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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Principles of method if other than guideline:
Method: other: comparable to guidelines established by OECD, USEPA, and USFDA
All tester strains were checked for the presence of the appropriate genetic markers. The S. typhimurium strains were obtained from Dr. Bruce Ames, University of California - Berkeley. The E. coli strain was obtained from Dr. M.H.L. Green, M.R.C. Cell Mutation Unit, University of Sussex, Falmer, England. For tests with metabolic activation, a reaction mixture was prepared with 8 mM of MgCl2, 33 mM KCl, 4 mM NADP, 5 mM glucose-6-phosphate, 100 mM Na2HPO4 (pH 7.4), and 6% (v/v) S9 Homogenate fraction. The S9 Homogenate was prepared at Pharmakon Research from Aroclor 1254-induced male Sprague-Dawley rat liver homogenate, according to the procedures of Maron and Ames (1983). The concentration of liver homogenate in the metabolic activation mixture was optimized for each lot of S9 prepared using 3 positive control articles (2-acetylaminofluorene, 2-anthramine, and benzo[a]pyrene). [Maron, D.M. and B.N. Ames. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Res. 113:173-215.] All required dilutions of the test substance were made with dimethyl sulfoxide (DMSO). Dilutions were prepared the day of the test and used immediately after preparation. A preliminary toxicity screen was conducted, using both liquid pre-incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100, and E. coli WP2 uvrA (treated with the test substance at doses of 50, 167, 500, 1670, and 5000 ug/plate), and a DMSO solvent control were prepared without metabolic activation. Following a 48-hour incubation at 37 deg. C, the background lawn and spontaneous revertants were scored for normal, inhibited, or no growth. Primary test cultures were prepared from working stock cultures in 25 mL of Oxoid nutrient broth #2, incubated for ~8 hours at 37 deg. C, and diluted 1:4 in de-ionized water. Optical densities were determined at 650 nm and cultures with optical densities of 0.4-0.6 (~1-2 x 10E9 cells/mL) were used for this study. The test substance was evaluated for mutagenicity using both liquid pre-incubation and plate incorporation treatment conditions. Triplicate cultures of all six test strains were prepared and evaluated at doses of 16.7, 50, 167, 500, 1670, and 5000 ug/plate, both with and without metabolic activation (S9). Positive and negative control articles (with and without activation) were prepared in triplicate for the primary mutagenicity testing:
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Methyltin tris (2-ethylhexylthioglycolate)
- Lot/batch No.: 1706-51
- Storage condition of test material: room temperature

Method

Target gene:
selected histidine loci
tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
exogenous metabolic activation system (S9)
Test concentrations with justification for top dose:
16.7, 50, 167, 500, 1670, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent giving the best solubility or suspension was selected.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
all tester strains plated with DMSO ±S9
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: 2-aminofluorene; 2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); liquid preincubation;

DURATION
- Preincubation period: approximately 30 minutes for those undergoing liquid pre-incubation
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate cultures


Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan- independent revertants, with at least one dose level inducing a revertant frequency that is two-fold the concurrent negative control value. Statistical analysis were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. If the tetst article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine or tryptophan-independent revertants. Inhibited growth was characterized by the absence of a confluent bacterial lawn nd /or the presence of pindot colonies.
Statistics:
Statistical analyses were conducted using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. A positive result was defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants, with at least one dose level inducing a revertant frequency that was two-fold the solvent control value. An equivocal result was declared if the test substance did not induce a statistically significant, dose-dependent increase in revertant frequency, but did induce a revertant frequency at one dose level that was two-fold the spontaneous control value. A negative result was defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan-independent revertants. Statistical analyses were only conducted when a 50% increase in revertant frequency (relative to the concurrent negative controls) was observed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none noted
- Water solubility: DMSO- solvent

RANGE-FINDING/SCREENING STUDIES:
Toxicity of the test article first was evaluated in a preliminary toxicity screen using both liquid pre-incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100 and WP2 urvA were treated with the test substance at doses of 50.0, 167, 500, 1670 and 5000 µg/plate, and the DMSO solvent control in the absence of S9 activation. Results of the pre-screen indicated that the test substance produced inhibited growth (characterised by the absence of a confluent bacterial lawn and /or the presence of pindot colonies) in both Salmonella tester strains at doses ≥500 µg/plate under liquid pre-incubation conditions. In addition, the test substance was found to be incompletely soluble at doses ≥500 µg/plate.
Preliminary test results showed that the test substance produced inhibited growth in both Salmonella tester strains at doses ≥500 µg/plate, under liquid pre-incubation conditions. Primary test results showed that, except for strain WP2 uvrA, inhibited growth was again observed for all test strain/S9/treatment combinations at the 3 highest dose levels (i.e., 500, 1670 and 5000 µg/plate). In addition, the test substance again was seen to be incompletely soluble at levels ≥500 µg/plate. Statistically significant increases in revertant frequencies (to ~1.6 -2.0 fold control values) were observed in strains TA1537 and TA1535 at a dose level of 16.7 µg/plate, without S9, under liquid pre-incubation and plate incorporation conditions, respectively. However, these increases were not determined to be dose dependent and the observed revertant frequencies approximated historical negative control values. Revertant frequencies for all other dose/test strain/S9/treatment condition combinations approximated or were less than control values. All positive and negative control values (in both assays) were within acceptable ranges. Therefore, the slight increases observed in strains TA1535 and TA1537 were considered statistical aberrations.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical data were provided for positive and negative controls. Six dose levels were evaluated in case of unacceptable toxicity and/or insolubility at the highest dose levels evaluated in the mutation assay. After a 48-hour incubation at 37 °C, all plates were scored for bacterial growth. Solvent and positive controls were scored first, and test substance-treated cultures were scored only if the average negative control values were within historical ranges.

Applicant's summary and conclusion

Conclusions:
Negative in the Ames/Salmonella-E.coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions of this study.
Executive summary:

The test article, methyltin tris(2­ethylhexylthioglycolate), was evaluated in the Ames/Salmonella­E. coli Reverse Mutation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102), and at the tryptophan locus in one Escherichia coli tester strain (WP2 uvrA), in the presence and absence of an exogenous metabolic activation system (S9).

Toxicity of the test article first was evaluated in a preliminary toxicity screen using both liquid pre­incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100 and WP2 uvrA were treated at doses of 50.0, 167, 500, 1670 and 5000 µg/plate, and the DMSO solvent control, in the absence of S9. Results of the pre-screen indicated the test material produced inhibited growth (characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies) in both Salmonella tester strains at doses ≥500 µg/plate under liquid pre­incubation conditions. In addition, the test article was found to be incompletely soluble at doses ≥500 µg/plate. 

The test article next was evaluated for mutagenicity using both treatment conditions. Based upon the results of the pre-screen, the test material was evaluated in triplicate cultures in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate with and without S9. Six doses were evaluated in the event of unacceptable toxicity and/or insolubility at the highest dose levels in the mutation assay. The S9 mixture included 6 % (v/v) Aroclor 1254­induced male Sprague­Dawley rat liver homogenate with the appropriate buffer and cofactors.

Except for strain WP2 uvrA, inhibited growth again was observed for all tester strain/S9/treatment combinations at the highest 1­3 doses evaluated. In addition, the test article again was found to be incompletely soluble at doses ≥500 µg/plate. Statistically significant increases in revertant frequencies, to approximately 1.6­ to 2.0­fold control values, were observed in strains TA1537 and TA1535 at a dose of 16.7 µg/plate without S9 under liquid pre­incubation and plate incorporation conditions, respectively. However, these increases were not dose dependent, and the observed revertant frequencies approximated historical negative control values. In addition, revertant frequencies for all other dose/tester strain/S9/treatment condition combinations approximated or were less than control values. All positive and negative control values in both assays were within acceptable ranges. Thus, the slight increases observed in strains TA1535 and TA1537 are considered to be statistical aberrations due to random fluctuation of the spontaneous revert frequencies.

Therefore, the results for the test article, methyltin tris(2­ethylhexylthio­glycolate), were negative in the Ames/Salmonella­E. coli Reverse Mutation Assay, using liquid pre­incubation and plate incorporation treatments, under the conditions of the study according to the criteria of the test protocol.