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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:
key study
Study period:
From July 24 to August 8, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected from 2011-06-28 to 2011-06-30/signed on 2011-08-17)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: colourless liquid
- Storage condition of test material: At ambient temperature in the dark

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from male Sprague-Dawley derived rats, dosed i.p. with phenobarbital sodium (30 mg/kg 4 days before killing and 60 mg/kg 1, 2 & 3 days before killing) and 5,6-benzoflavone (80 mg/kg 2 days before killing),
Test concentrations with justification for top dose:
Up to 5000 µg/plate (series of ca half-log10 dilutions)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was assessed at 50 mg/mL in water and in dimethyl sulphoxide (DMSO). It was found to be insufficiently soluble in or miscible with water, but was soluble in DMSO. DMSO (ACS spectrophotometric grade) was, therefore, used as the vehicle for this study.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
In the absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
In the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: First test : in agar (plate incorporation); second test : preincubation.

FIRST TEST DURATION
- Exposure duration: ca 72 hours at 37°C

SECOND TEST DURATION
- Preincubation period: 30 minutes at 37°C
- Exposure duration: ca 72 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates per treatment

DETERMINATION OF CYTOTOXICITY
- Method: substantial reduction in mean revertant colony counts or by a sparse or absent background lawn.

STERILITY CHECK
Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer.
Evaluation criteria:
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) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
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.
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. Biological importance will 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.
Statistics:
No statistical analysis is performed in case of clear positive results. If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, 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.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See attached background material
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: not expected (vapor pressure = 1.5 Pa at 25°C)
- Water solubility: not soluble in water, therefore DMSO was used.
- Precipitation: none seen

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.
[Cf Tables in attached background material]

ADDITIONAL INFORMATION:
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 109/mL in all cases, and therefore met the acceptance criteria.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The test material showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

In this in vitro assessment of the mutagenic potential of the test item, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a vehicle control. The study was conducted in compliance with OECD 471, EC B.13/14 and OPPTS 870.5100 test guidelines and with GLP.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

Concentrations of the test material up to, and including, 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series ofcahalf-log10dilutions of the highest concentration.

No signs of toxicity towards the tester strains were observed in either mutation test following exposure to the test material.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

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

This study is considered as acceptable and satisfies the requirement for the bacterial gene mutation endpoint.