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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 97%
- Source: Aldrich Chemical Company, Milwaukee, WI, USA
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DSM Engineering Plastics B.V. lotnumber SN0620140520
- Expiration date of the lot/batch: 2016.07.01
- Purity test date: 99.93%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in a cool area (IO-20°C). Containers that have been opened must be filled with dry nitrogen for at least 1min and then sealed. Be careful not to import any water or impurities during the filling and sealing course.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Easily soluble in cold water


FORM AS APPLIED IN THE TEST white waxy solid

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells:Molecular Toxicology Inc. US
Additional strain characteristics:
other: histidine deficient
Metabolic activation:
with and without
Metabolic activation system:
system(89 mix)
Test concentrations with justification for top dose:
5000, 1500, 500, 150, 50 and 15 pg/plate
Vehicle:
- Vehicle(s)/solvent(s) used:ultrapure water (H20)
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
other: Dexon (CAS-no. 140-56-7
Remarks:
Used in the tester strains TA97a, TA98 and TA102 in the absence of SQ mix (89-).
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used in the tester strains TA100 and TA1535 in 89-.
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene (2-AF)
Evaluation criteria:
CRITERIA OF POSITIVE RESULT
1) When there is a concentration-related increase over the range (two times greater than the corresponding solvent control in TA97a, TA98, TA100, TA102 and three times greater than the corresponding solvent control in TA1535) in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.
2) When there is a reproducible increase (two times greater than the corresponding solvent control in TA97a, TA98, TA100, TA102 and three times
greater than the corresponding solvent control in TA1535) at one or more concentrations in the mean number of revertant colonies in at least one strain
with or without metabolic activation system, the test item should be evaluated as positive.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test item was non-toxic to all the tester strains and soluble at the concentration of 50mg/ml in H20, so H20 was used as solvent, and six dose levels of 5000, 1500, 500, 150, 50 and 15 micro g/plate were set in the first test.

Any other information on results incl. tables

METABOLIC ACTIVATION SYSTEM

In this study, Aroclor 1254 induced rat liver 89 prepared in-house was used as

the metabolic activation system.

PREPARATION OF CULTURE MEDIAAND SOLUTIONS

PREPARATION OF POSITIVE CONTROLS

THE PRELIMINARY TEST

In the test, according to the solubility of the test item. H20 was used as solvent. The standard plate incorporation method was performed at four dose levels, including 5000, 1667.556 and 185ug/plate, in five tester strains of TA97a, TA98, TA100, TA102 and TA1535, with and without metabolic activation system. The dose volumes of each dose group and solvent control group were 0.1 ml/plate, in duplicate.

The results showed that there was no test item precipitate at all doses before and after the incubation.

Dose selection

The test item was non-toxic to all the tester strains and soluble at the concentration of 50mg/ml in H20, so H20 was used as solvent, and six dose levels of 5000, 1500, 500, 150, 50 and 15 microg/plate were set in the first test.

Enrichment culture

One day before the plate-incorporation, the stored tester strains were thawed and cultivated in nutrient broth medium for approximately 16 hours at 36.8~37.1°C.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the results both in the first test and the validation test were negative. Thus, the test item, Succinonitrile is considered
to be non-mutagenic in the bacterial reverse mutation assay using Salmonella
typhimurium tester strains.
Executive summary:

Introduction. The study was performed to evaluate the ability of Succinonitrile to induce reverse mutations in the genome of the Salmonella typhimurium tester strains with and without the metabolic activation

system(89 mix), and the method was designed to be compatible with the

OECD Guideline for the Testing of Chemicals No.471 “Bacterial Reverse Mutation Test" (July 21, 1997).

Method. Five histidine deficient (his-) mutant tester strains of Salmonella typhimurlum including TA97a, TA98, TA100, TA102 and TA1535 were treated with Succinonitrile using the standard plate incorporation method

and the preincubation method at six dose levels, in triplicate, with positive and solvent controls, both in the presence and absence of the cofactor—supplemented 89 (89 mix).

Based on the results of the preliminary test (initial toxicity test) that had been performed in this lab before, six dose levels in the first test were selected including 5000, 1500, 500, 150, 50 and 15 pg/plate, and sterile ultrapure water (H20) was used as solvent. Then the validation test was conducted using the same dose levels and solvent as the first test.

Results. In the first test, the results of the viable count showed that the density of the cultures for each tester strain was within 0.9~9>< 109 colony forming units (CFU)/m| and was considered acceptable. At the same time,

all results of positive and solvent controls met the requirements of this test, so the sensitivity of the assay and the efficacy of the 89 mix were validated.

In the first test, no test item precipitation was observed on the the surface of GM agar at each dose level before and after the incubation. In addition, no cytotoxicity was detected at any dose level in all tester strains.

In the first test, the mean number of the revertant colonies for any tester strain with any dose of test item, either with or without SQ‘mix, was less than two times (three times for TA1535) of the mean number in the

corresponding solvent control. In the validation test the same results were obtained as in the first test.

Conclusion. Under the conditions of this study, the results both in the first test and the validation test were negative. Thus, the test item, Succinonitrile is considered to be non-mutagenic in the bacterial reverse

mutation assay using Salmonella typhimurium tester strains.