2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP status not specified. No QA signature page or evidence of audits.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

156, 313, 625, 1250, 2500 and 5000 ug/plate

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Remarks:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Remarks:

Four

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furl)acrylamide

Details on test system and experimental conditions:

A dose-finding test was conducted first. The main test was conducted twice under identical conditions. Three plates were used for each dose in each of the main tests.

Evaluation criteria:

The test substance is judged to be positive if the following three criteria are met based on the average number of the revertant colonies on the plate inoculated with each dose:
1. On the plates treated with the test substance, the number of the revertant colonies is more than twice of that obtained with the negative control.
2. The number of the revertant colonies increases as the dose of the test substance increases.
3. According to the results of the main tests conducted twice, the increase in the number of the revertant colonies demostrates reproducibility.

Statistics:

None.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No bacterial growth inhibition was observed both in the direct and metabolic activation methods.
The precipitate that seems to be the test substance was observed on all the plates inoculated with 156 ug or higher after completion of incubation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The substance is not genotoxic in a Bacterial Reverse Mutation Assay, with and without metabolic (S9) activation.

Executive summary:

The reverse mutation study was performed by the preincubation method without (direct method) or with (metabolic activation method) S9 mix using Salmonella typhimuriumTA100, TA1535, TA98, TA1537 andEscherichia coliWP2uvrAas indicator strains to examine the presence or absence of the gene mutagenicity of 2,2’,6,6’-tetra-tert-butyl-4,4’-methylenediphenol.

As growth inhibition was not observed in any strains in the dose-finding study (preliminary study), the range of the doses was established between 156 and 5000 µg/plate (common ratio 2) both in the direct and metabolic activation methods.

The test was conducted twice and no increase in the number of the revertant colonies was observed in any strains with or without metabolic activation, nor was bacterial growth inhibition observed in any strains.

Based on these results, the gene mutagenicity of 2,2’,6,6’-tetra-tert-butyl-4,4’-methylenediphenol was determined to be negative in the bacteria under the experimental conditions.

Reference 2

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:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to recent guieline and under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

other: K-1 cell line

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix from male rats

Test concentrations with justification for top dose:

Experiment 1 with and without S9 mix:
0 (DMSO solvent), 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1 micro-g/ml
Experiment 2 with S9 mix:
0 (DMSO solvent), 300, 200, 150, 100, 75, 50, 30, 10, 5 micro-g/ml
Experiment 2 without S9 mix:
0 (DMSO solvent), 300, 200, 150, 100, 75, 50, 30, 10, 5, 2.5, 1.25 micro-g/ml

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: based on a solubility test wih solvents suitable for the test system. The test substance coudl be dissolved up to 5mg/ml in DMSO after sonification for 15 min at 40 deg. C

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

cyclophosphamide in the presence of S9, mitomycin c in the absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in tissue culture flasks with culture medium

DURATION
- Exposure duration:
Experiment 1 with and without S9: 4 h
Experiment 2:
with S9: 4 h
without S9: 18 h
- Expression time (cells in growth medium): 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 16 h (2 h before the end of the total incubation period)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 micro-g/ml)

NUMBER OF REPLICATIONS: 2 independent experiments, 2 cultures per concentration

NUMBER OF CELLS EVALUATED: 100 metaphases per culturr, 200 per concentration
Three concentrations of the test substance were selected for the evaluation based on the cytotoxicity (highest should reduce mitotiic index at least 50% but not more than 70%).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Positive response criteria: concentration relaed increase or reproducible increase in number of cells with structural chromosomal aberrations.

Equivocal response: percentage of cells with structural chromosomal aberrations is statistically significantly higher than that of the negative controls (0.05
A test substance is considered clasogenic if a concentration related increase of the percentage of cells with structural chromosomal aberrations over the concurrent control frequency is observed, or if a single positive test point is observed in both tests at approximately the same dose level.

A test substance is considered negative if it produces neither a dose rleated increase in the numberstructural chromosomal aberrations, nor a reproducible positive response at any of the test points.
Both statistical significance and biologicall relevance are consideed together in the evaluation.

Statistics:

Fisher's exact probability test (two-sided)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 300 and 200 micro-l/ml precipitation of the TS occurred in cultures with and without S9 mix. Turbidity of the medium was observed down to concentrations of 50 micro-g/ml
Test concentrations selected for CA scoring:
Exp. 1 with S9 mix:
DMSO control, cyclophosphamide control 5 micro-g/ml, TS: 200, 100, 50 micro-l/ml
without S 9 mix:
DMSO control, mitomycin c control 0.1 micro-g/ml, TS: 200, 100, 50 micro-l/ml
Experiment 2:
with S9 mix:
DMSO control, cyclophosphamide control 5 micro-g/m, TS: 300, 200, 100 micro-l/ml
without S 9 mix:
DMSO control, mitomycin c control 0.1 micro-g/ml, TS: 10, 5, 2.5 micro-l/ml
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1 with S9 mitotic index reduced: 71% of controls at 200 micro-g/ml, without S9: 54% at 200 and 59 at 100 micro-g/ml
Experiment 2: with S9 mitotic index reduced: 40% of controls at 300 micro-g/ml, 49% ot 200 micro-g/ml, 80% at 100 micro-g/ml, without S9: 47% at 10 and87% at 5 micro-g/ml, 91% at 2.5 micro-g/ml.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study the test substance was not clastogenic to Chinese Hamster Ovary cells

Executive summary:

The test substance AN-2 was examined for its potential to induce structural chromosomal aberrations in Chinese Hamster Ovary (CHO) cells, in both the absence and presence of a metabolic activation system (S9-mix). Dimethylsulfoxide (DMSO) was used as vehicle for the test substance. Two chromosomal aberration tests were conducted. In all instances, duplicate cultures were used. Dose levels, ranging from 1.25 to 300 micro-g/ml, were tested. Both solubility and cytotoxicity were used for dose level selection. Mitotic index reduction was used as indication for cell toxicity.

In the first chromosomal aberration test, in both the absence and presence of a metabolic activation system (S9-mix). treatment/harvesting time was 4/18 hours (pulse treatment) and three concentrations of the test substance (50, 100 and 200 micro-g/ml) were selected for chromosomal aberration analysis. In the pulse treatment group with metabolic activation (S9-mix), the highest concentration was weakly cytotoxic to the cells. In the pulse treatment group without metabolic activation (S9- mix), the two highest concentrations were clearly cytotoxic to the cells.

In the second chromosomal aberration test, in the presence of S9-mix. treatment/harvesting time was 4/18 hours (pulse treatment) and three concentrations of the test substance (100, 200 and 300 micro-g/ml) were selected for chromosomal aberration analysis. Dose-related cytotoxicity was induced by the test substance. In the absence of S9-mix, treatment/harvest time was 18/18 hours (continuous treatment) and three concentrations of the test substance (2.5, 5 and 10 micro-g/ml) were selected for chromosomal aberration analysis. The test substance was weakly cytotoxic to the cells at the two lowest concentrations and clearly cytotoxic to the cells at the highest concentration. At the next higher concentration (30 micro-g/ml), the test substance was too cytotoxic and therefore not selected for chromosomal aberration analysis.

In both the first and second chromosomal aberration lest, the test substance AN-2 did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed. when compared to the number of aberrant cells found in the vehicle (DMSO) control cultures. In both chromosomal aberration tests, the numbers of aberrant cells. found in the vehicle (DMSO) control cultures. were within the historical range and the positive control substances mitomycin C (in the absence of the S9·mix) and cyclophosphamide (in the presence of the S9-mix) induced the expected statistically significant increases in the incidence of structural chromosomal aberrations. This demonstrates the validity of the assay. Under the conditions used in this study. the test substance AN-2 was not clastogenic to Chinese Hamster Ovary (CHO) cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP status not specified. No QA signature page or evidence of audits.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung fibroblast (CHL/IU)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

250, 400 and 1000 ug/mL

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Remarks:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: 1-methyl-3-nitro-1-nitrosoguanidine

Remarks:

Also 3,4-benzo[a]pyrene

Details on test system and experimental conditions:

A preliminary cell proliferation inhibition test was conducted using doses of 15.6-1000 ug/mL and two dishes/dose.

Chromosome aberration test:
Based on the results of the cell proliferation inhibition test, the highest dose level of the test substance was established at 1000 µg/mL (upper limit of the preparable dose of the test solution) with a total of three doses including 500 and 250 µg/mL at a common ratio of two both in the short-term and continuous treatment methods. For the positive control, 2.5 µg/mL of MNNG and 10 µg/mL of B[a]P were used.

The highest dose level of the test solution (undiluted solution) was prepared by dissolving the test substance in acetone before use and then a part of the undiluted solution was diluted serially in acetone to obtain the test solutions of the test substance of the predetermined dose levels. For the positive control, 0.5 mg/mL of MNNG and 2.0 mg/mL of B[a]P were prepared as the test solutions.
To a round plastic petri dish of 6 cm in diameter (Becton Dickinson and Co.), 5 mL of the medium containing 4 x 103 cells/mL was added. After incubation for three days, the cells were treated as described below. Four petri dishes were used for incubation for each dose. Of them, two were used for preparation of chromosomal samples and the remaining two were used for measurement of the cell proliferation rate. However, as the cell proliferation rate of the positive control was not determined, only two petri dishes were used for preparation of chromosomal samples.
In the short-term treatment without S9 mix, the medium was removed from the petri dish with 3 mL of the medium left in each petri dish, to which 0.015 mL each of acetone, the test solutions of the test substance and MNNG were added and incubated. In the short-term treatment with S9 mix, the medium was removed from the petri dish with 2.5 mL of the medium left in each petri dish, to which 0.5 mL of S9 mix and then 0.015 mL each of acetone, the test solutions of the test substance and B[a]P were added. Both with and without S9 mix, after incubation for six hours, the medium was removed and 5 mL of the fresh medium was added and incubated further for 18 hours.
In the continuous treatment method, 0.025 mL each of acetone, the test solutions of the test substance and MNNG were added to each petri dish and incubated for 24 hours.
Both in the short-term and continuous treatment of each dose, immediately after adding the test solution of the test substance to a petri dish, the oil-droplet- or scale-like precipitate and floating substance was observed which remained until the completion of the predetermined incubation period.

Evaluation criteria:

The clastogenicity was judged to be positive when the frequency of the structural chromosomal aberration detected was 10% or more.

Statistics:

None.

Species / strain:

mammalian cell line, other: Chinese hamster lung fibroblast cell lines (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The clastogenicity of 2,2'6,6'-tetra-tert-butyl-4,4'-methylenediphenol was negative in CHL/IU cells under the experimental conditions detailed in the study report.

Executive summary:

In vitrochromosomal aberration study was performed using the Chinese hamster lung fibroblast cell lines (CHL/IU) to examine the presence or absence of the clastogenicity of 2,2’,6,6’-tetra-tert-butyl-4,4’
-methylenediphenol.

A cell proliferation inhibition test was conducted at the dose range between 15.6 and 1000 µg/mL to establish the doses for the chromosomal aberration study. No cell proliferation inhibition of more than 50% was observed both in short-term and continuous treatment methods.

Therefore, the doses for the chromosomal aberration study were established at 250, 500, and 1000 µg/mL both in short-term and continuous treatment methods.

According to the results of the study, no increase in the number of chromosomally aberrant cells was observed in the short-term treatment method with and without S9 mix. In the continuous treatment method, no increase in the number of chromosomally aberrant cells was also observed after 24 hour-treament.

Based on these results, the clastogenicity of 2,2’,6,6’-tetra-tert-butyl-4,4’-methylenediphenol was determined to be negative in CHL/IU cells under the experimental conditions.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Equivalent to OECD 481. GLP study.

Qualifier:

according to guideline

Guideline:

other: Zimmermann, F.K. (1977) and Davies, P.J., Evans, W.E. and Parry, J.M. (1975)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 481 (Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mitotic recombination assay with Saccharomyces cerevisiae

Species / strain / cell type:

other: Saccharomyces cerevisiae JD1

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction

Test concentrations with justification for top dose:

0.01, 0.1, 0.5, 1.0 or 2.5 mg/ml

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: Liquid suspension cultures of log-phase cells of Saccharomyces cerevisiae JD1 were dosed with solutions of the test substance in acetone to give final concentrations.


DURATION
After 18 hours incubation at 30 ºC in the absence of S9 fraction or 2 hours at 37 ºC followed by 16 hours at 30 ºC in the presence of S9 fraction, the cultures were seeded onto the appropriate culture media for the selection of prototrophic colonies. After 3 day incubation at 30 ºC the number of prototrophic colonies were counted.

Evaluation criteria:

Values of greater than twice the control values are considered to indicate a mutagenic response.

Species / strain:

other: Saccharomyces cerevisiae JD1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Exposure of log-phase cultures of the yeast, Saccharomyces cerevisiae JD1, did not result in any consistent increase in the rate of mitotic gene conversion either in the presence or in the absence of rat liver S9 fraction. No effects on cell viability were observed at concentrations up to 2.5 mg per ml.

Conclusions:

Interpretation of results (migrated information):
negative

The results showed that the test substance did not induce mitotic gene conversion in the yeast Saccharomyces cerevisiae JD1 under the experimental conditions described.

Executive summary:

The mutagenic activity of the test substane was investigated in liquid cultures of the yeast Saccharomyces cerevisiae JD1. Assays were performed both in the presence and in the absence of and S9 microsomal fraction obtained from a liver homogenate from rats pre-treated with Aroclor 1254. It was concluded that the test substance did not induce mitotic gene conversion in the yeast Saccharomyces cerevisiae JD1 under the experimental conditions described.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Equivalent to OECD 471. GLP study.

Qualifier:

according to guideline

Guideline:

other: Ames, B.N., McCann, J. and Yamasaki, E. (1975) and Venitt, S. and Crofton-Sleigh, C. (1981)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(triplicate plating should be used instead of duplicates. However, this deviation does not affect the outcome of the study)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction

Test concentrations with justification for top dose:

31.25, 62.5, 125, 250, 500, 1000, 2000 or 4000 µg per plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium TA1535 (with and without metabolic activation)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

S. typhimurium TA1538, TA98 and TA100 (with and without metabolic activation)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: potassium dichromate

Remarks:

E. coli WP2 uvrA pKM 101 (with and without metabolic activation)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: neutral red

Remarks:

S. typhimurium TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: 20 µl volumes of solutions of the test substance in acetone were added to top agar mix to give final concentrations.

DURATION
- Exposure duration: The cultures were incubated at 37 ªc for 48-72 hours before the revertant colonies were counted.


NUMBER OF REPLICATIONS: Two replicates

Evaluation criteria:

Reproducible dose-related increases or values of 2.5 x control values or greater are considered to indicate a mutagenic response.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound formed a flakey precipitate at amounts of 500 µg per plate and above in the top agar showing that it was not totally miscible at these amounts.

The addition of 2000 µg per ml test substance (equivalent to 4000 µg per plate) caused the pH of the medium to change from 7.49 to 7.45.

Microscopical evaluation of the background lawn showed no evidence of cytotoxicity at amounts up to 4000 µg per plate either in the presence or in the absence of rat liver S9 fraction.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that the test substance did not induce reverse gene mutation in the selected bacterial tester strains under the experimental conditions described.

Executive summary:

The mutagenic activity of the test substance was investigated in agar layer cultures of selected bacterial tester strains of Salmonella typhimurium and Escherichia coli. Assays were performed both in the presence and in the absence of and S9 microsomal fraction obtained from a liver homogenate from rats pre-treated with Aroclor 1254. It was concluded that the test substance did not induce reverse gene mutation in the selected bacterial tester strains under the experimental conditions described.

Reference 6

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:

2011-06-13 to 2012-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Modern guideline study under GLP

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

other: K1

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta-Naphthoflovone induced rat liver S9 mix from male rats

Test concentrations with justification for top dose:

Preliminary cytotoxicty test: 4.14 to 1060 micro-g/ml
Main study Experiment 1 and 2:
0, 1,2, 3, 4, 8, 16, 32 micro-g/ml withe and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone;
- Justification for choice of solvent/vehicle: Acetone was the solvent that gave the maximum solubility of the TS, but it still was limited to 212 mg/ml as the maximum achievable concentration.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

EMS poitive control without S9, DMBA positve control with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension; as impregnation on paper disk

DURATION

- Exposure duration: Experiment 1 4 h with and withoput S9 (2%), Experiment 2 24 h without S9, 4 h with S9 (1%)
- Expression time (cells in growth medium): 7 days with subcultures on days 3 to 4 to maintain logarithmic growth at 37 deg C, 5% CO2
- Selection time (if incubation with a selection agent): 6-thioguanidine 10 micro-g/ml, 14 days at 37 deg. C
- Fixation time (start of exposure up to fixation or harvest of cells): 21 days

SELECTION AGENT (mutation assays): 6-thioguanidine 10 micro-g/ml
STAIN : Giemsa

NUMBER OF REPLICATIONS: 2 independent experiments, 2 cultures per concentration, 6 concentrations, vehicle, positve controls. Triplicate plating per culture for the cytotxicity and one per culture for mutagenicity during expression. 5 replicates per group were grown in the selection media, and in three replicates cytotoxicity was determined via cloning efficiency.

NUMBER OF CELLS EVALUATED: Cytotxicity: 200 cells/ 25 cm2 flask, mutagenicity 2x 10exp5 cells per 75 cm2 flask

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A concentration-related or areproducible increase in mutant frequency compared to vehicle controls.

Statistics:

not performed

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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: investigated, no effect observed
- Effects of osmolality: investigated, no effect observed
- Evaporation from medium: no
- Water solubility: very low
- Precipitation:
Experiment 1: From 16 micro-g/ml and above cloudy precipitation was observed in the cultures with and without S9 mix and at 32 micro-g/ml it was seen as precipitatein the absence of S9 mix
Experiment 2: Cloudy pecipitate was observed at the end of the exposure period in the 24 h ex[posure group (without S9)at and above 16 micro-g/ml. and in the presence of S9 after the 4 h exposure period at 32 micro-g/ml. Precipitat was seen at 32 micro-g/ml in the absence of S9 and at and above 16 micro-g/ml with S9

RANGE-FINDING/SCREENING STUDIES:
A dose range of 4.14 to 1060 ~g/ml was used in the preliminary cytotoxicity test. The maximum dose tested was limited by formulation difficulties and the use of acetone as the solvent which was used at a 0.5% dosing concentration to reduce any effect on the cells. There was no marked reduction in the cloning efficiencies (CE) in any of the three exposure groups. In the 24-hour exposure group where there was a reduction in cloning efficiency of 34% at 66 ~g/ml , this was considered to be a spurious result and of no significance since it was not dose related and was only seen in the middle of the dose range.
Precipitate was noted at the end of the exposure period in all three exposure groups.
Cloudy precipitate was observed at and above 8.28 ~g/ml in the 4-hour exposure group in the absence of 89 with more defined precipitate being seen at and above 33.13 ~g/ml which became aggregated precipitate at 530 ~g/ml. In the 4-hour exposure in the presence of 89 precipitate was observed at the end of exposure period at and above 16.56 ~g/ml which became aggregated at 132.5 ~g/ml. Cloudy precipitate was also noted at and above 33.13 ~g/ml to 132.5 ~g/ml and at and above 530 ~g/ml in this exposure group. The 24-hour exposure group had cloudy precipitate observed at and
above 8.28 ~g/ml with precipitate at and above 33.13 ~g/ml and aggregated precipitate at 1 030 ~g/ml.
Therefore, with no marked toxicity being observed the maximum dose selected for Experiment 1 and 2 was limited to the lowest dose level where clearly defined precipitate was seen and was 32 ~g/ml for all exposure groups.

Conclusions:

Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Executive summary:

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells according to recent test guidelines and under GLP. The technique used is a plate assay using tissue culture flasks and 6·thioguanine (6-TG) as the selective agent. Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and limited by the precipitation of the test item.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system. The test item demonstrated no signficant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance was tested in the Ames S. typhimurium bacterial gene mutation assay (2 studies available), the invitro genemutation assay in mammalian cells (HPRT test in CHO cells) and thein vitrochromosomal aberration test in mammalian cells (CHO cells and CHL/IU cells). All test were performed in accordance with current guidelines and under GLP with and without a metabolic activation system. The test substance consistently did not show any genotoxic activity in these test systems and is therefore concluded to be non-genotoxic. This was also confirmed in a 2 -year rat carcinogenicity assay in which no test substance related increases in tumor incidences compared to controls were observed

Justification for selection of genetic toxicity endpoint
All referenced studies are relevant for the endpoint and need to be considered

Short description of key information:
The substance did not induce gene mutations or chromosomal; aberrations in any of the 3 standard valid in vitro genetic toxicity tests that were performed according to recent guidelines and under GLP. Two additional studies available in the public domain confirmed the negative results.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance was tested in the Ames S. typhimurium bacterial gene mutation assay (2 studies available), the invitro genemutation assay in mammalian cells (HPRT test in CHO cells) and thein vitrochromosomal aberration test in mammalian cells (CHO cells and CHL/IU cells). All test were performed in accordance with current guidelines and under GLP with and without a metabolic activation system. The test substance consistently did not show any genotoxic activity in these test systems and is therefore concluded to be non-genotoxic. This was also confirmed in a 2 -year rat carcinogenicity assay in which no test substance related increases in tumor incidences compared to controls were observed.

Consequently the substance is not classified for germ cell mutagenicity according to Regulation EC 1272/2008 and amendments.