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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
other: Comparable to guideline study conducted in compliance with GLP regulations

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988
Reference Type:
publication
Title:
Genetic Toxicology of Acrylic Acid.
Author:
McCarthy KL et al.
Year:
1992
Bibliographic source:
Fd. Chem. Toxic. 30: 505-515

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Acrylic acid
- Physical state: liquid
- Analytical purity: 99.92 %

Method

Target gene:
HGPRT (hypoxanthine-guanine phosphoribosyl transferase) gene
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Strain: 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
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without activation: Ethyl methanesulfonate; with activation: Benzo(a)pyrene
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.

Results and discussion

Test results
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
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative