Zinc acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Cells deficient in thymidine kinase (TK) due to the mutation TK+/- to TK-/- are resistant to the cytotoxic effects of trifluorothymidine (TFT). Thymidine kinase proficient cells (TK+/-) are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not able to proliferate.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus/TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

No data

Test concentrations with justification for top dose:

1.21-12.13 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Normal saline (1 %)
- Justification for choice of solvent/vehicle: Not reported

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

None

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium


DURATION
- Preincubation period: Not reported
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 d (at 37 °C in 5 % C02-95 % humidified air)



SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) - 4 μg/mL


NUMBER OF REPLICATIONS: Duplicate cell culture for test material treatment while triplicate plates were prepared for both survival and mutation frequency determinations for each of the 2 replicate cultures


NUMBER OF CELLS EVALUATED: Cells densities were 30000/mL (1 x 100,000/plate) for mutant selection and 15/mL (500/plate) for viability detrmination


DETERMINATION OF CYTOTOXICITY
- Method: Cell survival for each culture was the product of growth in suspension culture and cloning efficiency in soft-agar medium, each relative to solvent controls

OTHER: Cell culture contained 6 x 100,000 cells each in 10 mL test medium
Light exposure was minimal during treatment of cells.
Test conducted at 37 °C
For the recovery and mutant expression, all cells were maintained at 37 °C for 48 h in log phase growth after treatment with test material

Evaluation criteria:

Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT Resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates ie., large, were scored as mutants.

Statistics:

Not reported

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Not reported

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic.

Executive summary:

A study was conducted to assess the potential mutagenicity of the test substance in the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The mouse lymphoma cells (TK+/-) were treated with test substance at 1.21-12.13 µg/mL for 3 h. 48 h after treatment, cells were treated with 4 µg/mL trifluorothymidine (TFT) for 7 d. Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants. Under the study conditions, the test substance was found to be non-mutagenic (Amacher, 1980).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The clastogenic activity of zinc chloride was determined by studying chromosome aberrations in human dental pulp cells in vitro, both in the presence and absence of exogenous metabolic activation.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: Human dental pulp cells (D824 cells)

Details on mammalian cell type (if applicable):

- Dental pulp tissue obtained from a lower third molar extracted from a 22 years old woman
- Type and identity of media: α-minimum essential medium supplemented with 20 % fetal bovine serum (FBS), 100 µM L-ascorbic acid phosphate magnesium salt n-hydrate, 2 mM L-glutamine, 0.22% NaHCO3 and 100 µg/mL streptomycin

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

5% rat liver postmitochondrial supernatant (PMS) mixture

Test concentrations with justification for top dose:

30, 100 and 300 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (60mM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

None Migrated to IUCLID6: 50 µM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: Overnight
- Exposure duration: 3 h


NUMBER OF REPLICATIONS: Triplicate


NUMBER OF CELLS EVALUATED: 1.6×10 (5) cells/dish


DETERMINATION OF CYTOTOXICITY: Yes
- Method: The cytotoxicity of the test material was determined as the number of cells treated with the test material relative to the number of cells in the control cultures×100


OTHER EXAMINATIONS:
- Determination of polyploidy: Yes


OTHER: The pH range of the culture media containing the highest concentrations of test agents was approximately 7.2–7.5.

Evaluation criteria:

No data

Statistics:

χ2-test was used to assess the significance of the difference in the incidences of chromosome aberrations between control cultures and cultures treated with test agents. The level of significance in the statistical analysis was determined at p<0.05.

Key result

Species / strain:

other: Human dental pulp cells (D824 cells)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Table 1: Chromosome aberrations in D824 cells induced by treatment with ZnCl2 for 3 h and 30 h:

Test material	Time	Concentration	Relative cell number (%)	Number of metaphases scored	Type of structural aberrations(a) (%)								Aberrant metaphases (%)	Polyploidy and endoreduplication (%)
					ctg	csg	ctb	csb	cte	D	O	F
Control	3 h	0	100	500	0.8	0	0	0	0	0	0	0	0.8	2
Zinc chloride (µM)		30	91	100	0	0	0	0	0	0	0	0	0	0
		100	74	100	1	0	0	0	0	0	0	0	1	1
		300	77	100	0	0	0	0	0	0	0	0	0	1
Control	30 h	0	100	500	1	0	0	0	0	0	0	0	1	2
Zinc chloride (µM)		30	85	100	0	0	0	0	0	0	0	0	0	2
		100	77	100	1	0	0	0	0	0	0	0	1	2
		300	74	200	3.5	0	0.5	0	0	0	0	0	4	0

a = ctg, chromatid gaps; csg, chromosome gaps; ctb, chromatid breaks; csb, chromosome breaks; cte, chromatid exchanges; D, dicentric chromosomes; O, ring chromosomes; F, fragmentations.

 

Conclusions:

Under the study conditions, the test substance was considered to be non-clastogenic to human dental pulp cells in vitro.

Executive summary:

A study was conducted to investigate the ability of zinc chloride to induce chromosome aberrations in human dental pulp (D824) cells. Cells were treated with the test substance and evaluated for chromosome aberrations at up to 3 dose levels together with vehicle and positive controls. Rat liver (5%) post mitochondrial supernatant mixture was used as the exogenous metabolic activator. Ability to induce chromosome aberrations was examined in cells treated with test substance for 3 and30 h. The test substance failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by test substance. Under the study conditions, the test substance was considered to be non-clastogenic to human dental pulp cells in vitro (Someya, 1980).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, short report, few details but meets basic scientific principles.

Principles of method if other than guideline:

Method: According to "Ames BN, McCann J & Yamasaki E (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31: 347-364".

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fraction from Aroclor-pretreated rats

Test concentrations with justification for top dose:

At least 5 doses, up to 3,600 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium; test material was tested on 2 slightly different minimal media, the composition of which are as follows:

1. ZLM medium
- Tri-Na citrate·2H2O = 0.82 g/L
- K2HPO4·3H2O = 4.60 g/L
- KH2PO4 = 1.50 g/L
- (NH4)2SO4 = 1.00 g/L
- MgSO4·7H2O = 0.10 g/L
- Glucose = 17.0 g/L

2. Vogel-Bonner (VB) medium
The concentration of citrate was 3.5 times higher in VB medium than in ZLM medium. The concentrations of the other ions are up to 2-fold higher in VB medium.

Evaluation criteria:

Not available

Statistics:

Not available

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic.

Executive summary:

A study was conducted to determine the potential mutagenicity of the test substance using the bacterial reverse mutation assay (e.g. Ames test). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 strain were treated with the test substance in Vogel-Bonner (VB) and ZLM medium (modified minimal medium for E. coli) at a minimum of five dose levels, up to 3,600 µg/plate, both with and without activation by the S9 liver fraction from Aroclor-pretreated rats. The positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. No significant increases in the frequency of revertant colonies were observed for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Under the study conditions, the test substance was considered to be non-mutagenic (Gocke, 1981).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Dose level not up to cytotoxic range; neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Dose level not up to cytotoxic range; neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents

Principles of method if other than guideline:

The study was conducted according to the method described by Ames (1975) with minor modifications.
Ames BN et al. (1975). Mut. Res. 31: 347-364

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His -

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 mix

Test concentrations with justification for top dose:

3.1 - 1000 nL/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See remarks

Details on test system and experimental conditions:

Method of application: in medium; in agar (plate incorporation)
Replicates: 2-4

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Results

Mean number of revertants with metabolic activation:

Test substances	Dose / plate (µg)	TA1535	TA1537	TA98	TA98 TCPO*	TA100
Acrylic acid	3.3	23	24	51	79	117
	10.5	20	25	73	58	117
	33.2	23	23	58	58	124
	105	25	26	57	69	121
	331	20	25	44	61	116
	1050	20	22	64	63	111
Positive Control-1	3.15	-	-	245	308	387
	6.3	231	96	1298	-	1550
	10	519	58	-	-	1465
	20	890	-	-	-	-
Positive Control-2	10	21	150	599	781	520
Positive Control-3	90	22	85	800	676	3495
Positive Control-4	10	-	-	-	-	448
Vehicle Control		21	24	49	62	110

- Not done

Positive Control-1: 2-Aminoanthracene

Positive Control-2: Benzo(a)pyrene

Positive Control-3: 3-Methylcholanthrene

Positive Control-4: trans-N-Methyl-4-Aminostilbene

* 60 nL of epoxide hydratase inhibitor (1,1,1 Trichloro- propene-2,3-oxide) was added to each plate dissolved in 10 µL DMSO

Mean number of revertants without metabolic activation:

Test substances	Dose / plate (µg)	TA1535	TA1537	TA98	TA100
Acrylic acid	3.3	15	8	15	169
	10.5	18	10	17	160
	33.2	20	8	16	113
	105	22	10	17	113
	331	11	9	21	121
	1050	11	10	15	131
Positive Control-1	1	903	7	1913	232
	3.15	8500	31	29	4250
	10	31000	39	51	9250
Positive Control-2	2	17	572	3650	1060
Vehicle Control		19	8	15	117

- Not done

Positive Control-1: N-Methyl-N-Nitro-N-Nitrosoguanidine

Positive Control-2: Benzo(a)pyren-4-5-oxide

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance, acrylic acid (>99%) according to a method similar to OECD Guideline 471. The study was conducted according to the method described by Ames (1975) with minor modifications. Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 were used as test system, neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents. The test substance concentrations were from 3.1 to 1000 nL/plate (not up to cytotoxic level) in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, using the plate incorporation method (2-4 replicates), with and without metabolic activation (Aroclor 1254 induced rat liver S-9 mix). There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in study. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (BASF, 1977).

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From February 17, 1987 to August 30, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

The HGPRT assay was performed based on the procedure described by O'Neill et al. (1977) and Gupta and Sing (1982).
O'Neill et al. (1977). Mutat Res 45: 91-101
Gupta and Sing (1982). Mutat Res 94: 449-466

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Target gene:

HGPRT (hypoxanthine-guanine phosphoribosyl transferase) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (S9 mix) prepared from male Fischer rats that were induced by ip injection of Aroclor-1254 at 500 mg/kg bw

Test concentrations with justification for top dose:

Without metabolic activation: 0.3, 0.6, 1.0, 1.5, 1.9 µL/mL
With metabolic activation: 1.0, 1.5, 1.9, 2.4, 2.8 µL/mL

Vehicle / solvent:

Phosphate buffer

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Method of application: in medium

Duration
- Exposure duration: 5 h
- Expression time (cells in growth medium): 7-9 d
- Selection time (if incubation with a selection agent): 7-10 d

Selection agent (mutation assays): Hypoxanthine (Hx)

Number of replications:
Triplicate cultures were used for each treatment condition. Replicate plates were subcultured in F12FBS5 or F12FBS5-Hx throughout the experiment.
The mutation assay was repeated at the Sponsor's request with a confirmatory assay.

Determination of cytotoxicity
- Method: cloning efficiency

Evaluation criteria:

In the testing laboratory, the confidence interval for the CHO/HGPRT assay was set at 8.7/1 000 000 clonable cells. Therefore, the mutagenic response after treatment was considered significant only when the treatment mutant frequency was increased above that of the solvent control and the untreated control by at least 8.7 mutants/1 000 000 clonable cells and also was at least twice that of the solvent control and the untreated control.

Criteria for evaluation of a valid test:
1) The cloning efficiency of the solvent and untreated controls must be no less than 50 %.
2) The spontaneous mutant frequency in the solvent and untreated controls must fall within the range of 0-20 mutants per 1 000 000 clonable cells.
3) The positive control must induce a mutant frequency at least three times that of the solvent control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

all strains/cell types tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Additional information on cytotoxicity

- 1st trial:
Survival was 72, 95, 82, 85, and 86 % at 1.9, 1.5, 1.0, 0.6, and 0.3 µL/mL, respectively without, and 89, 71, 80, 86, and 91 % at 2.8, 2.4, 1.9, 1.5, and 1.0 µL/mL, respectively, with metabolic acitvation.

- 2nd trial (confirmatory):
Survival was 31, 38, and 37 % at 1.9 µL/mL, and 60, 52, and 65 % at 1.5 µL/mL, and 90, 72, and 82 % at 1.0 µL/mL, and 67, 80, and 78 % at 0.6 µL/mL, and 88, 91, and 102 % at 0.3 µL/mL, respectively, without metabolic activation.

Survival was 1, 2, and 3 % at 2.8 µL/mL, and 22, 26, and 23 % at 2.4 µL/mL, and 39, 31, and 36 % at 1.9 µL/mL, and 62, 63, and 51 % at 1.5 µL/mL, and 9, 89, and 103 % at 1.0 µL/mL, respectively with metabolic activation.

Results

In the first trial, the mutant frequencies of two and one of the test article tested groups without and with metabolic activation respectively were increased significantly above the controls. But increases were barely in excess of two-fold the highest concurrent background (untreated or solvent control) levels. In addition, no dose-response was apparent, and the non-activated assay had not achieved sufficient toxicity. Consequently, an additional mutation assay was performed. Triplicate cultures were independently subcultured throughout the repeat assay to provide confirmation of any significant, dose-dependent increases in mutant frequency.

Non-activated assay (confirmatory assay):

Treatment	Cloning efficiency*	Mutants/E+06 clonable cells*
Untreated	1.12	11.5
Solvent	1.03	1.6
1.9 µL/mL	1.03	5.0
1.5 µL/mL	1.08	5.8
1.0 µL/mL	1.02	16.7
0.6 µL/mL	0.82	1.2
0.3 µL/mL	0.88	11.7
EMS	1.10	126.6

* Means of triplicate cultures

 

 

 

Activated assay (confirmatory assay):

Treatment	Cloning efficiency*	Mutants/E+06 clonable cells*
Untreated	1.24	13.3
Solvent	1.28	8.2
2.4 µL/mL	0.96	4.2
1.9 µL/mL	1.15	2.2
1.5 µL/mL	0.97	1.3
1.0 µL/mL	1.16	7.9
BaP	0.98	243.5

* Means of triplicate cultures

In the confirmatory assay with and without metabolic activation the mutant frequencies of the test article treated groups were not increased significantly above the controls. The negative and positive controls fulfilled the requirements for a valid test. Under the conditions of the assay, acrylic acid should be considered negative in the CHO/HGPRT mutation assay.

Conclusions:

Under the study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in CHO cells.

Executive summary:

A study was conducted to determine the in vitro genotoxicity potential of the test substance, acrylic acid (99.2%), at the HGPRT locus in Chinese hamster ovary (CHO) cells, according to method similar to OECD Guideline 476, in compliance with GLP. The HGPRT assay was performed based on the procedure described by O’Neill et al. (1977) and Gupta and Sing (1982). Test substance concentrations (without metabolic activation: 0.3, 0.6, 1.0, 1.5, 1.9 µL/mL and with metabolic activation: 1.0, 1.5, 1.9, 2.4, 2.8 µL/mL) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five hour exposure was used both with and without S9-mix (rat liver homogenate prepared from male Fischer rats that were induced by i.p. injection of Aroclor-1254 at 500 mg/kg bw). In the first trial, the mutant frequencies of two and one of the test article tested groups without and with metabolic activation respectively were increased significantly above the controls. Hiowever, increases were barely in excess of two-fold the highest concurrent background (untreated or solvent control) levels. In addition, no dose-response was apparent, and the non-activated assay had not achieved sufficient toxicity. Consequently, an additional mutation assay was performed. Triplicate cultures were independently subcultured throughout the repeat assay to provide confirmation of any significant, dose-dependent increases in mutant frequency. In the confirmatory assay with and without metabolic activation the mutant frequencies of the test article treated groups were not increased significantly above the controls. The negative and positive controls fulfilled the requirements for a valid test. Under the study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in CHO cells (BAMM, 1988).

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From February 26, 1987 to January 15, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

other: hepatocytes: primary culture (rat)

Details on mammalian cell type (if applicable):

Primary rat liver cell cultures were derived from the livers of normal adult male Sprague-Dawley rats according to the procedure described by Williams et al. (In Vitro 13: 809-817, 1977):

Metabolic activation:

without

Test concentrations with justification for top dose:

0.01, 0.03, 0.06, 0.1, 0.2, 0.3, 0.4, 0.6 µL/mL

Vehicle / solvent:

Phosphate buffered saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

Method of application: in medium
Exposure duration: 18-20 h (with test article and 3H-thymidine)
Replications: 3
Number of cells evaluated: 25 cells in random areas on each of two coverslips per treatment
Determination of cytotoxicity
- Method: lactic acid dehydrogenase (LDH) activity

Evaluation criteria:

If the mean net nuclear count was increased by at least five counts over the control, the results for a particular dose level were considered significant. A test article was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the control. In the absence of a dose-response, a test article which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive.

Key result

Species / strain:

other: hepatocytes: Primary Rat Hepatocytes

Remarks:

all strains/cell types teste

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Result:

The test substance, acrylic acid, was tested in the Unscheduled DNA Synthesis Test using rat primary hepatocytes. The test substance was originally tested at nine dose levels ranging from 0.001 to 3.0 µL/mL and was fully evaluated at five dose levels of 0.01, 0.03, 0.1, 0.2 and 0.3 µL/mL. A repeat assay was conducted using eight dose levels ranging from 0.01 to 0.6 µL/mL and was fully evaluated at seven dose levels of 0.01, 0.03, 0.06, 0.1, 0.2, 0.3 and 0.4 µL/mL. The results of the original UDS assay indicate that under the test conditions, the test substance did cause a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the control), at the second highest dose level evaluated, 0.2 µL/mL. The remaining dose levels showed no increase in net nuclear counts above the control. Since an analysis of the raw data indicated that there had not been an actual increase in nuclear counts, only a decrease in cytoplasmic counts, a repeat assay was performed. In the repeat assay the test substance did not cause a significant increase in mean net nuclear grain counts at any dose level. Therefore, the test substance was considered to be negative in this study.

Summary of Repeat UDS Assay: 

Treatment [µL/mL]	Relative survival [%]	Average net grains/nucleus	% cells with 5 or more net nuclear grains
Acrylic acid 0.6	26	-	-
Acrylic acid 0.4	90	0.0 ± 1.6	0
Acrylic acid 0.3	94	-0.4± 1.6	0
Acrylic acid 0.2	96	0.1 ± 1.4	0
Acrylic acid 0.1	96	0.1± 0.9	0
Acrylic acid 0.06	97	0.1 ± 1.6	2
Acrylic acid 0.03	99	0.1 ± 1.0	0
Acrylic acid 0.01	100	-0.6 ± 1.9	0
DMBA 10 µg/mL	79	12.6 ± 4.8	100
DMBA 3 µg/mL	88	6.2 ± 3.2	66
DMSO	100	-0.7 ± 1.5	0
PBS	100	0.1 ±1.0	0
Media control	98	-0.2± 1.4	0

DMBA: 7,12- Dimethylbenzanthracene (positive control)

DMSO:Dimethylsulfoxide (vehicle control to positive control)

PBS: Phosphate buffered saline (vehicle control)

Conclusions:

Under the study conditions, the test substance was determined to be non-genotoxic without metabolic activation, in ray hepatocytes in vitro.

Executive summary:

A study was conducted to determine the in vitro genotoxic potential of the test substance, acrylic acid (99.8%), according to a method similar to OECD Guideline 482 (Unscheduled DNA Synthesis test in rat primary hepatocytes), in compliance with GLP. The substance was originally tested at nine dose levels (0.01, 0.03, 0.06, 0.1, 0.2, 0.3, 0.4, 0.6 µL/mL) and fully evaluated at the five dose levels of 0.01, 0.03, 0.1, 0.2 and 0.3 µL/mL. A repeat assay was conducted using eight dose levels ranging from 0.01 to 0.6 µL/mL and was fully evaluated at the seven dose levels of 0.01, 0.03, 0.06, 0.1, 0.2, 0.3 and 0.4 µL/mL. The results of the original UDS assay indicated that the substance did cause a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the control), at the second highest dose level evaluated, 0.2 µL/mL. The remaining dose levels showed no increase in net nuclear counts above the control. Since an analysis of the raw data indicated that there had not been an actual increase in nuclear counts, only a decrease in cytoplasmic counts, a repeat assay was performed. In the repeat assay, the test substance did not cause a significant increase in mean net nuclear grain counts at any dose level. The study met the validity criteria. Under the study conditions, the test substance was therefore determined to be non-genotoxic without metabolic activation in rat hepatocytes in vitro (BAMM, 1988).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 29, 1986 to August 05, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

not specified

Principles of method if other than guideline:

An in vivo bone marrow chromosomal aberration assay was conducted with rats. Chromosome aberrations were analyzed (5 animals per sex, 50 metaphases per animal) at 6, 12, and 24 h after oral gavage doses of 100, 333 or 1000 mg/kg bw.

GLP compliance:

yes

Type of assay:

other: Chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: 6-7 weeks of age
- Weight at study initiation: 142-177 g (males), 132-164 g (females)
- Housing: singly
- Diet: ad libitum
- Water: ad libitum
- Assigned to test groups randomly: yes
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal):

Duration of treatment / exposure:

One treatment by gavage

Frequency of treatment:

Once

Post exposure period:

At the time specified following dosing in the acute dosing regime, the rats received an intraperitoneal injection of colchicine (1.0 mg/kg bw) based on terminal weight to arrest mitosis. 2-4 hrs later the animals were sacrificed.

Remarks:

Doses / Concentrations: 100, 333, and 1000 mg/kg bw (in a total volume of 3 mL/kg bw)
Basis: actual ingested

No. of animals per sex per dose:

5 animals/sex/dose/sacrifice time

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 10 mg/mL

Tissues and cell types examined:

Bone marrow cells from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose levels for the in vivo cytogenetic assay were selected on the basis of body weight changes, gross observations, and mortality in a preliminary toxicity test. The results of the preliminary toxicity test led to the selection of 1000 mg/kg body weight as the maximum dose for the acute assay. Also a preliminary assessment of bone marrow cell cycle kinetics at 900 mg/kg bw indicated no significant cell cycle delay at 21 hr after dosing.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): at 6, 12, and 24 hours after dose administration


DETAILS OF SLIDE PREPARATION: At least three slides from each animal were prepared. Stain: Giemsa


METHOD OF ANALYSIS: 50 metaphase spreads for each animal were scored. The mitotic index for each animal was determined.
Each metaphase figure was scored for the following items:
1. Number of chromosomes in each metaphase figure.
2. Gaps - Achromatic region in chromatid no greater than the width of the chromatid.
3. Chromatid breaks - Achromatic region in the chromatid greater than the wigth of the chromatid or where the broken piece is misaligned with the rest of the arm.
4. Chromosome breaks - Achromatic region in both chromatids at the same locus with marked displacement of both distal fragments.
5. Fragments - Chromatid(s) not containing a centromere. May be seen in association or not in association with a parent chromatid.
6. Exchange figure - Chromatid interchange involving two or more chromosomes, with either symmetrical or asymmetrical distortion of the usual chromatid pattern.
7. Dicentric - Chromosome with two centromeres.
8. Ring - Chromosome whose ends have joined to form a double or single circle, with or without a centromere.
9. Polyploid - Increase in chromosome number in excess of the diploid and in multiple of the haploid number.
10. Pulverization - Extreme fragmentation of the chromatid material.
11. Severely damaged cell - Cell with ten or more abberations of any type or with pulverization.

Evaluation criteria:

The test substance is considered to induce a positive response when the number of aberrations per cell is significantly increased (p <= 0.05, Student's t-test) relative to the vehicle control. A significant increase at the high dose only with no dose-response also is considered positive. A significant increase at one dose other than the high dose with no dose-response is considered equivocal.

Statistics:

The t-test was used to compare pairwise the number of aberrations per cell of each treated group with that of the vehicle control. Each comparison was considered to be between two independent, random samples of unequal variance and a significant increase in the treatment mean relative to the vehicle control (one-sided) was sought.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Dose range: 0, 700, 900, 1100, and 1300 mg/kg bw (male); 0, 600, 800, 1000, and 1200 mg/kg bw (female)
- Solubility: completely soluble
- Clinical signs of toxicity in test animals: One female rat in the 1200 mg/kg bw dose group and one male rat in the 900 mg/kg bw group were found dead approximately 72 hours following dose administration. A reduction in body weight gain was observed in all test article treated rats 24 hours after dose administration and persisted up to 72 hours in male rats that received 1200 and 1000 mg/kg bw. Clinical signs of toxicity observed included lethargy, irregular breathing (including wheezing and sneezing), lacrimation, crusty eyes, excessive salivation, and nasal discharge. Based on these results and previous LD50 data, 1000 mg/kg bw was selected as high dose.
- Harvest times: 6, 12, and 24 hours

Mortality and Clinical signs:

A moderate reduction in weight gain was observed on day 1 after dose administration in male and female rats that received 1000 mg/kg bw of test substance. Two female animals, one that received 1000 mg/kg bw and one that received 333 mg/kg bw, were found dead prior to their scheduled sacrifice. Irregular breathing and wheezing were noted in three females from the 1000 mg/kg bw group. All other animals appeared normal over the course of the study period.

Chromosomal damage in bone marrow of male rats following acute exposure to acrylic acid:

 

Treatment [mg/kg bw]	Time [hr]	Total no. of cells		Incidence of aberrations^1[%]	Total no. of aberrations			Aberrations from severely damaged cells^3	Aberrations/cell^1
		evaluated	with aberrations^1		gaps	breaks^2	rearrangements
Water	6	250	0	0.0	3	0	0	0	0.000±0.000
(3 mL/kg)	12	250	1	0.4	0	1	0	0	0.004±0.009
	24	250	0	0.0	0	0	0	0	0.000±0.000
TS 1000	6	250	1	0.4	1	1	0	0	0.004±0.009
	12	250	0	0.0	0	0	0	0	0.000±0.000
	24	250	1	0.4	1	1	0	0	0.004±0.009
TS 333	6	250	2	0.8	2	2	0	0	0.008±0.011
	12	250	1	0.4	1	1	0	0	0.004±0.009
	24	250	0	0.0	1	0	0	0	0.000±0.000
TS 100	6	250	1	0.4	0	1	0	0	0.004±0.009
	12	250	1	0.4	0	1	0	0	0.004±0.009
	24	250	0	0.0	0	0	0	0	0.000±0.000
CP 30	24	250	98	39.2**	0	172	67	390	2.516±0.599**

¹: Excluding gaps.

²: Includes chromatid and chromosome breaks and fragments.

³: Cells having more than 10 aberrations were counted as 10.

* p<0.05; ** p<0.01

CP: cyclophosphamide

Chromosomal damage in bone marrow of female rats following acute exposure to acrylic acid:

 

Treatment [mg/kg bw]	Time [hr]	Total no. of cells		Incidence of aberrations^1[%]	Total no. of aberrations			Aberrations from severely damaged cells^3	Aberrations/cell^1
		evaluated	with aberrations^1		gaps	breaks^2	rearrangements
Water	6	250	1	0.4	0	1	0	0	0.004±0.009
(3 mL/kg)	12	250	1	0.4	2	1	0	0	0.004±0.009
	24	250	0	0.0	0	0	0	0	0.000±0.000
TS 1000	6	250	0	0.0	1	0	0	0	0.000±0.000
	12	250	0	0.0	1	0	0	0	0.000±0.000
	24	250	0	0.0	0	0	0	0	0.000±0.000
TS 333	6	250	1	0.4	1	1	0	0	0.004±0.009
	12	250	0	0.0	0	0	0	0	0.000±0.000
	24	250	0	0.0	0	0	0	0	0.000±0.000
TS 100	6	250	1	0.4	1	1	0	0	0.004±0.009
	12	250	1	0.4	0	1	0	0	0.004±0.009
	24	250	1	0.4	0	1	0	0	0.004±0.009
CP 30	24	250	83	32.2**	1	105	72	310^4	1.948±0.854**

¹: Excluding gaps.

²: Includes chromatid and chromosome breaks and fragments.

³: Cells having pulverization or > 10 aberrations of any type.

⁴: Includes 10 aberrations contributed by 1 pulverized cell.

* p<0.05; ** p<0.01

CP: cyclophosphamide

Conclusions:

Under the study conditions, the test substance was not considered to be genotoxic to rat by oral administration in chromosomal aberration assay.

Executive summary:

A study was conducted to determine the ability of the test substance to induce chromosomal aberrations in rats, according to a method similar to OECD Guideline, in compliance with GLP. The test substance doses were 100, 333, and 1000 mg/kg bw (in a total volume of 3 mL/kg bw) administered by oral gavage a single time. These doses were selected based on the range finding study. Cyclophosphamide positive control group received a single oral dose of mg/mL. A moderate reduction in weight gain was observed on Day 1 after dose administration in male and female rats that received 1000 mg/kg bw of test substance. Two female animals, one that received 1000 mg/kg bw and one that received 333 mg/kg bw, were found dead prior to their scheduled sacrifice. Irregular breathing and wheezing were noted in three females from the 1000 mg/kg bw group. All other animals appeared normal over the course of the study period. Of the 250 metaphase bone marrow cells examined from each animal, no significant increase in chromatid and chromosome breaks or structural rearrangements were noted for test substance. The results of the study indicate that test substance at these dose levels did not induce detectable chromosomal aberrations after oral administration. Under the study conditions, the test substance was not considered to be genotoxic to rat by oral administration in chromosomal aberration assay (Celanese, 1986).

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

A micronucleus test on mouse bone marrow was conducted to evaluate the mutagenic potential of test material.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S. Ivanovas GmbH and Co., Kisslegg/Allgau (Germany)
- Diet: Standard chow (Altromin GmbH, Lage, Germany), ad libitum
- Water: Ad libitum

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: No data

Details on exposure:

No data

Duration of treatment / exposure:

30 h

Frequency of treatment:

Twice, at 0 and 24 h

Post exposure period:

No data

Remarks:

Doses / Concentrations:
2 X 86.3, 2 X 57.5 and 2 x 28.8 mg/kg (treated twice, at 0 and 24 h)
Basis:
other: solution in 0.9% Sodium chloride

No. of animals per sex per dose:

2

Control animals:

yes

Positive control(s):

No data

Tissues and cell types examined:

Polychromatic erythrocytes

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Bone marrow smears were prepared at 30 h.


METHOD OF ANALYSIS: Slides were coded and 1000 polychromatic erythrocytes were scored per animal.

Evaluation criteria:

No data

Statistics:

Significance was calculated according to the Kastenbaum-Bowman tables.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Table 1: Results of the micronucleus test on mouse bone marrow

Compound	Surviving/treated mice	Dose		Route of application	Micronuclcated polychromatic erythrocytes (%)
		mg/kg	mmole/kg
Zinc sulphate	3/4	2 X 86.3	2 X 0.3	i.p.	2.9
	4/4	2 X 57.5	2 X 0.2	i.p.	1.9
	4/4	2 X 28.8	2 X 0.1	i.p.	1.7
	4/4	0	0	i.p.	1.9

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic.

Executive summary:

A micronucleus test in mouse bone marrow was conducted to evaluate the mutagenic potential of the test substance. Male and female NMRI mice were used for the study. Feed and water were provided ad libitum. 4 animals (2 male, 2 female) were used in each treatment and control groups. 86.3, 57.5 and 28.8 mg/kg doses were administered i.p. at 0 and 24 h. Bone marrow smears were prepared at 30 h. 1000 polychromatic erythrocytes were scored per mouse. Significance was calculated according to the Kastenbaum-Bowman tables. Under the study conditions, the test substance was found to be non-mutagenic (Gocke, 1981).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Zinc sulphate studies

A study was conducted to determine the potential mutagenicity of the test substance using the bacterial reverse mutation assay (e.g. Ames test). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 strain were treated with the test substance in Vogel-Bonner (VB) and ZLM medium (modified minimal medium for E. coli) at a minimum of five dose levels, up to 3,600 µg/plate, both with and without activation by the S9 liver fraction from Aroclor-pretreated rats. The positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. No significant increases in the frequency of revertant colonies were observed for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Under the study conditions, the test substance was considered to be non-mutagenic (Gocke, 1981).

Zinc chloride studies

Study 1:

A study was conducted to investigate the ability of zinc chloride to induce chromosome aberrations in human dental pulp (D824) cells. Cells were treated with the test substance and evaluated for chromosome aberrations at up to 3 dose levels together with vehicle and positive controls. Rat liver (5%) post mitochondrial supernatant mixture was used as the exogenous metabolic activator. Ability to induce chromosome aberrations was examined in cells treated with test substance for 3 and30 h. The test substance failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by test substance. Under the study conditions, the test substance was considered to be non-clastogenic to human dental pulp cells in vitro (Someya, 1980).

Study 2:

A study was conducted to assess the potential mutagenicity of the test substance in the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The mouse lymphoma cells (TK+/-) were treated with test substance at 1.21-12.13 µg/mL for 3 h. 48 h after treatment, cells were treated with 4 µg/mL trifluorothymidine (TFT) for 7 d. Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants. Under the study conditions, the test substance was found to be non-mutagenic (Amacher, 1980).

 

Acrylic acid studies

Study 1:

A study was conducted to determine the mutagenic potential of the test substance, acrylic acid (>99%) according to a method similar to OECD Guideline 471. The study was conducted according to the method described by Ames (1975) with minor modifications. Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 were used as test system, neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents. The test substance concentrations were from 3.1 to 1000 nL/plate (not up to cytotoxic level) in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, using the plate incorporation method (2-4 replicates), with and without metabolic activation (Aroclor 1254 induced rat liver S-9 mix). There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in study. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (BASF, 1977).

Study 2:

A study was conducted to determine the in vitro genotoxic potential of the test substance, acrylic acid (99.8%), according to a method similar to OECD Guideline 482 (Unscheduled DNA Synthesis test in rat primary hepatocytes), in compliance with GLP. The substance was originally tested at nine dose levels (0.01, 0.03, 0.06, 0.1, 0.2, 0.3, 0.4, 0.6 µL/mL) and fully evaluated at the five dose levels of 0.01, 0.03, 0.1, 0.2 and 0.3 µL/mL. A repeat assay was conducted using eight dose levels ranging from 0.01 to 0.6 µL/mL and was fully evaluated at the seven dose levels of 0.01, 0.03, 0.06, 0.1, 0.2, 0.3 and 0.4 µL/mL. The results of the original UDS assay indicated that the substance did cause a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the control), at the second highest dose level evaluated, 0.2 µL/mL. The remaining dose levels showed no increase in net nuclear counts above the control. Since an analysis of the raw data indicated that there had not been an actual increase in nuclear counts, only a decrease in cytoplasmic counts, a repeat assay was performed. In the repeat assay, the test substance did not cause a significant increase in mean net nuclear grain counts at any dose level. The study met the validity criteria. Under the study conditions, the test substance was therefore determined to be non-genotoxic without metabolic activation in rat hepatocytes in vitro (BAMM, 1988).

Study 3:

A study was conducted to determine the in vitro genotoxicity potential of the test substance, acrylic acid (99.2%), at the HGPRT locus in Chinese hamster ovary (CHO) cells, according to method similar to OECD Guideline 476, in compliance with GLP. The HGPRT assay was performed based on the procedure described by O’Neill et al. (1977) and Gupta and Sing (1982). Test substance concentrations (without metabolic activation: 0.3, 0.6, 1.0, 1.5, 1.9 µL/mL and with metabolic activation: 1.0, 1.5, 1.9, 2.4, 2.8 µL/mL) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five hour exposure was used both with and without S9-mix (rat liver homogenate prepared from male Fischer rats that were induced by i.p. injection of Aroclor-1254 at 500 mg/kg bw). In the first trial, the mutant frequencies of two and one of the test article tested groups without and with metabolic activation respectively were increased significantly above the controls. Hiowever, increases were barely in excess of two-fold the highest concurrent background (untreated or solvent control) levels. In addition, no dose-response was apparent, and the non-activated assay had not achieved sufficient toxicity. Consequently, an additional mutation assay was performed. Triplicate cultures were independently subcultured throughout the repeat assay to provide confirmation of any significant, dose-dependent increases in mutant frequency. In the confirmatory assay with and without metabolic activation the mutant frequencies of the test article treated groups were not increased significantly above the controls. The negative and positive controls fulfilled the requirements for a valid test. Under the study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in CHO cells (BAMM, 1988).

 

 

Genetic toxicity in vivo

Zinc sulphate studies

A micronucleus test in mouse bone marrow was conducted to evaluate the mutagenic potential of the test substance. Male and female NMRI mice were used for the study. Feed and water were provided ad libitum. 4 animals (2 male, 2 female) were used in each treatment and control groups. 86.3, 57.5 and 28.8 mg/kg doses were administered i.p. at 0 and 24 h. Bone marrow smears were prepared at 30 h. 1000 polychromatic erythrocytes were scored per mouse. Significance was calculated according to the Kastenbaum-Bowman tables. Under the study conditions, the test substance was found to be non-mutagenic (Gocke, 1981).

Acrylic acid studies

A study was conducted to determine the ability of the test substance to induce chromosomal aberrations in rats, according to a method similar to OECD Guideline, in compliance with GLP. The test substance doses were 100, 333, and 1000 mg/kg bw (in a total volume of 3 mL/kg bw) administered by oral gavage a single time. These doses were selected based on the range finding study. Cyclophosphamide positive control group received a single oral dose of mg/mL. A moderate reduction in weight gain was observed on Day 1 after dose administration in male and female rats that received 1000 mg/kg bw of test substance. Two female animals, one that received 1000 mg/kg bw and one that received 333 mg/kg bw, were found dead prior to their scheduled sacrifice. Irregular breathing and wheezing were noted in three females from the 1000 mg/kg bw group. All other animals appeared normal over the course of the study period. Of the 250 metaphase bone marrow cells examined from each animal, no significant increase in chromatid and chromosome breaks or structural rearrangements were noted for test substance. The results of the study indicate that test substance at these dose levels did not induce detectable chromosomal aberrations after oral administration. Under the study conditions, the test substance was not considered to be genotoxic to rat by oral administration in chromosomal aberration assay (Celanese, 1986).

Justification for classification or non-classification

Based on studies conducted on substances representative of its individual constituents, the test substance does not warrant classification for genetic toxicity according to EU CLP (1272/2008) criteria.