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

Genetic toxicity: in vitro

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 04-Apr-1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

416-140-4

EC Name:

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

Cas Number:

145650-60-8

Molecular formula:

C32H51 O3 P

IUPAC Name:

bis(2,4-di-tert-butyl-6-methylphenyl) ethyl phosphite

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene

Species / strain

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

Controls

Controls 1

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)

Controls 2

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.

Results and discussion

Test results

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'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative