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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non-mutagenic

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire PE28 4HS UK
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine for the S. typhimurium strains, and tryptophan for E. coli.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S-9 liver fraction (10% v/v), from male Sprague Dawley rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Doses are half-log intervals, with the high dose at the limit of 5000 µl/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
historical controls unless using a vehicle for which no data is present in the laboratory archives
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (first test) and preincubation (second test)
- Cell density at seeding (if applicable): 0.1 ml of a 10-h bacterial culture (having a density of at least 10E9/mL)

DURATION
- Preincubation period: 0.5 h
- Exposure duration: 48-72 h for plate incorporation; preincubation for 30 m before plating in top agar
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): cells not fixed

SELECTION AGENT (mutation assays): The Ames assay employs, as an indicator of mutation, observable (and countable) growth (colonies) in agar deficient in histidine. Colonies of bacteria represent mutants which have back-reverted to a histidine auxotroph. An automated colony counter is used.


NUMBER OF REPLICATIONS: 3 (triplicate)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: reduction in bacterial lawn

Rationale for test conditions:
The first plate incorporation test will include 7 concentrations, in triplicate. Plates will also be prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and phosphate buffer. All plates will be incubated at 34 to 39°C for 48-72 hours. After this period, the appearance of the background bacterial lawn will be examined and revertant colonies counted using an automated colony counter. Colonies may be counted manually if automated counting is not possible (e.g. if dense precipitate is present).
Any toxic effects of the test item will be detected as thinning or absence of the background lawn of non-revertant colonies, and/or reduction in revertant colony numbers to ≤ 50% of the concurrent vehicle control count. In the absence of any toxic effects the maximum concentration used in the second test will be the same as that used in the first. If toxic effects are observed at more than one concentration, a lower concentration may be chosen. A minimum of four non-toxic concentrations will be obtained. If this is not achieved then the first test is repeated using a more appropriate concentration range. If a negative or equivocal response is obtained a variation on the above procedure will be used, with a minimum of five concentrations of test item used.
If the required number of non-toxic concentrations is not obtained, the second test may be repeated using a more appropriate concentration range. It may also be repeated to confirm a positive response or to confirm a dose-response.
Evaluation criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range of the laboratory unless otherwise justified by the Study Director.
The positive control compounds must produce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls.
If exposure to a test item produces an increase in mean 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 will be considered to exhibit mutagenic activity in this test system. No statistical analysis will be performed.
If exposure to a test item does not produce an increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
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 will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989). Biological significance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below twice (three times in the case of strains TA1535 and TA1537) those of the concurrent vehicle controls (as described above) will not be considered biologically important.
Statistics:
The mean number and standard deviation of revertant colonies will be calculated for all groups. The means for all treatment groups will be compared with those obtained for the vehicle control groups.
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 will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989, Analysis of data from microbial colony assays in: Kirkland, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, pp.26-65.).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
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
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
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
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
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
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
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
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
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
Conclusions:
The substance was tested in a guideline Ames Assay (OECD 471, EC Method B.13/14, and OPPTS 870.5100) and found to be non-mutagenic. The substance does not meet the criteria for classification as a mutagen according to Regulation EC No. 1272, 2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable

Additional information

Negative (not mutagenic) in an OECD 471 compliant Ames Assay.

Justification for classification or non-classification

Non-mutagenic. The criteria for classification according to EC. No. 1272/2008 are not met.