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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The three in vitro recommended tests were performed on 4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid . The Ames test, HPRT test and in vitro micronucleus test showed negative results in presence and in absence of metabolic activation.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2013 -- 14 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains, in both experiments with and without S9 mix.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO), batch K48474850 145
- Justification for choice: according to solubility assays performed at CiToxLAB France, the highest dose-level of 5000 µg/plate was achievable using a test item solution at 100 mg/mL under the treatment volume of 50 µL/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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 agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
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 number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
no
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:
at 5000 µg/plate in the TA 1537 strain only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix
In both experiments, a moderate to strong precipitate and/or emulsion was observed in the Petri plates when scoring the revertants at dose-levels = 1666.7 µg/plate in the five tested strains. Except for a slight decrease in the number of revertants noted at 5000 µg/plate in the TA 1537 strain in the first experiment, no noteworthy toxicity was noted at any tested dose-levels, towards the five strains used. Slight increases in the number of revertants were noted in the first experiment at 555.6 µg/plate in the TA 98 strain, and at 61.7 and 185.2 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the second experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant. Slight increases in the number of revertants were noted in the second experiment at 20.6 and 61.7 µg/plate in the TA 1535 strain, and at 1666.7 µg/plate in the TA 1537 strain (up to 2.6-fold the vehicle control value). These increases did not exceed the positive threshold (3-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the first experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.

Experiments with S9 mix
No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.
In both experiments, a moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels = 1666.7 µg/plate in the five tested strains. Except for a moderate toxicity (thinning of the bacterial lawn) noted at 5000 µg/plate in the TA 1537 strain in both experiments and in the TA 102 strain in the second experiment only, no noteworthy toxicity was noted at any tested dose-levels towards the four other tested strains. A slight increase in the number of revertants was noted in the first experiment (direct plate incorporation method) at 5000 µg/plate in the TA 98 strain (1.6-fold the vehicle control value) and at 61.7 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value), and no dose-response relationship was observed. Furthermore, no
similar effect was noted in the second experiment, performed following the pre-incubation method. Consequently, these increases were not considered to be biologically relevant.
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 either in the presence or in the 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.

This study was conducted in compliance with OECD Guideline No. 471, Council Regulation (EC) No. 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex, Part B.13/14 p. 248 and the principles of Good Laboratory Practices.

 

Methods

 

A preliminary toxicity test was performed to define the dose-levels ofthe test itemto be used for the mutagenicity study. 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.

 

Both experiments were performed according to the direct plate incorporation method except for the second test 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.

 

The test item was dissolved indimethylsulfoxide (DMSO).

 

Results

 

The 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.

 

Since the test item was found to be poorly soluble and slightly toxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of precipitate and toxicity, according to the criteria specified in the international guidelines.

 

The treatment-levels were 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains, in both experiments with and without S9 mix.

 

Experiments without S9 mix

In both experiments, a moderate to strong precipitate and/or emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1666.7 µg/plate in the five tested strains.

 

Except for a slight decrease in the number of revertants noted at 5000 µg/plate in the TA 1537 strain in the first experiment, no noteworthy toxicity was noted at any tested dose-levels, towards the five strains used.

Slight increases in the number of revertants were noted in the first experiment at 555.6 µg/plate in the TA 98 strain, and at 61.7 and 185.2 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the second experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.

 

Slight increases in the number of revertants were noted in the second experiment at 20.6 and 61.7 µg/plate in the TA 1535 strain, and at 1666.7 µg/plate in the TA 1537 strain (up to 2.6-fold the vehicle control value). These increases did not exceed the positive threshold (3-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the first experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.

 

Experiments with S9 mix

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

 

In both experiments, a moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1666.7 µg/plate in the five tested strains.

Except for amoderate toxicity (thinning of the bacterial lawn) noted at 5000 µg/plate in the TA 1537 strain in both experiments and in the TA 102 strain in the second experiment only, no noteworthy toxicity was noted at any tested dose-levels towards the four other tested strains.

 

A slight increase in the number of revertants was noted in the first experiment (direct plate incorporation method) at 5000 µg/plate in the TA 98 strain (1.6-fold the vehicle control value) and at 61.7 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value), and no dose-response relationship was observed. Furthermore, no similar effect was noted in the second experiment, performed following the pre-incubation method. Consequently, these increases were not considered to be biologically relevant.

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 either in the presence or in the absence of a rat liver metabolizing system.

 

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2013 -- 28 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline No. 487 (In vitro mammalian cell micronucleus test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% (v/v) heat-inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatments with or without S9 mix were as follows:
- 6.25, 9.38, 12.5, 18.8, 25 and 50 µg/mL in the first experiment,
- 3.13, 6.25, 12.5, 18.8, 25 and 50 µg/mL in the second experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed at CiToxLAB France, the highest achievable dose-level was 1000 µg/mL, using a test item solution at 100 mg/mL and a treatment volume of 1% (v/v) in the culture medium.


Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),
-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
Acceptance criteria
Each main experiment was considered valid if the following criteria were met:
. the mean PD of the vehicle control had to be = 1 (indicating that cells have undergone mitotis),
. the mean frequency of micronucleated cells in the vehicle control should be consistent with (but not necessary within) control historical data of the Laboratory. In any case, this frequency should be = 5‰,
. a statistically significant increase in the frequency of micronucleated cells had to be obtained in the positive controls over the background frequency of the vehicle control cultures.
Since these criteria are not met for one of the test conditions, the corresponding results were invalidated and the experiment was repeated.

Evaluation criteria
The biological relevance of the results was considered first.
Evaluation of a positive response: a test item was considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
. a dose-related increase in the frequency of micronucleated cells was observed,
. for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
. a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.
Evaluation of a negative response: a test item was considered negative if none of the criteria for a positive response were met.
Statistics:
For each experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the ¿2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix
Cytotoxicity: Following the 3- and 24-hour treatments, a severe toxicity was induced at the highest tested dose-level of 50 µg/mL as shown by a 89 to 100% decrease in the PD (Tables 2 and 4).
Micronucleus analysis : The dose-levels selected for micronucleus analysis were as follows 12.5, 18.8 and 25 µg/mL for the 3- and 24-hour treatments. No significant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments (Tables 3 and 5).

Experiments with S9 mix
Cytotoxicity : No noteworthy toxicity (decrease in the PD) was noted at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the PD (Tables 6 and 8).
Micronucleus analysis: The dose-levels selected for micronucleus analysis were as follows: 18.8, 25 and 50 µg/mL for both experiments.
No significant increase in the frequency of micronucleated cells was noted after the 3-hour treatment in either experiment (Tables 7 and 9).
Conclusions:
Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y. This study was conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

 

Methods

 

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration).

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

 

The mean PD and mean frequencies of micronucleated cells for the vehicle control cultures were as specified in the acceptance criteria. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of precipitate and/or cytotoxicity, according to the criteria specified in the international guidelines.

 

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatments with or without S9 mix were as follows:

- 6.25, 9.38, 12.5, 18.8, 25 and 50 µg/mL in the first experiment,

- 3.13, 6.25, 12.5, 18.8, 25 and 50 µg/mL in the second experiment.

 

A precipitate was observed at the end of the treatment periods at the highest tested dose-level of 50 µg/mL, in all experiments.

Experiments without S9 mix

Cytotoxicity

Following the 3- and 24-hour treatments, a severe toxicity was induced at the highest tested dose-level of 50 µg/mL as shown by a 89 to 100% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows: 12.5, 18.8 and 25 µg/mL for the 3- and 24-hour treatments.

 

No significant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments.

Experiments with S9 mix

Cytotoxicity

No noteworthy toxicity (decrease in the PD) was noted at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows: 18.8, 25 and 50 µg/mL for both experiments.

 

No significant increase in the frequency of micronucleated cells was noted after the 3-hour treatment in either experiment.

 

Conclusion

 

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March 2013 to 19 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk+/- (3.7.2°C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 ± 1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9), prepared from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Positive controls
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no metabolic activation
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL with metabolic activation

Cytotoxicity Range-Finder Experiment: 31.25, 62.50, 125.0, 250.0, 500.0 and 1000 µg/mL in the presence and absence of S-9
Experiment 1: 5, 10, 20, 25, 30, 35, 40, 45 and 50 µg/mL in the absence of S-9 and 100, 125, 150, 175, 200 and 225 µg/mL in the presence of S-9
Experiment 2: 10, 20, 30, 35, 40, 42.5, 45, 47.5, 50 and 55 µg/mL in the absence of S-9 and 50, 100, 125, 150, 175, 200 and 225 µg/mL in the presence of S-9
Vehicle / solvent:
DMSO diluted 100-fold in the treatment medium.
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
For positive control concentrations see test concentrations section
Details on test system and experimental conditions:
DURATION
- Preincubation period: Not applicable
- Exposure duration: 3-hour exposure period followed by 7-day expression period
Evaluation criteria:
For valid data, the test substance was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS Guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 31.25 to 1000 µg/mL (limited by solubility in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (250 to 1000 µg/mL). Following the 3-hour treatment incubation period, precipitate was observed at the highest two concentrations tested in the absence of S-9 (500 and 1000 µg/mL) and the highest concentration tested in the presence of S-9 (1000 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence of S-9 was retained and the higher concentration was discarded. All cultures were retained in the presence of S-9. The highest concentrations to give > 10% RS were 31.25 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 60% and 102% RS, respectively.
No marked changes in osmolality or pH were observed in the Range-Finder at any concentration analysed, compared to the concurrent vehicle controls.
In Experiment 1, twelve concentrations, ranging from 5 to 75 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 25 to 400 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest seven concentrations tested in the presence of S-9 only (150 to 400 µg/mL) but no precipitate was observed in the absence or presence of S-9 following the 3-hour treatment incubation period. Seven days after treatment, the highest three concentrations tested in the absence and presence of S-9 (55 to 75 µg/mL and 250 to 400 µg/mL, respectively) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest two concentrations tested in the presence of S-9 (25 and 50 µg/mL) were not selected as there were sufficient concentrations available to determine the toxicity profile of the test article. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed in the absence of S-9 (50 µg/mL) and in the presence of S-9 (225 µg/mL) gave 6% and 20% RS, respectively. In the absence of S-9, no concentration gave 10 - 20% RS due to steep concentration-related toxicity. Cultures treated at 45 and 50 µg/mL gave 34% and 6% RS, respectively, therefore both concentrations were analysed for viability and 6TG resistance.
In Experiment 2, twelve concentrations, ranging from 5 to 65 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 300 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations tested in the presence of S-9 only (125 to 300 µg/mL) but no precipitate was observed in the absence or presence of S-9 following the 3-hour treatment incubation period. Seven days after treatment, the highest concentration tested in the absence of S-9 (65 µg/mL) and the highest three concentrations tested in the presence of S-9 (250 to 300 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition the lowest concentration tested in the absence of S-9 (5 µg/mL) was not selected as there were sufficient concentrations available to determine the toxicity profile of the test article. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed in the absence of S-9 (55 µg/mL) and in the presence of S-9 (225 µg/mL) each gave 6% RS. Steep concentration-related toxicity was observed under both treatment conditions with cultures treated at 50 and 55 µg/mL (-S-9) giving 34% and 6% RS, respectively and cultures treated at 200 and 225 µg/mL (+S-9) giving 37% and 6% RS, respectively. Under both treatment conditions, both concentrations were analysed.
Conclusions:
It is concluded that 4,4' lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid did not induce biologically relevant increases in mutant frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolising system (S-9).
Executive summary:

4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test substance was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3-hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 31.25 to 1000 µg/mL (limited by solubility in culture medium). The highest concentrations to give>10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 60% and 102% RS, respectively.

In Experiment 1, twelve concentrations, ranging from 5 to 75 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 25 to 400 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 50 µg/mL in the absence of S-9 and 225 µg/mL in the presence of S-9, which gave 6% and 20% RS, respectively. In the absence of S-9, no concentration gave 10-20% RS due to steep concentration-related toxicity. Cultures treated at 45 and 50 µg/mL gave 34% and 6% RS, respectively, therefore both concentrations were analysed for viability and 6TG resistance.

In Experiment 2, twelve concentrations, ranging from 5 to 65 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 300 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 55 µg/mL in the absence of S-9 and 225 µg/mL in the presence of S-9, which each gave 6% RS. In the absence and presence of S-9, no concentration gave 10-20% RS. Steep concentration-related toxicity was observed under both treatment conditions with cultures treated at 50 and 55 µg/mL (-S-9) giving 34% and 6% RS, respectively and cultures treated at 200 and 225 µg/mL (+S-9) giving 37% and 6% RS, respectively. Under both treatment conditions, both concentrations were analysed.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In the absence of S-9 in Experiment 1, a statistically significant increase in mutant frequency (MF) in the data set was observed at the highest concentration analysed (50 µg/mL), but this concentration gave 6% RS and there was no statistically significant linear trend. In Experiment 2, no statistically significant increases in MF were observed at any concentration analysed, but there was a weakly significant linear trend. The highest concentration analysed in Experiment 2 (55 µg/mL) gave 6% RS. This maximum concentration was higher than the one analysed in Experiment 1 (50 µg/mL, also giving 6% RS), therefore, the only statistically significant increase in MF was observed at a concentration giving <10% RS in a single experiment. As the observation was not reproduced in a second experiment, in which slightly higher concentrations were analysed, and there was no statistically significant linear trend in Experiment 1, the isolated increase observed in Experiment 1 was not considered to be biologically relevant.

In the presence of S-9, no statistically significant increases in MF were observed at any concentration tested in Experiments 1 and 2. A weak but statistically significant linear trend was observed in Experiment 1 but in the absence of any statistically significant increases in MF in this experiment, the observation was considered not biologically relevant.

It is concluded that 4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid did not induce biologically relevant increases in mutant frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolising system (S-9).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay (Brient 2013):

The objective of this study was to evaluate the potential of the test item to induce reverse mutations inSalmonella typhimurium (OECD 471). After the preliminary tests, 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.Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).Five strains of bacteriaSalmonella typhimuriumwere 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). The test item was dissolved indimethylsulfoxide (DMSO).The treatment-levels were 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains, in both experiments with and without S9 mix.

Experiments without S9 mix:In both experiments, a moderate to strong precipitate and/or emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1666.7 µg/plate in the five tested strains.Except for a slight decrease in the number of revertants noted at 5000 µg/plate in the TA 1537 strain in the first experiment, no noteworthy toxicity was noted at any tested dose-levels, towards the five strains used.Slight increases in the number of revertants were noted in the first experiment at 555.6 µg/plate in the TA 98 strain, and at 61.7 and 185.2 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the second experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.Slight increases in the number of revertants were noted in the second experiment at 20.6 and 61.7 µg/plate in the TA 1535 strain, and at 1666.7 µg/plate in the TA 1537 strain (up to 2.6-fold the vehicle control value). These increases did not exceed the positive threshold (3-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the first experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.

 Experiments with S9 mix:No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.In both experiments, a moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1666.7 µg/plate in the five tested strains.Except for amoderate toxicity (thinning of the bacterial lawn) noted at 5000 µg/plate in the TA 1537 strain in both experiments and in the TA 102 strain in the second experiment only, no noteworthy toxicity was noted at any tested dose-levels towards the four other tested strains.A slight increase in the number of revertants was noted in the first experiment (direct plate incorporation method) at 5000 µg/plate in the TA 98 strain (1.6-fold the vehicle control value) and at 61.7 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value), and no dose-response relationship was observed. Furthermore, no similar effect was noted in the second experiment, performed following the pre-incubation method. Consequently, these increases were not considered to be biologically relevant.

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimuriumeither in the presence or in the absence of a rat liver metabolizing system.

In vitro mammalian cell micronucleus test (Brient 2013):

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y (OECD 487). After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration).The test item was dissolved in dimethylsulfoxide (DMSO).

Experiments without S9 mix:Following the 3- and 24-hour treatments, a severe toxicity was induced at the highest tested dose-level of 50 µg/mL as shown by a 89 to 100% decrease in the PD.The dose-levels selected for micronucleus analysis were as follows: 12.5, 18.8 and 25 µg/mL for the 3- and 24-hour treatments.No significant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments.

Experiments with S9 mix: No noteworthy toxicity (decrease in the PD) was noted at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the PD.The dose-levels selected for micronucleus analysis were as follows: 18.8, 25 and 50 µg/mL for both experiments.No significant increase in the frequency of micronucleated cells was noted after the 3-hour treatment in either experiment.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

 

In vitro mammalian cell gene mutation assay (Lloyd 2014):

4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test substance was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3-hour treatment incubation period was used for all experiments.

In the absence of S-9 in Experiment 1, a statistically significant increase in mutant frequency (MF) in the data set was observed at the highest concentration analysed (50 µg/mL), but this concentration gave 6% RS and there was no statistically significant linear trend. In Experiment 2, no statistically significant increases in MF were observed at any concentration analysed, but there was a weakly significant linear trend. The highest concentration analysed in Experiment 2 (55 µg/mL) gave 6% RS. This maximum concentration was higher than the one analysed in Experiment 1 (50 µg/mL, also giving 6% RS), therefore, the only statistically significant increase in MF was observed at a concentration giving <10% RS in a single experiment. As the observation was not reproduced in a second experiment, in which slightly higher concentrations were analysed, and there was no statistically significant linear trend in Experiment 1, the isolated increase observed in Experiment 1 was not considered to be biologically relevant.

In the presence of S-9, no statistically significant increases in MF were observed at any concentration tested in Experiments 1 and 2. A weak but statistically significant linear trend was observed in Experiment 1 but in the absence of any statistically significant increases in MF in this experiment, the observation was considered not biologically relevant.

It is concluded that 4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid did not induce biologically relevant increases in mutant frequency at thehprtlocus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations in two independent experiments in the absence and presence ofa rat liver metabolising system(S-9).

Justification for classification or non-classification

Based on the negative results in all three regulatory in vitro genotoxicity tests, no classification for 4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid is required for genotoxicity according to the Regulation EC n°1272/2008.