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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 14th to 16th, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Blue REg 6080
IUPAC Name:
Blue REg 6080

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions of rats, induced with phenobarbital/ beta-naphthoflavone
Test concentrations with justification for top dose:
Concentration range in the main test (with and without metabolic activation): 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
DMSO, chosen because of solubility and nontoxicity to bacteria
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Remarks:
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:
with metabolic activation
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation in the overlay agar: 1000 µg/plate and above.
Each concentration, including the controls, was tested in triplicate in a plate incorporation test.
Rationale for test conditions:
A pre-experiment on toxicity was run on TA98 and TA100.
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.
Evaluation criteria:
A test item is considered mutagenic if in strains TA98, TA100, TA102 the numebr of reversions is at least twice as high and in strains TA1535, TA1537 at least three times higher as comparede 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 test item regardeless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA1537, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1535, TA102
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Substantial and dose dependent increases in revertant colony numbers were observed following treatment with test item in strains TA 1537, TA 98, and TA 100 in the presence and absence of metabolic activation (S9 mix) and in strains TA 1535 and TA 102 in the presence of metabolic activaiton. The number of colonies exceeded the threshold of twice (strains TA 98, TA 100, and TA 102) and thrice (strains TA 1535 and TA 1537) the number of the corresponding solvent control. In the presence of metabolic activation, the number of revertant colonies was reduced at higher concentrations. This effect may either be based upon slight toxic effects of the test item or upon the heavy precipitation of the test item interfering with bacterial colony growth.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
Therefore, test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.