Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-01-26 to 2005-03-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 21 July, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
8 June 2000
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vivo Mammalian Micronucleus Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
mouse
Strain:
other: Crl:CD-1™(ICR)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 20 - 27 g
- Assigned to test groups randomly: yes
- Housing: in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Diet (e.g. ad libitum): ad libitum (Certified Rat and Mouse Diet Code 5LF2, BCM, IPS Limited, London, UK)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Arachis oil
- Justification for choice of solvent/vehicle: required for a suspension at the appropriate concentrations.
- Concentration of test material in vehicle: 25, 30, 40 mg/mL for the range finding toxicity test and 5, 7.5, 15 and 30 mg/mL for the main test
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.
Duration of treatment / exposure:
24 h in the range finding toxicity test
24 h (in one vehicle control and in the 75, 150, 300 and positive control groups) and 48 h (at the dose level of 300 mg/kg bw and in the vehicle control) in the main test
Frequency of treatment:
single exposure
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
main test
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
main test
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
range finding toxicity test
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
range finding toxicity test and main test
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
range finding toxicity test
No. of animals per sex per dose:
2 animals (1 female and 1 male) for 250 mg/kg bw, 2 animals (2 males) for three 300 mg/kg bw groups and 9 male animals for 400 mg/kg bw in the range finding test
7 in the main test
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test.
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw / 5 mg/mL

Examinations

Tissues and cell types examined:
2000 premature erythrocytes were evaluated after termination (i.e. 24 or 48 hours following dosing) of the exposure. Both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose level selected should ideally be 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 sexes or males only. It was considered to be unnecessary to investigate either the intraperitoneal route of administration or dose levels above 400 mg/kg based on the results of preliminary tests.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): The test material was administered orally to 7 male mice (main study) each group, the animals were observed for 24 or 48 h and then killed by cervical dislocation

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

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCB-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

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 non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to norrnochromatic 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 using Student's t-test (two tailed) and any significant results were confirmed using the one-way analysis of variance.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 300 and 400 mg/kg bw
- Clinical signs of toxicity in test animals: Clinical signs were observed at and above 250 mg/kg and included lethargy, ptosis, decreased respiratory rate, hunched posture and ataxia.
- Rationale for exposure: existing toxicology data

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): No increase in micronucleated PCEs observed as compared to the concurrent vehicle control
- Appropriateness of dose levels and route: The dose levels were determined in a preliminary dose range finding test

Any other information on results incl. tables

Table 1: Relative Group Frequency Categories of Micronuclei Per 1000 PCEs

48-Hour Control Group (60 Groups)

Frequency Categories

Groups

0.0-0.4

21

 

35%

0.5-0.9

18

 

30%

1.0-1.4

14

23%

1.5-2.0

7

 

12%

2.1-2.5

0

0%

Table 2: Relative Group Frequency Categories of Micronuclei Per 1000 PCEs

24-Hour Control Group (60 Groups)

Frequency Categories

Groups

0.0-0.4

15

25%

0.5-0.9

25

42%

1.0-1.4

14

23%

1.5-2.0

3

12%

2.1-2.5

3

0%

Combined 24 and 48-Hour Groups (120 Groups)

Frequency Categories

Groups

0.0-0.4

36

30%

0.5-0.9

43

36%

1.0-1.4

28

23%

1.5-2.0

10

8%

2.1-2.5

3

3%

Table 3

Micronucleus Test - Summary of Group Mean Data

 

Treatment Group

Number of PCE with

Micronuclei per 2000 PCE

PCE/NCE Ratio

Group Mean

SD

Group Mean

SD

l.

Vehicle Control

10 ml/kg

48-hour Sampling Time

2.0

3.2

1.45

0.46

2.

Vehicle Control

10 ml/kg

24-hour Sampling Time

1.3

1.0

1.32

0.36

3.

Positive Control

50 mg/kg

24-hour Sampling Time

 58.4**

23.5

1.32

0.39

4.

1 ,4-dithane-2,5-diol

300 mg/kg

48-hour Sampling Time

3.3

2.1

0. 89 *

0.34

5.

1 ,4-dithane-2,5-diol

300 mg/kg

24-hour Sampling Time

2.0

1.5

2.21

0.49

6.

1 ,4-dithane-2,5-diol

150 mg/kg

24-hour Sampling Time

1.9

1.2

1.70

0.50

 7.

1 ,4-dithane-2,5-diol

75 mg/kg

24-hour Sampling Time

1.0

1.2

1.60

0.58

PCE: Polychromatic erythrocytes

NCE: Normochromatic erythrocytes

SD: Standard deviation

* = p < 0.05

** = p < 0.001

Applicant's summary and conclusion

Conclusions:
The present test was conducted according to OECD guideline 474 (adopted 21 July 1997). Each 7 male mice of the Crl:CD-1™(ICR)BR strain were orally administered 50, 75, 150 or 300 mg/kg bw of the test material. After 24h or 48h bone marrow smear was prepared from both femur of each animal. Micronuclei from polychromatic, premature erythrocytes were scored. Based on the obtained results the test substance is considered non-genotoxic under the conditions of the test. Thus, the test item does not need to be classified according to (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) with respect to genotoxicity.