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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:
Gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 20, 1990 to November 13, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Qualifier:
according to guideline
Guideline:
other: Japan 1984, Toxicity Test Guideline, "Reversion Test with Bacteria"
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: FAT 40'406/A
Batch No: Vers. Nr. SR 6947/14
Stability: Pure: stable for at least 2 years; In solvent: > 48 hours in water, DMSO, and DMF
Storage condition of test material: room temperature
Expiration date of the lot/batch: July 1995
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 6947/14
- Expiration date of the lot/batch: July, 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: > 48 h in H20, DMSO, and DMF

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Details on mammalian cell type (if applicable):
The bacterial strains were obtained from Dr. Heinz Träger, Knoll AG, D-6700 Ludwigshafen, F.R.G.
Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Metabolic activation:
with and without
Metabolic activation system:
aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
Experiment 1: 10, 100, 333.3, 1000, and 5000 µg/plate.
Experiment 2: 100, 333.3, 1000, 2500 and 5000 µg/plate.
Vehicle / solvent:
water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100), with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strains: TA 1535, TA 100, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Strains: TA 1537, TA 1538, TA 98, without metabolic activation
Details on test system and experimental conditions:
Method of application: in agar

Duration: After solidification the plates were incubated upside down for 72 h at 37°C in the dark.

Determination of cytotoxicity: Toxic effects were evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

Number of replications: Two independent experiments performed in triplicate.
Evaluation criteria:
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows: A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method is available.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Strain TA 1537 without S9 mix and TA 1538 with S9 mix both at the highest investigated dose.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- To evaluate the toxicity of the test article, a pre-study was performed with strains TA 98 and TA 100 exposed to 1, 3.3, 10, 33.3, 100, 333.3, 1000 and 5000 µg/plate. The plates with the test article showed normal background growth up to 5000 µg/plate in strain TA 98 and TA 100.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 without S9 mix and TA 1538 with S9 mix both at the highest investigated dose in experiment I. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Applicant's summary and conclusion

Conclusions:
FAT 40406/A did not show any mutagenic activity in any Salmonella typhimurium strains.
Executive summary:

In a GLP compliant Ames test, performed according to OECD guideline 471, 5 Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98, and TA 100) were used to the test the mutagenic potential of the test substance (10, 100, 333.3, 1000, 2500, 5000 µg per plate), both with and without metabolic activation.

Cytotoxicity was observed in strain TA 1537 without S9 mix and TA 1538 with S9 mix at the highest concentration tested (5000 µg/plate) in experiment 1.

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Therefore, it was concluded that FAT 40406/A did not show any mutagenic activity in any Salmonella typhimurium strains.

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