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Genetic toxicity: in vitro

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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:
08 August 2013 - 06 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name: PREPOLYMER D
Chemical Name: Reaction mass of Bis (2-chloroethoxy)methane and 1,15-dichloro-3,5,8,11,13-pentaoxa-pentadecane and 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane
Batch/Lot Number: 030613
Purity: 100% (multi constituent substance)
Appearance: slightly brownish liquid

Method

Target gene:
histidine/tryptophan operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: see table 1
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: see table 1
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the solubility test, 100 mg/mL stock formulation was prepared in the vehicle, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method (5.6.4) was used.

Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared from the test item with DMSO, which was diluted by serial dilutions in six steps to obtain seven dosing solutions for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See table 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: In the Range Finding Test and in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine/tryptophan

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: Positive with and without metabolic activation in TA1535

The test item had slight mutagenic activity in Salmonella typhimurium TA1535 strain under the test conditions of this study. No mutagenic activity was observed in the other four examined bacterium strains under the test conditions of the study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the available information, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

In the Initial Mutation Test (using the plate incorporation method) and Confirmatory Mutation Tests (using the pre-incubation method), a clear repeatable positive effect of the test item was obtained in Salmonella typhimurium TA1535 strain as a dose-related increase in the number of revertants occurred and the observed revertant colony numbers were above or near the respective biological threshold value.

No insolubility was detected in the main tests. Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate concentration without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate concentration with metabolic activation.

The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show (see Appendix 2 to 5) that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item had slight mutagenic activity in Salmonella typhimurium TA1535 strain under the test conditions of this study. No mutagenic activity was observed in the other four examined bacterium strains under the test conditions of the study.