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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:
16 June 1995 - 02 February 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A bacterial strain with an AT base pair at the primary reversion site (e.g. S. typhimurium TA102 or E. coli WP2uvrA) was not included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
The study report does not state the study was conducted in accordance with a particular published test guideline, but the methodology used is largely consistent with the 1997 OECD TG 471 (plate incorporation method), except that a strain with an AT base pair at the primary reversion site (e.g. S. typhimurium TA102 or E. coli WP2uvrA) was not included.
2-aminoathracene was used as the sole indicator of the efficacy of the S9 mix in the assay. It is not clear whether prior to assay initiation, the S9 underwent any other characterisation with a mutagen that requires activation by microsomal enzymes.
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):
- Substance type: Complex reaction product.
- Physical state: Liquid
- Analytical purity: The test material was considered to be "pure".
- Impurities (identity and concentrations): NA
- Composition of test material, percentage of components: NA
- Isomers composition: NA
- Purity test date: NDA
- Lot/batch No.: NDA
- Expiration date of the lot/batch: April 2000.
- Stability under test conditions: NDA
- Storage condition of test material: Room temperature.
- Other:

Method

Target gene:
The target genes in the Salmonella strains control the synthesis of histidine.
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
Metabolic activation:
with and without
Metabolic activation system:
rat S9 (Arochlor 1254 pretreated Sprague Dawley rats)
Test concentrations with justification for top dose:
50, 100, 500, 1000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone (Lot No. E41620) was used for the test substance.
DMSO (Lot No. 06935PZ) was used for the positive control substances.

- Justification for choice of solvent/vehicle:
A solubility test was performed to assess the solubility of the test substance in tetrahydrofuran (THF), methyl sulfoxide (DMSO), water, and acetone. Solubiity testing indicated the test substance was soluble in DMSO, and toxicity and initial assays were performed using DMSO as the vehicle. The results from the initial concentration verification were received after these assays were performed and indicated that the test substance was not completely soluble in DMSO. Therefore, the toxicity and initial assays were repeated using acetone as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
used with TA1537 without S9

Migrated to IUCLID6: Lot No. 96F05641
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (Lot No. 04507JV)
Remarks:
used with all strains with S9
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine (Lot No. 08029JG)
Remarks:
used with TA100 and TA1535 without S9
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
used with TA98 and TA1538 without S9

Migrated to IUCLID6: Lot No. 01703EV
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


EXPOSURE DURATION: 2 days.


NUMBER OF REPLICATIONS: 3 (triplicate).


DETERMINATION OF CYTOTOXICITY
- Method: size of background lawn, and/or >50% reduction in the mean number of revertant colonies when compared to the vehicle control.
Evaluation criteria:
An individual dose was considered positive if the mean revertant count on the test plates was equal to or greater than three times the mean number of spontaneous revertants on the vehicle control plates. A positive result for the assay was defined as a dose-related increase in the mean number of revertant colonies over at least three concentrations of test substance including at least one positive dose. A lack of response in the positive controls or spontaneous revertant frequencies which were not in keeping with historical laboratory values would render that portion of the test invalid.

Toxicity was defined as a notable reduction in the background lawn and/or a greater than 50% reduction in the mean number of revertant colonies when compared to the vehicle control.

A positive result in this assay indicates that, under the test conditions, the test substance induced point mutations by base changes or frameshifts in the genome of Salmonella typhimurium. Negative results indicate that, under the test conditions, the test substance was not mutagenic in Salmonella typhimurium.
Statistics:
The mean revertant colony count and standard deviation were determined for each dose point.

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
Remarks:
at highest concentration of 5000 µg/plate in strains TA100 and TA1527
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material was beaded on all plates at 5000 µg/plate in all tester strains with and without metabolic activation in both the initial and repeat assays.


RANGE-FINDING/SCREENING STUDIES: Prior to initiation of the initial assay, a rangefinding test was performed to determine the doses used for both assays. Toxicity, a notable reduction in the background lawn and/or a greater than 50% reduction in the mean number of revertant colonies when compared to the vehicle control, was observed at 5000 µg/plate without metabolic activation. At the 1000, 2000 and 5000 µg/plate dose levels (with and without metabolic activation) beading was observed increasing with increasing dose levels. Based on the results of the rangefinding test, the doses selected for the mutagenicity assay were 50, 100, 500, 1000 and 5000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA: The nontreated and vehicle controls responded in a manner consistent with data from previous assays.


ADDITIONAL INFORMATION ON CYTOTOXICITY: A greater than 50% reduction in the mean number of revertant colonies when compared with the vehicle (acetone) control was observed in the initial assay in TA100, TA1537, and TA1538 without metabolic activation at 5000 µg/plate. In the repeat assay, a greater than 50% reduction in the mean number of revertant colonies was observed in TA100, and TA1538 with and without metabolic activation at 5000 µg/plate. Pinpoint colony background was observed in several plates in TA1537 and TA1538 with and without metabolic activation at 5000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study, the test material was not mutagenic in any strain of Salmonella typhimurium, w hich included at least one dose that was toxic and above the solubility of the test substance (5000 ug/plate).
Executive summary:

The test material was assessed for mutagenic potential in a bacterial reverse mutation (Ames) assay conducted in accordance with GLP. The study was largely consistent with the 1997 OECD Test Guideline 471 (plate incorporation method), except that a bacterial strain with an AT base pair at the primary reversion site was not included. Mutagenic potential was assessed in five strains of Salmonella typhimurium - TA98, TA100, TA1535, TA1537 and TA1538 - in both the presence and absence of S9 metabolic activation. Five concentrations of test material were evaluated: 50, 100, 500, 1000 and 5000 ug/plate; the highest concentration being above the solubility of the test substance and also showing cytotoxicity. The assay positive and negative (vehicles and nontreated) control groups responded appropriately. The test substance did not induce a significant increase in revertant colonies (equal to or greater than three times the vehicle control) in any strain at any dose level, with or without metabolic activation, in either the initial or repeat assays. It is concluded that the test substance was not mutagenic under the conditions of this test.