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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:
17 Dec 1992 - 05 Apr 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given. Only four strains tested.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted September 19, 1984
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dimer Acid
- Physical state: viscous liquid
- Analytical purity: no data
- Storage condition of test material: ambient conditions in the dark
- Other: golden color

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate from Sprague-Dawley rats
Test concentrations with justification for top dose:
Toxicity assay: 0.5, 5, 50, 500, 5000 μg/plate
Mutation assay:
50, 150, 500, 1500, 5000 μg and 1000, 2000, 3000, 4000, 5000 μg/plate
Vehicle / solvent:
95 % ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9: 2-aminoanthracene (1 and 2 µg/plate); -S9: 2-nitrofluorene (5 µg/plate), sodium azide (2 µg/plate), 9-aminoacridine (20 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: triplicate performed at least twice

DETERMINATION OF CYTOTOXICITY
- Method: microcolony formation method


Evaluation criteria:
The average number of mutant colonies per plate was compared with the average number of spontaneous revertants in the control. A dose-related increase in the number of colonies which reaches at least a doubling of the control values is usually considered to be a positive response.

Results and discussion

Test results
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For tables of results refer to attached background material.

Preliminary toxicity assay

 

For all strains tested, the top dose level of 5000 µg/plate could not be scored as the bacterial lawn was obscured by precipitate. However, this dose was included in the mutagenicity test as it was considered unlikely that the precipitate would interfere with scoring of revertant colonies. No evidence of significant toxicity was seen in the presence or absence of metabolic activation at the lower doses.

 

Mutagenicity assay

 

The mutagenicity assay was performed twice both with and without addition of S9 for all four strains. Third, fourth and fifth assays were performed for TA1537 and TA98 as the results of the first two assays were contradictory.

 

A significant increase in revertant colonies was also seen in a fourth assay with TA1537 at 2000 µg/plate with 30% S9. However, this was not dose-related or reproduced in two other assays at this dose level. Therefore, this increase was also considered to be spurious. In the first assay with strain TA98, a significant increase in the number of revertants was seen at 5000, 1500, and 500 µg/plate with 30% S9 and at 5000, 1500, 500 and 150 µg/plate with 10% S9. This was not reproduced in a further four assays and was also thought to be a spurious result.   In the first and fourth assays with strain TA1537 the positive control mutagen at 30% S9 mix did not induce a doubling of revertant colonies over the contr No increase in the number of revertant colonies was seen with strains TA1535, or TA100 in either assay in the presence or absence of S9. Strain 1537 showed a significant increase in revertant colonies in the first assay at 5000 µg/plate and 30% S9 only. However, this response was not reproducible in four further assays and is thought to be a spurious result. No increase was seen in the presence of 10% S9 or in the absence of S9. ol value. As high S9 levels are known to reduce the potency of 2-aminoanthracene, a positive response was indicated at the 10% S9 level for the first assay and the number of revertant colonies was noticeably increased for the fourth assay, these results are considered acceptable.   The reason for the spurious results seen with the test substance in the presence of metabolic activation with strains TA1537 and TA98 are unclear. The test substance is a viscous liquid which forms a precipitate at high dose levels on the agar plates used in this study. The precipitate closely resembles bacterial colonies and great care was needed to differentiate between precipitate and revertant colonies. It was possible that some precipitate was scored as revertant colonies in some assays.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

As some significant increases seen in revertant colonies were irreproducible, it is concluded that under the conditions of this study Dimer Acid did not induce mutations in Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98.