4-hydroxybutyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybutyl acrylate

EC Number:

219-606-3

EC Name:

4-hydroxybutyl acrylate

Cas Number:

2478-10-6

Molecular formula:

C7H12O3

IUPAC Name:

4-hydroxybutyl acrylate

Test material form:

liquid

Method

Target gene:

his, trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from male Wistar rats livers (induces with phenobarital and ß-naphthoflavone)

Test concentrations with justification for top dose:

1st experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (all strain; SPT with and without S9 mix)
2nd experiment: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate (all strian; PIT with and without S9 mix)
No mutagenicity was observed in the standard plate test. Due to toxicity, the doses were adjusted in the preincubation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (ultrapure water)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine

Remarks:

Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
: 3 test plates per dose or per control

Standard plate test: The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test: The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

Acceptance criteria:Generally, the experiment was considered valid if the following criteria were met:
The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
The sterility controls revealed no indication of bacterial contamination.
The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria: The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

TOXICITY: A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 333 μg/plate.

Decreased revertant numbers were observed at following concentrations (μg/plate):

S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
Without	1000 - 5000	5000	1000 - 5000	2500 - 5000	-
With	2500 - 5000	5000	5000	1000 - 2500	-
Without	2500	-	1000 - 2500	1000 - 2500	1000
with	1000 - 2500	-	1000 - 2500	1000 - 2500	333 - 2500

- = no adverse effects observed

Reduced background growth was observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	5000	5000	5000	5000	5000
1st-SPT	With	5000	5000	5000	5000	5000
2nd-PIT	Without	2500	2500	2500	2500	-
2nd-PIT	with	-	-	-	-	-

- = no adverse effect observed

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance 4-Hydroxybutyl Acrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.