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Diss Factsheets

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:
Nov. 19, 1996 to Jan. 27, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
"Japanese Guidelines for Screening Toxicity Testings of Chemicals:Testing Methods for New Chemical Substances, enacted July 13, 1974, amended December 5, 1986
GLP compliance:
yes (incl. QA statement)
Remarks:
German GLP
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
FAT45'176/A
IUPAC Name:
FAT45'176/A
Test material form:
other: solid
Details on test material:
Code number: FAT45'176/A
Batch No.: WP2/96
Purity: ca. 60 %
Appearance: orange-brown solid
Storage: room temperature
Expiration date: 11/2004
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WP2/96
- Expiration date of the lot/batch: November 30, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: pure: stable under storage conditions
in solvent: 48 h in H2O, saline, polyethylene glycol, and CMC
- Solubility of the test substance in the solvent/vehicle: dissolved in deionised water

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
deionised water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
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:
TA 1537, TA 98: Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, WP2: Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, WP2 : With metabolic activatior
Details on test system and experimental conditions:
The bacterial strains were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
Evaluation criteria:
Evaluation of Results
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration is induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, WP2, and its uvrA derivative the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible 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 statistical evaluation of the data is required.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
FAT 45176/A is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed with FAT 45176/A according to OECD 471 and EU B.14 guideline to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 and WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate.

No toxic effects evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Based on the study results, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, FAT 45176/A is considered to be non-mutagenic.