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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
Study period:
28 September 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
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 gene mutation tests using the thymidine kinase gene

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
EC Number: 948-020-7
Specific details on test material used for the study:
-Purity: > 99% (UVCB)
-Description: Translucent blackish yellow liquid
-Storage conditions:Room temperature, protected from light

Method

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:
In the intial preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL. Visible precipitate was observed at a concentration of ≥1250 µg/mL by the end of treatment. Due to excessive toxicity in all treatment conditions, the preliminary toxicity assay was repeated with the following concentrations 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL.

In the preliminary toxicity retest, no visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 36, 8 and 78% at concentrations of 5 µg/mL (4-hour treatment with S9), 2.5 µg/mL (4-hour treatment without S9) and 1.25 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 0.38, 0.75, 1.5, 2.5, 4.5, 6 and 8 µg/mL (4-hour treatment with S9), and 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 µg/mL (4 and 24-hour treatments without S9).
Vehicle / solvent:
Tetrahydrofuran
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
L5178Y/TK+/- cells were exposed to the vehicle alone in duplicate cultures and nine to ten concentrations of test substance using single cultures. The maximum concentration evaluated was based on toxicity of the test substance in the vehicle. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Osmolality of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured at the beginning of treatment for the A1. Precipitation was determined with the unaided eye at the beginning and end of treatment. Dose levels for the definitive assay were based upon post-treatment cytotoxicity (growth inhibition relative to the vehicle control).
Evaluation criteria:
The cytotoxic effects of each treatment condition were expressed relative to the vehicle-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency for each treatment condition was calculated by dividing the mean number of colonies on the TFT-plates by the mean number of colonies on the VC-plates and multiplying by the dilution factor (2 x 10 4), and was expressed as TFT-resistant mutants/106 surviving cells. The induced mutant frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant frequency of the vehicle control cultures. The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response at an IMF of ≥90 mutants/106 clonable cells at the Aberdeen meeting in 2003 (Moore et al., 2006).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
Definitive re-test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test material 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 material was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cellsin the presence and absence of an exogenous metabolic activation system. Tetrahydrofuran (THF) was used as the vehicle.

In the intial 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 ≥313 µg/mL at the beginning of treatment and at concentrations ≥1250 µg/mL by the end of treatment. Due to excessive toxicity in all treatment conditions, the preliminary toxicity assay was repeated with the following concentrations 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL.

In the retest of preliminary toxicity assay, no visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 36, 88 and 78% at concentrations of 5 µg/mL (4-hour treatment with S9), 1.25 µg/mL (4-hour treatment without S9) and 1.25 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 0.38, 0.75, 1.5, 2.5, 4.5, 6 and 8 µg/mL (4-hour treatment with S9), and 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 µg/mL (4 and 24-hour treatments without S9).

In the initial definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 0.38, 1.5, 2.5, 4.5 and 6 µg/mL (4-hour treatment with S9) exhibited 12 to102 % RSG, and were cloned. Relative total growth of the cloned cultures ranged from 10 to 94% (4‑hour treatment with S9). No increases in induced mutant frequency ≥90 mutants/106clonable cells were observed under 4-hour treatment with S9.   

Cultures treated at 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 10 µg/mL had no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 0.25, 0.5, 1 and 2 µg/mL (4-hour treatment without S9) and 0.25, 0.5, 1 and 2 µg/mL (24-hour treatment without S9) exhibited 30 to 91% and 38 to 85% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 33 to 102% (4-hour treatment without S9) and 41 to 81% (24‑hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/106clonable cells were observed under any treatment condition. 

These results indicate Reaction products of 2,5-dimercapto-1,3,4-thiadiazole, sodium salt, with 1-octanethiol and hydrogen peroxide 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.