Registration Dossier

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 phases of the study were performed between 21 October 2010 and 11 December 2010
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:
2011
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): S196439
- Physical state: Black solid
- Analytical purity: 87.2%
- Lot/batch No.: NBZ 6411/22/B
- Date received: 16 September 2010
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
The test animals were albino Hsd: ICR (CD-1®) strain mice were obtained from Harlan UK. At the start of the main test the male mice weighed 24 to 30g and were approximately five to eight 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 2014 Rodent Pelleted Diet) was allowed throughout the study.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Distilled water for animals dosed intraperitoneally with test substance.
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 bodyweight at the time of dosing. Animals were observed between 0.5 and 4 hours after dosing as required 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 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 20, 10 or 5 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 20 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 (distilled water) 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 including vehicle and positive controls.
Control animals:
yes, concurrent vehicle
other: positive 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.

A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.

If these criteria were not fulfilled, then the test material was considered to be nongenotoxic under the conditions of the test.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent 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:
Clinical signs seem at and above 5 mg/kg
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Any other information on results incl. tables

Range-Finding Toxicity Test

Other than black coloured faeces no evidence of toxicity was observed in animals dosed with test item via the oral route and, therefore systemic absorption could not be confirmed using this dose route. Animals dosed with test item via the intraperitoneal route were killed in extremis at and above 250 mg/kg due to the severity of the clinical signs observed: hunched posture, ptosis, lethargy, ataxia, splayed gait, extremities black in colour, pilo-erection, decreased respiratory rate, laboured respiration, prostration, and tip-toe gait.

In animals dosed at and above 30 mg/kg premature deaths and the following clinical signs were observed: hunched posture, ptosis, ataxia, and extremities black in colour. Therefore, with evidence of excessive toxicity, the main test was performed using the maximum tolerated dose level of 20 mg/kg, with 10 and 5 mg/kg as the two lower dose levels. The test item showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test.

Micronucleus Test

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test item at and above 5 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: testes and extremities black in colour, hunched posture, ptosis, and pilo-erection.

Although not statistically significant, a modest decrease in the PCE/NCE ratio was observed in the 24-hour 20 mg/kg test item group when compared to the vehicle control group. This decrease, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. It was considered that the extended post-dosing period of the animals of the 48-hour 20 mg/kg group had resulted in them recovering from the initial toxicity observed in the 24-hour 20 mg/kg group. There were no statistically significant increases in the frequency of micronucleated PCE in any of the test item dose groups 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.

The test item was found not to produce a significant increase In the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

See attached background material section for a summary table of group mean results.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic 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 20, 10 or 5 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 20 mg/kg was killed after 48 hours. A vehicle control (distilled water) group and a postive control (cyclophosphamide) group were also included in the study.

Results

Although not statistically significant, a modest decrease in the PCE/NCE ratio was observed in the 24-hour 20 mg/kg test item group when compared to the vehicle control group. This decrease, together with 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.