2,2'-isopropylidenebis(p-phenyleneoxy)diethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genotoxic tests were performed with 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol

-   Gene mutation in bacteria (Ames test according to OECD 471)

-   Cytogenicity in mammalian cells (In vitro Mammalian Cell Micronucleus Test according to OECD 487)

-   Gene mutation in mammalian cells (Mouse Lymphoma Assay according to OECD4 476)

All these tests were negative with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 June 2015 - 17 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Target gene:

Histidine operon

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

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.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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.

Conclusions:

egative 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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 September 2015 -- 12 November 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 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
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

see Executive summary

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: based on available solubility data, the maximum dose-level to be tested was 2000 µg/mL, since it was excepted to produce precipitation in the culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth, relative suspension growth.

Evaluation criteria:

IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

A trend test was performed to assess the linear trend between the mutation frequency and the dose-levels tested. This statistical analysis was performed using SAS Enterprise Guide software.
Only individual mutation frequencies obtained from cultures showing an Adj. RTG = 10% were used in this analysis.

Key result

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

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, 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 mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/- mouse lymphoma cells.

 

The study was performed according to international guidelines (OECD guideline and Council Regulation No. 440/2008) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary cytotoxicity test, the test item, dissolved in DMSO, was tested in two independent experiments with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵ cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 10⁵ cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a +37°C, 5% CO₂ humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

 

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

Results

 

Since the test item was found 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 cytotoxicity, according to the criteria specified in the international guidelines.

 

The Cloning Efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%.The study was therefore considered to be valid.

 

Experiments without S9 mix

The selected dose-levels were as follows:

. 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL for the 3-hour treatment,

. 6.25, 12.5, 25, 50, 75, 100, 150 and 300 µg/mL for the 24-hour treatment.

 

Cytotoxicity

Following the 3-hour treatment, severe cytotoxicity was induced at 250 µg/mL, as shown by a 100% decrease in Adj. RTG.

Following the 24-hour treatment, slight to severe cytotoxicity was induced at dose-levels = 50 µg/mL, as shown by a 53 to 100% decrease in Adj. RTG.

 

No precipitate remaining in the culture medium was noted at the end of the treatment periods.

Mutagenicity

Following the 3-hour treatment, only a few minimal increases in the IMF were observed. None of the obtained IMF reached the GEF and no dose-response relationship was demonstrated.

No dose-level showing an Adj. RTG between 10 and 20% was observed, but there was no evidence of mutagenicity at dose-level up to 125 µg/mL, showing Adj. RTGs of 73%.

Following the 24-hour treatment, there was no increase in Induced Mutation Frequency (IMF) that met the criterion of the GEF up to 150 µg/mL. Furthermore, there was no significant dose-response relationship demonstrated in this experiment.

 

Despite no dose-level showing Adj.RTG between 10 and 20% was obtained in the first experiment, no experiment revealed a dose-response relationship or any noteworthy increase in the IMF, even up to a dose-level inducing more than 90% decrease in Adj. RTG following the 24-hour treatment (Adj. RTG of 1% at 150 µg/mL). The overall results without S9 mix were therefore considered to meet the criteria of a negative response.

Experiments with S9 mix

The selected dose-levels were as follows:

. 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL for the first experiment,

. 12.5, 25, 50, 100, 150, 200 and 250 µg/mL for the second experiment.

 

Cytotoxicity

Following the first experiment, severe cytotoxicity was induced at 250 µg/mL, as shown by a 100% decrease in Adj. RTG.

Following the second experiment, marked to severe cytotoxicity was induced at dose-levels = 200 µg/mL, as shown by 81 to 100% decrease in Adj. RTG.

 

No precipitate remaining in the culture medium was noted at the end of the treatment periods.

 

Mutagenicity

Following the first experiment, there was no increase in Induced Mutation Frequency (IMF) that met the criterion of the GEF up to 125 µg/mL. The next tested dose-level of 250 µg/mL was excessively cytotoxic (Adj RTG 0%). Furthermore, there was no significant dose-response relationship demonstrated in this experiment.

No dose-level showing an Adj. RTG between 10 and 20% was observed, but there was no evidence of mutagenicity at dose-levels up to 125 µg/mL, showing an Adj. RTG of 75%.

 

Following the second experiment, there was no increase in Induced Mutation Frequency (IMF) that met the criterion of the GEF up to 200 µg/mL, which shown the acceptable level of cytotoxicity (Adj. RTG between 10 and 20%). While the statistical test for trend was significant (P<0.05), the highest IMF observed (55 x 10^-6 mutants; dose-level of 200 µg/mL) remained substantially below the GEF of +126 x 10^-6. The dose-response relationship was thus considered as meaningless and the overall results were considered to meet 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 mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 June 2015 -- 11 September 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.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro micronucleus)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

other: mouse lymphoma L5178Y TK+/- cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% 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 treatment were as follows:
- 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the 3-hour treatment in the first experiment without S9 mix,
- 12.5, 25, 30, 33.3, 40, 44.4, 50 and 100 µg/mL for the 24-hour treatment in the first experiment without S9 mix,
- 12.5, 25, 27.3, 30, 33.3, 40, 50 and 100 µg/mL for the 3-hour treatment in the second experiment without S9 mix,
- 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 µg/mL for the experiments with S9 mix.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: according to solubility assays performed at CiToxLAB France, the highest recommended dose-level of 2000 µg/mL was achievable using a test item solution at 200 mg/mL under a treatment volume of 1% (v/v) in the culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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
Without S9 mix 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h recovery

Second experiment
Without S9 mix 3 h treatment + 24 h recovery
With S9 mix None


NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was considered first.

Evaluation of a positive response: a test item is considered to have clearly clastogenic and/or aneugenic potential, if all the following criteria were met:
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels,
- a dose-response relationship is demonstrated by a statistically significant trend test,
- any of the results are above the historical data range of the corresponding vehicle control.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response were met.

Statistics:

For each condition of the main experiments, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the Khi2 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. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose-levels. This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

other: L5178Y TK+/- mouse lymphoma 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

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, either in the absence or presence of a rat liver metabolizing 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 TK^+/-.

 

Methods

 

After a preliminary toxicity test, the test item, dissolved in DMSO, was tested in two independent experiments, with or 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	Second experiment
Without S9 mix	3 h treatment + 24 h recovery 24 h treatment + 0 h recovery	3 h treatment + 24 h recovery
With S9 mix	3 h treatment + 24 h recovery	None

 

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.

Then, after the final cell counting, the cells were washed and fixed. Cells from three dose-levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). 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 dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Results

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

 

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

 

Experiments without S9 mix

The dose-levels used for treatment were as follows:

. 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the 3-hour treatment in the first experiment,

. 12.5, 25, 30, 33.3, 40, 44.4, 50 and 100 µg/mL for the 24-hour treatment in the first experiment,

. 12.5, 25, 27.3, 30, 33.3, 40, 50 and 100 µg/mL for the 3-hour treatment in the second experiment.

 

No precipitate was observed in the culture medium at the end of the treatment periods.

Cytotoxicity

In the 3-hour treatment (first experiment), a marked to severe cytotoxicity was induced at dose-levels = 50 µg/mL, as shown by a 69 to 100% decrease in the PD.

In the 24-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels = 33.3 µg/mL, as shown by a 40 to 100% decrease in the PD.

In the 3-hour treatment (second experiment), a slight to severe cytotoxicity was induced at dose-levels = 27.3 µg/mL, as shown by a 25 to 100% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

. 6.25, 12.5 and 25 µg/mL for the first 3-hour treatment, higher dose-levels being too cytotoxic,

. 25, 30 and 33.3 µg/mL for the 24-hour treatment, the latter inducing 40% decrease in the PD and higher dose-levels being too cytotoxic,

. 25, 27.3 and 30 µg/mL for the second 3-hour treatment, higher dose-levels being too cytotoxic.

 

No noteworthy increases in the frequency of micronucleated cells were noted in either treatment periods.

It is to be noted that none of the analyzed dose-levels reached the targeted level of cytotoxicity (i.e. 55 ± 5% cytotoxicity). However, the test item was tested in two independent experiments performed following a 3-hour treatment period, and in an experiment using a long period of exposure (24-hour treatment + 0-hour recovery). All these experiments revealed clear negative results using narrower ranges of dose-levels.

Consequently, the absence of analyzed dose-levels showing the targeted level of cytotoxicity did not prejudice the conclusion in favour of the absence of mutagenicity effect without S9 mix.

 

Experiments with S9 mix

The dose-levels used for treatment were as follows: 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 µg/mL.

No precipitate was observed in the culture medium at the end of the treatment period.

 

Cytotoxicity

A moderate to severe cytotoxicity was induced at dose-levels = 50 µg/mL, as shown by a 50 to 100% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows: 12.5, 25 and 50 µg/mL, the latter inducing the recommended level of cytotoxicity (i.e. 50% decrease in the PD).

 

Dose-related increases in the frequency of micronucleated cells were observed at the three analyzed dose-levels (p < 0.05). However, the values obtained remained within the range of the corresponding vehicle control historical data. Furthermore, these increases were not statistically significant.

As a consequence, the dose-response relationship observed in this experiment was meaningless in terms of mutagenicity. The overall results were considered negative.

 

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol does not need to be classified as mutagenic in accordance with the criteria outlined and Annex I of 1272/2002/EC.