4,4'-Isopropylidenediphenol, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic potential of the target substance 4,4-isopropylidenediphenol, ethoxylated is assessed in a weight-of-evidence approach based on several in vitro studies (OECD 471, OECD 490 and OECD 487) with the target substance itself.

The mutagenicity potential of 4,4-isopropylidenediphenol, ethoxylated (BPA-4EO) in Salmonella typhimurium (strain: TA 1535, TA 100, TA 1537 and TA 98) and Escherichia coli (strain: E. coli WP2 uvrA) was determined in accordance with the OECD Guideline for Testing of Chemicals 471 in the absence and the presence of metabolic activation (S9 mix). The test substance was non-mutagenic in both bacterial reverse mutation tests regardless of metabolic activation. 

The mutagenicity potential of 4,4’-Isopropylidenediphenol, ethoxylated (Grade 4EO), on the TK +/- locus in L5178Y cells was assessed in an OECD TG 490 study (mammalian cell gene mutation using the Thymidine kinase gene). The substance did not induce any increases in the mutant frequency that exceeded the Global Evaluation Factor (GEF), consequently, it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.

In addition, 4,4-isopropylidenediphenol, ethoxylated (BPA-4EO) potential to induce chromosome aberrations was assessed in an in vitro micronucleus study performed according to OECD TG 487. Under the conditions of this study, the test substance induced a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of metabolic activation. The substance was therefore considered to be clastogenic and /or aneugenic to human lymphocytes in vitro.

Therefore, to conclude on the genotoxic potential of ethoxylated 4,4-isopropylidenediphenol, a testing proposal for an in vivo micronucleus study according to OECD TG 474 is submitted.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

FROM 16 OCT 2020 to 19 MAY 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Source and lot/batch number of test material: I0348349AW
- Expiration date of the lot/batch: 27 March 2024
- Purity test date: UVCB (tested as supplied)

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: non-smoking volunteer lymphocytes

For lymphocytes:

- Sex, age and number of blood donors:
Preliminary Toxicity Test: female, aged 28 years
Main Experiment: female, aged 34 years
Main Experiment Repeat I: male, aged 23 years
Main Experiment Repeat II: male, aged 32 years
Main Experiment Repeat III: female, aged 35 years

- Whether whole blood or separated lymphocytes were used: whole blood

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 deg. with 5% CO2 in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 Microsomal Enzyme Fraction was purchased from Moltox and Lot no 4222 with the expiry date of 12 March 2022, and Lot no 4272 with expiry date of 16 July 2022, were used in this study.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9: metabolic capability

Test concentrations with justification for top dose:

Main Experiment:
4-hour without S9 and with S9 (2%): 0, 40, 80, 160, 180, 200, 240, 280 or 320 µg/mL
24-hour without S9: 0, 20, 40, 60, 80, 100, 120, 140 or 160 µg/mL

Main Experiment Repeat I:
4-hour without S9 and with S9 (2%): 0, 40, 80, 160, 240, 280, 320, 360, 400, 440, 480, 560 or 600 µg/mL

Main Experiment Repeat II:
4-hour without S9: 0, 80, 160, 240, 280, 320, 340, 360, 380, 400, 420 or 480 µg/mL
4-hour with S9 (2%): 0, 80, 160, 240, 280, 320, 360, 380, 400, 420, 440 or 480 µg/mL

Main Experiment Repeat III:
4-hour without S9: 0, 80, 160, 240, 280, 320, 340, 360, 380, 400, 420 or 480 µg/mL

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Experiments with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Experiments without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Experiments without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate lymphocyte cultures (quadruplicate for the solvent) were established for each dose level.
- Number of independent experiments: 4

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 hours
- Exposure duration/duration of treatment: 4 hours with S9 mix, 4 hours without S9 mix and 24 hours without S9 mix

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: At the end of each exposure period, the washed cell cultures were incubated for a further 24 hours in the presence of Cytochalasin B at a final concentration of 4.5 µg/mL.

- Methods of slide preparation and staining technique used including the stain used: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

- Number of cells spread and analysed per concentration: Minimum of approx. 500 cells per culture

- Criteria for scoring micronucleated cells:
The micronucleus frequency in 1000 binucleated cells was analysed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the solvent controls). Cells with 1, 2 or more micronuclei were recorded and included in the total. Following consultation with the Sponsor, an additional 1000 cells were scored for the vehicle control and test item dose levels in the 4-hour exposure group repeat III in the absence of metabolic activation due to the statistically significant increases observed after the first 1000 binucleate cells were scored. Scoring additional cells increases the statistical power of the data and is considered to be acceptable under the OECD 487 Guideline. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: cytokinesis-block proliferation index

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test item is then considered to be unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase
compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated
with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative
control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent solvent control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if considered appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Species / strain:

lymphocytes: humans

Metabolic activation:

without

Genotoxicity:

positive

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:

lymphocytes: humans

Metabolic activation:

with

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:

RANGE-FINDING/SCREENING STUDIES (if applicable): Yes
The concentrations used for the Preliminary Toxicity Test were 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 or 5000 μg/mL. The maximum dose level was the maximum recommended dose level of 5000 μg/mL.
Precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 1250 μg/mL in all three of the exposure groups.
Hemolysis was observed following exposure to the test item at and above 625 μg/mL in the 4-hour exposure groups, and at and above 312.5 μg/mL in the 24-hour exposure group.
Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. A reduced cell pellet was also noted at the end of the exposure period at and above 625 μg/mL in all three exposure groups and is considered to be indicative of toxicity to the whole cell population.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present up to 312.5 μg/mL in all three of the exposure groups. The test item induced evidence of marked toxicity in all three of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based test item-induced toxicity.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
All solvent (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

- Micronucleus test in mammalian cells:
For more details, see the section "Any other information on results, including tables" below.

HISTORICAL CONTROL DATA
For more details, see the section "Any other information on results, including tables" below.

Table 1: Summary of results

Exposure  Condition	Treatment/  Concentration  (μg/mL)	Mean  Cytostasis  (%)	% Binucleated cells containing  micronuclei (a)
			Mean	p -value (b)	Trend test  p -value (d)
4-Hour-S9   (Repeat II)	Vehicle   (DMSO)	0	0.38	-	0***
	160	12	0.35	-
	360	21	0.85	0.172*
	380	35	0.85	0.172*
	400	44	1.10	0.0007***
	MMC 0.2	35	3.80	4.41E-24***	-
4-hour -S9   (Repeat III)	Vehicle†   (DMSO)	0	0.25	-	0.015*
	160†	17	0.18	-
	240†	15	0.33	0.4596
	340†	36	1.13	7.46E-10***
	360†	65	1.60	6.18E-17***
	MMC 0.2†	50	3.25	3.43E-44***	-
4-hour +S9   (Repeat II)	Vehicle   (DMSO)	0	0.55	-	0.217
	160	15	0.55	-
	280	40	0.45	-
	320	64	0.35	-
	CP 6	41	1.95	3.47E-07***	-
24-hour -S9	Vehicle   (DMSO)	0	0.20	-	0.052
	40	0‡	0.20	-
	80	20	0.40	0.1567
	160	51	0.65	0.0054**
	DC 0.075	51	3.80	4.65E-29***	-

(a) The percentage of micronucleated cells determined in a sample of 2000 binucleate cells (4000 for solvent)

(b) p-values are for comparison with the control using Chi-square test 

(d) Trend test p-values using Linear regression model applied to control and test item concentrations

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MMC Mitomycin C 

CP Cyclophosphamide 

DC Demecolcine 

DMSO Dimethyl sulphoxide 

† 1000 additional binucleate cells scored from each replicate 

* P<0.05 

** P<0.01 

*** P<0.001

 

Table 2: Historical range for solvent control cultures

	4-hour exposure without S9	4-hour exposure with S9	24-hour exposure without  S9
	% binucleate cells with  micronuclei	% binucleate cells with  micronuclei	% binucleate cells with  micronuclei
Minimum	0.13	0.13	0.03
Maximum	0.78	1.05	1.00
Mean	0.40	0.39	0.35
Standard   Deviation	0.16	0.21	0.17
95% Control   Limits	0.08 – 0.72	0 - 0.81	0.01 – 0.69
Number of   Experiments	40	40	40

Table 3: Historical range for positive control cultures

	4-hour exposure without S9  (MMC)	4-hour exposure with S9  (CP)	24-hour exposure without  S9 (DC)
	% binucleate cells with  micronuclei	% binucleate cells with  micronuclei	% binucleate cells with  micronuclei
Minimum	1.75	1.30	2.00
Maximum	6.80	3.90	10.65
Mean	3.71	2.18	4.31
Standard   Deviation	1.25	0.61	1.75
95% Control   Limits	1.21 – 6.21	0.96 – 3.40	0.81 – 7.81
Number of   Experiments	40	40	40

MMC Mitomycin C 

CP Cyclophosphamide 

DC Demecolcine

Conclusions:

Under the conditions of this study, the test item induced a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of metabolic activation. The test item was therefore considered to be clastogenic and /or aneugenic to human lymphocytes in vitro.

Executive summary:

In an in vitro micronucleus assay (according to OECD TG 487) human lymphocyte cultures were exposed to 4,4'-Isopropylidenediphenol, ethoxylated (Grade 4EO) in DMSO at concentrations up to 600 µg/mL in three exposure conditions for the study using 4-hour exposure in the presence and absence of a standard metabolising system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation.  

The test item was tested up to cytotoxic concentrations. The test item was toxic, and optimum toxicity was difficult to achieve due to the steep nature of the toxicity curve. Close to optimum toxicity was eventually achieved in all three exposure groups following repeated experiments.

In the 4-hour exposure group in the absence of metabolic activation (S9), the test item did induce statistically significant increases in the frequency of binucleate cells with micronuclei. The initial 4-hour exposure in the absence of S9 scored for micronuclei in the binucleate cells (repeat II) demonstrated statistically significant increases in micronuclei which exceeded the 95% control limits of the vehicle historical control data. This exposure was repeated with a revised dose range (repeat III) in an effort to achieve closer to optimum toxicity. Although optimum toxicity was not achieved, statistically significant increases in micronuclei that exceeded the 95% control limits of the vehicle historical control data range were observed at a dose level that achieved less than optimum toxicity. There was a statistically significant concentration-related increase in micronuclei in both these exposures. The 4-hour exposure group in the absence of metabolic activation, therefore, meets the criteria for a positive result. The 4-hour exposure group in the presence of S9 did not demonstrate any statistically significant increases in micronuclei in the binucleate cells using a dose range that included a dose level which, again, marginally exceeded optimum toxicity. The 24-hour exposure group achieved optimum toxicity and did demonstrate a statistically significant increase in the frequency of binucleate cells at 160 µg/mL. However, because the increase was within the 95% control limits for a vehicle and not part of a concentration-related response it is considered to be of little biological relevance. Vehicle and positive controls induced the appropriate responses.

Thus, the test item induced a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of a metabolising system. Therefore, under the conditions of this study 4,4'-Isopropylidenediphenol, ethoxylated (Grade 4EO) was considered to be clastogenic and/or aneugenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: Histidine gene
Escherichia coli: Tryptophan gene

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 mix from induced rats

Test concentrations with justification for top dose:

Standard Plate Test (SPT): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Preincubation Test (PIT): 0, 10, 33, 100, 333, 1000 and 2500 µg/plate

Vehicle / solvent:

Dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his(-)) to histidine prototrophy (his(+)) was determined in the Salmonella typhimurium strains. The rate of induced back mutations from tryptophan auxotrophy (trp(-)) to tryphtophan independence (trp(+)) was determined in the Escherichia coli strain.

The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of stock solution. The test substance was dissolved in dimethylsulphoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration. The stability of the test substance in the vehicle DMSO was not determined analytically. A standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation (S9 mix) were carried out.

Evaluation criteria:

Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative vehicle.
Titre: The titre is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
Toxicity: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his(-) or trp(-) background growth), or reduction in the titre, was recorded for all test groups both with and without S9 mix in all experiments.
Solubility: Precipitation of the test material was recorded.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preincubation Test

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preincubation Test

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preincubation Test

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preincubation Test

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preincubation Test

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A bacteriotoxic effect (reduced his(-) background growth, decrease in the number of his(+) or trp(+) revertants, reduction in the titre) was observed in the standard plate test depending on the strain and test conditions from about 2500 µg/plate onward. In the preincubation assay bacteriotoxicity (reduced his(-) background growth, decrease in the number of his(+) or trp(+) revertants, reduction in the titre) was observed depending on the strain and test conditions from about 1000 µg/plate onward. In addition, no test substance precipitation was found with and without S9 mix.

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (according to OECD TG 471), strains of S. typhimurium TA 1535, TA 100, TA 1537 and TA 98 and Escherichia coli (strain: E. coli WP2 uvrA) were exposed to the test item in DMSO at a concentration range of 33 µg - 5000 µg/plate first in a standard plate test (SPT) and then at a concentration range of 10 µg – 2500 µg/plate in a preincubation test (PIT) in the presence and absence of mammalian metabolic activation (S9). The positive controls induced the appropriate responses in the corresponding strains.   A biologically relevant increase in the number of his(+) or trp (+) revertants was not observed in the STP or in the PIT, both with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 1000 µg/plate onward. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 27 OCT 2020 to 24 MAY 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch number: I0348349AW
- Purity, including information on contaminants, isomers, etc.: UVCB (tested as supplied)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was formulated in DMSO at 500 mg/ml, serially diluted, and dosed at 1% to give the maximum recommended dose level of 5000 μg/mL of test item in the cultures.

OTHER SPECIFICS
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

For cell lines:
- Mycoplasma:
absent in master stock

- Methods for maintenance in cell culture:
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air.

- doubling time:
12 hours

MEDIA USED

- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
RPMI 1640 with 20% donor horse serum (R20), 10% donorhorse serum (R10), and without serum (R0), are used during the course of the study.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- source of S9 - Rat liver

- method of preparation of S9 mix: The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G-6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration.

- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was pre-tested for acceptability by the supplier prior to purchase and were supplied with the relevant “Quality Control & Production Certificate” which are presented in Annex 1 of the attached report.

Test concentrations with justification for top dose:

0, 19.5, 39, 78, 156, 182, 208, 234, 260, 286, 312 ug/mL.
The maximum dose levels in the Mutagenicity Test were limited by test item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 490 guideline.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item was immiscible in culture medium at 50 mg/mL but was fully miscible in DMSO at 500 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration:
Duplicate

- Number of independent experiments
One

METHOD OF TREATMENT/ EXPOSURE:
The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
Expression period of 2 days

- Selection time (if incubation with a selective agent):
12 days incubation

- Method used:
96 well microwell plates

- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure:
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well plates.

- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
2000 cells/well, MTT

- Criteria for small (slow growing) and large (fast growing) colonies:
Large colonies are defined as those that cover approximately 1⁄4 to 3⁄4 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutation Frequency per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

Evaluation criteria:

In place of statistical analysis generally used for other
tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above
the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, i.e. the
mutant frequency of the concurrent solvent control plus 126, which is based on the analysis
of the distribution of the solvent control MF data from participating laboratories.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The dose range used in the preliminary toxicity test was 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 μg/mL.

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
- Data on pH:
No marked change in pH as a result of test substance

- Data on osmolality:
Osmolality did not increase by more than 50 mOsm by test substance

- Water solubility:
immiscible in culture medium at 50 mg/mL

- Precipitation and time of the determination:
At and above 1250 μg/mL in both the absence and presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES (if applicable):
The dose range used in the preliminary toxicity test was 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 μg/mL for both of the exposure groups. Test concentration in the main experiment were decided on the basis of toxicity in the screening test.

STUDY RESULTS
- Concurrent vehicle control data
The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD 490 guideline.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
There was evidence of marked toxicity, in both the absence and presence of metabolic activation, following exposure to the test item, as indicated by the %RSG and RTG values. There was no evidence of any reductions in cloning efficiency (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the RTG values observed, optimum levels of test item-induced toxicity were achieved in both the absence and presence of metabolic activation.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures:
treated- 1 x 106 cells/mL in 10 mL aliquots in R10 medium
After treatment: resuspended in R20 medium at a cell density of 2 x 105 cells/mL and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL. On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for cloning efficiency (%V) in non-selective medium.

Details on large and small colonies: Table 3 and 5 of the attached report

HISTORICAL CONTROL DATA
Appendix 1 of the attached report

Table 1: Results summary

Concentration (ug/mL)	4-hours-S9			Concentration (ug/mL)	4-hours+S9
	% RSG	RTG	MF		% RSG	RTG	MF
0	100	1	82.52	0	100	1	140.37
19.5	96			19.5	95
39	94	0.93	105.01	39	100	1.27	124.08
78	107	1.08	96.78	78	100	1.17	129.57
156	91	0.94	88.87	156	85	0.96	162.19
182	74	0.71	91.19	182	59	0.72	122.34
208	45	0.45	70.68	208	31	0.35	149.50
234	7	0.10	46.97	234	9	0.14	82.39
260	2			260	3
286	3			286	2
312	2			312	2
MF threshold for a positive response			208.52	MF threshold for a positive response			266.37
Positive Control				Positive Control
EMS				CP
400	74	0.81	601.44	1.5	78	0.56	1058.44

Conclusions:

The test item, 4,4’-Isopropylidenediphenol, ethoxylated (Grade 4EO), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B67 of Commission Regulation (EC) No. 440/2008 of 26 September 2019, and the US EPA OPPTS 870.5300 Guideline.

 

One main Mutagenicity Tests was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at ten dose levels in duplicate, together with solvent (dimethyl sulfoxide (DMSO)), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9). The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose levels in the Mutagenicity Test were limited by test item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 490 guideline. The dose levels selected for the Main Experiment were 0, 19.5, 39, 78, 156, 182, 208, 234, 260, 286, 312 ug/mL (with and without S9).

 

The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD 490 guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test item did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that achieved optimum levels of toxicity in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

 

The test item, 4,4’-Isopropylidenediphenol, ethoxylated (Grade 4EO), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Due to the positive result of the in vitro micronucleus test according to OECD TG 487, it is proposed to perform an in vivo micronucleus test (according to OECD TG 474) according to the ECHAs recommendations for the assessment of genotoxic effects of the target substance.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
4,4'-Isopropylidenediphenol, ethoxylated (EC# 500-082-2)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies:
The potential of 4,4'-Isopropylidenediphenol, ethoxylated to induce genotoxic effects was tested in a suitable in vitro test battery, conducted under GLP. The target substance was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471 and in a In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene according to OECD 490. In an in vitro micronucleus test (OECD 487), 4,4'-Isopropylidenediphenol, ethoxylated did induce chromosomal damage. No additional GLP studies were identified on 4,4'-Isopropylidenediphenol, ethoxylated to comply with the requirements outlined in Annex IX of REACH regarding the Mammalian Erythrocyte Micronucleus Test.

- Available non-GLP studies:
No non-GLP studies were identified on 4,4'-Isopropylidenediphenol, ethoxylated to comply with the requirements outlined in Annex IX of REACH regarding the Mammalian Erythrocyte Micronucleus Test.

- Historical human/control data
No historical human/control data were identified on 4,4'-Isopropylidenediphenol, ethoxylated to comply with the requirements outlined in Annex IX of REACH regarding the Mammalian Erythrocyte Micronucleus Test.


- (Q)SAR
The outcome of a QSAR assessment will not overrule the positive results of an in vitro micronucleus test.

- In vitro methods
Further in vitro testing will not be sufficient to provide information to draw conclusion on this endpoint.

- Weight of evidence
There is not sufficient data available on 4,4'-Isopropylidenediphenol, ethoxylated and read-across substances that could be used in a weight-of-evidence approach in order to comply with the requirements outlined in Annex IX of REACH regarding the the toxicity to reproduction potential of the substance.


- Grouping and read-across:
No further experimental data on potential analogues are available.

- Substance-tailored exposure driven testing [if applicable]
Omitting testing based on absence or no significant exposure is not applicable for the substance based on the available exposure scenarios.


- Approaches in addition to above [if applicable]
Not applicable

- Other reasons [if applicable]
Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- In accordance to ANNEX IX of the REACH Regulation 1907/2006, column 2, "If there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant."

According to ECHA R7a Guidance Table R.7.7-5, in case of a negative bacterial mutagenicity and a positive cytogenic assay, adequate somatic cell in vivo test to investigate structural or numerical chromosome aberrations should be selected. Hence, it is necessary to propose an appropriate in vivo somatic cell genotoxicity test (OECD 474).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant]
According to ECHA R7a Guidance Table R.7.7-5, "If systemic availability cannot be ascertained reliably, it should be investigated before progressing to in vivo tests". For the test substance, blood parameter changes observed in the repeat dose oral toxicity study would suggest that absorption does take place orally. In acute oral exposures, only mild systemic effects reported at extremely high dose. For example, at 5000 mg/kg bw 5/10 females died within 48 hours. In addition, at acute exposures of 3200-6400 mg/kg bw, Immediately after administration quiet behaviour, reddened eyes, staggering, compulsive mastication, salivation, scrubby fur were observed and the male rats showed squatting posture and irregular respiration, whereas the females showed abdominal-lateral-back position, apathy and wet fur. Overall, evidence suggest that absorption of test substance takes place. However, no data in mice is available. Hence, a dose range finding study is proposed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Route of administration:

oral: gavage

Details on exposure:

Single dose. The animals will be sacrificed 24 and 48 hours after treatment.

Executive summary:

The potential of 4,4'-Isopropylidenediphenol, ethoxylated to induce genotoxic effects was tested in a suitable in vitro test battery, conducted under GLP. The target substance was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471 and in a In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene according to OECD 490. In an in vitro micronucleus test (OECD 487), 4,4'-Isopropylidenediphenol, ethoxylated did induce chromosomal damage. No additional GLP studies were identified on 4,4'-Isopropylidenediphenol, ethoxylated to comply with the requirements outlined in Annex IX of REACH regarding the Mammalian Erythrocyte Micronucleus Test.

In accordance to ANNEX IX of the REACH Regulation 1907/2006, column 2, "If there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant."

According to ECHA R7a Guidance Table R.7.7-5, in case of a negative bacterial mutagenicity and a positive cytogenic assay, adequate somatic cell in vivo test to investigate structural or numerical chromosome aberrations should be selected. Hence, it is necessary to propose an appropriate in vivo somatic cell genotoxicity test (OECD 474)

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Two studies are reported to assess the genetic toxicity of the substance. These studies were considered reliable as they were conducted on the registered substance according to OECD Testing Guidelines 471. The test substance was not mutagenic in both bacterial reverse mutation tests in the absence and the presence of metabolic activation.

At the request of ECHA request (decision number CCH-D-2114489551-41-01/F) two further studies were commissioned, the in vitro mammalian cell micronucleus test (OECD 487) and the in vitro mammalian cell gene mutation test (OECD 490). 

In the in vitro mammalian cell gene mutation test (according to OECD TG 490), the substance did not induce any increases in the mutant frequency on the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic as a result of this study (mammalian cell gene mutation using the Thymidine kinase gene) in which all acceptability criteria were met.

In the in vitro micronucleus test (according to OECD TG 487), the test substance induced a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of metabolic activation. The substance was therefore considered to be clastogenic and /or aneugenic to human lymphocytes in vitro.

Thus, to conclude on the genotoxic potential of ethoxylated 4,4-isopropylidenediphenol, a testing proposal for an in vivo micronucleus study according to OECD TG 474 is submitted.

 

Justification for classification or non-classification

Ethoxylated 4,4-isopropylidenediphenol induces structural and/or numerical chromosomal damage in the absence of metabolic activation in an in vitro micronucleus test with human lymphocytes (OECD 487). Without additional information, it is not possible to classify the target substance for mutagenicity. Therefore, an in vivo micronucleus test according to OECD 474 is proposed.