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Toxicological information

Genetic toxicity: in vivo

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
All animals used on this study were Crl: CD(SD) rats and were sourced from Charles River UK Limited, Margate, Kent, England. Animals
used on the study weighed between:

Preliminary toxicity test: Males weighed between 194g to 205g.
Females weighed between 164g to 181g.
Micronucleus test: Males weighed between 174g to 224g

Animal age on despatch and on Day 1 of dosing were:

Preliminary toxicity test: On despatch Males and females ca 42 days old.
Day 1 Males and females ca 48 days old.
Micronucleus test: On despatch Males ca 42 days old.
Day 1 Males ca 49 days old.

Each group was kept, with the sexes separated, in cages and maintained in a controlled environment, with the thermostat and relative humidity target ranges set at 19 to 23°C and 40 to 70% respectively. Temperature and humidity were within range throughout the study. The room was
illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance
diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum.

Food, chew blocks and tap water are routinely analysed for quality at source. All animals were given small soft white untreated wood (ASPEN) chew blocks and a red plastic shelter for environmental enrichment, were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Stability and homogeneity of the test substance and of the test substance in the vehicle were not determined in this test and remain the responsibility of the Sponsor. Chemical analysis of dosing formulations for achieved concentration was not performed in this study.
Suspensions of the test substance were prepared in Corn oil obtained from Sigma, batch number MKBF6012V and MKBG9425V.

Cyclophosphamide obtained from Sigma, batch number 120M1253V was used as the positive control compound. A solution was prepared using purified water at a concentration of 2 mg/mL just prior to administration.

All animals were dosed orally by gavage. The vehicle control and test substance groups were dosed using a dose volume of 2 mL/kg. The positive control group were dosed using a dose volume of 10 mL/kg.
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
daily for 2 consecutive days
Doses / concentrations
Remarks:
Doses / Concentrations:
125/62.5, 250/125, 500/250
Basis:
nominal conc.
No. of animals per sex per dose:
6 males at 125 mg/kg/day
6 males at 250 mg/kg/day
8 males at 500 mg/kg/day (due to toxicity)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (single dose at 20 mg/kg/day, 2 mg/mL)

Examinations

Tissues and cell types examined:
femur bone marrow
Details of tissue and slide preparation:
The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of the femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.

The resulting cell suspensions were centrifuged at 1000 rpm (150 ¿ g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).

Fixation and slide staining:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water
Evaluation criteria:
A positive response is normally indicated by a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes for the treatment group compared with the vehicle control group (p<0.01); individual and/or group mean values should exceed
the laboratory historical control range (Morrison and Ashby 1995).

A negative result is indicated where individual and group mean incidences of micronucleated polychromatic erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent vehicle control group and where these values fall
within the historical control range.

An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant decrease in the proportion of polychromatic erythrocytes (p<0.01).
Statistics:
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.

For micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.

Statistical significance was declared at the 1% level for all tests.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
OS 1600 did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD(SD) rats when administered orally by gavage in this in vivo test procedure.