Bis(2,4-di-tert-butyl-6-methylphenyl)ethyl phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in the Ames test in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA, tested both in the absence and presence of S9 mix (Ciba-Geigy 1992). No genotoxicity was observed in a HPRT test (Ciba-Geigy 1994) and in a cytogenetic Test with Chinese Hamster Cells (Ciba-Geigy 1993).

Link to relevant study records

Reference

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:

1993-08-30 to 1994-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study (OECD Guideline 476)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted on 04-Apr-1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F10 Medium supplemented with 10% pre-tested foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin
- 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:

Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Cytotoxicity test
Range with and without metabolic activation:
0.73 to 1500 µg/mL

Mutagenicity test (original and confirmatory experiment):
Range with and without metabolic activation:
55.56 to 1500 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

With metabolic activation

Migrated to IUCLID6: 1.0 µL/mL (DMN)

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Migrated to IUCLID6: 0.3 µL/mL (EMS)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Exposure duration: with S9: 5 hours; without S9: 27 hours.
- Expression time (cells in growth medium): seven to eight days

SELECTION AGENT: The selection medium was growth medium to which 6-thioguanine (6-TG) was added to a final concentration of 8 µg/mL.

NUMBER OF REPLICATIONS: two

DETERMINATION OF CYTOTOXICITY
cloning efficiency

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.

Criteria for a positive response. The test substance will be considered to be mutagenic if:
• The assay is valid (see assay acceptance criteria)
• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.
• There is a significant dose-relationship as indicated by the linear trend analysis.
• The effects described above are reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- CYTOTOXICITY TEST
- Highest concentration: 1500 µg/mL
- Higher concentrations produced non tolerable precipitates in the culture medium. In the part with metabolic activation, at the highest concentration of 1500 µg/mL an acute growth inhibiting effect of 47.14% could be seen, while the next lower concentration inhibited 27.66%. Without metabolic activation treatment with the test substance revealed an inhibition of 25.79 % at the highest concentration. The next lower concentration revealed an acute inhibition of growth of 28.25%. Accordingly, 1500 µg/mL with and without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

MUTAGENICITY TEST WITH METABOLIC ACTIVATION
-Highest concentration: 1500 µg/mL
- Higher concentrations produced non tolerable precipitates in the culture medium
- Mean growth inhibiting values after treatment and expression: 5.05% and 2.83%.

MUTAGENICITY TEST WITHOUT METABOLIC ACTIVATION
- Highest concentration: 1500 µg/mL
- Higher concentrations produced non tolerable precipitates in the culture medium
- Mean growth inhibition values after treatment and expression: 1.12% and 10.33%

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Remarks on result:

other: strain/cell type: As described above

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames Test

The mutagenic potential of the test substance was investigated in a GLP-compliant Ames test (Ciba-Geigy 1992) according to OECD 471, performed on Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9 mix). None of the tested concentrations ( 312.5 to 5000 µg/plate) led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control either with or without metabolic activation. This result was confirmed in a second independent experiment. Therefore, based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Chromosome Aberration Test

In a chromosome aberration test (Ciba-Geigy 1993) according to OECD 473 and in compliance with GLP, the substance was investigated for clastogenic effects on Chinese hamster ovary cells in vitro. In studies performed without metabolic activation using 18 and 42 hours incubation no biologically significant increase in the number of specific chromosome aberrations was observed. In three experiments performed in the presence of a metabolic activation system (3 hours treatment and 15 or 39 hours recovery time), no biologically significant increase in the number of specific chromosome aberrations was observed. The number of chromosome aberrations was within the historical control range at all doses assessed. Therefore, it is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro.

HPRT Test

In a mammalian gene mutation test (Ciba-Geigy 1994) the test article was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiment without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment. In all experiments performed comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies. Based on this result and under the given experimental conditions, it is concluded that the test article and its metabolites did not show any mutagenic activity in this forward mutation system.

Justification for selection of genetic toxicity endpoint
The most recent GLP-compliant guideline study was selected, although all three studies are required for assessment.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.