4,4'-Isopropylidenediphenol, ethoxylated

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

1

Reference substance name:

4,4'-Isopropylidenediphenol, ethoxylated

EC Number:

500-082-2

EC Name:

4,4'-Isopropylidenediphenol, ethoxylated

Cas Number:

32492-61-8

Molecular formula:

C15H16O2.(C2H5O)n.(C2H5O)m sum of n+m: >1 - <4.5 moles EO

IUPAC Name:

2-[4-[2-[4-(2-hydroxyethoxy)phenyl]propan-2-yl]phenoxy]ethanol

Test material form:

liquid: viscous

Details on test material:

Chemical name : 4,4'-isopropylidenediphenol, ethoxylated (1 – 4.5 moles ethoxylated)
EC number : 500-082-2

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agree by each registrant.

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.

Method

Target gene:

TK

Species / strain

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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.