Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
29 October 2003 to 11 December 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted in accordance with the standardised testing guideline OECD 474 and in line with GLP with no deviations thought to affect the quality of the presented data. The study was reported to a high standard. The test material DOTO (di-n-octyltin oxide) is in the same category of substances as the registration substance and it is therefore considered to be acceptable to use a read-across approach to address this endpoint.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

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): Dioctyloxostannane, Dioctyltin oxide, DOTO
- Physical state: powder, white
- Storage condition of test material: <-18 °C in the dark

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adult
- Weight at study initiation: Approximately 34 g
- Fasting period before study: 2 hours and 45 minutes in the range finding test and 3 hours and 30 minutes in the main test.
- Diet: ad libitum
- Water: Tap water, ad libitum delivered in polypropylene bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): At least 30 % not exceeding 70 %
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

DOSE-RANGE FINDING TEST IN-LIFE DATES: From: 4 November 2003 To: 7 November 2003
MAIN TEST IN-LIFE DATES: From: 25 November 2003 To: 28 November 2003

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 100, 50 and 25 mg/mL
- Amount of vehicle: 20 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The day before dosing, the test material was suspended in corn oil at a stock concentration of 100 mg/mL and stirred overnight on a magnetic stirrer. The two lower concentrations of 50 and 25 mg/mL were prepared prior to dosing on day 0.
Duration of treatment / exposure:
One treatment
Frequency of treatment:
A single exposure was used
Post exposure period:
At 24 hours after treatment, 5 animals of each dose level of the test material, 5 negative control animals and 5 positive control animals were sacrificed by cervical dislocation. After 48 hours the remaining animals of the high dose and and the negative control group were sacrificed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 (A), 500 (B), 1000 (C) and 2000 (D) mg/kg bw
Basis:
nominal conc.
MAIN TEST
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
Range Finding Test
No. of animals per sex per dose:
In the range finding test, 2 males and 2 females were used in each dose group to determine the toxicity of the test material.
In the main test, 10 male animals were used in each of the high dose group and the negative control group and 5 in all other dose groups and the positive control.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Route of administration: Intraperitoneal injection
- Doses / concentrations: 0.75 mg/kg bw (10 mL/kg bw) in saline

Examinations

Tissues and cell types examined:
From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears. Two smears per animal were prepared.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels and sex chosen for the main test were based on the clinical signs observed during the dose range finding test. The range-finding test demonstrated an absence of toxicity and no sex difference, therefore, the main test was carried out with male mice only and graded dose levels up to the limit dose of the guideline were used.

DETAILS OF SLIDE PREPARATION: The smears were air-dried and fixed in methanol. One smear per animal was stained with May-Grünwald Giemsa solution. The pther slide was stored as a reserve slide.

METHOD OF ANALYSIS: The slides were read by moving from the begining of the smear to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE were scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was therefore recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.
Evaluation criteria:
The study was considered to be valid if the positive controls gave a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were in the historical range.
A response was considered positive if the mean number of MPE/2000 PE was statistically significantly higher in comparison to the vehicle controls.
The test material was considered to cause chromosomal damage and/or damage to the mitotic apparatus, if a clear dose-related increase in the mean numbers of MPE/2000 PE was observed, when compared to the vehicle controls.
A test material was considered to be negative if it produced no positive response at any of the doses and time points analysed.
The test material or its metabolites was considered to have reached the general circulation and thereby the bone marrow, if the test material statistically reduced the mean number of PE/E or caused systemic toxicity.
Both statistical significance and biological relevance were considered when evaluating the responses.
Statistics:
At 24 hours, data on MPE and PE were evaluated using One Way Anova with a factor group (A, B, C and D). If the Anova demonstrated a significant effect (p<0.05), pooled error variance t-tests were performed, or if the variances were not homogenous, separate variance t-tests. These t-tests were applied to the negative control group (A) versus treatment groups B (500), C (1000) and D (2000). Furthermore, the positive control group E and the negative control group A were compared using the same process. A linear trend test (orthogonal contrasts) was also applied across groups A-D.
At 48 hours after administration, for treatment groups A and D, data on MPE and PE were analysed using pooled error variance t-tests, or if variances were not homogenous, separate variance t-tests.
All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000 and 2000 mg/kg bw, dosed as 20 mL/kg bw
- Clinical signs of toxicity in test animals: Female 3 died 24 hours post dosing due to a failed oral administration. No severe clinical signs were observed as a results of test material administration.

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: At both sacrifice times, the two way ANOVA did not demonstrate a statisitically significant effect for MPE and PE. There was no observable genotoxicity or clastogenicity up to a dose of 2000 mg/kg bw. At 24 hours, the incidence of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the vehicle control. The test system was therefore considered to be valid.

Any other information on results incl. tables

Table 1: Results of range finding test

Test material concentration

1 h post administration

4 h post administration

24 h post administration

48 h post administration

Animal number and sex

500 mg/kg bw

sl; se; br

sl; se; p; br; †

Male 2

Male 4

Female 1

Female 3

1000 mg/kg bw

Male 6

Male 8

Female 5

Female 7

2000 mg/kg bw

Male 10

Male 12

Female 9

Female 11

Empty cells = no clinical signs observed

sl = Sluggishness

se = slit-eyes

p = piloerection

br = irregular breathing

† = animal died

 

Table 2: Results of the main test

Group

Bodyweight (g)

Mouse no.

Observation time (h)

PE

NE

MPE

MNE

A

36.1

2

24

87

113

2

0

32.2

4

24

74

126

2

0

35.7

6

24

93

107

3

0

37.6

8

24

113

87

1

0

32.7

10

24

79

121

2

0

34.5

12

48

98

102

1

1

36.0

14

48

96

104

2

0

35.6

16

48

93

107

2

0

32.8

18

48

79

121

4

0

35.1

20

48

74

126

2

0

34.83 ± 0.56

Mean ± S.D.

24

89.2 ± 15.2

 

2.0 ± 0.7

48

88.0 ± 10.8

 

2.2 ± 1.1

B

33.1

22

24

101

99

3

0

34.7

24

24

63

137

1

0

33.8

26

24

89

111

2

0

35.6

28

24

81

119

4

0

34.7

30

24

92

108

3

0

34.38 ± 0.43

Mean ± S.D.

 

85.2 ± 14.3

 

2.6 ± 1.1

C

34.6

42

24

77

123

2

0

32.8

44

24

90

110

2

0

35.7

46

24

109

91

2

0

33.9

48

24

77

123

2

0

33.6

50

24

102

98

1

0

34.12 ± 0.49

Mean ± S.D.

 

91.0 ± 14.4

 

1.8 ± 0.4

D

36.3

62

24

66

134

2

0

34.7

64

24

111

89

2

0

31.5

66

24

81

119

1

0

33.6

68

24

99

101

1

1

33.7

70

24

103

97

1

0

37.8

72

48

83

117

1

0

33.8

74

48

77

123

2

0

33.2

76

48

73

127

2

0

33.3

78

48

74

126

2

0

34.3

80

48

69

131

0

0

34.22 ± 0.55

Mean ± S.D.

24

92.0 ± 18.2

1.4 ± 0.5

48

75.2 ± 5.2

1.4 ± 0.9

E

33.8

82

24

81

119

50

0

34.1

84

24

78

122

64

0

32.3

86

24

66

134

57

1

34.4

88

24

74

126

40

0

33.9

90

24

86

114

41

0

33.70 ± 0.36

Mean ± S.D.

 

77.0 ± 7.6

50.4 ± 10.3*

 

A = Vehicle control (corn oil)

B = 500 mg/kg bw test material

C = 1000 mg/kg bw test material

D = 2000 mg/kg bw test material

E = Mitomycin C 0.75 mg/kg bw in saline i.p. injection

Body weight = Bodyweight prior to dosing

PE = number of PE scored per 200 E scored

NE = number NE scored per 200 E scored

MPE = number of MPE scored per 2000 PE scored

MNE = number MNE scored per number of NE scored

* P<0.001 (t-tests) n = 5

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the study, the test material was found not to produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow cells of mice.
Executive summary:

The potential genotoxicity and clastogenicity of the test material to the bone marrow cells of Swiss male mice in vivo was assessed in a study conducted in accordance with OECD 474 and to GLP. At oral doses up to 2000 mg/kg bw (the limit dose recommended by the guideline) no chromosomal damage or damage to the mitotic spindle apparatus was noted. Under the conditions of the study the test material was found to be non-genotoxic and non-clastogenic.