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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted per standard procedures for this assay and GLP guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Published methods including those of Ames (1975) and Green (1976)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Base pair substitution mutations at hisG locus - TA1535 & TA100 salmonella strains; frameshift mutations at the hisD locus - TA1538 & TA98 Salmonella strains; frame shift mutations at hisD locus - TA1537 strain; base pair substitution mutations at trp locus - E. coli WP2 uvrA strain
Test concentrations with justification for top dose:
Ranged from 0.167 to 167 ug/plate. Test article appeared to be partially insoluble >50 ug/plate
Vehicle / solvent:
DMSO was used as it solubilized the test substance to a satisfactory degree, and is commonly used in the Ames assay.
Details on test system and experimental conditions:
Preincubation procedure was used. This entailed mixing aliquot of tester strain, media, and test article - with or without rat liver S9 enzyme system. This combination was incubated at 30 degrees C for 30 minutes. The mixture was then added to molten top agar, and added to minimal glucose plates. Plates were then incubated for 48 hr at 37 degrees C for colony development. Colonies were counted with use of Artek electronic colony counter. Cultures were run in triplicate. Mutations were expressed in the absence of tryptophan (E. coli) or histidine (Salmonella) in incubation media .

A toxicity prescreen was performed in the absence of S9 only in TA 1538, TA100 & E. coli strains. Levels of test material were 50, 167, 500, 1670 & 5000 ug/plate. Test material was not toxic up to level of 167 ug/plate in E. coli. Severe toxicity was seen in Salmonella at 500 ug/plate and at 167 in TA100. Slight to moderate toxicty was seen at 50 ug/plate as judged by reduced background lawn or presence of pindot colonies.
Evaluation criteria:
A positive effect was defined as statistically significant dose-dependent increase in # of revertants with at least one dose level inducing frequency two-fold of spontaneous (solvent) control value. Assay results must be replicated to confirm positive results.
Statistics:
Mean values were calculated for triplicate determinations, and standard deviations calculated. Criteria for positive mutagenic activity requires determination of whether results for test chemical exceed 2x control levels.

Results and discussion

Additional information on results:
POTENTIAL CONFOUNDING FACTORS None apparent. It was recognized that test cmpd has low water solubility, and that precipitation was noted & reported in high exposure samples. The range-finding test showed significant cytotoxicity and insolubility at 50 ug/plate or above in 2 of 3 bacterial strains tested . 50 ug/plate was then chosen as high dose for all assays.
Remarks on result:
other: Details on the test results is presented in section "Any other information on results"

Any other information on results incl. tables

Bacterial Mutation Assay 1 ( # revertant)
Dose Level S9 TA1535 TA1537 TA1538 TA98 TA100  E.coli
Solvent  -  5 5 6 21 88 4
0.167  - 6 7 10 22 99  -
0.5  - 5 5 7 19 97  -
1.67  - 7 3 7 14 92  -
5  - 6 6 3 19 90 3
16.7  - 7 a/b 7 a 4 a/b 13 a/b 70 a/b 3
50  - 3 b/c 2 a/b 5 a/b 21 a/b 57 b 4
167  -  -  -  -  -  - 2 a
500  -  -  -  -  -  - 4 a
1000  -  -  -  -  -  - 4 b/c
Solvent  + 5 9 16 26 104 4
0.167  + 7 7 20 30 118  -
0.5  + 5 7 15 27 115  -
1.67  + 7 9 15 22 105  -
5  + 10 6 15 36 99 4
16.7  + 7 11 37 a 47 a 119 a 3
50  + 9 a 14 a/b 94 a 80 a 102 a/b 5
167  +  -  -  -  -  - 3
500  +  -  -  -  -  - 3 a/b
1000  +  -  -  -  -  - 5 a/b
a/b/c - slight/moderate/severe toxicity

Bolded/italics - value 2x control value

Bacterial Mutation Assay - 2 (# revertants

Dose Level

S9 TA1535 TA1537 TA1538 TA98 TA100  E.coli
Solvent  -  8 5 7 23 86 4
0.167  - 11 6 7 22 95  -
0.5  - 8 5 6 24 101  -
1.67  - 5 10 5 17 80  -
5  - 8 5 6 18 86 3
16.7  - 5 a/b 7 a 3 a/b 16 a 48 a 3
50  - 5 b/c 4 a/b 4 a/b 18 a/b 35 b/c 4
167  -  -  -  -  -  - 3
500  -  -  -  -  -  - 3 a
1000  -  -  -  -  -  - 3 a/b
Solvent  + 13 13 13 31 99 3
0.5  + 8 3 18 25 110  -
1.67  + 9 9 16 25 86  -
5  + 13 8 19 23 108 4
16.7  + 9 10 27 32 111 3
50  + 15 9 57 a/b 57 a 103 a 4
167  + 8 a/b 21 a/b 152 a/b 185 a/b 88 a/b 5
500  +  -  -  -  -  - 4
1000  +  -  -  -  -  - 4 a/b
a/b/c - slight/moderate/severe toxicity

Bold italics - 2x control level

Applicant's summary and conclusion

Conclusions:
The test material is considered to induce frameshift gene mutations in an Ames (Salmonella) assay in the presence of an enzyme activation system.
Executive summary:

The test substance was subjected to the Ames tests according to standard practices. Through use of positive controls, the bacterial strains used were shown to be active. Increased numbers of revertants were seen in TA1538 & TA98 strains in replicate assays, suggesting mutations were frameshift. This activity was observed only in the presence of S9 activating enzymes. The results for this test substance met criteria for a positive response by virtue of (a) exhibiting dose-responsiveness for # of revertants, (b) one or more concentrations of test chemical induced # of revertants in excess of 2-fold above control values, and (c) results were duplicated in a second assay.