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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
A key study was available for bacterial reverse mutation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 strains and Escherichia coli WP2uvrA strain in the presence and absence of metabolic activation, demonstrating that the test substance has no mutagenic potential under the test conditions. Various supporting studies were available in which Ethyl trichloroacetate was also tested negative either in Salmonella typhimurium strains TA98, TA100 and TA1535or Saccharomyces cerevisiae strains D7 and XV185-14C without and with metabolic activation.
Link to relevant study records
Reference
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:
other: The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification.”
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
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
Target gene:
Histidine (Salmonellla typhimurium)
Tryptophan (Escherischia 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:
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.
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
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
(SAZ), 2 μg, Salmonella TA100 and TA1535, -S9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
(9AA), 50 μg, Salmonella TA1537, -S9
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
(MMS), 2 μL, E.coli WP2 uvrA, -S9
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
2 μg, all of Salmonella strains, +S9
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.

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'.
Conclusions:
Interpretation of results (migrated information):
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A key study for bacterial reverse mutation with Ethyl trichloroacetate was 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 coliWP2uvrA) in the presence and absence of metabolic activation (Vértesi, 2012). Based on the results of the solubility and preliminary Range Finding study, concentrations solved in DMSO were 5000, 1581, 500, 158, 50 and 15.8µg/plate. 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 in the initial and confirmatory assay.

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.

The reported data of this mutagenicity assay showed 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.

Supporting studies were further available:

- Mutagenicity of ozonated humic substances was evaluated in Salmonalla TA 98 & 100 strains; Ethyl trichloroacetate was negative for mutagenicity with and without metabolic activation (Matsuda et al., 1992).

- Mutagenicity of ozonated and chlorinated p-hydoxybenzaldehyde identified in ether extract was investigated in Salmonalla TA 98 & 100 strains; Ethyl trichloroacetate was negative for mutagenicity with and without metabolic activation (Matsuda et al., 1991)

- Mutagenicity of spent chlorination liquor from the bleaching of softwood craft pulp extracted with ether was evaluated in Salmonella TA 1535 without the addition of liver microsomes (S-9 mix, metabolic activiation) according to the Ames test, carried out in the direct plating mode. Ethyl trichloroacetate was negative without metabolic activation. (Knut et al. 2008)

- Compounds found in effluents of pulp and paper mills were screened for genetic activity in growing cells using Saccharomyces cerevisiae strains D7 and XV185-14C without and with S9. Ethyl trichloroacetate was negative for D7 gene conversion and XV185-14C reversion. (Nestmann and Lee 1985)

- Mutagenicity of soil humic substances chlorinated in solution and extracted with ether was evaluated in Salmonella TA100 strains. Ethyl trichloroacetate was negative for mutagenicity with and without metabolic activation (Sato et al. 1985).


Justification for selection of genetic toxicity endpoint
Key study, performed according to regulatory standards.

Justification for classification or non-classification

Based on the negative results for bacterial reverse muatation, classification for mutagenicity is not warranted according to EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008).