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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay

Test material

1
Reference substance name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)
EC Number:
500-607-5
EC Name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)
Cas Number:
161278-17-7
Molecular formula:
unspecified
IUPAC Name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)
Test material form:
liquid

Method

Target gene:
thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Non-activated 4 hours 0.38, 0.75, 1.5, 3, 6, 8 and 10
Non-activated 24 hours 0.38, 0.75, 1.5, 3, 6, 8 and 10
S9-activated 4 hours 2.19, 4.38, 8.75, 17.5, 35, 36, 37, 38 and 40

Based upon the results of the preliminary toxicity assay
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
The preparation and addition of the test substance dose formulations was carried out under filtered lighting during the exposure period. Treatment was carried out by combining 100 μL of test substance dose formulation, vehicle or positive control dose formulation and F0P medium or S9 mix (as appropriate) with 6 x 10^6 L5178Y/TK+/- cells in a total volume of 10 mL. All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9-activated 10-mL culture contained 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.

For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post-treatment, with cell concentration adjustment on the first day. After a 24-hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.

For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal, and 2 and 3 days after treatment.
Evaluation criteria:
• A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of ≥90 mutants/10^6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/10^6 clonable cells, a doubling of mutant frequency over the vehicle would also be required (Mitchell et al., 1997).
• A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants/10^6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, EK195 was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.
Executive summary:

The test substance, EK195, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥625 μg/mL at the beginning and end of treatment. Relative suspension growth (RSG) was 97% at concentrations of 19.5 μg/mL (4-hour treatment with S9). RSG was 0% at all higher concentrations using all treatment conditions. Based upon the steep toxicity profile observed, the concentrations chosen for the repeat of preliminary toxicity assay were 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 μg/mL (4-hour treatment with S9) and 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 and 20 μg/mL (4-hour treatment without S9 and 24-hour treatment without S9).

In the repeat of preliminary toxicity assay, no visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 19, 41 and 24% at concentrations of 36 μg/mL (4-hour treatment with S9), 5 μg/mL (4-hour treatment without S9) and 5 μg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 2.19, 4.38, 8.75, 17.5, 35, 36, 37, 38 and 40 μg/mL (4-hour treatment with S9), 0.38, 0.75, 1.5, 3, 6, 8 and 10 μg/mL (4-hour treatment without S9) and 0.38, 0.75, 1.5, 3, 6, 8 and 10 μg/mL (24-hour treatment without S9).

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 2.19, 4.38, 8.75 and 17.5 μg/mL (4-hour treatment with S9), 0.38, 0.75, 1.5 and 3 μg/mL (4-hour treatment without S9) and 0.38, 0.75, 1.5 and 3 μg/mL (24-hour treatment without S9) exhibited 42 to 93%, 66 to 103% and 60 to 108% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 40 to 86% (4-hour treatment with S9), 62 to 126% (4-hour treatment without S9) and 48 to 95% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed under any treatment condition.

These results indicate EK195 was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.