Lithium bis(oxalato)borate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015/11/27 - 2016/04/11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of GLP compliance in the study report but no certificate attached

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

Lithium bis(oxalato)borate

EC Number:

456-990-3

EC Name:

Lithium bis(oxalato)borate

Cas Number:

244761-29-3

Molecular formula:

C4BLiO8 (Hill Empirical Formula) C4BO8.Li (CAS Empirical Formula)

IUPAC Name:

boron(3+) lithium(1+) dioxalate

Test material form:

solid: particulate/powder

Details on test material:

Identification: Lithium-bis(oxalate) borate (LiBOB)
Appearance: White powder
Purity/Composition: > 99%
Test item storage: At room temperature, desiccated
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Stable
Chemical name (IUPAC), synonym or trade name: Lithium -bis[oxalate(2-)O,O]borate
CAS Number: 244761-29-3
Molecular formula: C4BLiO8 (Hill Empirical Formula), C4BO8.Li (CAS Empirical Formula)
Molecular weight 194.0

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

These mice are recommended by international guidelines (e.g. OECD, EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 30.5 ± 1.5 g (27 - 34 g).
- Assigned to test groups randomly: yes
- Fasting period before study: 3-4 h
- Housing: The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height 180 mm, length 380 mm and width 220 mm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). A shelter (disposable paper corner home, MCORN 404, Datesand Ltd, USA) and paper bedding (Envirodri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet
- Water (e.g. ad libitum): ad libitum, tap-water
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range: 21.1 - 22.3°C)
- Humidity (%): 40 - 70% (actual range: 33 - 55%)
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: propylene glycol
- Amount of vehicle (if gavage or dermal): 5 mL/kg bw

Details on exposure:

The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Lithium-bis(oxalate) borate (LiBOB). The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use. Feed was withheld 3 - 4 hours prior to dosing until 0.5 - 2 hours after administration of Lithium-bis(oxalate) borate (LiBOB). The dosing volume was 5 ml/kg body weight. Lithium-bis(oxalate) borate (LiBOB) concentrations were prepared on the day of administration.

Frequency of treatment:

Two treatments were performed, administered at a 24-hour interval.

Post exposure period:

24 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral intubation
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow erythrocyte

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study to find out the maximum tolerated dose. The test procedure and conditions were similar to those applied in the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Lithium-bis(oxalate) borate (LiBOB). Two treatments were performed, administered at a 24-hour interval. Sampling was performed 48 h after the first treatment

DETAILS OF SLIDE PREPARATION: Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, the Netherlands). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The
Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

METHOD OF ANALYSIS: To prevent bias, all slides were randomly coded before examination. An adhesive label with the study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
A test item is considered positive in the micronucleus test if:
a) At least one of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) All results are within the 95% control limits of the negative historical control data range.

Statistics:

see 'Evaluation criteria'

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: mortality at doses >= 375 mg/kg bw, at 375 mg/kg bw: ataxia; lethargy; hunched posture; rough coat

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow
- Ratio of PCE/NCE (for Micronucleus assay): The groups that were treated with 62.5 and 125 mg Lithium-bis(oxalate) borate (LiBOB)/kg body weight showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with 250 mg Lithium-bis(oxalate) borate (LiBOB)/kg body weight and cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Applicant's summary and conclusion

Conclusions:

It is concluded that this test is valid and that Lithium-bis(oxalate) borate (LiBOB) is not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 250 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described. In the highest dose tested the ratio of polychromatic to monochromatic erythrocytes was significantly decreased, indicating that the test item had reached the bone marrow. Test item exposure of the target tissue was also confirmed by the analytical verification of the test substance in the serum of mice treated at the highest dose level.

Executive summary:

Cytogenicity of the test item in mice bone marrow erythrocytes was determined according to OECD Guideline 474 following GLP. As a result, Lithium-bis(oxalate) borate (LiBOB) was not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 250 mg/kg bw (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions. In the highest dose tested the ratio of polychromatic to monochromatic erythrocytes was significantly decreased, indicating that the test item had reached the bone marrow. Test item exposure of the target tissue was also confirmed by the analytical verification of the test substance in the serum of mice treated at the highest dose level.