Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 August to 24 October, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD 474 and EU method B.12. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1998

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
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): OS 2200 (546-97A).
- Physical state: Clear to light yellow liquid.
- Analytical purity: 92.72%.
- Lot/batch No.: AT3205-78C.
- Expiration date of the lot/batch: July 1998.
- Storage condition of test material: room temperature in the dark, under nitrogen.

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England.
- Weight at study initiation: males 28-30g, females 22-24g.
- Assigned to test groups randomly: yes.
- Fasting period before study: no.
- Housing: sexes separated, in plastic disposable cages.
- Diet (e.g. ad libitum): ad libitum, RMl(E)SQC standard laboratory pelleted rodent diet.
- Water (e.g. ad libitum): ad libitum, tap water.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25ºC
- Humidity: 42-58%
- Air changes (per hr): 20 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours artificial light.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: dried corn oil
- Justification for choice of solvent/vehicle: an initial preliminary toxicity test employed DMSO as the vehicle but, because of the toxic effect of this vehicle using the intraperitoneal route, the vehicle was amended to corn oil.
- Concentration of test material in vehicle: 16, 32, 64 mg/mL
- Lot/batch no. (if required): 8/8/97/3B
Details on exposure:
Suspensions of the test substance were freshly prepared on the morning of the test (using identical methods for each phase of the test) and were diluted to the concentrations shown overleaf in dried corn oil.
The test substance and negative control were dosed intraperitoneally by injection using a standard dose volume of 10 mg/mL, and Mitomycin C, the positive control compound, was administered orally by intragastric gavage using a dose volume of 20 mL/kg. The intraperitoneal route was selected for use in this test to maximise potential absorption of the test substance.
Duration of treatment / exposure:
Single dose.
Frequency of treatment:
Single dose.
Post exposure period:
24 and 48 h.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 160, 320 , 640 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Test item: 5 animals per sex for the 160 and 320 mg/kg doses and 10+1* animals per sex for the 640mg/kg dose.
*= additional animals, dosed concurrently, to replace any that might die.
Vehicle control: 10 animals per sex.
Positive control: 5 animals per sex.
Control animals:
yes, concurrent vehicle
Positive control(s):
-Positive control: Mitomycin C (0.6mg/mL in water solution).
- Justification for choice of positive control(s): Mitomycin C cause large, highly, significant increases in the frequency of micronucleated immature erythrocytes.
- Route of administration: Intragastric gavage.
- Doses / concentrations: 12 mg/kg bw.

Examinations

Tissues and cell types examined:
Erythrocytes from the bone marrow of the femur.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary toxicity test had previously shown that 640 mg/kg was expected to be approximately the maximum tolerated dose; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 5 mice per sex from the negative control, test groups and the positive control group were sacrificed 24 hours after dosing. 5 mice per sex from the negative control and high level treatment group were sacrificed 48 hours after dosing. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum using a 2 ml disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum.

DETAILS OF SLIDE PREPARATION:
A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner.At least three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of Gurr's improved R66 Giemsa (BDH) with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6 4 , the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear. Micronuclei were identified. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.

OTHER:
Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded.
Evaluation criteria:
A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent 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 immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P<0.01) and where these values fall within the historical range.
Statistics:
The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation. Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected. For individual inter-group comparisons this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The range-finding study was carried out in 2 phases, the first one was used to give an approximate indication of the maximum tolerated dose, and the second phase refined the results:
- Dose range:
Phase 1: 250, 500, 1000, 2000 mg/kg
Phase 2: 270, 360, 480, 640 mg/kg
Results: The preliminary toxicity test showed that 640 mg/kg was expected to be approximately the maximum tolerated dose; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The test substance did not cause any statistically significant increases in the incidence of micronucleated immature or mature erythrocytes or any substantial decrease in the proportion of immature erythrocytes.

OTHER: CLINICAL SIGNS AND MORTALITIES: One female died after treatment with the substance at the high dose level. This animal was replaced by an animal from the concurrently treated satellite group. At post mortem examination, this animal showed no signs of mis-dosing. The severity of the clinical signs for the high level group and the single mortality obtained were consistent with the maximum tolerated dose having effectively been achieved. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.

Any other information on results incl. tables

Table 1 . Summary of results and statistical analysis

Sampling time

Treatment

Dose (mg/kg)

% ie/(ie+me)

Incidence mie (mean)

Incidence mme (mean)

24 Hours

Vehicle control

-

46

1.7

0.0

OS 2200

160

43

1.3

1.3

320

47

1.6

0.7

640

47

2.1

2.0

Mitomycin C

12

46

26.2**

2.3

48 Hours

Vehicle control

-

50

1.2

0.3

OS 2200

640

46

1.2

0.7

% ie/(ie+me): proportion of immature erythrocytes
mie: Number of micronucleated cells observed per 2000 immature erythrocyte examined.     
mme: Number of micronucleated cells observed pero 2000 mature erythrocyte examined.

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

**P < 0.001 (highly significant)
*P < 0.0 1(significant)
otherwise P> 0.01 (not significant)

Mitomycin C caused large, highly significant increases [P<0.001] in the frequency of micronucleated immature erythrocytes. Since a relatively high variation in the positive control mie values occurs between studies, and because the value of 26.2 mie per 2000 erythrocytes examined falls within the historic control range, the response obtained in this study is considered to be fully acceptable.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that OS-2200 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.
Executive summary:

An in-vivo mammalian erythrocyte micronucleus test was performed with test item according to EU method B.12 and OECD guideline 474 under GLP conditions. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 0 (control) 160, 320 and 640 mg/kg bw (based on a preliminary toxicity test results) in dry corn oil. A positive control group was dosed orally, by intragastric gavage, with Mitomycin C at 12 mg/kg bw. Bone marrow smears were obtained from 5 animals per sex in the negative control group, test substance groups and the positive control group 24 hours after dosing. In addition, bone marrow smears were obtained from 5 animals per sex in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept. Mice treated with the test substance did not show any significant increase in the frequency of micronucleated immature erythrocytes at either sampling time. There was no significant decrease in the proportion of immature erythrocytes after treatment of the animals with the test substance. Since the test substance did not cause any significant increase in the incidence of micronucleated immature erythrocytes or any substantial decrease in the proportion of immature erythrocytes, it was concluded that the test item did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in-vivo to mice.