2-ethylbutyric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2-Ethylbutyric acid was found not to be mutagenic at up to 5000 µg/plate with and without S9-mix metabolic activation following a bacterial reverse mutation assay (Ames test) in Salmonella typhimurium and Escherichia coli. At 1200 and 1600 µg/plate of 2-ethylbutyric acid, structural chromosomal aberration was observed in Chinese hamster lung (CHL/IU) cells after 24-hours of continuous treatment without metabolic activation. Based on this outcome, the registered substance can be regarded as having the capacity to induce genetic toxicity in mammalian cells. A conservative approach should be adopted with respect to potential genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

T31-18: Chemical Substance Control Law of Japan

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0, 313, 625, 1250, 2500, 5000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 ug/plate with metabolic activation

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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:

At 5000 ug/plate with metabolic activation

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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:

At 5000 ug/plate with metabolic activation

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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:

At 5000 ug/plate with metabolic activation

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No gene mutation was observed in any Salmonella typhimurium or Escherichia coli strain, however, microbial toxicity was observed at 5000 µg/plate in Salmonella typhimurium TA1535, TA1537, TA98, and TA100 with metabolic activation.

Conclusions:

2-Ethylbutyric acid was concluded not to be mutagenic with and without metabolic activation in Salmonella typhimurium and Escherichia coli at ≤5000 µg/plate.

Executive summary:

A bacterial reverse mutation assay (Ames test) was performed to determine the genetic toxicity of 2-ethylbutyric acid at 0, 313, 625, 1250, 2500, 5000 µg/plate. The following species and strains were exposed for the purpose of the experiment, with and without metabolic activation (S9-mix): Salmonella typhimurium TA1535, TA1537, TA98, and TA100 and Escherichia coli WP2 urvA. Positive and negative (dimethyl sulfoxide solvent) control groups were prepared and run in parallel. The experiment was undertaken according to Good Laboratory Practise (GLP), OECD Guideline 471 (Bacterial Reverse Mutation Assay), and T31-18: Chemical Substance Control Law of Japan.

The positive and negative controls demonstrated that the test system was valid. Microbial toxicity was observed at 5000 µg/plate in Salmonella typhimurium TA1535, TA1537, TA98, and TA100 with metabolic activation, however, no genetic mutation was observed in any strain with and without metabolic activation between 0 - 500 µg/plate. 2-Ethylbutyric acid was subsequently concluded not to be mutagenic at ≤5000 µg/plate.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

T31-18: Chemical Substance Control Law of Japan

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Test concentrations were between 0 and 1600 ug/ml. The maximum concentration was established based on an initial growth inhibtion test, in which 50 % growth inhibition was observed at 1002 ug/ml with and without metabolic activation for the 6-hour short-term treatment and 1272 ug/mL without metabolic activation for the 24-hour continuous treatment.

Details on test system and experimental conditions:

DURATION
- Exposure duration: 6-hour experiment was carried out with and without S9-mix and a long-term 24-hour continuous experiment was performed without S9-mix.

Key result

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Remarks:

6-hour

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Remarks:

24-hour

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Structural chromosomal aberration at 1200 and at 1600 ug/ml without S9-mix

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Polyploidy was not observed in any treatment group. Structural chromosomal aberrations were noted in Chinese hamster lung (CHL/IU) cells at 1200 and 1600 ug/ml without metabolic activation.

Conclusions:

2-Ethylbutyric acid was observed to induce structural chromosomal aberration in Chinese hamster lung (CHL/IU) cells following continuous 24-hour treatment without metabolic activation. The registered substance can be regarded as having the capacity to induce genetic toxicity.

Executive summary:

An in vitro experiment was performed to determine the capacity of 2-ethylbutyric acid to induce structural chromosomal aberration in Chinese hamster lung (CHL/IU) cells according to Good Laboratory Practise (GLP), OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), and T31-18: Chemical Substance Control Law of Japan. A 6-hour experiment was carried out with and without S9-mix metabolic activation and an additional 24-hour continuous exposure experiment carried out without S9-mix at several dose levels between 0 and 1600 ug/ml. Polyploidy was not observed in any treatment group, however, structural chromosomal aberration was reported at 1200 and 1600 ug/ml in the 24-hour continuous treatment without metabolic activation. This genetic toxicity was believed to be a result of the clastogenic effect of 2-ethylbutyric acid as opposed to pH. Under the conditions of this test, 2-ethylbutyric acid can be regarded as comprising the potential to induce structural chromosomal aberration in mammalian cells. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

It has been concluded from an in vivo mammalian erythrocyte micronucleus assay that although a decrease in the polychromatic erythrocyte (PCE) to normo-chromatic erythrocyte (NCE) ratio was observed in male mice, which is suggestive of bone marrow toxicity, the results of the experiment did not support 2-ethylbutyric acid as being mutagenic in vivo. This is predominantly because no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was found across any treatment group.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

T37-15: Chemical Substances Control Law of Japan

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: T23-48:BDF1 (C57BL/6 x DBA/2)

Sex:

male

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: Corn oil.

Duration of treatment / exposure:

48-hour experimental period

Frequency of treatment:

Twice at 0 and 24 hours (24-hour interval)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five male animals per dose

Control animals:

yes, concurrent no treatment

yes, historical

Positive control(s):

Positive control based on historical data for mitomycin C (2 mg/kg i.p. single administration).

Tissues and cell types examined:

Mouse bone marrow cells

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See additional information.

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At 1000 and 2000 mg/kg bw/day, the ratio of polychromatic erythrocytes (PCE) to total erythrocytes (%) observed in mice significantly decreased. One animal died at 2000 mg/kg bw/day.

Conclusions:

2-Ethylbutyric acid at ≤2000 mg/kg bw/day was determined not to be mutagenic as part of an in vivo mammalian erythrocyte micronucleus assay using mouse bone marrow cells.

Executive summary:

An in vivo mammalian erythrocyte micronucleus assay for 2-ethylbutyric acid was undertaken in line with Good Laboratory Practise (GLP), OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test), and T37-15: Guideline for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan). The purpose of the experiment was to determine the mutagenic potential of the registered substance in male mice (strain: T23-48:BDF1 (C57BL/6 x DBA/2)) following administration of 0 (untreated control), 500, 1000, and 2000 mg/kg bw/day in a corn oil vehicle via oral gavage twice with a 24-hour interval. The experiment ran for a 48-hour period. A dose-finding study was conducted initially to obtain the concentrations for the main test. Historical positive (mitomycin C 2 mg/kg i.p. single administration) and negative control data from 1986 - 2002 were referred to.

The study was reported to have satisfied validity criteria. That one animal died at 2000 mg/kg bw/day, and that a significant decrease in the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) (%) was observed at 1000 and 2000 mg/kg bw/day, is suggestive of bone marrow toxicity induced by the substance. However, 2-ethylbutyric acid was ultimately concluded not to be mutagenic to mammalian cells in vivo given that there was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in all treatment groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)