Ethyl trichloroacetate

Genetic toxicity: in vitro

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1) (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

514-84-4

IUPAC Name:

514-84-4

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Ethyl trichloroacetate
- Substance type: Organic, monoconstituent substance
- Physical state: Colourless, liquid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): see confidential details
- Composition of test material, percentage of components: see confidential details
- Purity test date: CoA, dated October 04,2012
- Lot/batch No.: DEFM114019
- Expiration date of the lot/batch: 2014-09-07
- Stability under test conditions: stable
- Storage condition of test material: Room temperature

Method

Target gene:

Histidine (Salmonellla typhimurium)
Tryptophan (Escherischia coli)

Species / strain

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:

S9-Mix

Test concentrations with justification for top dose:

Range Finding Test: 5, 15.8, 50, 158, 500, 1581 and 5000 μg/plate.
Initial and Confirmatory Mutation Tests: 15.8, 50, 158, 500, 1581 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:solubility
The behaviour of the test item solutions with the solution of top agar (5.4.4 and 5.4.5) and phosphate buffer (5.5.3) was determined in the preliminary Solubility Test. Dimethyl sulfoxide (DMSO) was found as suitable vehicle for preparing the test item suspensions, solutions.
All dilutions of test item were made in the testing laboratory using DMSO.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO for Test Item, NPD, 9AA and 2AA ; Ultrapure water for SAZ and MMS

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

4 μg, Salmonella TA98, -S9

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(SAZ), 2 μg, Salmonella TA100 and TA1535, -S9

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(9AA), 50 μg, Salmonella TA1537, -S9

Controls 4

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(MMS), 2 μL, E.coli WP2 uvrA, -S9

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

2 μg, all of Salmonella strains, +S9

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

50 µg, E.coli WP2 uvrA, +S9

Evaluation criteria:

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary Range Finding Test the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and TA100 tester strains under activation and non-activation conditions (in presence and absence of metabolic activation (S9 Mix)) with appropriate positive and negative controls.
In the test the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the range finding test the concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
In comparison with the revertant colony numbers of the vehicle control plates sporadic changes, slightly lower revertant colony counts were observed in S. typhimurium TA98 at the concentration of 1581 μg/plate, without and with addition of metabolic activation (±S9 Mix). These obtained changes were considered as reflecting the biological variability of the applied test system.
The experimental results (revertant colony numbers per plate, mutation factors, standard deviations) are summarised in Table 7 (Appendix I) and in Tables 10-11 (Appendix II).


COMPARISON WITH HISTORICAL CONTROL DATA:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

negative with metabolic activation
negative without metabolic activation

The test item Ethyl trichloroacetate has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item Ethyl trichloroacetate was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimrium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli ( Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphtoflavone-induced rats.

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

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000, 1581, 500, 158, 50 and 15.8µg/plate.

The revertant colony numbers of vehicle control (dimethyl sulfoxide, DMSO) plates with and without S9 Mix were mostly within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Ethyl trichloroacetate at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item Ethyl trichloroacetate has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.