Registration Dossier

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:
10 August 2010 – 02 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD, EC and US EPA and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The study report included a current certificate of GLP compliance for the test facility, issued by the MHRA.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Light brown powder
- Storage condition of test material: aprox. -20 degrees in the dark.

Method

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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
First test: 5, 15, 50, 150, 500, 1500, and 5000 µg/plate
Second test: 50, 150, 500, 1500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The sponsor indicated that the test substance was soluble in DMSO.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 µg/plate for TA100 and TA1535

Migrated to IUCLID6: In the absence of S9 Mix
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 µg/plate for TA1537

Migrated to IUCLID6: In the absence of S9 Mix
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
2 µg/plate for TA98

Migrated to IUCLID6: In the absence of S9 Mix
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
2 µg/plate for WP2 uvrA (pKM101)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
In the presence of S9-mix. 5 µg/plate for TA100 and TA1535; 10 µg/plate for WP2 uvrA (pKM101)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98 and TA1537.

Migrated to IUCLID6: In the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (first test); in agar with preincubation (second test)


DURATION
- Preincubation period: 30 minutes at 37°C (second test only).
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: Three plates per concentration per strain

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn.
Evaluation criteria:
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups. If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
Statistics:
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

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:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No evidence of toxicity was obtained following exposure to the test substance in the first test. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the vehicle controls were within or close to the 99%
confidence limits of the current historical control range of the laboratory
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

A bacterial reverse mutation test was performed by Huntingdon Life Sciences, UK, to assess the potential of the test substance to cause gene mutation. The study was conducted to OECD, EC and US EPA was performed in compliance with GLP. In each of two tests, no evidence of cytotoxicity was observed, and no increase in the number of revertant colonies relative to the controls were seen. It was concluded that the test substance showed no evidence of mutagenic activity in the bacterial system under the test conditions employed.