lithium 3-((3,4-dicyanophenyl)sulfonyl)propane-1-sulfonate)

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:

The experimental phase of the study was performed between 13 May 2013 and 03 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as reliable without restriction according to Klimisch et al (1997).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

lithium 3-((3,4-dicyanophenyl)sulfonyl)propane-1-sulfonate)

EC Number:

700-777-2

Cas Number:

667889-11-4

Molecular formula:

C11H9N2O5S2Li

IUPAC Name:

lithium 3-((3,4-dicyanophenyl)sulfonyl)propane-1-sulfonate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): S193308
- Physical state: White powder
- Analytical purity: 98.9%
- Lot/batch No.: 422 (Bag 3)
- Date received: 29 April 2013
- Storage condition of test material: Room temperature, in the dark.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The test animals were albino Hsd: ICR (CD-1®) strain mice obtained from Harlan UK. At the start of the main test the mice weighed 23 to 30g and were approximately six to ten weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.

The animals were housed in groups of up to seven, by sex, in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Arachis oil for animals dosed intraperitoneally with test susbtance.

Details on exposure:

Range-Finder Test

A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. The criteria for dose level selection is ideally the maximum tolerated dose level, or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The range-finding toxicity test was also used to determine if the main test was to be performed using both of the sexes or males only.

All animals were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its body weight at the time of dosing. Animals were observed 1, 3 and 4 hours after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed. As it was not possible to confirm systemic absorption by the oral route following dosing at 2000 mg/kg, further dosing was undertaken via the intraperitoneal route.

Micronucleus Test

Groups, each of seven male mice, were dosed once only via the intraperitoneal route with the test item at 800, 400 or 200 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 800 mg/kg was killed after 48 hours. In addition, two further groups of male mice were included in the study; one group (seven mice) was dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a second group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test. The vehicle and positive control group animals were killed 24 hours following dosing.

Frequency of treatment:

Once only

No. of animals per sex per dose:

Seven male mice were used per dose group and vehicle control. The postive control group consisted of five males.

Control animals:

yes, concurrent vehicle

other: Positve control

Positive control(s):

Cyclophosphamide dosed orally was used as the positive control.

Examinations

Tissues and cell types examined:

Bone Marrow.

Details of tissue and slide preparation:

Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a square root of (x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

one death and clinical signs observed in animals dosed via the intraperitoneal route at 1000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Range- Finder Toxicity Test

In animals dosed with the test item at 2000 mg/kg via the oral route, no clinical signs were observed throughout the 48 hour exposure period. The route of administration was therefore changed to the intraperitoneal route to maximise exposure to the test item.

In animals dosed with the test item at 1000 mg/kg via the intraperitoneal route, a premature death occurred and the following clinical signs were observed: hunched posture, lethargy, ataxia, splayed gait, elevated tail, clonic convulsions, and tonic convulsions. These indicated that systemic absorption had been achieved. In animals dosed with the test item at 300 mg/kg, clinical signs were not observed. In animals dosed with the test item at 600 and 800 mg/kg, the clinical signs hunched posture and ptosis was observed. Therefore, with evidence of excessive toxicity at 1000 mg/kg, the maximum dose level in the main test was set at 800 mg/kg, the considered maximum tolerated dose level, with 400 and 200 mg/kg as the two lower dose levels using the intraperitoneal route of administration.

The test item showed no marked difference in its toxicity to male or female mice; therefore the main test was performed using male mice only.

Micronucleus Test

There were no premature deaths seen in any of the dose groups. The clinical signs hunched posture and ptosis were observed in animals dosed with the test item at 800 mg/kg in both the 24 and 48-hour dose groups.

A statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour 200 mg/kg dose group and, although not statistically significant, a modest decrease was also observed in the 24-hour 400 mg/kg dose group. However, the reductions were part of an inverse dose related response and, therefore, may be considered to be artefactual and of no toxicological significance.

The observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

See attached background material section for tables of results.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy. The method was designed to meet the requirements of the following guideline:

OCED Guideline for Testing of Chemicals No 474 " Mammalian Erthrocyte Micronucleus Test (adopted 21st July 1997)

Method

Groups of seven male mice, were dosed once only via the intraperitoneal route with the test item at 800, 400 or 200 mg/kg. One group of mice from each dose level was killed 24 hours following treatment and a second group dosed with test item at 800 mg/kg was killed after 48 hours. A vehicle control (arachis oil) group and a postive control (cyclophosphamide) group were also included in the study.

Results

A statistically significant decrease in the PCE/NCE ratio was observed in the 24 -hour 200 mg/kg test item group and although not statistically significant, a modest increase was also observed in the 24 -hour 400 mg/kg dose group. However, the reductions were part of an inverse dose related response and considered to be artifactual and of no toxicological significance. The observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positve control group responded as expected.

Conclusion

The test material was considered to be non-genotoxic under the conditions of the test.