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Toxicological information

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:
26 June 2015 - 17 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
EC Number:
212-985-6
EC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Cas Number:
901-44-0
Molecular formula:
C19H24O4
IUPAC Name:
2-(4-{2-[4-(2-hydroxyethoxy)phenyl]propan-2-yl}phenoxy)ethan-1-ol
Test material form:
liquid: viscous

Method

Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
The selected dose-levels were the following:Without S9 mix: 125, 250, 500, 1000, 2000 and 4000 µg/plate in all strains for both mutagenicity experiments.With S9 mix:- 125, 250, 500, 1000, 2000 and 4000 µg/plate for the first experiment, and in the TA 98, TA 100 and TA 102 strain for the second experiment,- 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535 and TA 1537 strains in the second experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide- Justification for choice: test item was found to be soluble in the vehicle at 100 mg/mL
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agarDURATION- Preincubation period: 60 minutes- Incubation time: 48 to 72 hours.DETERMINATION OF TOXICITY- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.The test item is considered to have shown mutagenic activity in this study if:- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,- and/or a reproducible dose-response relationship is evidenced. The test item is considered to have shown no mutagenic activity in this study if:- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels, - nor any evidence of a dose-response relationship is noted.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
For all strains, a moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted in both experiments without S9 mix, at dose-levels = 2000 µg/plate. A moderate to strong toxicity (thinning of bacterial lawn) was noted in both experiments with S9 mix, at dose-levels from 500 to 4000 µg/plate, depending on the strain.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Negative under the experimental conditions of the study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

 

The study was performed according to the international guidelines (OECD No. 471 and Council Regulation No. B.13/14).

 

Methods

A preliminary toxicity test was performed to define the dose-levels of the test item dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Results

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

The test item was found toxic and not freely soluble in the preliminary test. The selection of the highest dose-level to be used in the main experiments was based on the first limiting factor (i.e. level of toxicity in this case), according to the criteria specified in the international guidelines.

 

The selected dose-levels were the following:

Without S9 mix: 125, 250, 500, 1000, 2000 and 4000 µg/plate in all strains for both mutagenicity experiments.

 

With S9 mix:

.            125, 250, 500, 1000, 2000 and 4000 µg/plate for the first experiment, and in the TA 98, TA 100 and TA 102 strain for the second experiment,

.            31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535 and TA 1537 strains in the second experiment.

 

A strong precipitate (preventing the scoring of revertants) was observed in the Petri plates at dose-level of 4000 µg/plate in the five strains, both with and without S9 mix.


A moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted in the five strains tested in both experiments without S9 mix, at dose-levels = 2000 µg/plate.

A moderate to strong toxicity (thinning of bacterial lawn) was noted in the five tested strains tested in both experiments with S9 mix, at dose-levels from 500 to 4000 µg/plate, depending on the strain.

 

No noteworthy increase in the number of revertants was noted in any of the five tested strains, either with or without S9 mix. These results met thus the criteria of a negative response.

Conclusion

Under the experimental conditions of the study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.