Esterification products of acrylic acid and 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:

3 August 2012 to 15 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Esterification products of acrylic acid and 4,4'-isopropylidenediphenol ethoxylated

EC Number:

701-362-9

Molecular formula:

(C2 H4 O)x (C2 H4 O)y C21 H20 O4

IUPAC Name:

Esterification products of acrylic acid and 4,4'-isopropylidenediphenol ethoxylated

Details on test material:

SID Change
Previous CAS: 64401-02-1
Previous EC: 613-584-2

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9), prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range finding test: six concentrations were tested in the presence and absence of S-9 ranging from 31.25 to 1000 µg/mL
Experiment 1: eleven concentrations ranging from 6 to 60 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9
Experiment 2: twelve concentrations ranging from 5 to 50 µg/mL in the absence of S-9 and ten concentrations ranging from 50 to 275 µg/mL in the presence of S-9

Positive controls:
4-nitroquinoline 1 oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no S-9 present
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL, S-9 present

Vehicle / solvent:

DMSO diluted 100-fold in the treatment medium

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment area

True negative controls:

no

Positive controls:

yes

Remarks:

4-nitroquinoline-N-oxide without metabolic activation and benzo(a)pyrene with metabolic activation) Positive control substance 4-nitroquinoline-N-oxide without metabolic activation and benzo(a)pyrene with metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: NA
- Exposure duration: 3 Hours
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): 7 Days

NUMBER OF REPLICATIONS: 9

DETERMINATION OF CYTOTOXICITY
- Method: Osmolality

Evaluation criteria:

For valid data, the test article 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.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes
- Effects of osmolality: No marked changes
- Evaporation from medium: Not reported
- Water solubility: Preliminary solubility data indicated that Ethoxylated bisphenol A diacrylate (CAS Number 64401-02-1) was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 500.0 mg/mL. The solubility limit in culture medium was approximately 156.3 to 312.5 µg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 3 hours after test article addition
- Precipitation: no precipitate was observed in the absence and presence of S-9 following the 3 hour treatment incubation period
- Other confounding effects: Not Applicable

Details of results :
In Experiment 1 eleven concentrations, ranging from 6 to 60 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the time of treatment at the highest nine concentrations (200 to 500 µg/mL) in the presence of S-9 but no precipitate was observed in the absence and presence of S-9 following the 3-hour treatment incubation period. Seven days after treatment, the highest two concentrations in the absence of S-9 (50 and 60 µg/mL) and the highest seven concentrations in the presence of S-9 (300 to 500 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentration analysed in the absence of S-9 was 40 µg/mL, which gave 21% RS and was considered acceptably close to the target toxicity range of 10-20% RTG. In the presence of S-9, steep concentration-related toxicity was observed between 200 and 250 µg/mL, which gave 44% and 2% RS, respectively. Marked heterogeneity was observed at 250 µg/mL but the concentration was included in the analysis for comparative purposes.
In Experiment 2 twelve concentrations, ranging from 5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 275 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the time of treatment at the highest eight concentrations in the presence of S-9 (150 to 275 µg/mL) but no precipitate was observed in the absence and presence of S-9 following the 3-hour treatment incubation period. Seven days after treatment the highest concentration in the absence of S-9 (50 µg/mL) and the highest five concentrations in the presence of S-9 (220 to 275 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, concentrations of 5 and 15 µg/mL in the absence of S-9 were not selected as there were sufficient non-toxic concentrations to define the toxicity profile. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 45µg/mL in the absence of S-9 and 210 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively.

Any other information on results incl. tables

Table1: RS Values - Range-Finder Experiment

Treatment (µg/mL)	-S-9 % RS	+S-9 % RS
0	100	100
31.25	13	190
62.5	0	154
125	0	127
250	0 P	72 P
500	0 P, PP	0 P
1000	NP P, PP	0 P, PP

% RS                      Percent relative survival adjusted by post treatment cell counts

P                             Precipitation observed at time of treatment

PP                          Precipitation observed following treatment incubation period

NP                          Not plated for viability due to precipitation

 

Table 2: Summary of mutation data

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

Treatment (mg/mL)		-S-9				Treatment (mg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	2.55			0		100	5.11
6		93	3.24		NS	50		114	6.12		NS
12		85	4.19		NS	100		106	4.21		NS
16		85	4.33		NS	200	P	44	4.95		NS
20		74	4.08		NS	250	P,$	2	3.95		NS
24		62	5.78		NS
28		50	6.69		NS
32		12	5.28		NS
36		25	6.60		NS
40		21	1.68		NS
Linear trend				NS		Linear trend				NS
NQO						B[a]P
0.15		61	47.14			2		70	43.33
0.2		44	52.31			3		52	71.30

 

Experiment 2 (3 hour 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	9.15		0		100	10.71
10		107	8.86	NS	50		112	1.90	NS
20		76	1.80	NS	100		105	3.98	NS
25		68	5.56	NS	150	P	71	2.33	NS
30		50	10.67	NS	200	P	21	7.64	NS
33		41	7.43	NS	210	P	14	6.57	NS
36		35	6.10	NS
39		33	3.56	NS
42		20	3.97	NS
45		15	1.92	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		58	61.92		2		50	27.37
0.2		57	101.97		3		28	47.20

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

% RS                      Percent relative survival adjusted by post treatment cell counts

P                             Precipitation observed at the time of treatment

$                             Marked heterogeneity observed for mutation, but included in analysis for comparative purposes

NS                          Not significant

 

Applicant's summary and conclusion

Conclusions:

It is concluded that Ethoxylated bisphenol A diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

Ethoxylated bisphenol A diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for all experiments. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 31.25 to 1000 µg/mL (limited by solubility in culture medium). The highest concentrations to survive treatment were 31.25 µg/mL in the absence of S-9 and 250 µg/mL in the presence of S-9, which gave 13% and 72% relative survival (RS), respectively. Accordingly, for Experiment 1 eleven concentrations, ranging from 6 to 60 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentration analysed in the absence of S-9 was 40 µg/mL, which gave 21% RS and was considered acceptably close to the target toxicity range of 10-20% RTG. In the presence of S-9, steep concentration related toxicity was observed between 200 and 250 µg/mL, which gave 44% and 2% RS, respectively. Marked heterogeneity was observed at 250 µg/mL but the concentration was included in the analysis for comparative purposes. In Experiment 2 twelve concentrations, ranging from 5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 275 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed were 45 µg/mL in the absence of S-9 and 210 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively. Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within acceptable 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). Therefore the study was accepted as valid. In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Ethoxylated bisphenol A diacrylate (CAS Number 64401 -02-1) at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. It is concluded that Ethoxylated bisphenol A diacrylate (CAS Number 64401-02-1) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).