Ethyl 3-hydroxypropanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-03-02 ~ 2022-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-hydroxypropanoate

Cas Number:

623-72-3

Molecular formula:

C5H10O3

IUPAC Name:

ethyl 3-hydroxypropanoate

Test material form:

liquid

Method

Target gene:

Without and with S9 mix : (TA98, TA100, TA1535, TA1537, WP2uvrA )

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

Test concentrations with justification for top dose:

1. Concentration range-finding test
According to the guidelines, 5000 μg/plate was the maximum concentration and the following concentrations were set to common ratio of about √10.
Test strain : TA100, TA1535, TA98, TA1537, WP2uvr A
With and without S9 mix concentration (μg/plate) : 50, 150, 500, 1500, 5000

2. In the concentration range-finding test, microbial toxicity was not observed at the any concentration in the presence and absence of S9 mix. Precipitation was not observed on the
agar plates at the any concentration in the presence and absence of S9 mix. Based on the above result, main test were conducted at the following concentrations.
Test strain : TA100, TA1535, TA98, TA1537, WP2uvr A
With and without S9 mix concentration (μg/plate) : 313, 625, 1250, 2500, 5000

Vehicle / solvent:

1. Negative control substance
In the preliminary test for the selection of the negative control, the test substance was dissolved at 50 mg/mL in DMSO. No heat, discoloration or foaming was observed in the preparation using DMSO. Based on this result, DMSO was selected as the vehicle for the test substance and used as the negative control substance in this study.

2. Positive control substance
These chemicals are widely used in reverse mutation assays using bacteria and are recommended in the OECD guidelines for the testing of chemicals, section 4, test No. 471.
Furylfuramide (AF-2), Sodium azide(NaN3), 2aminoanthracene (2AA), 9-Aminoacridine (9AA), Benzo(a)pyrene

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO.

True negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

benzo(a)pyrene

furylfuramide

other:

Details on test system and experimental conditions:

1. Storage of the test strains
- Composition
Bacterial suspension (0.8 mL) of each test strain, which was cultured with shaking for 10 hours
at 37°C in the liquid growth medium, was mixed with 0.07 mL of DMSO (Wako pure chemical industries, Ltd.,).
- Storage : Divided portions (0.5 mL) were frozen
- Storage conditions : below −70 ℃
- Storage date : 2021-06-30 (Concentration range finding test), 2022-03-08 (main test)
- Expiration date :2022-06-29 (Concentration range finding test) 2023-03-07 (main test)

2. Pre-culture
- Pre-culture temperature: 37 ℃
- Pre-culture time: 10 hours
- Pre-culture method: Reciprocal shaking (Shaking frequency: 120 times/minute)
- Pre-culture vessel: 50 mL Erlenmeyer flask.
- Media: Nutrient broth 15 mL
- Strain and volume of inoculate: A frozen stock suspension of the test strain will be thawed
and 0.01 mL will be inoculated.

Rationale for test conditions:

Reason for selection of the test strains
These strains are widely used in reverse mutation assays using bacteria and are recommended in the OECD guidelines for the testing of chemicals, section 4, test No. 471.

Evaluation criteria:

The test strains were chosen because they are very sensitive to mutagens and widely used in mutagenicity studies with bacteria.
This study was conducted in compliance with the methods described in OECD Guidelines for the testing of chemicals, Section 4, TG. No. 471 'Bacterial Reverse Mutation Test' (Adopted : 1997-07-21, Corrected : 2020-06-26).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 2

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:

not valid

Untreated negative controls validity:

not valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 3

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:

not valid

Untreated negative controls validity:

not valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 4

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:

not valid

Untreated negative controls validity:

not valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 5

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:

not valid

Untreated negative controls validity:

not valid

True negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

1. Main test
Microbial toxicity was not observed at any concentration in the presence and absence of S9 mix. Precipitation was not observed on the agar plates at the any concentration in the presence and absence of S9 mix.
The number of revertant colonies in the test substance-treated groups was less than twice that in the corresponding negative control in any test strain regardless of the presence or absence of S9 mix.

2. Sterility test
Microbial contamination was not observed in the highest concentration of the test substance solution and S9 mix in the sterility test group in concentration range finding test and main test.

Applicant's summary and conclusion

Conclusions:

Therefore, it was concluded that Ethyl 3-hydroxypropanoate was not mutagenic (negative) under the conditions employed in the present study.

Executive summary:

Based on the results of the concentration range-finding test, main test, the number of revertant colonies in the test substance-treated groups was less than twice that in the
corresponding negative control in any test strain regardless of the presence or absence of S9 mix. The reproducibility of the test results were confirmed with the concentration range-finding test and main tests.
The numbers of revertant colonies in the negative control and positive control groups in the present study were within the acceptable ranges stipulated at the testing facility (Appendix 1).
The positive controls used in the assays with the presence or absence of S9 mix showed positive responses in every test strains, as evidenced by the number of revertant colonies
being greater than 2-fold of the respective negative control value. In each test, there were 4 or more concentrations did not show microbial toxicity and at least 5 concentrations in all
strains were analyable both in the presence and absence of S9 mix. Consequently, the validity of the present study was confirmed.
Therefore, it was concluded that Ethyl 3-hydroxypropanoate was not mutagenic (negative) under the conditions employed in the present study.