Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
april 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his and trp
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from aroclor induced rats and syrien hamster
Test concentrations with justification for top dose:
Range finder and first experiment 2.4, 12, 60, 300, 1500 micro g per plate, with and without S9
second experiment 93.75, 187.5, 375, 750, 1500 micro g per plate, with and without S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
other: 2-aminoanthracene: one strain, for standard S9 mix, with S9, 5 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), second experiment with pre-incubation 30 min at 30°C

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: in triplicate

OTHER: The S-9 used for the 'standard' plate-incorporation treatments was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 used for the 'Prival' treatments was prepared from uninduced male Golden Syrian hamsters.

range finder: tested in TA 100, in triplicate +/- S9 mix, with positive and solvent control, incorporation method

counting: Seescan Colony Counter (Seescan pic), Manual counts were performed for all treatments performed at 1500 /micro g/plate, as precipitation of the test agent on these plates prevented an accurate automated count being obtained.
Evaluation criteria:
The test article was considered to be mutagenic if:
1) the assay was valid
2) Dunnett's test gave a significant response (p < 0.01) and the data set showed a significant dose correlation
3) the positive responses described in 2) were reproducible.
Statistics:
The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis

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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 1500 micrgram/plate

RANGE-FINDING/SCREENING STUDIES: yes, see above under study details

COMPARISON WITH HISTORICAL CONTROL DATA:

the second experiment was done twice due to an error in the first trial (induced instead of uninduced hamster liver)

treatments of strain TA100 in Experiment 1 produced a small increase in revertant numbers (1.3 fold over the negative solvent control counts) in the presence of metabolic activation, that was however statistically significant at the 1% levelwhen the data were analysed using Dunnett's test. However, this increase was not reproduced in the second experiment (either with 'standard' or 'Prival' S-9 mix) and was therefore not attributed to mutagenic activity, but was considered to have been a chance occurrence. This study was therefore considered to have provided no clear evidence of mutagenic activity.

Applicant's summary and conclusion

Conclusions:
It was concluded that the test item did not induce mutation when tested in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and one strain of Escherichia coli (WP2 uvrA) under the conditions employed for this study, which included treatment up to 1500 /µg/plate (a precipitating dose, approaching and/or extending into the toxic range), both in the absence and in the presence of metabolic activation systems (both 'standard' and 'reductive' S-9 mixes).