Potassium isobutyrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In accordance with Annex VII of REACH, one study is avaiable to assess mutagenicicty in bacteria. The stusy was performed according to an appropriate OECD guideline (OECD 471) and under the conditions of GLP.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jan 2019 - 01 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21. Jul. 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / 18110908G
- Expiration date of the lot/batch: 31 Oct 202


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5 °C); Keep away from humidity
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:

Stability in solvents H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone.
The test item is soluble in demin. water.
Based on the non-GLP pre-test, demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the first and the second experiment, a stock solution containing 50 g/L of the test item in demin. water was prepared. The test item solution was not sterile filtrated before use.
- Final dilution of a dissolved solid, stock liquid or gel:
The stock solution was used to prepare the geometric series of the concentrations to be tested.
The following nominal concentrations were prepared for the first experiment:
5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate
The following nominal concentrations were prepared for the second experiment:
5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate

FORM AS APPLIED IN THE TEST (if different from that of starting material) In solution

Species / strain / cell type:

other: Salmonella typhimurium LT2: TA97A, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem GmbH, Gießen
- method of preparation of S9 mix: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the first experiment:
5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate
The following nominal concentrations were prepared for the second experiment:
5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
replicants

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: nitro-phenylendiamine

Negative solvent / vehicle controls:

yes

Remarks:

Demineralised water

Positive controls:

yes

Positive control substance:

sodium azide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two experiments

First Experiment
Date of treatment 08. Jan. 2019
Concentrations tested 5000 / 1500 / 500 / 150 / 50 μg/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method

Second Experiment
Date of treatment 30. Jan. 2019
Concentrations tested 5000 / 2500 / 1250 / 625 / 313 / 156 μg/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): minimum 10E9 cells.mL
- Test substance addedP:
in agar (plate incorporation) - experiment 1;
preincubation - experiment 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 2 days


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: background growth inhibition

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

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

Positive controls validity:

valid

First Experiment 

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

 

Solubility and Toxicity

In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.

No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not reduced.

 

Mutagenicity

No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

To verify this result, a further experiment was performed.

 

Second Experiment

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

 

Solubility and Toxicity

In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.

No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not reduced.

 

Mutagenicity

No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

Conclusions:

The test item Potassium isobutyrate showed no increase in the number of revertants in all bacteria strains in both experiments.
All negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabol-ic activation system functioned properly.
Based on the results of this study it is concluded that Potassium isobutyrate is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experi-mental conditions in the present study.

Executive summary:

SUMMARY

Title of Study:Determination of the mutagenic potential of Potassium isobutyrate with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14

Findings and Results:

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EU guidelines.

The test item Potassium isobutyrate was tested in theSalmonella typhimuriumreverse mutation assay with five strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment, the test item (dissolved in demin. water) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the results of the first experiment, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-relatedincrease in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that Potassium isobutyrate is not mutagenic in theSalmonella typhimuriumstrains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

 

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The data provided in accordance with Annex VII of Regulation (EC) No.1907/2006 (REACH) does not indicate genotoxicity.