Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 July - 21 August, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to appropriate Guidelines and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium Isobutyrate Solution

IUPAC Name:

Sodium Isobutyrate Solution

Constituent 2

Reference substance name:

Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate

EC Number:

939-200-6

IUPAC Name:

Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate

Constituent 3

Reference substance name:

Reaction mass of lithium 3-hydroxy-2,2,4-trimethyl-pentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate

IUPAC Name:

Reaction mass of lithium 3-hydroxy-2,2,4-trimethyl-pentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate

Test material form:

other: semi liquid

Details on test material:

- Name of test material (as cited in study report): Sodium Isobutyrate Solution
- Physical state: Light yellow semi liquid
- Analytical purity: 86.5 %
- Composition of test material, percentage of components:
lithium 3-hydroxy-2,2,4-trimethylpentanoate: 13.1 %
lithium isobutyrate: 26.2 %
sodium 3-hydroxy-2,2,4-trimethylpentanoate: 14.0 %
sodium isobutyrate: 33.2 %
- Lot/batch No.: W195_02
- Expiration date of the lot/batch: 17 October 2013
- Storage condition of test material: Controlled room temperature

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: Stable karyotype
- Periodically "cleansed" against high spontaneous background: low background aberrations

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial S9 fraction

Test concentrations with justification for top dose:

The examined concentrations of the test item were 5000, 3750, 2500, 1250, 625 and 312.5 μg/mL. Treatment concentrations for the mutation assay were selected based on the results of two short preliminary tests.

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

water

Controls 2

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

ethylmethanesulphonate

Controls 3

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: In Assay 1 and 2, 3-hour treatment with metabolic activation and 20-hour treatment without metabolic activation. In Assay 3, 3-hour treatment with metabolic activation and 3-hour treatment without metabolic activation.
- Expression time (cells in growth medium): In Assay 1 and 3, sampling was performed 20 hours after the beginning of the treatment both with and without metabolic activation. In Assay 2, sampling was performed 28 hours after the beginning of the treatment both with and without metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): Harvesting was performed after 20 hours or 28 hours from the beginning of treatment.2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine.
-Stain: The slides were stained with 5 % Giemsa solution.

NUMBER OF REPLICATIONS: Duplicate cultures were used for each test item concentration or controls.

NUMBER OF CELLS EVALUATED: 200 cells examined per concentration

DETERMINATION OF CYTOTOXICITY
- Method: For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time,, the number of surviving cells was determined using a haemocytometer.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells was scored
- Determination of endoreplication: the number of endoreduplicated cells was scored
- Other: Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality can was also performed at the end of the treatment period

Evaluation criteria:

The assay is considered valid, if the following criteria are met:
- The negative (solvent) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.
The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Sodium Isobutyrate Solution induced a marginally significant level of chromosome aberrations in the performed experiments without metabolic activation, but no clastogenic activity was observed in the experiments with metabolic activation.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in the pH.
- Effects of osmolality: Large changes were detected in osmolality at 5000 μg/mL concentration with and without metabolic activation.
- Evaporation from medium: Not reported
- Water solubility: no insolubility was detected
- Precipitation: no insolubility was detected
- Other confounding effects: Not reported

RANGE-FINDING/SCREENING STUDIES:
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 3-hour treatment with metabolic activation or 20-hour treatment without metabolic activation, 28-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous aberration frequencies of the negative (solvent) controls in the performed experiments were within the historical control range of the testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity tests, single cultures were tested and positive controls were not included. Following treatments, cell concentrations were determined using a haemocytometer. Precipitation of the test item in the final culture medium was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
A total of eight test concentrations between 5000 and 39.06 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.

Remarks on result:

other: other: Not applicable

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No further data.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

Sodium Isobutyrate Solution induced a marginally significant level of chromosome aberrations in the performed experiments without metabolic activation, but no clastogenic activity was observed in the experiments with metabolic activation under the conditions of the study. Therefore, Sodium Isobutyrate Solution is considered clastogenic in this test system.

Executive summary:

Sodium Isobutyrate Solution was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Distilled water and was examined at doses up to cytotoxic concentrations and/or the recommended maximum concentration according to the OECD guidelines. In the independent Chromosome Aberration Assays which were performed using duplicate cultures, at least 200 well-spread metaphase cells were analysed for each test item treated, negative (solvent) and positive control sample.

All concentrations and dose-levels in the report are expressed as the total of active ingredients.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000, 3750, 2500, 1250, 625 and 312.5 μg/mL.

In Assay 1, no insolubility was detected at the end of the treatment period in the final treatment medium with and without metabolic activation. There were no large changes in the pH; but large changes were detected in osmolality at 5000 μg/mL concentration with and without metabolic activation. No marked cytotoxicity was observed in this assay. Therefore, concentrations of 3750, 2500 and 1250 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation. However, based on the observed scoring results of Assay 1, the first main test was considered invalid and terminated. An additional main test (Assay 3) was performed to obtain valid and interpretable data.

the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000, 3750, 2500, 1875, 1250, 625 and 312.5 μg/mL without metabolic activation; and 5000, 3750, 2500, 1250, 625 and 312.5 μg/mL with metabolic activation.

In Assay 2, similarly to the first experiment, no insolubility was detected at the end of the treatment period in the final treatment medium with and without metabolic activation. No large changes in the pH were detected, but large changes were detected in osmolality at 5000 μg/mL concentration with and without metabolic activation. Marked cytotoxicity was observed in this assay at 5000 and 3750 μg/mL concentrations without metabolic activation (relative survival values of 25 and 39%, respectively). Therefore, concentrations of 3750, 2500 1250 and 625 μg/mL (a total of four) were chosen for evaluation in the experiment without metabolic activation; and concentrations of 3750, 2500 and 1250 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation.

In Chromosome Aberration Assay 3, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000, 3750, 2500, 1250, 625 and 312.5 μg/mL.

In Assay 3, no insolubility was detected at the end of the treatment period in the final treatment medium with and without metabolic activation. There were no large changes in the pH; but large changes were detected in osmolality at 5000 μg/mL concentration with and without metabolic activation. No marked cytotoxicity was observed in this assay. Therefore, concentrations of 3750, 2500 and 1250 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.

None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations when compared with the appropriate negative (solvent) control values using the short treatments and 20-hour harvesting period (Assay 3). However, using the longer harvesting period of 28-hour (Assay 2), a marginally significant increase in the number of aberrant cells was observed at 3750 μg/mL concentration without metabolic activation and at 1250 μg/mL concentration with metabolic activation. In case of the experiment with metabolic activation, there was no dose response and the effect was not repeatable between assays. However, in case of the long treatment without metabolic activation a clear dose-response was observed, and the effect was reproducible between replicate cultures. It should be noted that Assay 2 overall had a higher than normal level of endoreduplicated cells, an indicator of cell cycle perturbation which can lead to the development of aberrations without genotoxicity.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1-6) and endoreduplicated metaphases (1-3) were found in some cases in the negative (solvent) control, positive control or test item treated samples in the performed experiments.

The negative (solvent) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, Sodium Isobutyrate Solution induced a marginally significant level of chromosome aberrations in the performed experiments without metabolic activation, but no clastogenic activity was observed in the experiments with metabolic activation under the conditions of the study. Therefore, Sodium Isobutyrate Solution is considered clastogenic in this test system.