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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Feb. 10, 1986 to Feb. 14, 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No additional analyses of the composition and stability of the test sample in the solvent or test system were performed
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
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:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254 induced, rat-liver homogenate (S9
Test concentrations with justification for top dose:
Preliminary Toxicity study: 0.001, 0.003, 0.01, 0.03, 1, 3, 5 and 10 mg/plate
Definitive study: 0.0003, 0.001, 0.003, 0.01 and 0.03 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test material was soluble in acetone
Untreated negative controls:
yes
Remarks:
Acetone
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine, 9-aminoacridine, sodium azide and 2-aminoanthracene
Remarks:
with and without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Expression time: 48-72 h


NUMBER OF REPLICATIONS: Three


DETERMINATION OF CYTOTOXICITY
- Method: Growth inhibition of the background lawn or a reduction in the number of spontaneous mutants
Evaluation criteria:
If the mean number of revertant colonies was not increased to two or three times higher than the concurrent solvent control value, then the test chemical was not considered to be a bacterial mutagen.
Statistics:
No data
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
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
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Test chemical at concentration range of 0.001-10 mg/plate was evaluated for cytotoxicity using strain TA100. Toxicity assessed at 24-48 h by observations for either growth inhibition of the backgroud lawn or a reduction in the number of spontaneous mutants.


COMPARISON WITH HISTORICAL CONTROL DATA: Yes


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity in the definitive test was defined as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn.
- In the test without metabolic activation, dose-related toxicity was observed in all strains except TA1535 at 0.03 mg /plate.
- In the test with metabolic activation, some degree of toxicity was observed at 0.03 mg/plate with all strains except TA98 and TA1535.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Preliminary study: Test material at concentration of 0.03 mg/plate allowed only sparse growth of the background lawn while a slightly higher dose of 0.1 mg/plate and higher dose levels produced complete absence of the background lawn and were considered to be cytotoxic.

Conclusions:
Under the test conditions, the test material was considered to be non-mutagenic in the Salmonella/microsome bacterial mutagenicity assay.

Executive summary:

A study was conducted to evaluate the potential mutagenic activity of the test substance using the Salmonella/microsome bacterial mutagenicity assay (Ames test). The study was conducted according to OECD Guideline 471, in compliance with GLP. Test doses were chosen from the data obtained in a preliminary study which indicated that a concentration of 0.03 mg/plate allowed only sparse growth of the background lawn while 0.1 mg/plate and higher dose levels produced complete absence of the background lawn and were considered to be cytotoxic. The test substance was studied with and without metabolic activation using triplicate cultures at concentration range of 0.03-0.0003 mg/plate. Mutagenic activity was not observed with any of the five bacterial strains (S. typhimurium TA 1538, 1535, TA 1537, TA 98 and TA 100) with or without metabolic activation. Under the test conditions, the test substance was considered to be non-mutagenic in the Salmonella/microsome bacterial mutagenicity assay (Guzzie and Morabit, 1986).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
The test material was tested with and without activation in the following Salmonella strains: TA 98, 100, 1535 and 1537, and E. coli WP-2uvrA- up to the highest dose level of 1,000 µg/plate using the disc test and plate test.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable

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:
No data
Test concentrations with justification for top dose:
Highest tested dose level: 1000 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate test and disc test

Key result
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:
not specified
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the test conditions, the test material was considered to be non-mutagenic in the Ames assay.
Executive summary:

A study was conducted to evaluate the potential mutagenic activity of the test substance using the Ames assay. The test substance was tested with and without activation in the following tester strains: S. typhimurium TA 98, 100, 1535 and 1537, and E. Coli WP-2uvrA- up to the highest dose level of 1,000 µg/plate using the disc test and plate test. Under the test conditions, the test substance was considered to be non-mutagenic in the assay (Koschier, 1981).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Nov. 22, 2002 to Mar. 27, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoys media, supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum 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 applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1:
- Without S9: 0, 2.5, 5, 10, 15, 20 and 40 µg/mL
- With S9: 0, 2.5, 5, 10, 20, 30 and 40 µg/mL

Experiment 2:
- Without S9: 0, 2.5, 5, 10, 15, 20 and 40 µg/mL
- With S9: 0, 1.25, 2.5, 5, 10, 15 and 20 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 5.0 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.1 and 0.05 µg/mL for the 4 and 24-hour cultures respectively
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium


DURATION
- Preincubation period: 16 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 20 h (in Experiment 1, ±S9 and Experiment 2, +S9) and 24 h (in Experiment 2, -S9)


STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: Two


NUMBER OF CELLS EVALUATED: 1,000 cells


DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index
Evaluation criteria:
Not reported

Statistics:
No data
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 19.1 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- Precipitate of the test material was seen ≥ 52.69 µg/mL
- Metaphase cells were present at dose levels up to 19.1 µg/mL in the 4(20)-h with S9-treatment case and at up to 38.17 µg/mL in the 4(20)-h without S9-treatment case. The maximum dose with metaphases present in the 24-hour continuous exposure case was 19.1 µg/mL.
- The mitotic index data indicated that approximately 50 % inhibition was achieved at 19.1 µg/mL in all three exposure groups
Remarks on result:
other: strain/cell type: CHO-WBL cell line
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the test conditions, the test material was considered to be non-clastogenic to CHO cells in vitro.
Executive summary:

A study was conducted to determine the genotoxic potential of the test substance in CHO cells. The study was performed according to OECD Guideline 473 and EU method B10 in compliance with and OECD GLP standards. Duplicate cultures of Chinese Hamster Ovary (CHO) cells treated with the test substance were evaluated for chromosome aberrations at up to 4 dose levels, together with vehicle and positive controls. Four exposure conditions were used for this study. In experiment 1, 4 h exposure with and without metabolic activation (2 % S9) with a 20 h expression period were used. In experiment 2, 4 h exposure with and without metabolic activation (1 % S9) followed by 24 h exposure time in absence of activation. Vehicle control cultures had frequencies of cells with aberrations within the range expected for the CHO cell line. Both of the positive control substances induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any significant increases in the frequency of cells with aberrations, in either of two separate experiments, either with or without metabolic activation. Under the test conditions, the test substance was considered to be non-clastogenic to CHO cells in vitro (Durward and Jenkinson, 2003).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 Apr., 2010 to 28 Jun., 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
- Experiment 1: The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material (0.16 to 7.5 μg/ml in the absence of metabolic activation, and 0.63 to 12.5 μg/ml in the presence of metabolic activation), vehicle and positive controls.

- Experiment 2: 0.31 to 10 μg/ml in both the absence and presence of metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
* Experiment 1: 4 h, with and without S9
* Experiment 2: 4 h with S9, 24 h without S9

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cell culture counts
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Remarks on result:
other: strain/cell type: L5178Y TK+/- 3.7.2c mouse lymphoma cell line
Remarks:
Migrated from field 'Test system'.

Preliminary Toxicity Test

The dose range of the test material used in the repeat of the Preliminary Toxicity Test was 0.16 to 20 μg/ml. In all three of the exposure groups there were marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle control groups. The toxicity curve of the test material was very steep in all three of the exposure groups. Precipitate of test material was not observed at any of the dose levels. In the subsequent mutagenicity test the maximum dose level was limited by test material-induced toxicity.

Mutagenicity Test

* Experiment 1

There was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation. The levels of toxicity observed were very similar to those of the Preliminary Toxicity Test. There was also evidence of a reduction in viability (%V) in the presence of metabolic activation, therefore indicating that some residual toxicity had occurred in this exposure group. Optimum levels of toxicity were achieved in the absence of metabolic activation, and near optimum levels of toxicity were achieved in the presence of metabolic activation. In the presence of metabolic activation, a dose level that marginally exceeded the optimum level of toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. It was therefore considered that with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including the dose levels that approached or exceeded the optimum level of toxicity in the presence of metabolic activation, or in the absence of metabolic activation where optimum levels of toxicity were achieved, the test material had been adequately tested. The excessive levels of toxicity observed at 7.5 μg/ml in the absence of metabolic activation, and at 12.5 μg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

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 test material induced very modest statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in both the absence and presence of metabolic activation (Tables 3 and 6). A statistically significant increase in mutant frequency was observed at one individual dose level in the presence of metabolic activation, a dose level that exceeded the usual upper limit of acceptable toxicity. It was therefore considered that the slightly elevated mutant frequency value observed at this dose level was due to a cytotoxic mechanism rather than a true genotoxic response. It should also be noted that the GEF was not exceeded at any of the individual dose levels in either the absence or presence of metabolic activation, there was no evidence of a marked increase in the absolute numbers of mutant colonies in either of the exposure groups, and the mutant frequency values observed in both of the exposure groups would have been considered acceptable for vehicle controls.

Therefore, the responses observed were considered to be spurious and of no toxicological significance. Precipitate of test material was not observed at any of the dose levels.

*Experiment 2

There was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 9 and 12). The levels of toxicity observed were once again very similar to those of the Preliminary Toxicity Test. There was evidence of reductions in viability (%V) in the presence of metabolic activation, therefore indicating some residual toxicity had once again occurred in this exposure group. Optimum levels of toxicity were achieved in the presence of metabolic activation, and near optimum levels of toxicity were achieved in the absence of metabolic activation.

In the absence of metabolic activation, a dose level that marginally exceeded the optimum level of toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. It was therefore considered that with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including the dose levels that achieved or exceeded the optimum level of toxicity in the presence of metabolic activation, or in the absence of metabolic activation where optimum levels of toxicity were approached or exceeded, the test material had been adequately tested. The excessive levels of toxicity observed at 10 μg/ml in the absence of metabolic activation, and at and above 8 μg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Both positive controls induced acceptable levels of toxicity.

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no effect on the toxicity of the test material. 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. A very modest statistically significant dose related linear-trend response was observed in the absence of metabolic activation. However, a statistically significant increase in mutant frequency was only observed at one individual mid-range dose level, there was no evidence of any marked increases in absolute numbers of mutant colonies, the GEF value was not exceeded, and the mutant frequency values observed were within the acceptable range for vehicle controls. The response observed was therefore considered to be spurious and of no toxicological significance. A more marked statistically significant dose related linear-trend response was observed in the presence of metabolic activation. Statistically significant increases in mutant frequency were observed at two individual dose levels, one of which at the very limit of acceptable toxicity and the other exceeding the upper limit of acceptable toxicity. The increases in mutant frequency observed at these dose levels only marginally exceeded the GEF at the dose level that exceeded the upper limit of toxicity. However, there was no evidence of any marked increases in absolute numbers of mutant colonies, the GEF value was not exceeded at dose levels with acceptable levels of toxicity, and the mutant frequency values observed would have been considered acceptable for vehicle controls. It is therefore considered that the responses observed in the presence of metabolic activation were once again due to a cytotoxic mechanism rather than a true genotoxic response and, therefore, were considered to be of no toxicological significance. No precipitate of test material was observed at any of the dose levels.

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

A study was conducted to assess the potential mutagenicity of m-TMXDI on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD Guideline 476. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test substance at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test substance was selected following the results of a preliminary toxicity test and for the first experiment was 0.16 to 7.5 μg/ml in the absence of metabolic activation, and 0.63 to 12.5 μg/ml in the presence of metabolic activation. For the second experiment the dose range was 0.31 to 10 μg/ml in both the absence and presence of metabolic activation. The maximum dose level used in the mutagenicity test was limited by test substance-induced toxicity. Precipitate of test substance was not observed at any of the dose levels in the mutagenicity test. 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 substances induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test substance was considered to be non-mutagenic to L5178Y cells under the conditions of the test (Flanders, 2010).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to evaluate the potential mutagenic activity of the test substance using the Salmonella/microsome bacterial mutagenicity assay (Ames test). The study was conducted according to OECD Guideline 471, in compliance with GLP. Test doses were chosen from the data obtained in a preliminary study which indicated that a concentration of 0.03 mg/plate allowed only sparse growth of the background lawn while 0.1 mg/plate and higher dose levels produced complete absence of the background lawn and were considered to be cytotoxic. The test substance was studied with and without metabolic activation using triplicate cultures at concentration range of 0.03-0.0003 mg/plate. Mutagenic activity was not observed with any of the five bacterial strains (S. typhimurium TA 1538, 1535, TA 1537, TA 98 and TA 100) with or without metabolic activation. Under the test conditions, the test substance was considered to be non-mutagenic in the Salmonella/microsome bacterial mutagenicity assay (Guzzie and Morabit, 1986).

A study was conducted to evaluate the potential mutagenic activity of the test substance using the Ames assay. The test substance was tested with and without activation in the following tester strains: S. typhimurium TA 98, 100, 1535 and 1537, and E. coli WP-2uvrA- up to the highest dose level of 1,000 µg/plate using the disc test and plate test. Under the test conditions, the test substance was considered to be non-mutagenic in the assay (Koschier, 1981).

A study was conducted to determine the genotoxic potential of the test substance in CHO cells. The study was performed according to OECD Guideline 473 and EU method B10 in compliance with and OECD GLP standards. Duplicate cultures of Chinese Hamster Ovary (CHO) cells treated with the test substance were evaluated for chromosome aberrations at up to 4 dose levels, together with vehicle and positive controls. Four exposure conditions were used for this study. In experiment 1, 4 h exposure with and without metabolic activation (2 % S9) with a 20 h expression period were used. In experiment 2, 4 h exposure with and without metabolic activation (1 % S9) followed by 24 h exposure time in absence of activation. Vehicle control cultures had frequencies of cells with aberrations within the range expected for the CHO cell line. Both of the positive control substances induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any significant increases in the frequency of cells with aberrations, in either of two separate experiments, either with or without metabolic activation. Under the test conditions, the test substance was considered to be non-clastogenic to CHO cells in vitro (Durward and Jenkinson, 2003).

A study was conducted to assess the potential mutagenicity of m-TMXDI on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD Guideline 476. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test substance at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test substance was selected following the results of a preliminary toxicity test and for the first experiment was 0.16 to 7.5 μg/ml in the absence of metabolic activation, and 0.63 to 12.5 μg/ml in the presence of metabolic activation. For the second experiment the dose range was 0.31 to 10 μg/ml in both the absence and presence of metabolic activation. The maximum dose level used in the mutagenicity test was limited by test substance-induced toxicity. Precipitate of test substance was not observed at any of the dose levels in the mutagenicity test. 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 substances induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test substance was considered to be non-mutagenic to L5178Y cells under the conditions of the test (Flanders, 2010).

Justification for classification or non-classification

m-TMXDI did not demonstrate any mutagenic or clastogenic activity in guideline-compliant in vitro studies. Based on these results, m-TMXDI is considered to be non-genotoxic. No classification is therefore required for this endpoint.