Registration Dossier

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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
mouse lymphoma assay using L5178Y cell line as described by Clive et al., 1979.

Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.M. (1979). Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutat. Res 59:61-108
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
EPON 828 (25068-38-6)

Method

Target gene:
thymidine kinase gene (TK+/-)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
The S-9 fraction was obtained from the livers of male Sprague-Dawley rats that were dosed with 500 mg/kg body weight of Aroclor 1254 and killed five days later.
Test concentrations with justification for top dose:
0.0018-0.013 mg/mL nonactivated; 0.032-2.4 mg/L activated
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
no
Remarks:
Ethylmethane sulfonate without S9 and Benzo(a)pyrene with S9
Positive control substance:
other: Ethylmethane sulfonate without S9 and Benzo(a)pyrene with S9
Details on test system and experimental conditions:
Cells were treated with various test material concentrations, a negative control (vehicle) and a positive control (Ethylmethane sulfonate) concurrently for toxicity and mutagenicity.

Cells were counted on test day 2 for colony density. Cultures were selected for cloning for treated and both control groups, plated, and cultured for 10 days to determine viability. Mutant counts were recorded, and tutant frequency was calculated.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity was noted in suspension growth at 0.18 mg/ml and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Chemical analyses were performed on samples of EPON 828 used to dose the cells. The nominal concentration of both solutions was 100 mg/ml and the actual amount, as determined by analysis, was 104 +/- 1 mg/ml for Assay II (without S9) and 103 +/- mg/ml for Assay III (with S9). No anal;ysis was done on samples from Assay I as this was used as a preliminary assay to determine toxicity and establish dose levels for the mutagenicity assays (II and III).



In the absence of S-9 and EPON 828, there was a toxic effect on both suspension growth and soft agar growth and an increase in the mutant frequency at concentrations of 0.003 mg/ml and higher. The assay was repeated (Assay II) with 0.0075 mg/ml as the highest concentration and an additional five concentrations in decreasing 1/8 log10 dilutions. The results were similar to those obtained in the preliminary assay. There was evidence of a toxic effect on suspension growth and on soft agar growth commencing at 0.0024 mg/ml and a dose-related increase in mutants at all concentrations in the absense of S-9.



When EPON 828 was exposed to the cells in the presence of metabolic activation, no toxicity was noted on suspension growth or soft agar growth in Assay I at concentrations up to 0.1 mg/ml and there was no increase in the mutant frequency at any concentrations. Therefore in the repeat assay (III) a higher concentration, 0.24 mg/ml, was used and the other doses were set at 1/8 log 10 dilutions therefrom. Toxicity was apparent in suspension growth at 0.18 mg/ml and higher and in soft agar growth at 0.24 mg/ml. No increase in mutant frequency which exceeded 2-fold the background was noted at any concentration tested in the presence of S-9 metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Chemical analyses were performed on samples of EPON 828 used to dose the cells. The nominal concentration of both solutions was 100 mg/ml and the actual amount, as determined by analysis, was 104 +/- 1 mg/ml for Assay II (without S9) and 103 +/- mg/ml for Assay III (with S9). No anal;ysis was done on samples from Assay I as this was used as a preliminary assay to determine toxicity and establish dose levels for the mutagenicity assays (II and III).

In the absence of S-9 and EPON 828, there was a toxic effect on both suspension growth and soft agar growth and an increase in the mutant frequency at concentrations of 0.003 mg/ml and higher. The assay was repeated (Assay II) with 0.0075 mg/ml as the highest concentration and an additional five concentrations in decreasing 1/8 log10 dilutions. The results were similar to those obtained in the preliminary assay. There was evidence of a toxic effect on suspension growth and on soft agar growth commencing at 0.0024 mg/ml and a dose-related increase in mutants at all concentrations in the absense of S-9.

When EPON 828 was exposed to the cells in the presence of metabolic activation, no toxicity was noted on suspension growth or soft agar growth in Assay I at concentrations up to 0.1 mg/ml and there was no increase in the mutant frequency at any concentrations. Therefore in the repeat assay (III) a higher concentration, 0.24 mg/ml, was used and the other doses were set at 1/8 log 10 dilutions therefrom. Toxicity was apparent in suspension growth at 0.18 mg/ml and higher and in soft agar growth at 0.24 mg/ml. No increase in mutant frequency which exceeded 2-fold the background was noted at any concentration tested in the presence of S-9 metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

EPON 828 was a direct acting mutagen in the mouse lymphoma gene mutation assay and such activity was eliminated by the addition of a metabolizing enzyme fraction derived from rat liver.
Executive summary:

EPON 828 was tested in the mouse lymphoma cell mutagenicity assay for its ability to induce gene mutation at the thymidine kinae locus in the absence and presence of activation by a rat liver microsome (S-9) fraction. Cells were exposed to concentrations of EPON 828 up to and including those which resulted in toxicity to the cells.

In the absence of activation, EPON 828 in DMSO produced a dose-related cytotoxic effect at concentrations of 0.0024 mg/ml and higher and a dose-related increase in the mutant frequency at concentrations from 0.0018 to 0.0056 mg/ml. The positive control (EMS, 0.8 mg/ml) produced a mutant frequency approximately 20 times that of the untreated control. In the presence of activation EPON 828 produced cytotoxic effects at concentrations of 0.18 mg/ml and higher; however, there was no increase in mutant frequency at any concentration tested. The positive control (P[a]P, 0.005 mg/ml) produced mutant frequency values greater than twofold that of the untreated control.

It can be concluded that APON 828 is a direct acting mutagen in the mouse lymphoma gene mutation assay and that this activity is removed by the addition of a rat liver metabolizing enzyme fraction.