4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an in vitro Ames assay with the test substance and in vitro mouse lyphoma assays with the read across substance BADGEDA, the test substance is not expected to have mutagenic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 March, 2004 to 26 March, 2004

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

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor-1254 induced rat liver S9-mix).

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Mutation assay: 33, 100, 333, 1000 and 3330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was soluble in DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: sodium azide (5 µg/plate in saline for TA 1535)

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine (60 µg/plate in water for TA1537)

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: daunomycin (4 µg/plate in saline for TA 98)

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (650 µg/plate in DMSO TA100)

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide (10 µg/plate in DMSO for WP2uvrA)

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar

DURATION:
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

COLONY COUNTING: The revertant colonies (histidine independent C.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of the bacterial background lawn, increase in the size of the microcolonies and reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test substance on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding test: The test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100,333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
-Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate.
Toxicity: A reduction in the number of revertants equal to the minimal value of the historical control data range is not considered biologically relevant and, therefore, will not be considered cytotoxic. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutation assay: Based on the results of the dose range finding test, the test substance was tested up to concentrations of 3330 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
-Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation on the plates was observed at the start and end of the incubation period at the concentration of 3330 µg/plate.
Toxicity: In both mutation assays, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Number of revertants: In the first mutation experiment an exceedingly low (3 revertants/plate vs historical mean of 7 revertants/plate) solvent control value resulted in a non-dose-related increase in responses observed at all test levels in tester strain TA1537 without metabolic activation. However, no similar 2-fold increase or any dose-related increases in revertants/plate were observed in TA1537 without metabolic activation in an independently repeated experiment

All other bacterial strains showed negative responses over the entire dose range, i.e. no dose related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Refer to attached document under 'Attached background material' for details on results.

Conclusions:

Under the test conditions, the substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance '4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride' according to OECD Guideline 471 and EU Method B.13/14 (Ames test), in compliance with GLP. Tester strains TA98, TA100, TA1535, TA1537 of Salmonella typhimurium and WP2uvrA of Escherichia coli were exposed to the test substance. The test was performed in two independent experiments in the presence and absence of Aroclor-1254 induced rat liver S9-mix. In the dose range finding test, the substance was tested up to concentrations of 5,000 µg/plate in the strains TA100 and WP2uvrA. The test substance precipitated as of 3,330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at 33 to 3,330 µg/plate in the absence and presence of 5% (v/v) S9-mix in TA1535, TA1537 and TA98. In the second mutation assay, it was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance precipitated on the plates at 3,330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations and no decrease in the number of revertants was observed. The test substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in WP2uvrA both in the absence and presence of S9-metabolic activation, as confirmed in an independently repeated experiment. In this study, the negative and strain-specific control values were within laboratory historical control data ranges, indicating that the conditions were adequate and the metabolic activation system functioned properly. Based on the results, it is concluded that the test substance is not mutagenic in theSalmonella typhimurium and the Escherichia coli reverse mutation assays (Verspeek, 2004).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 14 December 2009 to 24 February 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the read across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 Jul 1997

Deviations:

yes

Remarks:

during viability scoring, erroneous counts noted in single culture (with S-9, in experiment 1) due to dilution error

GLP compliance:

yes

Type of assay:

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

Target gene:

Thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Type and identity of media: RPMI 1640 medium
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes; each batch was purged of TK- mutants

Metabolic activation:

with and without

Metabolic activation system:

2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Range-finder experiment: 0, 46.88, 93.75, 187.5, 375 or 750 μg/mL (with and without S-9)
Main study:
- Experiment 1: Without S-9: 0, 0.0977, 0.1953, 0.3906, 0.7813, 1.563, 3.125, 6.25, 12.5, 25 or 50 μg/mL; with S-9: 0, 3.75, 7.5, 15, 30, 35, 40, 45, 50, 60 or 75 μg/mL
- Experiment 2: Without S-9: 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 μg/mL; with S-9: 0, 15, 30, 40, 50, 60, 70, 80, 90, 100 or 125 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test substance was soluble in anhydrous DMSO, with the aid of warming at 80 °C, at concentrations up to at least 545.5 mg/mL; solubility limit in the culture medium: 340.9-681.8 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzo(a)pyrene 2 or 3 µg/mL

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide 0.10, 0.15, 10 or 15 µg/mL

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 d, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 12-13 d, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Duplicates (single cultures only used for positive control treatments)

NUMBER OF CELLS EVALUATED: 20000 cells/well plated for survival, viability and 6TG resistance

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

OTHER: Cell viability were identified by eye using background illumination and counted; cell densities were determined using a coulter counter.

Evaluation criteria:

For valid data, the test substance was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05)
3. The effects described above were reproducible.

- Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis

Statistics:

The experimental data was analysed using UKEMS recommended statistical guidelines.

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: No data; solubility limit in the culture medium: 340.9-681.8 μg/mL
- Precipitation: Yes; in the range-finding study, precipitation was observed at concentrations of 93.75-1500 μg/mL tested in the absence and presence of S-9

RANGE-FINDING/SCREENING STUDIES: Six concentrations were tested with and without S-9 ranging from 46.88-1500 μg/mL (limited by solubility in culture medium).
- Precipitation was observed at concentrations of 93.75 to 1500 μg/mL tested with and without S-9. Lowest precipitating concentration was retained and the higher concentration was discarded.
- Without S-9: Complete toxicity was observed at all concentrations; with S-9: highest concentration providing > 10% RS was 46.88 μg/mL
- See table 1 for more details


COMPARISON WITH HISTORICAL CONTROL DATA: Yes; historical mean mutant frequencies for the vehicle controls were 4.34 without S-9 and 4.22 with S-9

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding study, complete toxicity was observed at all concentrations in the absence of S-9. In the presence of S-9, highest concentration providing > 10% RS was 46.88 μg/mL which gave 21% RS.

Remarks on result:

other: strain/cell type: TK+/- (3.7.2C) cells

Remarks:

Migrated from field 'Test system'.

Table 1: RS values - range-finder experiment:

Treatment (µg/mL)	-S-9 %RS	+S-9 %RS
0	100	100
46.88	0	21
93.75 P	0	0
187.5 P	0	0
375 P	0	0
750 P, PP	0	0

% RS: Percentage Relative Survival

P: Precipitation observed at time of treatment

PP: Precipitation observed following treatment incubation period

Table 2: Summary of mutation data:

Experiment 1: (3 h treatment in the absence and presence of S-9)

Treatment (µg/mL)	-S-9			Treatment (µg/mL)	+S-9
	%RS	MF§			%RS	MF§
0	100	2.21		0	100	1.34
0.0977	91	4.25	NS	3.75	103	NE
0.1953	84	2.3	NS	7.5	113	NE
0.3906	105	NE		15	107	NE
0.7813	90	NE		30	98	3.53	NS
1.563	84	NE		35	92	1.72	NS
3.125	79	3.81	NS	40	77	NE
6.25	75	2.24	NS	45	81	3.45	NS
12.5	58	6.66	*	50	83	NE
25	4	2.97	NS	60	61	9.53	*
50	0	NE		75	45	5.23!	NS
Linear trend		NS		Linear trend		**
NQO				B[a]P
0.1	61	45.16		2	30	97.71
0.15	47	49.83		3	11	190.6

Experiment 2: (3 h treatment in the absence and presence of S-9)

Treatment (µg/mL)	-S-9 %			Treatment (µg/mL)	+S-9
	%RS	MF§			%RS	MF§
0	100	2.89		0	100	3.56
3	101	4.05	NS	15	86	5.71	NS
6	71	4.69	NS	30	63	3.61	NS
9	76	7.85	*	40	29	4.24	NS
12	57	3.38	NS	50	21	6.69	NS
15	59	3.15	NS	60	6	4.67	NS
18	27	3.66	NS	70	4	NE
21	18	6.87	NS	80	3	NE
24	5	NE		90	1	NE
27	2	NE		100	0	NE
30	0	NE		125	0	NE
Linear trend		NS		Linear trend		NS
NQO				B[a]P
10	34	29.68		2	70	85.43
15	40	52.00		3	25	61.29

§: 6TG resistant mutants/106 viable cells 7 days after treatment

% RS: Percent relative survival adjusted by post treatment cell counts

* Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

NS: Not significant

*, **, *** Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively

NE: Not evaluated for mutant frequency due to excessive toxicity. Presented for information only

! Based on one replicate only

Conclusions:

Based on the results of the read across study, the test substance is not considered mutagenic in an in vitro mammalian cell gene mutation test.

Executive summary:

An in vitro mammalian cell gene mutation test was conducted with the read-across substance ‘4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid’ according to OECD Guideline 476, in compliance with GLP. Mouse lymphoma L5178Y TK+/-(3.7.2C) cells were exposed to test substance in DMSO at concentrations of 0, 46.88, 93.75, 187.5, 375 or 750 μg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test. In the main study, two experiments were performed. Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a) pyrene (with S-9). In Experiment 1, statistically significant increases in mutant frequency were seen at 12.5 (without S-9) and 60 µg/mL (with S-9). A significant linear trend occurred in the presence of S-9 only. In Experiment 2, a statistically significant increase in mutant frequency was observed at 9 µg/mL in the absence of S-9 only. However, no significant increases were seen following treatment with S-9 and there were no significant linear trends in either the absence or presence of S-9. Further, in both experiments, the increases in mutant frequency at the significant concentrations were all within three times the historical mean value and the increases were not considered concentration related or biologically relevant (Stone, 2010). Based on the results of the read across study, the test substance is not considered mutagenic in an in vitro mammalian cell gene mutation test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the results of the in vivo micronucleus assay with the read across substance BADGEDA in mice, the test substance is not expected to have clastogenic potential.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 3, 2007 to August 7, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the read across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: CD-1® (ICR) BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, USA
- Age at study initiation: 8 wk
- Weight at study initiation: Preliminary study: 32.9-39.4 g (males) or 25.8-29.4 g (females); Main study: 32.1-40.0 g (males)
- Assigned to test groups randomly: Yes, animals randomized using a computer program
- Housing: Individually housed in sanitary polycarbonate cages
- Diet (e.g. ad libitum): PMI Certified Rodent Diet® #5002, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 64-79 °F
- Humidity: 30-70%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: April 12, 2007 To: April 19, 2007

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: 25 or 50 mg/mL
- Amount of vehicle (if gavage): 20 mL/kg
- Lot/batch no. (if required): 12-455
- Source: Welch, Holme, & Clarke
- Storage: Refrigerated at 0-10 °C

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was emulsified with corn oil to give stock solution which was then diluted further to give the desired dose levels; formulations were held in a waterbath at ~60 to 70 °C prior to dosing and stirred during the dosing procedure to prevent emulsion breakdown.

Duration of treatment / exposure:

24 or 48 h

Frequency of treatment:

Once for dose levels of 500 and 1000 mg/kg bw, and twice, approximately 1 h apart, for the dose level of 2000 mg/kg bw (1000 mg/kg/dose, BID).

Remarks:

Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

-Range-finding study: 3 mice/sex/dose
-Main study: Five male mice/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Lot no.: 076K1050
- Storage: Refrigerated at 0-10 °C
- Route of administration: Oral (gavage)
- Doses / concentrations: 80 mg/kg bw

Tissues and cell types examined:

Hind limb bones (tibias) were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and total erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dose selection was based on a preliminary range-finding test conducted on 3 mice/sex/dose at 500, 1000 and 2000 mg/kg bw. No mortality and/or toxic signs were recorded after 2 d of exposure.

TREATMENT AND SAMPLING TIMES: Hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group at 24 or 48 h after exposure.

DETAILS OF SLIDE PREPARATION: Bone marrow cells extracted, preparations spread on slides and air-dried. The slides were fixed in methanol, stained with May-Grunwald solution and Giemsa and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, PCE:NCE ratio was determined in a population of 500 erythrocytes/animal.

Evaluation criteria:

- Criteria for a positive response: Detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response.
- Test substance that does not induce both of these responses is considered negative.
- Biological relevance of the results should be considered first.

Statistics:

- Statistical analysis was performed using ANOVA/Program Trend computer system.
- ANOVA followed by Dunnett’s t-test performed on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-2000 mg/kg bw
- Solubility: Solubility limit in corn oil: 50 mg/mL
- Clinical signs of toxicity in test animals: No
- Evidence of cytotoxicity in tissue analyzed: Not examined
- Rationale for exposure: No appropriate toxicity data were available

RESULTS OF DEFINITIVE STUDY
- Frequency of micronuclei in polychromatic erythrocytes and PCE:NCE ratio: See table 1

Table 1: Micronucleus Assay - Summary Table

Treatment	Dose	Harvest Time (h)	% Micronucleated PCEs Mean of 2000 per Animal ± S.E.(Males)	Ratio PCE:NCE Mean ± S.E.(Males)
Controls
Vehicle	Corn Oil 20 mL/kg	24	0.04 ± 0.02	0.51 ± 0.06
		48	0.03 ± 0.02	0.35 ± 0.01
Positive	CP 80 mg/kg bw	24	2.77 ± 0.28*	0.55 ± 0.1
Test substance	500 mg/kg bw	24	0.04 ± 0.03	0.43 ± 0.05
	1000 mg/kg bw	24	0.06 ± 0.02	0.32 ± 0.07**
	2000 mg/kg bw^a	24	0.02 ± 0.01	0.59 ± 0.06
		48	0.05 ± 0.02	0.4 ± 0.08

* Significantly greater than the corresponding vehicle control, p ≤ 0.01.

** Significantly less than the corresponding vehicle control, p ≤ 0.05.

CP = Cyclophosphamide; PCE = Polychromatic erythrocyte; NCE = Normochromatic erythrocyte

^aDue to solubility limit, the high dose was dosed at 1000 mg/kg BID (~1 h apart) to achieve a dose of 2000 mg/kg bw.

Conclusions:

Based on the results of the read across study, the test substance is not considered as mutagenic in an in vivo bone marrow micronucleus test.

Executive summary:

An in vivo bone marrow micronucleus test was performed with read across substance 'bisphenol A diglycidyl ether diacrylate' according to OECD Guideline 474, in compliance with GLP. CD-1® (ICR) BR male mice (5/dose) were given a single oral (gavage) dose of the test substance in corn oil at concentrations of 500 and 1000 mg/kg bw and twice, approx 1 h apart, at concentration of 2000 mg/kg bw (1000 mg/kg/dose, BID). Bone marrow was extracted after 24 or 48 h of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, the number of PCEs and normochromatic erythrocytes (NCEs) in a population of 500 erythrocytes was determined as a measure of cytotoxicity. A preliminary range-finding test was also conducted on 3 mice/sex/dose at 500, 1000 and 2000 mg/kg bw and animals were observed for 2 d. In this previous test, no mortality and/or toxic signs were recorded. No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. A statistically significant decrease in the PCE:NCE ratios at a dose level of 1000 mg/kg bw was observed but was not considered biologically significant as the highest dose of 2000 mg/kg bw did not show a similar trend (Yong, 2007). Based on the results of the read across study, the test substance is not considered as mutagenic in an in vivo bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

An in vitro bacterial reverse mutation assay was conducted to determine the mutagenic potential of the test substance '4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride' according to OECD Guideline 471 and EU Method B.13/14 (Ames test), in compliance with GLP. Tester strains TA98, TA100, TA1535, TA1537 of Salmonella typhimurium and WP2uvrA ofEscherichia coliwere exposed to the test substance. Two independent experiments were conducted in the presence and absence of Aroclor-1254 induced rat liver S9-mix. In the dose range finder, the substance was tested up to concentrations of 5,000 µg/plate in the strains TA100 and WP2uvrA. The substance precipitated as of 3,330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations and no biologically relevant decrease in the number of revertants was observed. Based on the results of the dose range finding test, the substance was tested in the first mutation assay at 33 to 3,330 µg/plate in the absence and presence of 5% (v/v) S9-mix in TA1535, TA1537 and TA98. In the second mutation assay, it was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in TA1535, TA1537, TA98, TA100 and WP2uvrA. The substance precipitated on the plates at 3,330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations and no decrease in the number of revertants was observed. The substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in WP2uvrA both in the absence and presence of S9-metabolic activation, as confirmed in an independently repeated experiment. In this study, the negative and strain-specific control values were within laboratory historical control data ranges, indicating that the conditions were adequate and the metabolic activation system functioned properly. Based on the results, it is concluded that the test substance is not mutagenic in theSalmonella typhimurium and theEscherichia colireverse mutation assays (Verspeek, 2004).

An in vitro mammalian cell gene mutation test was conducted with the read-across substance ‘4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid’ according to OECD Guideline 476, in compliance with GLP.Mouse lymphoma L5178Y TK+/-(3.7.2C) cells were exposed to test substance in DMSO at concentrations of 0, 46.88, 93.75, 187.5, 375 or 750 μg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test. In the main study, two experiments were performed. Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a) pyrene (with S-9). In Experiment 1, statistically significant increases in mutant frequency were seen at 12.5 (without S-9) and 60 µg/mL (with S-9). A significant linear trend occurred in the presence of S-9 only. In Experiment 2, a statistically significant increase in mutant frequency was observed at 9 µg/mL in the absence of S-9 only. However, no significant increases were seen following treatment with S-9 and there were no significant linear trends in either the absence or presence of S-9. Further, in both experiments, the increases in mutant frequency at the significant concentrations were all within three times the historical mean value and the increases were not considered concentration related or biologically relevant (Stone, 2010).Based on the results of the read across study, the test substance is not considered mutagenic in an in vitro mammalian cell gene mutation test.

An in vitro cytogenicity study in mammalian cells was not conducted since in vivo testing with a read-across substance was available (mouse micronucleus study).

In vivo

An in vivo bone marrow micronucleus test was performed with read across substance 'bisphenol A diglycidyl ether diacrylate' according to OECD Guideline 474, in compliance with GLP.CD-1® (ICR) BR male mice (5/dose) were given a single oral (gavage) dose of the test substance in corn oil at concentrations of 500 and 1000 mg/kg bw and twice, approx 1 h apart, at concentration of 2000 mg/kg bw (1000 mg/kg/dose, BID). Bone marrow was extracted after 24 or 48 h of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, the number of PCEs and normochromatic erythrocytes (NCEs) in a population of 500 erythrocytes was determined as a measure of cytotoxicity. A preliminary range-finding test was also conducted on 3 mice/sex/dose at 500, 1000 and 2000 mg/kg bw and animals were observed for 2 d. In this previous test, no mortality and/or toxic signs were recorded.No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. A statistically significant decrease in the PCE:NCE ratios at a dose level of 1000 mg/kg bw was observed but was not considered biologically significant as the highest dose of 2000 mg/kg bw did not show a similar trend (Yong, 2007).

Overall, based on the available results from thein vitroAmes assay with the test substance andin vitromouse lymphoma andin vivomouse micronucleus assays with the read across substance indicates that the test substance is not expected to have genotoxic potential.

Justification for classification or non-classification

The test substance and the read across substance BADGEDA were not mutagenic in an bacterial reverse mutation assay and mouse lymphoma assay respectively. The read across substance was also not clastogenic in an in vivo micronucleus assay. Based on the overall weight of evidence, the test substance is not required to be classified for genotoxicity according to EU CLP (EC 1272/2008) criteria.