Esterification products of 4,4'-isopropylidenediphenol, ethoxylated and 2-methylprop-2-enoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2018 - 07 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid

EC Number:

935-411-2

Cas Number:

not available

Molecular formula:

C23H24O4 (C2H4O)n

IUPAC Name:

Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid

Test material form:

liquid

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found poorly soluble in the final treatment medium in the preliminary test and toxic only at dose levels close to the highest recommended one, the selection of the highest dose level to be used in the main experiments was based on the level of emulsion or was 5000 µg/plate, according to the criteria specified in the international guidelines.
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.

Experiments without S9 mix
Selected dose levels
-6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the five strains in both mutagenicity experiments, except for the TA 1537 strain in the 2nd experiment,
-6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the 2nd experiment.
A moderate to strong emulsion and/or precipitate was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

Experiments with S9 mix
Selected dose levels
-20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the 5 strains in both mutagenicity experiments, except for the TA 1537 strain in the 2nd experiment,
-6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the 2nd experiment.
A moderate to strong emulsion or precipitate was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the 5 strains, in either experiment. These results met thus the criteria of a negative response.

Vehicle / solvent:

According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control plates was dimethylsulfoxide (DMSO).

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 (without S9 mix) 2-Anthramine, Benzo(a)pyrene (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
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).

DURATION
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn.

NUMBER OF REPLICATIONS: three plates/dose level

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.

Statistics:

no

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Emulsion and/or precipitate

RANGE-FINDING STUDY:
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate with and without S9 mix.
A moderate toxicity (thinning of the bacterial lawn) was noted at dose levels = 1666.7 µg/plate in the three strains used in the presence of S9 mix. In the absence of S9 mix, no noteworthy toxicity was noted at any dose levels.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
Since the test item was found poorly soluble in the final treatment medium in the preliminary test and toxic only at dose levels close to the highest recommended one (i.e. 5000 µg/plate), the selection of the highest dose level to be used in the main experiments was based on the level of emulsion or was 5000 µg/plate, according to the criteria specified in the international guidelines.

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.

Experiments without S9 mix
The selected dose levels were:
- 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the five strains in both mutagenicity experiments, except for the TA 1537 strain in the second experiment,
- 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the second experiment.

A moderate to strong emulsion and/or precipitate was observed in the Petri plates when scoring the revertants at dose levels ¿ 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment. These results met thus the criteria of a negative response.

Experiments with S9 mix
The selected dose levels were:
- 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains in both mutagenicity experiments, except for the TA 1537 strain in the second experiment,
- 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the second experiment.

A moderate to strong emulsion or precipitate was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment. These results met thus the criteria of a negative response.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached document.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this 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 Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose levels of the test item, diluted 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 at least 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.

Thetreatment of the TA 1537 strain in the second experiment with and without S9 mix was performed at the test site.

Results

Since the test item was found poorly soluble in the final treatment medium in the preliminary test and toxic only at dose levels close to the highest recommended one (i.e. 5000 µg/plate), the selection of the highest dose level to be used in the main experiments was based on the level of emulsion or was 5000 µg/plate, according to the criteria specified in the international guidelines.

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 selected dose levels were:

. 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the five strains in both mutagenicity experiments without S9 mix, except for the TA 1537 strain in the second experiment,

. 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains in both mutagenicity experiments with S9 mix, except for the TA 1537 strain in the second experiment,

. 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the second experiment with and without S9 mix.

 

A moderate to strong emulsion and/or precipitate was observed in the Petri plates when scoring the revertants at dose levels =555.6 µg/plate in both experiments with and without S9 mix.

No noteworthy toxicity was noted at any dose levels, in any strains or test conditions.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment with or without S9 mix. These results met thus the criteria of a negative response.

Conclusion

Under the experimental conditions of this 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.