Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2010 - 03 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
no
Remarks:
The study was reported throughly and the lack of GLP certification is considered not to effect the reliability of the study result.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Storage conditions: Room temperature
CPTC ID No.: MID-4853.01

Method

Target gene:
Histidine locus
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomes (S9)
Test concentrations with justification for top dose:
Test sample: 5.0, 1.0, 0.5, 0.1, 0.05 mg/plate (with and without metabolic activation).
Vehicle:
- Solvent: 2-Propanol
- Justification: The solubility ofthe test sample was tested in different solvents at 50 mg/mL concentration and found to
be soluble in 2-Propanol and was the solvent used to dissolve this test sample in the study.
Controls
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
mitomycin C
other: 2-aminoanthracene (w/S9), ICR 191 Acridine, Daunomycin
Details on test system and conditions:
The bacterial reverse mutation assay was used to evaluate the mutagenic potential of the test sample at 5 concentrations of the test sample: 5.0, 1.0, 0.5, 0.1 and 0.05 mg. Testing was done with the appropriate solvent control and positive controls were plated with overnight cultures of the test systems (TA 97a, TA 98, TA 100, TA102, TA 1535) on selective minimal agar in the presence and absence ofAroclor-induced rat liver S9. All dose levels of the test sample, solvent controls and positive controls were plated in triplicate. (Refer to attachment A: Protocol M1 0-4853 for detailed test procedure).
Rationale for test conditions:
The Bacterial Reverse Mutation Assay is widely used to evaluate the mutagenic properties of chemicals.The test is based on the work of Dr. Bruce Ames and his coworkers and is commonly referred to as the Ames Test. Their studies involved the development of select histidine auxotrophs of S. typhimurium that are normally growth arrested due to mutations in a gene needed to produce the essential amino acid Histidine. in the absence of an external histidine source, the cells cannot grow to form colonies unless a reversion of the mutation occurs which allows the production of histidine to be resumed. As might be expected, Spontaneous reversions occur with each of the strains. However, chemical agents can induce a mutagenic response so that the number of revertant colonies is substantially higher than the spontaneous background reversion level. The test involves the analysis of the number of revertant colonies that are obtained with each strain in the presence and absence of the test chemical. Since the mutagenic response of a formulation could vary with the concentration, test articles are routinely dosed over an appropriate concentration range. in this-protocol, a complete set of positive and negative controls is included with each assay, and is plated routinely with all of the tester strains. AroclorTM 1254 induced rat liver microsomes are included to mimic the in viva activity of the liver enzymes in activating some pro-mutagens to mutagenic status.
Evaluation criteria:
Negative (solvent) Control Counts
The colonies that grew on the Minimal Glucose Agar plates developed from single cells that had regained their ability to grow in the absence of added histidine. The genetic reversion, from histidine auxotrophy to prototrophy, that enabled those cells to grow in the absence of exogenous histidine might have arisen spontaneously or as the result of a mutation induced by the treatments (see Maron & Ames, p. 181). It is important to realize that some of the colonies that arose in the positive control plates would have grown in the absence of treatment; they arose spontaneously. Accordingly, the negative (solvent) control colony counts constitute an important baseline in your evaluation of the test results. Unfortunately, the spontaneous reversion frequencies for the various tester strains can be quite variable - nevertheless, large deviations form the "normal" range of spontaneous reversion values may signal systematic problems with the assay.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
The results in Tables 1 and 2 show that the test strains were sensitive to the positive control mutagens and had a spontaneous reversion rate well within the accepted values of each strain, indicating that under the test conditions, the strains were sensitive to the detection of potentially genotoxic agents. Test sample M10-4853.01 was not cytotoxic to the test system. The metabolic activation using the S9 activation mixture shows an active microsomal preparation.
Using the same test conditions, there was no detectable genotoxic activity associated with the five concentrations (5.0, 1.0, 0.5, 0.1, 0.05 mg) of test sample M10-4853.01 (Hetester HCA Lot #: G30074 Tables 1 and 2) either in the presence or absence of S9 enzyme activation.

Applicant's summary and conclusion

Conclusions:
The test material failed to induce reverse mutation in any of the test concentrations and cell strains (Salmonella typhimurium) tested with and without metabolic activation.
Executive summary:

In this guideline (OECD 471) study, conducted without GLP certification, the test material did not induce mutagenic effects in any of the test concentrations or cell strains used, with and without metabolic activation. The test was conducted at 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate. Salmonella typhimurium was chosen as the model using strains 97, 98, 100, 102, 1535 with and without metabolic activation (Aroclor induced rat liver S9 mix).