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 vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 20 January, 2014

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

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

Test material form:

other: 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

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 35.3 – 36.8 g (males, preliminary experiment), 29.0 – 31.7 g (females, preliminary experiment), 31.5 – 36.7 g (males, main test)
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Group caging (5 animals/cage or 2 animals/cage) and with deep wood sawdust bedding.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete diet for mice and rats – breeding and maintenance" ad libitum
- Water (e.g. ad libitum): tap water from municipal supply ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 – 25.0°C
- Humidity (%): 30 – 68 %
- Air changes (per hr): 15 – 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 15 January 2014 To: 17 January 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Not applicable
- Concentration of test material in vehicle: 200, 100 and 50 mg/mL.
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
- Lot/batch no. (if required): 0790713

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask; the appropriate amount of vehicle (water) was added and stirred to obtain homogenous formulations.

Duration of treatment / exposure:

One dose with examination 24 or 48 hours later

Frequency of treatment:

One dose with examination 24 or 48 hours later

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 males/dose for the 500 and 1000 mg/kg bw
15 males/dose for the 2000 mg/kg bw
The main test was performed using male animals only because the toxic effect of the test item was similar in both sexes in the preliminary toxicity test.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide monohydrate
- Justification for choice of positive control(s): Not reported
- Route of administration: intraperitoneal injection
- Doses / concentrations: 6.0 mg/ml (60 mg/kg bw/day)

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the preliminary toxicity test, 2000 mg/kg body weight (the maximum recommended dose) was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (1000 and 500 mg/kg body weight) were also included in the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): One dose was administered and sampling was carried out 24 or 48 hours later.

DETAILS OF SLIDE PREPARATION: The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (at least 2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.
Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.

METHOD OF ANALYSIS: Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. Fewer than 2000 PCEs may be counted in the event of a clear positive effect.
The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs). During this process, the number of micronuclei was recorded in mature erythrocytes (NCEs) as well.

Evaluation criteria:

Criteria for Identification of Micronucleated Erythrocytes:
- A bluish mauve strongly coloured uniform round or oval particle in the cell.
- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
- During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.

Criteria for a positive response:
The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related. Historical control data are taken into consideration when evaluating the biological significance of small increases.
Criteria for a negative response:
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical negative control values.
Equivocal response:
Results which do not meet the criteria for a positive or negative response are considered to be equivocal. Further investigations or scoring of additional cells may be necessary in case of an equivocal result.

Statistics:

Kruskal Wallis test

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 2000 mg/kg bw
- Solubility: Not reported
- Clinical signs of toxicity in test animals: None observed
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: Not reported
- Harvest times: Not applicable
- High dose with and without activation: Not applicable

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei in bone marrow erythrocytes was observed
- Ratio of PCE/NCE (for Micronucleus assay): The MNPCE/2000 PCE and the PCE-100 PCE+NCE ratios were reported
- Appropriateness of dose levels and route: Based on the results of the preliminary toxicity test, dose levels of 2000, 1000 and 500 mg/kg body weight were selected for the micronucleus test.
- Statistical evaluation: Kruskal Wallis test

Any other information on results incl. tables

There was no treatment related effect on the body weight of the mice. No mortality or signs of systemic toxicity were observed during the study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
No induction of micronuclei in bone marrow erythrocytes was observed following administration of Sodium Isobutyrate Solution to mice at up to and including 2000 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Executive summary:

A study was conducted to determine whether Sodium Isobutyrate Solution test item caused genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated mice in accordance with the OECD guideline “Mammalian Erythrocyte Micronucleus Test”, No. 474 (1997).

In the preliminary toxicity test, groups of two male and two female mice were treated with the test item formulated in Distilled water at 2000, 1000, 500 and 250 mg/kg body weight by oral gavage. No treatment related effect was observed in the preliminary experiment. Therefore, the highest dose level selected for the main test was 2000 mg/kg body weight which is the maximum recommended dose level for materials of low toxicity. Only male mice were used in the main test as observations in the preliminary test showed that there was no substantial difference in the toxicity of the test item between the sexes.

In the main test, groups of male mice were treated with the vehicle (Distilled water) or the test item at 2000, 1000 and 500 mg/kg body weight by oral gavage or the positive control item (Cyclophosphamide dissolved in physiological saline) at 60 mg/kg body weight administered by intraperitoneal injection (five replacement animals were also treated in the high dose group). Five mice from each group were examined 24 hours after dosing, and a further five mice dosed with the vehicle or test item at 2000 mg/kg body weight were examined 48 hours after dosing. Bone marrow smears were prepared on glass slides for each of the mice, stained, and scored. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.

There was no treatment related effect on the body weight of the mice in the main test. No mortality or signs of systemic toxicity were observed during the study. Piloerection was observed at some time points for four animals in the high dose group, for two animals in the mid dose group and for one animal in the low dose group. The animals in the negative (vehicle) and positive control groups were symptom-free during the whole observation period.

No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in mice treated with the test item, compared to the negative (vehicle) control values at any of the sampling time points. The positive control treatment caused a strong, clearly positive response demonstrating the sensitivity of the test system.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Sodium Isobutyrate Solution to mice at up to and including 2000 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.