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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
Study period:
1993-07-02 to 1993-08-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
2-Aminoanthracene was used as a positive control for all stains with S9 (OECD 471 notes that 2-AA should not be used as the sole indicator of the efficacy of the S-9 mix), however, the S9 mix was evaluated with other positive controls prior to testing (non-concurrent). Not all of the tests had 5 concentrations that were not cytotoxic, however, there was no indication that an increase in revertant colonies would have been seen even if more non-cytotoxic doses had been included.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Minor deviations relating to positive control used with-S9 and the number of non-toxic dose levels
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: amber visous liquid
- Storage condition of test material: room temperature

Method

Target gene:
Histidine locus in Salmonella, tryptophan locus in E.coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor S9
Test concentrations with justification for top dose:
0, 0.5, 1.67, 5.0, 16.7, 50.0, 167 ug/plate in the preliminary test for all strains
0, 0.05, 0.167, 0.5, 1.67, 5.0, 16.7 ug/plate in test 1 for all salmonella strains
0, 0.167, 0.5, 1.67, 5.0, 16.7, 50.0 ug/ml in test 1 for E.coli
0, 0.05, 0.167, 0.5, 1.67, 5.0, 16.7 ug/plate in a confirmatory test salmonella strain TA1538
0, 1.67, 5.0, 16.7, 50.0, 167, 500 ug/plate in a confirmatory test for the other salmonella strains and E.coli
0, 0.5, 1.67, 5.0, 16.7, 50.0, 100 ug/plate in a third test in salmonella strains TA1535, TA1537, TA98 and TA100 with S9 only
0, 0.167, 0.5. 1.67, 5.0, 16.7, 50.0, 100 ug/plate in a third test in E.coli with S9 only
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article was soluble in DMSO but insoluble in water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 10.0 ug/plate TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: 150 ug/plate TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9

Migrated to IUCLID6: 5.0 ug/plate TA98 and TA1538
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9

Migrated to IUCLID6: 2.0 ug/plate E.coli WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine 2.5 ug/plate for Salmonella, 80 ug/plate for E. coli
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation for 30 minutes prior to plating

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates per dose level

NUMBER OF CELLS EVALUATED: Approximately 1 x 10E8 cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of agar plates for reduced growth of the background lawn, pindot colnies

Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance estalished at the 95% confidence limit. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan-independent revertants.
Statistics:
Snee RD and JD Irr (1981) Design of a statistical method for the analysis of mutagenesis at the hypoxanthine guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells, Mutation res., 85:77-93.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In an initial preliminary toxicity test the test material precipitated at 500, 1670 and 5000 ug/plate but these were also toxic dose levels.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The data tables from the study report are attached as a Word file. For some strains in some tests, either with or without S9, there were fewer than four dose levels without toxicity. However, in most cases the toxicity was limited to a modest effect on the growth of the background lawn of bacteria and the frequency of revertant colonies was not affected.

2-Aminoanthracene was used as a positive control for all strains with S9, whereas OECD 471 notes that 2-AA should not be used as the sole indicator of the efficacy of the S-9 mix, however, the S9 mix was evaluated with other positive controls prior to testing (non-concurrent), which provides confidence in the results with-S9.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results for the test item were negative in the Ames/Salmonell-E.coli Liquid Pre-incubation assay under the conditions, and according to the criteria, of the test protocol.
Executive summary:

Test Guidance

OECD Guideline 471

Method and Materials

The test item was evaluated in the Ames/Salmonella-E.coli liquid Pre-incubation assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella (TA1535, TA1537, TA1538, TA98 and TA100) and at the tryptophan locus in one Escherichia coli strain (WP2 uvrA), in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of the test material was initially evaluated in a prescreen by treating duplicate cultures of strains TA1538, TA100 and WP2 uvrA with the test article at doses of 50, 167, 500, 1670 and 5000 ug/plate in the absence of S9. Results of the pre-screen indicated the test material produced inhibited growth (characterised by a reduced background lawn and/or the presence of pindot colonies) or complete toxicity in all three tester strains at all doses evaluated. In addition, the test article was incompletely soluble at dose >= 500 ug/plate. Therefore, the test item was re-evaluated under identical conditions at doses of 0.5, 1.67, 5.0, 16.7, 50 and 167 ug/plate. The test item again produced inhibited growth or complete toxicity in strains TA1538 and TA100 at dose >= 5.0 ug/plate, and in strain WP2 uvrA at doses >= 16.7 ug/plate.

Based upon these findings, the test item was evaluated in triplicate cultures in all five Salmonella strains at doses of 0.05, 0.167, 0.5, 1.67, 5.0, 16.7 ug/plate, and in strain WP2 uvrA at doses of 0.167, 0.5, 0.67, 5.0, 16.7 and 50 ug/plate, in the presence and absence of S9. A further confirmatory test using modified dose ranges was performed both in the presence and absence of S9 and a third test was performed with all strains in the presence of S9 only.

Results

Toxicity was observed that varied both between strains and between the exposure conditions in the presence and absence of S9. However, overall a satisfactory number of analysable dose levels was achieved in most cases, both in the presence and absence of S9. There were no statistically significant and dose-related increases in the frequency of revertant colonies seen for any strain of bacteria, either in the presence or absence of S9.

Conclusion

Therefore, the results for the test item were negative in the Ames/Salmonella-E.coli Liquid Pre-incubation assay under the conditions, and according to the criteria, of the test protocol.