Acrylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acrylic acid did not induce gene mutations in Salmonella typhimurium or CHO cells (HGPRT locus) but was positive in the mouse lymphoma assay and in the in vitro chromosomal aberration test. Since in the mouse lymphoma assay preferentially small colonies were induced, the mutagenic potential of acrylic acid seems to be limited to clastogenicity.

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:

key study

Study period:

17 Feb 1987 - 30 Aug 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 using the Hprt and xprt genes)

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:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Physical state: liquid
- Analytical purity: 99.92 %

Target gene:

HGPRT (hypoxanthine-guanine phosphoribosyl transferase) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

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:

- Vehicle(s)/solvent(s) used: phosphate buffer

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 5 hrs
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 7-10 days


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:
The cloning efficiency of the solvent and untreated controls must be no less than 50 %. 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. 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)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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.

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 nonactivated 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.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb 1987 - 15 Jan 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)

GLP compliance:

yes

Type of assay:

other: Unscheduled DNA synthesis (UDS)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: > 99.8 %
- Source: Hoechst Celanese Company, Somerville, NJ, USA

Species / strain / cell type:

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:

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

DURATION
- Exposure duration: 18-20 hrs (with test article and 3H-thymidine)

NUMBER OF 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:

hepatocytes: Primary Rat Hepatocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.4 µL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

The test article, acrylic acid, was tested in the Unscheduled DNA Synthesis Test using rat primary hepatocytes. The test article 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 article 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 article did not cause a significant increase in mean net nuclear grain counts at any dose level. Therefore, the test article 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)

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Physical state: liquid
- Analytical purity: > 99 %
- Impurities (identity and concentrations): approx. 0.4 % (w/w) propionic acid, approx. 0.3 % (w/w) acetic acid; stabilized by 200 ppm hydrochinone monomethylether

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:

benzo(a)pyrene

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: trans-N-Methyl-4-Aminostilbene

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Methyl-N'-Nitro-N-Nitrosoguanindin

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyren-4,5-oxide

Remarks:

without S9 mix

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 TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

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

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

According to Haworth et al. Environ. Mutagen. 5 (Suppl.1): 3-142, (1983).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: 97.9 %
- Vendor purity: 99 %
- Supplier: Aldrich

Target gene:

Histidin auxotrophy

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 liver S-9 mix from the Sprague-Dawley rat and male Syrian hamster.

Test concentrations with justification for top dose:

up to 3333 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix (TA1535 and TA 100)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix (TA 1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9 mix (TA98)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3333 µg/plate (TA100, TA1535, TA98)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3333 µg/plate (TA100, TA1535, TA98)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3333 µg/plate (TA100, TA1535, TA98)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3333 µg/plate (TA100, TA1535, TA98)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Mean Number of Revertants:

Dose (µg/plate)	TA100			TA1535
	NA	10% HLI	10% RLI	NA	10% HLI	10% RLI
0	106	136	135	6	9	9
3.3	-	-	-	-	-	-
10	-	-	-	-	-	-
33	89	152	139	6	8	12
100	93	148	132	7	7	10
333	75	152	127	6	8	6
1000	71	118	93	4	9	6
3333	t	t	t	0	t	t
POS	1394	2580	1387	468	423	213

Dose (µg/plate)	TA1537			TA98
	NA	10% HLI	10% RLI	NA	10% HLI	10% RLI
0	7	7	7	13	20	21
3.3	8	-	-	-	-	-
10	10	10	11	15	-	-
33	9	13	12	14	23	26
100	6	11	12	11	21	15
333	4	10	11	14	19	16
1000	-	9	12	10	17	16
3333	-	-	-	-	t	t
POS	570	244	208	317	1476	769

RLI: Rat Liver S9
HLI: Hamster Liver S9
t: Toxic (complete clearing of background lawn)

- : not done

Reference 5

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:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

The mutagenicity assay was performed according to the procedure described by Clive and Spector (1975). The results were interpreted as described by Clive et al. (1979).

Clive and Spector (1975). Mutat Res 31: 17-29
Clive et al. (1979). Mutat Res 59: 61-108

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: no data
- Source: no data

Target gene:

L5178Y TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Strain: L5178Y TK+/-3.7
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 homogenate (prepared from male Sprague-Dawley rats after injection with Aroclor 1254 at 500 mg/kg bw)

Test concentrations with justification for top dose:

Without metabolic activation: 1.62E-03, 2.65E-03, 3.53E-03, 4.56E-03, 5.44E-03 M
With metabolic activation: 4.41E-03, 1.01E-02, 1.62E-02, 2.21E-02, 2.65E-02 M

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 3-Methylcholanthrene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension


DURATION
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 10-12 days


SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: duplicate cultures, three plates/culture


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without: 64% relative total growth at 1.62E-03 M; With: 55.5 % relative total growth at 1.62E-02 M

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Under the conditions of this study, acrylic acid was positive in the L5178Y TK+/- Mouse Lymphoma in both the presence and the absence of metabolic activation. With S9, cytotoxicity for the chemical decreased, but mutagenic activity increased. The mutant frequency with respect to colony size was not determined for acrylic acid.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 May 1986 - 28 July 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): CJP-60
- Source: Hoechst Celanese Company (according to McCarthy et al., 1992)
- Analytical purity: >99.8 % (according to McCarthy et al., 1992)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (S9 mix) prepared from Sprague-Dawley rats that were induced by ip injection of Aroclor-1254 as described by Ames et al. (Mutation Research 31: 347 - 364, 1975)

Test concentrations with justification for top dose:

692, 923, 1231, 1615, 2154, and 2846 nL/mL with metabolic acitvation;
692, 923, 1231, 1615, 2154, 2846, 3769, and 5000 nL/mL without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclohexylamine

Remarks:

with S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: approx. 24 hrs
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 16 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hrs


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF CELLS EVALUATED: 100 metaphase spreads for each dose


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Each metaphase spread was scored for the following parameters:
- Number of chromosomes in each metaphase figure.
- Gaps - Achromatic region in chromatid no greater than the width of the chromatid.
- Chromatid breaks - Achromatic region in the chromatid greater than the width of the chromatid or where the broken piece is misaligned with the rest of the arm.
- Chromosome breaks - Achromatic region in both chromatids at the same locus with marked displacement of both distal fragments.
- Fragments - Chromatid(s) not containing a centromere. May be seen in association or not in association with a parent "chromatid".
- Exchange figure - Chromatid interchange involving two or more chromosomes, with either symmetrical or asymmetrical distortion of the usual chromatid pattern.
- Dicentric - Chromosome with two centromeres.
- Ring - Chromosome whose ends have joined to form a double or single circle, with or without a centromere.
- Polyploid - Increase in chromosome number in excess of the diploid and in multiple of the haploid number.
- Pulverization - Extreme fragmentation of the chromatid material.
- Severly damaged cell - Cell with ten or more abberations of any type or with pulverization.

Statistics:

Chi square analysis using a 2 x 2 contingency table was used to ascertain significant differences between the number of cells with aberrations in the treatment and control groups.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test based upon cloning efficiency after treatment relative to the solvent control.

The frequency of cells with structural aberrations was significantly increased above that of the solvent control in two of the test article treated groups in the absence of metabolic activation. In the presence of S-9 mix, the frequency of cells with structural aberrations was significantly increased in a dose-dependent manner above that of the solvent control at all doses scored. The types of aberrations after treatment in vitro with acrylic acid were primarily chromatid breaks and exchanges (clastogenic effect).

Under the conditions of the presented cytogenicity assay, acrylic acid induced a dose-responsive, significant increase in chromosome aberrations when tested both in the presence and absence of metabolic activation.

Cytogenetic analysis of CHO cells treated with acrylic acid in the absence of metabolic activation:

Treatment	Cells scored	Rel. cloning efficiency [%]	Number and type of aberrations^2										Structural aberrations/cell^3,4	Cells with structural aberrations [%]^3	Polyploid cells
			G	Tb	Sb	F	D	R	Ex	Pu	SDC	PP
Untreated	100	-	0	0	0	0	0	0	0	0	0	5	0	0	5
Water	100	100	0	1	0	0	1	0	0	0	0	2	0.02	2	2
5000 nL/mL	100	42	4	30	0	0	2	1	19	1	4	9	1.02	30**	9
3769 nL/mL	100	62	2	10	0	0	0	0	5	0	2	8	0.35	11*	8
2846 nL/mL	100	78	1	2	0	0	0	0	0	0	0	7	0.02	2	7
TEM 0.75 µg/mL	100	48^1	6	27	0	5	1	1	37	0	0	4	0.71	40**	4

 

 

Cytogenetic analysis of CHO cells treated with acrylic acid in the presence of metabolic activation:

Treatment	Cells scored	Rel. cloning efficiency [%]	Number and type of aberrations^2										Structural aberrations/cell^3,4	Cells with structural aberrations [%]^3	Polyploid cells
			G	Tb	Sb	F	D	R	Ex	Pu	SDC	PP
Untreated	100	-	3	0	0	0	0	0	0	0	0	4	0	0	4
Water	100	100	2	1	0	0	0	0	0	0	0	4	0.01	1	4
3769 nL/mL	0	15	-	-	-	-	-	-	-	-	-	-	-	-	-
2846 nL/mL	100	35	3	34	0	4	0	0	24	1	5	6	1.22	28**	6
2154 nL/mL	100	54	2	19	0	1	0	2	24	0	2	5	0.66	20**	5
1615 nL/mL	100	68	2	10	0	0	0	1	15	0	0	3	0.26	9*	3
CP 35 µg/mL	100	35^1	13	56	2	14	0	2	59	0	2	7	1.53	63**	7

 

¹calculated relative to untreated cells

²G: gaps; Tb: chromatid breaks; Sb: chromosome breaks; F: chromatid fragments; D: dicentric; R: ring; Ex: exchange figure; Pu: pulverization; SDC: severely damaged cell; PP: polyploid cell

³gaps not included

⁴SDC and Pu counted as 10 aberrations

* p< 0.05, ** p< 0.01

Reference 7

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:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

L5178Y/TK+- cells were treated without exogenous activation for 4 h according to the procedures of Clive et al (1979). Slides for cytogenetic analysis were prepared according to Perry and Wolff (1974).

Clive et al. (1979). Mutat Res. 59: 61-108
Perry and Wolff (1974). Nature. 251: 156-158

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: no data

Target gene:

not applicable

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Test concentrations with justification for top dose:

300, 450, and 500 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

TEST PROCEDURE
Treatment of the cells were initiated by diluting the test substance in DMSO solution. The cells were treated without exogenous activation for 4 hr according to procedures described previously (Clive et al., 1979).

Cytogenetic Analysis: Following cell treatment and wash, 10 μM BrdUrd was added to those cultures used for cytogenetic analysis. The cells were incubated for 14 hr, with Colcemid (0.1 μg/ml) present for the last 2 hr. Cells were treated with hypotonic KCl (0 .075 M) and fixed in acetic acid:methanol (1:3). Slides were made, stained using the fluorescence-plus-Giemsa method [Perry and Wolff, 1974 ; Goto et al, 1978], and blind coded. For each dose, 100 metaphase spreads that satisfied the following criteria were analyzed for aberrations: 1) well spread, 2) near normal centromere count (40 ± 3), and 3) first-division cell (as indicated by the absence of BrdUrd-induced differential staining). Aberrations were classified as chromatid breaks, deletions, and fragments (grouped as chromatid type breaks); triradials, quadraradials, and complex rearrangements (chromatid type rearrangements); chromosome breaks, deletions, fragments, and minutes (chromosome type breaks); and dicentrics, rings, and translocations (chromosome type rearrangements). A special category was used for metaphases with ten or more aberrations. For these cells, aberrations were not characterized because of extreme amounts of damage. Chromatid and chromosome gaps were recorded but not included as aberrations.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Analysis of Aberrations (100 cells analyzed)

Dose (µg/mL)	Chromatid*		Chromosome**		Total No. aberrations	% Cells with aberrations
	Breaks	Rearrangements	Breaks	Rearrangements
0	0	0	1	3	5	4
300	2	0	3	2	7	7
450	10	7	6	3	26	17
500	15	17	9	1	42	21

*chromatid breaks include breaks, deletions, and fragments. Chromatid rearrangements include triradials, quadriradials, and complex rearrangements

** Chromosome breaks include breaks, deletions, fragments, and minutes. Chromosome rearrangements include dicentrics, rings, and translocations .

Reference 8

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test procedure was conducted based on the method published by Clive et al. (1979).

Clive et al. (1979). Mutat Res 59: 61-108

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: no data
- Supplier: Aldrich, Milwaukee

Target gene:

TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK+/- -3.7.2C heterozygote of L5178Y mouse lymphoma cells

Metabolic activation:

without

Test concentrations with justification for top dose:

100, 200, 300, 400, 450, 500, 550, 600 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hr
- Expression time (cells in growth medium): 48 hr
- Selection time (if incubation with a selection agent): 9-11 d


SELECTION AGENT (mutation assays): TFT selection (trifluorothymidine)


NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: other: suspension growth and plating efficiency


OTHER EXAMINATIONS: Small and large mutant colonies were counted separately. Accordingly, a separate small-colony TK and large-colony TK mutant frequency was calculated.

Evaluation criteria:

Small-colony TK and large-colony TK mutant frequency

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not specified

Acrylic acid was mutagenic without exogenous activation in two separate experiments. A 500 μg/mL concentration of acrylic acid induced a mutant frequency over background of 245 and 325/1E+6 survivors (b.g . = 61 and 80 mutants/1E+6 survivors, respectively) in these two experiments. Induced mutant frequencies above 450/1E+6 survivors were observed at the 550 and 600 μg/mL concentrations; however, the survival was below 10%. The majority of the TFT-resistant colonies was small. The maximum induced large-colony mutant frequency was 26/1E+6 survivors (b.g. = 22/1E+6 survivors) and was observed at both the 300 and 500 μg/mL concentrations (colony sizing was performed for selected concentrations in one experiment).

Reference 9

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Measurement of unscheduled DNA synthesis (UDS) was carried out as described by Schiffermann D. et al. Canc. Lett. 23: 297-305, 1984 and Tsutsui T. et al. Cancer Res. 44: 184-189, 1984.

GLP compliance:

not specified

Type of assay:

other: unscheduled DNA synthesis (UDS)

Specific details on test material used for the study:

- Name of test material (as cited in study report): acrylic acid
- Delivered by Huels AG, Marl
- Analytical purity: no data

Species / strain / cell type:

other: Syrian hamster embryo fibroblasts (SHE)

Metabolic activation:

without

Test concentrations with justification for top dose:

1 - 300 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Tertiary cultures (1.7 x 10E5 cells) were incubated with arginine-free medium (including 2.5 % fetal bovine serum) for 48 h. After change of medium 3H-thymidine and 10 mM hydroxyurea were added and the cultures were incubated with various concentrations (1 - 300 µg/mL) of the test compound (solvent: DMSO). After an incubation time of 5 h the cells were washed (phosphate-buffered saline) and solubilized (2 % SDS). Subsequently the cells were precipitated (20 % trichloroacetic acid) and the precipitate was washed with ethanol. Subsequently the precipitate was incubated with tissue solubilizer (6 h, 50°C) and measurement of radioactivity (liquid scintillation) was carried out.

Key result

Species / strain:

other: Syrian hamster embryo fibroblasts (SHE)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Reference 10

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was investigated for its ability to induce micronuclei according to the method described by Schmuck et al. (Mutat Res. 203(6): 397-404, 1988).

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Name of test material (as cited in study report): acrylic acid
- Delivered by Huels AG, Marl
- Analytical purity: no data

Species / strain / cell type:

other: Syrian hamster embryo fibroblasts (SHE)

Metabolic activation:

without

Test concentrations with justification for top dose:

0.5 - 10 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Diethylstilbestrol

Details on test system and experimental conditions:

Tertiary cultures (1.5 x 10E5 cells) were incubated for 24 h at 37° C in a humified atmosphere in 12 % CO2 in air. The cultures were treated with various concentrations (0.5 - 10 µg/ml) of the test compound (solvent: DMSO). After incubation time of 5 h the compound was removed by medium change. After further incubation for 18 h cells were fixed, stained and scored for micronuclei. Only structures smaller than one third of the nucleus were counted in order to avoid confusion with dividing cells. For each concentration the number of cells containing single and multiple micronuclei was determined among a population of 2000 cells.

Key result

Species / strain:

other: Syrian hamster embryo fibroblasts (SHE-cells)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo, acrylic acid did not induce mutagenic effects in either rat bone marrow cells or mouse germ cells after oral administration. Based on the present results, it is unlikely that acrylic acid is mutagenic 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:

key study

Study period:

29 Apr 1986 - 05 Aug 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)

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:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): CJP-60
- Source: Hoechst Celanese Company (according to McCarthy et al., 1992)
- Analytical purity: >99.8 %

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.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

in a total volume of 3 mL/kg bw

Dose / conc.:

333 mg/kg bw/day (actual dose received)

Remarks:

in a total volume of 3 mL/kg bw

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

in a total volume of 3 mL/kg bw

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 article 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 examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- 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

 

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Apr 1986 - 05 Aug 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

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 exposure to 2000 or 5000 ppm acrylic acid in drinking water (corresponding to approx. 180 and 450 mg/kg bw) for 5 days.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): CJP-60
- Source: Hoechst Celanese Company (according to McCarthy et al., 1992)
- Analytical purity: >99.8 %

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: drinking water

Vehicle:

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

Duration of treatment / exposure:

5 days

Frequency of treatment:

continuously

Post exposure period:

On day six approximately 4 hours after the fifth dosing in the subchronic 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.

Dose / conc.:

2 000 ppm

Remarks:

approx.180 mg/kg bw, nominal in water

Dose / conc.:

5 000 ppm

Remarks:

approx. 450 mg/kg bw, nominal in water

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes, concurrent no treatment

Positive control(s):

none

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. 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. For the repeated dosing regimen, the upper dose level of 5000 ppm in drinking water was selected on the basis of the minimal effect level in a 3-month drinking water study (DePass et al., 1983).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 4 hours after the 5th dosing on day six

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 article 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:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Mortality and Clinical signs:

Male rats in the 5000 ppm group showed a moderate weight loss from day 1 to day 2 and from day 2 to day 3 but began gaining weight by day 4. A very small weight loss (1 gram) was observed for the female rats in the 2000 ppm group from day 1 to day 2. A slower rate of weight gain over the study period was observed in the test article treated groups as compared to the vehicle control. One male rat from the 2000 ppm dose group appeared to be wheezing with irregular breathing on the first day the test article treated water was made available but had returned to normal by day 2. All other animals in the subchronic assay appeared normal throughout the study.

Chromosomal damage in bone marrow of male and female rats following five-day exposure to acrylic acid in drinking water:

Treatment [ppm]	Sex	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	M	250	0	0.0	1	0	0	0	0.000±0.000
	F	250	0	0.0	0	0	0	0	0.000±0.000
TS 5000	M	250	0	0.0	0	0	0	0	0.000±0.000
	F	250	1	0.4	1	1	0	0	0.004±0.009
TS 2000	M	250	1	0.4	0	1	0	0	0.004±0.009
	F	250	0	0.0	0	0	0	0	0.000±0.000

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

 

 

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted according to the method described by McCarthy K. L.et al., Food Chem. Toxic. 30: 505-515 (1992).

GLP compliance:

yes

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Physical state: clear liquid
- Analytical purity: > 99.8 % (as given in McCarthy et al.)

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Raleigh, NC or Portage, MI.
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: Males, 27-38 g
- Housing: The animals were housed in an AAALAC-accredited facility. Male mice were housed up to five per cage in plastic autoclavable cages with filter tops. Female mice were housed up to 25 per cage in large plastic autoclavable cages with metal lids.
- Diet (ad libitum): Certified laboratory rodent chow
- Water (ad libitum): Tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 50 ± 2
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For dose level selection, male animals were randomly assigned to three groups of ten males each for each of three toxicity tests (32, 108, 324 mg/kg bw) and were dosed by oral gavage with a single administration at a rate of 10 ml/kg bw. Animals were observed after each dose administration for clinical signs of chemical effect.

Duration of treatment / exposure:

Single administration

Frequency of treatment:

once

Post exposure period:

Immediately after the last dose administration, each male mouse was mated with 2 virgin female mice. Females were checked for vaginal plugs each morning. Each mated female was replaced with a virgin female. The mating process was repeated until the males had mated for a total of 8 days in order to evaluate the effect of treatment on fertility.

Dose / conc.:

32 mg/kg bw/day (actual dose received)

Dose / conc.:

108 mg/kg bw/day (actual dose received)

Dose / conc.:

324 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 males / 40 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Uterine contents (number of live and dead implants were recorded)

Evaluation criteria:

Dominant lethality (DL) was calculated according to the following formula:

DL = 1 – [Live embryos / pregnant female treated : live embryos / pregnant female control]

Parameters which also were evaluated statistically on the basis of four day mating intervals include:

- The fertility index. This paramter measures the number of fertile matings.

- The total implantations per pregnant female. A reduction in litter size is not critical evidence of mutation; however, the parameter may provide useful information regarding the effect of the test article.

- Dead implants per pregnant female (early deaths). A statistically significant increase in this parameter is reported to be the most reliable indicator of a dominant lethal effect.

- The proportion of pregnant females with one or more dead implants and with two or more dead implants. These parameters give an indication of the severity of fetal damage.

- Dead implants per total implants (early deaths). This index adjusts the dead implants per pregnant female by the total implants for that female because of the positive correlation between the two. The index is a reliable indicator of dominant lethality.

- Live implants per pregnant female. This parameter reflects the combined effects of preimplantation losses and dead implants.

Statistics:

The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: t-test, Chi-square analysis, the Freeman-Tukey arcsine transformation.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

During or shortly after dose administration, several mice were found to exhibit swelling in the shoulder region. Upon observation, it was determined that the swellings were abscesses most likely caused by bite wounds from fighting during the time that the males were group housed. Two mice died shortly after dose administration; one mouse receiving 324 mg/kg died within 3 hours of dose administration. Additional mice were found dead during the first mating week and included 2 mice in the water control, 1 mouse receiving 324 mg/kg, 1 mouse receiving 32 mg/kg. These deaths did not appear to be test substance related. Many of the males that died were those with the shoulder swellings observed earlier. It is possible that the wounds from fighting were irritated by holding the mice for the gavage procedure and the animals succumbed to infection. Necropsies were not performed. Other than the swollen shoulders, all test substance-treated mice appeared normal at about 2 to 3 hours after dose administration and on the day following dose administration in this study.

 

- LD: The dominant lethal indices were calculated for the test substance treated groups. No statistical evaluation could be made on this index.

Dominant Lethal Index
Interval (days)	Test substance
	32 mg/kg bw	108 mg/kg bw	324 mg/kg bw
1 – 4	0.10	0.06	0.03
5 - 8	0.08	0.20	0.01
9 – 12	0.08	0	0.04
13 – 16	0.11	0.01	0.03
17 – 20	0.03	0.01	0.04
21 – 24	0.01	0.12	0.06
25 – 28	0.09	0.07	0.05
29 – 32	0.01	0.03	0.08
33 – 36	0.03	0.02	0.03
37 – 40	0.08	0.02	0.04
41 - 46	0.01	0.04	0.01

- The fertility index: The fertility index was calculated by dividing the number of pregnant mice by the number of animals mated. No difference was found between the test article-treated and vehicle control animals.

Fertility Index
Interval (days)	Water 10 ml/kg bw	Test substance			CP 50 mg/kg bw
		32 mg/kg bw	108 mg/kg bw	324 mg/kg bw
1 – 4	0.85 (22/26)	0.87 (20/23)	0.88 (28/32)	0.79 (19/24)	0.80 (24/30)
5 - 8	0.89 (25/28)	0.96 (26/27)	0.90 (19/21)	0.97 (28/29)	0.92 (23/25)
9 – 12	0.91 (20/22)	0.95 (21/22)	0.95 (19/20)	1.00 825/25)	1.00 (13/13)
13 – 16	0.86 (12/14)	0.95 (19/20)	0.80 (8/10)	0.88 (15/17)	0.82 (9/11)
17 – 20	0.80 (16/20)	0.96 (24/25)	1.00 (27/27)	1.00 (15/15)	1.00 832/32)
21 – 24	0.94 (17/18)	0.92 (23/25)	1.00 (27/27)	0.92 (23/25)	0.95 (18/19)
25 – 28	0.96 (25/26)	0.97 (31/32)	0.86 (24/28)	0.91 (31/34)	0.95 (21/22)
29 – 32	0.71 (27/38)	0.91 (31/34)	0.97 (37/38)	0.93 (41/44)	0.90 (26/29)
33 – 36	0.74 (28/38)	0.90 (37/41)	0.85 (28/33)	0.93 (25/27)	0.97 833/34)
37 – 40	0.72 (18/25)	0.96 (20/21)	0.88 (22/25)	0.95 (18/19)	0.93 825/27)
41 - 46	0.91 (41/45)	0.89 (49/55)	0.85 856/66)	0.97 (57/59)	0.93 (54/58)

- No effect on total implantations was seen.

 

- Mean live implants per pregnant female: The live implants, presented in the table below, were not significantly reduced in the test substance-treated groups. CP markedly reduced the number of live implants at mating intervals 1-4 and 13-16.

Mean live implants per pregnant female
Interval (days)	Water 10 ml/kg bw	Test substance			CP 50 mg/kg bw
		32 mg/kg bw	108 mg/kg bw	324 mg/kg bw
1 – 4	12.18	11.00	11.46	11.79	10.54^*
5 - 8	12.04	11.12	9.68	11.89	11.43
9 – 12	11.35	10.43	11.37	11.84	11.38
13 – 16	12.42	11.11	12.25	12.80	9.56^**
17 – 20	11.81	11.50	11.74	12.27	10.97
21 – 24	11.24	11.09	12.63	11.86	10.44
25 – 28	10.96	9.97	10.21	11.55	10.38
29 – 32	11.48	11.32	11.14	10.59	11.62
33 – 36	11.64	11.30	11.86	11.32	10.55
37 – 40	10.94	11.79	10.68	11.33	11.00
41 - 46	10.85	10.92	10.41	10.93	10.98

 

CP: Cyclophosphamide

^*Significantly increased relative to vehicle control (p≤0.05)

 

- Mean dead implants per pregnant female: A statistically significant increase in the mean number of dead implants per pregnant female was observed at 324 and 108 mg/kg in the 5-8 day mating interval and at 324 mg/kg at 33-36 days. A number of female mice removed from males during the 5-8 day mating interval appeared dehydrated at the time of sacrifice. The cause of the dehydration is not known but the animals did have free access to water at all times. These mice had an increased number of dead implants which were probably due to the dehydration and not to test substance effect. The significance observed at days 33-36 was a reflection of the low vehicle control rather than markedly elevated values in the test article-substance group. CP significantly increased the number of dead implants at intervals 1-4, 5-8, 9-12, 13-16, 17-20, 33-36, 37-40, and 41-46.

Mean dead implants per pregnant female
Interval (days)	Water 10 mL/kg bw	Test substance			CP 50 mg/kg bw
		32 mg/kg bw	108 mg/kg bw	324 mg/kg bw
1 – 4	0.36	0.60	0.54	0.79	0.88^*
5 - 8	0.56	1.00^a	2.21^*a	1.07^*a	1.17^*
9 – 12	0.55	1.19	0.79	0.48	1.69^*
13 – 16	0.42	0.63	0.50	0.27	2.89^*
17 – 20	0.38	0.79	0.52	0.47	0.84^*
21 – 24	1.18	0.57	0.41	0.77	0.83
25 – 28	0.64	1.81	1.54	0.48	0.52
29 – 32	0.52	0.52	0.38	0.71	0.54
33 – 36	0.29	0.51	0.43	0.60^*	1.12^*
37 – 40	0.39	0.33	0.55	0.56	0.88^*
41 - 46	0.46	0.53	0.54	0.46	0.80^*

CP: Cyclophosphamide

^*Significantly increased relative to vehicle control (p≤0.05)

^aSome pregnant females were dehydrated at sacrifice

 

- The proportion of pregnant females with one or more dead implants or with two or more dead implants was not increased in the test article-treated groups. CP significantly increased the proportion of pregnant females with one or more dead implants at days 9-12, 13-16, and 41-46 and with two or more dead implants at days 13-16.

 

- The mean number of dead implants per total implants was observed to be significantly increased at days 5-9 at 108 mg/kg and at days 33-36 at 324 mg/kg. The increase observed at days 5-8 is probably due to dehydration described above. The increase seen at days 33-36 is probably due to the low concurrent control at this time point since the test substance-treated values are not unlike those at other weeks. CP significantly increased the dead implants per total implants at mating intervals 9-12, 13-16, 17-20, 33-36, 37-40 and 41-46.

Reference 4

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted according to the method described by McCarthy K. L.et al., Food Chem. Toxic. 30: 505-515 (1992).

GLP compliance:

yes

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Physical state: clear liquid
- Analytical purity: > 99.8 % (as given in McCarthy et al.)

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Raleigh, NC or Portage, MI.
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: Males, 27-38 g
- Housing: The animals were housed in an AAALAC-accredited facility. Male mice were housed up to five per cage in plastic autoclavable cages with filter tops. Female mice were housed up to 25 per cage in large plastic autoclavable cages with metal lids.
- Diet (ad libitum): Certified laboratory rodent chow
- Water (ad libitum): Tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 50 ± 2
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For dose level selection, male animals were randomly assigned to three groups of ten males each for each of three toxicity tests (16, 54, 162 mg/kg bw) and were dosed by oral gavage with 5 daily administrations at a rate of 10 ml/kg bw. Animals were observed after each dose administration for clinical signs of chemical effect.

Duration of treatment / exposure:

Five days

Frequency of treatment:

once per day

Post exposure period:

Immediately after the last dose administration, each male mouse was mated with 2 virgin female mice. Females were checked for vaginal plugs each morning. Each mated female was replaced with a virgin female. The mating process was repeated until the males had mated for a total of 6 days in order to evaluate the effect of treatment on fertility. The subchronic mating period was reduced to 6 days due to depletion of available virgin female mice for mating.

Dose / conc.:

16 mg/kg bw/day (actual dose received)

Dose / conc.:

54 mg/kg bw/day (actual dose received)

Dose / conc.:

162 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 males / 40 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Uterine contents (number of live and dead implants were recorded)

Evaluation criteria:

Dominant lethality (DL) was calculated according to the following formula:

DL = 1 – [Live embryos / pregnant female treated : live embryos / pregnant female control]

Parameters which also were evaluated statistically on the basis of four day mating intervals include:

- The fertility index. This paramter measures the number of fertile matings.

- The total implantations per pregnant female. A reduction in litter size is not critical evidence of mutation; however, the parameter may provide useful information regarding the effect of the test article.

- Dead implants per pregnant female (early deaths). A statistically significant increase in this parameter is reported to be the most reliable indicator of a dominant lethal effect.

- The proportion of pregnant females with one or more dead implants and with two or more dead implants. These parameters give an indication of the severity of fetal damage.

- Dead implants total implants (early deaths). This index adjusts the dead implants per pregnant female by the total implants for that female because of the positive correlation between the two. The index is a reliable indicator of dominant lethality.

- Live implants pregnant female. This parameter reflects the combined effects of preimplantation losses and dead implants.

Statistics:

The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: t-test, Chi-square analysis, the Freeman-Tukey arcsine transformation.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

During or shortly after dose administration, several mice were found to exhibit swelling in the shoulder region. Upon observation, it was determined that the swellings were abscesses most likely caused by bite wounds from fighting during the time that the males were group housed. Mice were found dead during the first mating week and included 4 mice receiving water, 3 mice receiving 162 mg/kg bw, 1 mouse receiving 54 mg/kg bw, 2 mice receiving 16 mg/kg bw and 4 mice receiving 20 mg CP/kg/bw/d. These deaths did not appear to be test substance related. Many of the males that died were those with the shoulder swellings observed earlier. It ispossible that the wounds from fighting were irritated by holding the mice for the gavage procedure and the animals succumbed to infection. Necropsies were not performed. Other than the swollen shoulders, all test substance-treated mice appeared normal at about 1.5 to 2 hours after each daily administration.

 

- LD: The dominant lethal indices were calculated for the test substance treated groups. No statistical evaluation was made on this index.

Dominant Lethal Index
Interval (days)	Test substance
	16 mg/kg bw	54 mg/kg bw	162 mg/kg bw
1 – 4	0.04	0.04	0.06
5 - 8	0.06	0.09	0.07
9 – 12	0.11	0.01	0.04
13 – 16	0.04	0.04	0.05
17 – 20	0	0.11	0.05
21 – 24	0.02	0.05	0.03
25 – 28	0.03	0.11	0.03
29 – 32	0.06	0.07	0.07
33 – 36	0.23	0.02	0.06
37 – 40	0.06	0.01	0.05
41 - 46	0.07	0.07	0

- The fertility index was calculated by dividing the number of pregnant mice by the number of animals mated. No difference was found between the test substance-treated and vehicle control animals.

Fertility Index
Interval (days)	Water 10 ml/kg bw	Test substance			CP 20 mg/kg bw
		16 mg/kg bw	54 mg/kg bw	162 mg/kg bw
1 – 4	0.93 (25/27)	0.86 (25/29)	0.74 (20/27)	0.88 (30/34)	0.70 (14/20)
5 - 8	0.96 (23/24)	1.00 (29/29)	0.93 (26/28)	0.94 (30/32)	0.95 (19/20)
9 – 12	0.90 (9/10)	0.89 (16/18)	0.95 (20/21)	0.90 (28/31)	1.00 (8/8)
13 – 16	1.00 (9/9)	1.00 (18/18)	0.93 (14/15)	0.94 (15/16)	0.90 (9/10)
17 – 20	0.90 (19/21)	1.00 (19/19)	0.96 (24/25)	1.00 (18/18)	0.88 (14/16)
21 – 24	0.97 (32/33)	0.94 (31/33)	0.89 (33/37)	0.90 (28/31)	0.90 (26/29)
25 – 28	0.94 (30/32)	0.97 (30/31)	0.94 (29/31)	1.00 (29/29)	0.96 (25/26)
29 – 32	0.81 (26/32)	0.82 (32/39)	0.72 (23/32)	0.90 (28/31)	0.89 (25/28)
33 – 36	0.90 (18/20)	1.00 (26/26)	1.00 (17/17)	0.92 (22/24)	0.86 (19/22)
37 – 40	0.91 (20/22)	0.90 (19/21)	0.89 (25/28)	0.91 (20/22)	0.96 (23/24)
41 - 46	0.96 (46/48)	0.95 (40/42)	0.96 (48/50)	1.00 (55/55)	0.96 (47/49)

- Total implantations (live and dead) per pregnant female: A reduction in total implantations was observed at 54 mg/kg/bw/d at mating intervals 17-20, 25-28 and 41-46 and at 16 mg/kg/day at 9-12 days. CP significantly reduced the total implantations at days 1-4, 5-8, 9-12, 17-20 and 21-24.

Mean implantations (live and dead) per pregnant female
Interval (days)	Water 10 ml/kg bw	Test substance			CP 20 mg/kg bw
		16 mg/kg bw	54 mg/kg bw	162 mg/kg bw
1 – 4	11.64	11.72	11.90	12.07	9.79^*
5 - 8	12.78	12.00	12.81	12.27	11.26^*
9 – 12	13.11	12.06^*	13.10	12.61	11.50^*
13 – 16	12.00	12.33	12.36	12.73	11.78
17 – 20	12.74	12.26	10.96^*	12.72	10.57^*
21 – 24	12.09	11.74	12.58	11.71	11.12^*
25 – 28	12.53	12.00	11.03^*	11.72	11.92
29 – 32	12.27	12.09	11.52	11.61	11.60
33 – 36	11.72	13.85	12.12	12.36	12.26
37 – 40	12.05	11.21	11.96	11.50	11.70
41 - 46	12.00	11.28	10.96^*	11.95	11.87

CP: Cyclophosphamide

^*Significantly increased relative to vehicle control (p≤0.05)

 

 

- Mean dead implants per pregnant female: A statistically significant increase in the mean number of dead implants per pregnant female was observed at 54 mg/kg/day in the 5-8 day mating interval and at 16 mg/kg at 29-32 days. A number of female mice removed from males during the 5-8 day mating interval appeared dehydrated at the time of sacrifice. The cause of the dehydration is not known but the animals did have free access to water at all times. These mice had an increased number of dead implants which were probably due to the dehydration and not to test substance effect. The increase seen days 33-36 was probably due to the low concurrent control at this time point since the test article-substance value was not unlike those at other weeks. CP significantly increased the number of dead implants at intervals 1-4, 5-8, 9-12, 13-16, and 21-24.

Mean dead implants per pregnant female
Interval (days)	Water 10 mL/kg bw	Test substance			CP 20 mg/kg bw
		16 mg/kg bw	54 mg/kg bw	162 mg/kg bw
1 – 4	0.72	0.32	1.40	0.53	2.71^*
5 - 8	0.61	0.62	1.77^*a	0.97^a	3.58^*
9 – 12	0.44	0.75	0.50	0.50	4.25^*
13 – 16	0.44	0.28	0.36	0.60	2.11^*
17 – 20	0.79	0.26	0.33	0.17	0.86
21 – 24	0.59	0.42	0.42	0.57	1.38^*
25 – 28	0.70	0.50	0.55	0.28	0.84
29 – 32	0.27	0.78^*	0.39	0.43	0.56
33 – 36	0.78	0.35	0.94	0.73	1.42
37 – 40	0.70	0.53	0.52	0.70	0.48
41 - 46	0.52	0.60	0.31	0.49	0.38

CP: Cyclophosphamide

^*Significantly increased relative to vehicle control (p≤0.05)

^aSome pregnant females were dehydrated at sacrifice

 

- Mean live implants per pregnant female: The live implants were significantlyreduced at 16 mg/kg/bw/day during mating interval 9-12 and at 54 mg/kg/bw/day during mating interval 25-28. CP markedly reduced the number of live implants at mating intervals 1-4, 5-8, 17-20 and 21-24.

 

- The proportion of pregnant females with one or more dead implants was increased at 16 mg/kg/day at days 29-32. The proportion of pregnant females with two or more dead implants was not increased in the test substance-treated groups. CP significantly increased the proportion of pregnant females with one or more dead implants at days 1-4, 5-8, 9-12, and 21-24 and with two or more dead implants at days 1-4, 5-8, and 9-12.

 

- The mean number of dead implants per total implants was observed to be significantly increased at days 5-8 at 54 mg/kg/bw/day and at days 29-32 at 16 mg/kg/bw/day. The increase observed at days 5-8 is probably due to dehydration described above. The increase seen days 33-36 was probably do to the low concurrent control at this time point since the test substance-treated value was not unlike those at other weeks. CP significantly increased the dead implants per total implants at mating intervals 1-4, 5-8, 9-12, 13-16, and 21-24.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

In vitro gene mutation assays in bacteria

Acrylic acid was negative in several assays with and without metabolic activation. Concentrations between 3.15 and 1000 nL/plate were tested in Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 in the absence and presence of S-9 mix. No bacteriotoxic effect was observed up to the highest concentration tested (BASF AG, 1977). In the same 4 Salmonella typhimurium tester strains at concentrations up to 3333 µg/plate was no increase in the mutant frequency caused by the compound. Only at the highest concentration tested a bacteriotoxic effect was found (Zeiger et al., 1987). A bacterial mutation test with Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 was negative in concentrations up to 5000 µg/plate with and without S-9 mix. Doses from 1000 or 3333 µg/plate upwards induced bacteriotoxic effects, depending on the strain (Cameron et al., 1991). In a further study according to Ames et al, the test item was negative in concentrations up to 1000 µg/plate (Lijinski, 1980).

In vitro tests in mammalian cells

A mammalian cell gene mutation test with CHO cells (HGPRT locus) was negative at doses up to 1.9 µL/mL (2000 µg/mL) without S-9 mix and up to 2.4 µL/mL (2500 µg/mL) with S-9 mix. Survival at the highest concentrations was 35% and 24%, respectively (BAMM, 1988, McCarthy et al., 1992).

In the mouse lymphoma assay increases of mutation frequencies were found in two independent investigations. Cameron et al. (1991) reported a positive mouse lymphoma assay with and without metabolic activation: without S-9 mix 3-fold to 6-fold dose-related increases of mutant frequencies were induced in the dose range 2.65 to 4.56 mmol/L (191 to 329 µg/mL); with S-9 mix 3-fold to 6-fold increases were found in the dose range 16.2 to 22.1 mmol/L (1167 to 1593 µg/mL). The relative total growth at the concentrations that gave a positive response in the absence of S-9 was 36 -15 % and in the presence of S-9 was 55.5 - 20 %, respectively. At concentrations which resulted in a relative total growth greater than 55 %, no increases in mutant frequencies were observed. The colony size distribution was not determined in this study.

Acrylic acid was mutagenic without exogenous activation in another mouse lymphoma assay comprising of two separate experiments (Moore et al., 1988). A 500 μg/mL concentration of acrylic acid induced a mutant frequency over background of 245 and 325/10E6survivors (background = 61 and 80 mutants/106survivors, respectively) in these two experiments. Induced mutant frequencies above 450/10E6survivors were observed at the 550 and 600 μg/mL concentrations; however, the survival was below 10%. The majority of the TFT-resistant colonies was small indicating a clastogenic effect. The maximum induced large-colony mutant frequency was 26/10E6survivors (background = 22/10E6 survivors) and was observed at both the 300 and 500 μg/mL concentrations (colony sizing was performed for selected concentrations in one experiment). In a further mouse lymphoma assay the test substance produced positive response in the presence and absence of exogenous metabolic activation at concentrations which reduced total growth to less than 20% (Procter&Gamble, 1986). In another mouse lymphoma assay the test substance induced a positive response with and without S9 mix (NIH, 1982). 

Positive effects were described also for in vitro chromosomal aberration assays. Celanese Corp. (1986) reported a positive chromosomal aberration assay in CHO cells with and without metabolic activation. Without S-9 mix, doses of 3.8 and 5.0 µL/mL (approx. 3960 and 5250 µg/mL) induced 11 % and 30 % aberrant cells (2 % in the vehicle control); with S-9 mix, in the dose range 1.6 to 2.8 µL/mL (1700 to 2990 µg/mL) aberration frequencies of 9 % to 28 % were found in a dose-related manner (1 % in the vehicle control). The positive response was not bound exclusively to cytotoxic effects (42 % - 62 % in the absence and 35 % - 68 % relative cloning efficiency in the presence of metabolic activation, respectively) or to decreases in pH (pH was adjusted to pH 7.0) (Celanese Corp. 1986;McCarthy et al.,1992).

More recent studies have indicated that there is an association between chromosomal aberrations and cytotoxicity at exposure concentrations which reduce cell growth to less than 50% of the control value (Galloway, 2000 and references cited therein). These data suggest that the increase in mutagenicity reported in the mouse lymphoma TK and chromosomal aberration assays with acrylic acid may be an artefact of the experimental method. However, at a concentration of 1.6 µL/mL (approx. 1700 µg/mL) in the presence of metabolic activation a significantly elevated frequency of cells with aberrations (9 % versus 1 % in the solvent control) and 68 % survival were reported. Thus, acrylic acid showed clastogenic potential in in vitro assays in mammalian cells.

In primary rat hepatocytes acrylic acid did not induce unscheduled DNA synthesis (UDS) in non-toxic doses up to 0.4 µL/mL (420 µg/mL). Higher doses could not be analysed due to high cytotoxicity (BAMM, 1988; McCarthy et al., 1992).

The test substance was investigated for its ability to induce micronuclei in syrian hamster embryo fibroblasts without the addition of metabolic activation. No genotoxicity was reported (Weigand, 1989A). Measurement of unscheduled DNA synthesis (UDS) was carried out in syrian hamster embryo fibroblasts and showed no gentotoxic effects (Weigand, 1989B)

In vivo Studies

Two in vivo bone marrow chromosomal aberration assays with rats gave negative results (Celanese Corp., 1986; McCarthy et al., 1992). Chromosome aberrations were analyzed (5 animals per sex, 50 metaphases per animal) at 6, 12, and 24 h after oral doses of 100, 333 or 1000 mg/kg bw or after exposure to 2000 or 5000 ppm acrylic acid in drinking water (corresponding to approx. 180 and 450 mg/kg bw) for 5 days. In the acute as well as in the subacute regimens the highest doses led to reduced body weight gains. Mitotic activity of bone marrow cells was not influenced by the treatments.

Two dominant lethal assays in CD-1 mice led to negative results. Male mice were given single oral doses (gavage) up to 324 mg/kg bw or five daily oral doses up to 162 mg/kg bw. Immediately after dose administration male mice were mated. Females were checked for vaginal plugs each morning. Each mated female was replaced with a virgin female. The mating process was continued for 46 days. An analysis of the uterine contents of female mice was made 12-15 days after observation of the vaginal plug (McCarthy et al., 1992). Acrylic acid did not induce a statistically significant increase in dead implants accompanied by a significant reduction in live implants at the same dose and time point in either the acute or repeated dosing studies. Likewise, the percentage of dominant lethals was not markedly increased in any treatment group. In contrast, the positive control cyclophosphamide induced an increase in the percentage of dead implants and dominant lethals in both the acute and repeated dosing regimens. Consequently, acrylic acid did not induce a dominant lethal effect.

In a Drosophila SLRL assay treatment of males of Drosophila melanogaster via feed or injection (2% acrylic acid) did not induce genotoxic effects (Celanese Corp, 1987).

Conclusion

Acrylic acid did not induce gene mutations in Salmonella typhimurium or CHO cells (HGPRT locus) but was positive in the mouse lymphoma assay and in the in vitro chromosomal aberration test. Since in the mouse lymphoma assay small colonies were induced preferentially, the mutagenic potential of acrylic acid seems to be limited to clastogenicity. In vivo, acrylic acid did not induce mutagenic effects in either rat bone marrow cells or mouse germ cells after oral administration. Based on the present results, it is unlikely that acrylic acid is mutagenic in vivo.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.