species-independent.

Additional information

Justification for classification or non-classification

All available test results for gene mutation and chromosome aberrations (micronucleus test) in vitro are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified.