Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD 471 and in accordance with the Principles of GLP
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
; This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines.
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-ethylhexanoic acid
- Molecular formula: C8H16O2
- Molecular weight: 144.2
- Physical state: liquid
- Analytical purity: > 99%
- Impurities (identity and concentrations): not specified in the report
- Composition of test material, percentage of components: not specified in the report
- Isomers composition: not specified in the report
- Purity test date: not specified in the report
- Lot/batch No.: Lot # 10312TN
- Expiration date of the lot/batch: not specified in the report
- Stability under test conditions: not specified in the report
- Storage condition of test material: not specified in the report

Method

Target gene:
Histedine Locus for Salmonella and trp for E. coli strains
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA1535, TA1537, TA98 and TA100, Escherichia coli WP2uvrA
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: The DNA repair mutation (uvrA/B) eliminates excision repair, a repair pathway for DNA damage from UV light and certain mutagens. The presence of the uvrA/B mutation makes the strains more sensitive to the test articles that induce damage in this manner. T
Metabolic activation:
with and without
Metabolic activation system:
S-9 metabolic activation system (Rat liver homogenate)
Test concentrations with justification for top dose:
50-5000 µg/ml
Vehicle / solvent:
Stock solutions of the test article and positive control chemicals were prepared fresh just prior to treatment. Due to the limited aqueous solubility, the test material was first dissolved in DMSO by mixing on a vortex mixer on the day of each experiment. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 1/16 inch pellets with a nominal pore diameter of 4A.
Controls
Untreated negative controls:
yes
Remarks:
Dimethylsulfoxide (DMSO, Sigma), the solvent used to dissolve the test material, was used as the negative control treatment
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO, Sigma), the solvent used to dissolve the test material, was used as the negative control treatment
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9:ENNG: 3 pg/plate for TA100, 5 pg/plate for TA1535 and 2 pg/plate for WP2uvrA; 9AA: 80 pg/plate for TA1537; 4NQO: 0.2 pg/plate for TA98 +S9: 2AA at 1 pg/plate for TA100; 2AA at 2 pg/plate for TA1535 and TA1537; BP at 5 pg/plate for TA98; 2AA at
Remarks:
none
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: Trplicate

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: By back ground lawn


Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures were in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value was at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 1997 are presented in Appendix I.
A minimum of four non-toxic dose levels were achieved.
No evidence of excessive contamination.

Statistics:
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls both with and without metabolic activation were calculated

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium TA1535, TA1537, TA98 and TA100, Escherichia coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test material caused a visible reduction in the growth of the bacterial lawn and/or a significant decrease in the frequency of revertant colonies to all of the tester strains at 5000 pg/plate. The toxic response was observed predominantly to the plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified in the report
- Effects of osmolality: not specified in the report
- Evaporation from medium: not specified in the report
- Water solubility: limited
- Precipitation: not specified in the report
- Other confounding effects: not specified in the report

RANGE-FINDING/SCREENING STUDIES: was conducted
The dose range of the test material used in the preliminary toxicity study was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 pg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: valid, within the range

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Study

The dose range of the test material used in the preliminary toxicity study was0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 pg/plate. The testmaterial was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

The number of revertant colonies for the toxicity assay wer

With (+)/without

(-)mix

Strain

Dose tug/plate)

 

0.15

0.5

1.5

5

15

50

150

500

1500

5000

+

TA100

152

157

143

150

154

145

148

147

137

133

112

-

TA100

121

125

128

153

138

140

143

134

150

118

140

+

WP2uvrA

21

31

32

26

23

21

29

32

17

21

21

-

WP2uvrA

23

15

17

22

20

26

20

22

26

17

15

 

Mutation Study

Prior to use, the master strains were checked for characteristics, viability andspontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The test material caused a visible reduction in the growth of the bacterial lawn and/or a significant decrease in the frequency of revertant colonies to all ofthetester strains at 5000 pg/plate. The toxic response was observed predominantlyto the plates dosed with S9-mix.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mixand the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that 2-ethylhexanoic acid did not induce a mutagenic response in the bacterial reverse mutation assay under the experimental conditions used.
Executive summary:

2 -Ethylhexanoic acid [2 -alpha-ethylcaproic acid] was evaluated in the bacterial reverse mutation assay. Salmonella typhimurium strains TA1535, TM 537, TA98, TA100 and Escherichia colistrain WP2uvrA were treated with the test material using the Ames plate incorporationmethod at five dose levels, in triplicate, both with and without the addition of a ratliver homogenate metabolising system (10% liver S9 in standard co-factors).

This method conforms to the guidelines for bacterial mutagenicity testing published by themajor Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 microg/plate inthe first experiment. The experiment was repeated on a separate day using the samedose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus,the sensitivity of the assay and the efficacy of the S9-mix were validated.The test material caused a visible reduction in the growth of the bacterial lawn and/or a significant decrease in the frequency of revertant colonies to all of the tester strains at 5000 pg/plate. The toxic response was observed predominantly to the plates dosedwith S9-mix. The test material was tested up to the maximum recommended dose of5000 microg/plate. No significant increases in the frequency of revertant colonies were recorded for anyof the bacterial strains, with any dose of the test material, either with or withoutmetabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

It was concluded that 2-ethylhexanoic acid did not induce a mutagenic response in the bacterial reverse mutation assay under the experimental conditions used.