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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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
October 26nd, 1998 to November 18th, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OECD Guideline For Testing Of Chemicals, 474 Genetic Toxicology, Mammalian Erythrocyte Micronucleus Test, Adopted 21st, July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 -156
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
U.S. EPA: OPPTS 870.5395 Health Effects Test Guidelines; Mammalian Erythrocyte Micronucleus Test, August 1998
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Solubility: suspension in Tylose HEC 4000 (0.5% w/v)
Stability and homogeneity in the vehicle: confirmed over 5 hours in Tylose HEC 4000 (0.5% w/v), signed October 26th, 1998
Concentration of stock suspension: 200 mg/ml

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Species of animals: mouse
Strain of animals: HsdWin:NMRI

Micronuclei are small secondary nuclear structures resulting from either chromosomal breakage or malfunction of the spindle apparatus of the cell which regulates the distribution of chromosomes during mitosis. The micronucleus test originally described by Schmid is a suitable in vivo method for investigating the clastogenic potential of substances and their ability to affect the mitotic spindle mechanism. Micronuclei rarely occur in normal dividing cells. The mouse has been chosen for this study since it provides a convenient in vivo mammalian model.
Sex:
male/female
Details on test animals and environmental conditions:
Species of animals: mouse
Strain of animals: HsdWin:NMRI
Origin (supplier) of animals: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
Animal identification: fur marking with KMnO4 and cage numbering
Body weight at start of study
male animals
mean = 33.5 g (=100%)
min = 32.0 g (- 4.5 %)
max = 36.0 g (+ 7.5 %)
n =15

female animals
mean = 27.1 g (=100%)
min = 24.0 g (-11.4%)
max = 29.0 g (+ 7.0 %)
n =15

Age at the start of study male/female animals approximately 7 weeks
Randomization procedure randomization schemes 98.0890 and 98.0891

Animal maintenance: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
Room temperature: 22 ± 3 °C
Relative humidity: 50 ± 20 %
Lighting times: 12 hours daily
Acclimatization: 5 days under study conditions
Food: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff® GmbH, Postbox 2039, 59480 Soest
Water: tap water in plastic bottles, ad libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle used: Tylose HEC 4000 (0.5% w/v)
Solubility in the vehicle: Suspension in Tylose HEC 4000 (0.5% w/v)
Stability and homogeneity in the vehicle: confirmed over 5 hours in Tylose HEC 4000 (0.5% w/v)
Concentration of test item in vehicle: 200 mg/ml
Amount of vehicle: 10 ml/kg body weight
Details on exposure:
Formulation of test compound: On the days of administration the test substance was suspended in Tylose HEC 4000 (0.5% w/v) at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.
Formulation of reference Compound: CPA dissolved in distilled water on the second day of experiment; final concentration: 0.5 % (w/v)
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Twice at an interval of 24 hours
Post exposure period:
24 hours after last administration.
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per dose group (5 male/5 female).
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
Name or number of compound (I.N.N, or U.S.A.N): cyclophosphamide
Synonyms: Endoxan®
Formula of the compound: C7H15CI2N2P-H2O
CAS-Register number: 50-18-0
Supplier of reference compound: ASTA Medica AG, Weismüllerstr. 45, D-60314 Frankfurt, Germany
Batch number: 603575B
Certificate of analysis: certified by the supplier, Quality Control, Dr. Milsmann dated January 10th, 1996

Examinations

Tissues and cell types examined:
polychromatic erythrocytes
Details of tissue and slide preparation:
Test procedure
The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg per kg body weight. The vehicle, Tylose HEC 4000 (0.5% w/v), was administered in the same way to the negative control groups. The study included a concurrent positive control using Endoxan®, which was administered once orally by gavage at a dose of 50 mg per kg body weight.
Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.

Preparation and staining
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grünwald's solution
- brief rinsing twice in distilled water
- 1 0 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan®

Evaluation
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment. No signs of toxicity were observed.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.
The incidence of micronudeated polychromatic erythrocytes in the dose group of C. I. Disperse Red 82 was within the normal range of the negative control groups. No statistically significant increase of micronudeated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Any other information on results incl. tables

Summary tables and statistics

Test compound: C.I. Disperse Red 82

Sex

Dose

mg/kg b.w.

Killing time

Number

Poly/Ery

Poly/Ery

SD

Poly with MN

Poly with MN

[%]

Poly with MN

SD

of animals

Poly counted

Mean

Mean

Mean

Mean

Mean

Male

Male

Male

0 – Control

2000

50 – Endoxan

24 h

24 h

24 h

5

5

5

2000

2000

2000

0.48

0.48

0.42

0.07

0.04

0.04

1.6

2.2

60.6

0.08

0.11

3.03

0.03

0.11

0.69

Female

Female

Female

0 –Control

2000

50 – Endoxan

24 h

24 h

24 h

5

5

5

2000

2000

2000

0.54

0.53

0.44

0.05

0.06

0.05

1.2

2.4

53.8

0.06

0.12

2.69

0.07

0.06

0.59

 

Sex

Dose

mg/kg b.w.

Killing time

Number

Poly/Ery

Poly/Ery

SD

Poly with MN

Poly with MN

[%]

Poly with MN

SD

Mut I.

of animals

Poly counted

Mean

Mean

Mean

Mean

Mean

Pooled

Pooled

Pooled

0 – Control

2000

50 – Endoxan

24 h

24 h

24 h

10

10

10

2000

2000

2000

0.51

0.51

0.43

0.07

0.06

0.04

1.40

2.30

57.20*

0.1

0.1

2.9

0.05

0.09

0.63

1.0

1.6

40.9

Mut I. = Mutagenic index

Control = Vehicle Tylose HEC 4000 (0.5% w/v)

* = significantly different from control (p < 0.05)

 

Table of individual date

Test compound: C.I. Disperse Red 82

Group

Animal number

Sex

Dose

mg/kg b.w.

Poly/200 Ery

Poly

with MN

Poly/Ery

Poly

with MN [%]

1

1

1

1

1

1

1

1

1

1

1

2

3

4

5

6

7

8

9

10

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

114

78

97

102

91

111

99

123

97

113

2

2

1

2

1

0

2

1

0

3

0.57

0.39

0.49

0.51

0.46

0.56

0.50

0.62

0.49

0.57

0.10

0.10

0.05

0.10

0.05

0.00

0.10

0.05

0.00

0.15

2

2

2

2

2

2

2

2

2

2

11

12

13

14

15

16

17

18

19

20

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

2000

2000

2000

2000

2000

2000

2000

2000

2000

2000

89

99

109

91

90

100

107

91

122

113

2

2

0

6

1

2

3

4

2

1

0.45

0.50

0.55

0.46

0.45

0.50

0.54

0.46

0.61

0.57

0.10

0.10

0.00

0.30

0.05

0.10

0.15

0.20

0.10

0.05

3

3

3

3

3

3

3

3

3

3

21

22

23

24

25

26

27

28

29

30

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

73

96

83

79

84

80

82

79

100

95

79

71

56

49

48

58

361

33

60

57

0.37

0.48

0.42

0.40

0.42

0.40

0.41

0.40

0.50

0.48

3.95

3.55

2.80

2.45

2.40

2.90

3.05

1.65

3.00

2.85

 

Historical control values

 

Based on OECD 474, adopted 1997:

 

Micronucleated PCE / 2000 PCE

PCE / Ery (200 Ery counted)

Vehicle

Sex

N

Mean

STD

Min

Med

Max

N

Mean

STD

Min

Med

Max

DMA / PEG / citrate buffer

Female

5

2.4

0.89

1

3

3

5

0.43

0.06

0.37

0.42

0.51

Male

5

3.8

2.49

2

2

7

5

0.45

0.07

0.35

0.45

0.52

Deionized water

Female

25

1.88

1.36

0

2

6

25

0.50

0.06

0.41

0.51

0.66

Male

25

1.64

1.19

0

1

5

25

0.45

0.07

0.33

0.46

0.59

Sesame oil

Female

5

2

1.58

0

2

4

5

0.50

0.08

0.40

0.51

0.60

Male

5

3

1.58

1

3

5

5

0.48

0.10

0.34

0.47

0.59

 

Total

Female

35

1.97

1.32

0

2

6

35

0.49

0.07

0.37

0.50

0.66

Male

35

2.14

1.65

0

2

7

35

0.46

0.07

0.33

0.46

0.59

 

Based on OECD 474, adopted 1983:

 

Micronucleated PCE / 2000 PCE

PCE / Ery (200 Ery counted)

Vehicle

Sex

N

Mean

STD

Min

Med

Max

N

Mean

STD

Min

Med

Max

NaCl 0.9% +

1 N sodium hydroxid

Female

5

1.2

2.17

0

0

5

5

1.12

0.13

0.94

1.11

1.28

Male

5

0.6

0.55

0

1

1

5

1.00

0.10

0.87

1.01

1.11

Beriate buffer

Female

5

1.0

1.0

0

1

2

5

1.48

0.32

1.17

1.31

1.85

Male

5

1.0

1.0

0

1

2

5

1.09

0.56

0.65

0.85

2.01

DMSO

Female

5

0.2

0.45

0

0

1

5

0.98

0.15

0.79

1.00

1.14

Male

5

1.4

0.89

0

2

2

5

0.89

0.17

0.63

0.88

1.06

Dextrose 5%

Female

5

1.4

0.89

0

2

2

5

0.68

0.05

0.62

0.68

0.74

Male

5

1.4

0.55

1

1

2

5

0.96

0.24

0.70

0.96

1.26

Eluat Tecegen S

Female

5

1.4

1.52

0

1

3

5

1.08

0.07

1.01

1.08

1.19

Male

5

0.8

0.84

0

1

2

5

0.83

0.18

0.55

0.91

1.01

Deionized water

Female

55

0.87

0.90

0

1

3

55

0.92

0.17

0.57

0.91

1.36

Male

55

0.95

0.89

0

1

4

55

0.87

0.21

0.50

0.89

1.39

Lecithin emulsion

Female

5

1.0

0.71

0

1

2

5

0.80

0.014

0.69

0.73

1.02

Male

5

1.4

1.52

0

1

3

5

0.91

0.12

0.80

0.89

1.12

Methyl cellulose 1%

Female

15

1.2

1.08

0

1

3

15

0.89

0.20

0.61

0.84

1.35

Male

15

1.27

0.96

0

1

3

15

0.78

0.20

0.41

0.78

1.10

NaCl 0.9%

Female

15

1.27

1.10

0

1

4

15

1.01

0.21

0.62

0.94

1.33

Male

15

1.73

1.22

0

2

4

15

0.85

0.11

0.64

0.85

1.03

Insulin-placebo

Female

5

1.4

1.67

0

1

4

5

1.52

0.37

0.90

1.62

1.88

Male

5

2.0

1.41

1

1

4

5

2.10

0.34

1.53

2.24

2.35

Sesame oil

Female

50

1.36

1.10

0

1

4

50

0.93

0.19

0.52

0.93

1.25

Male

50

1.32

1.04

0

1

4

50

0.80

0.13

0.55

0.79

1.14

Starch mucilage

Female

120

1.13

1.02

0

1

4

120

0.97

0.18

0.53

0.99

1.38

Male

120

1.43

1.16

0

1

5

120

0.87

0.19

0.48

0.89

1.38

Tylose 0.1%

Female

10

0.9

0.99

0

1

3

10

0.93

0.20

0.67

0.89

1.24

Male

10

1

0..94

0

1

2

10

0.89

0.20

0.60

0.95

1.11

H2O + Na2CO3(2.24% w/v)

Female

5

0.6

0.55

0

1

1

5

0.65

0.25

0.39

0.57

1.04

Male

5

1.2

0.84

0

1

2

5

1.02

0.32

0.62

1.15

1.40

 

Total

Female

340

1.147

1.07

0

1

5

340

0.94

0.23

0.39

0.93

1.88

Male

340

1.31

1.09

0

1

6

340

0.87

0.25

0.41

0.85

2.35

PCE: Polychromatic Erythrocytes

NCE: Normochromatic Erythrocytes

Ery: Total Erythrocytes (PCE + NCE)

Applicant's summary and conclusion

Conclusions:
The results lead to the conclusion that C. I. Disperse Red 82 did not lead to a substantial increase of micronudeated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.
Executive summary:

The micronucleus test was carried out with C. I. Disperse Red 82. The test compound was suspended in Tylose HEC 4000 (0.5% w/v) and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study).

According to the test procedure the animals were killed 24 hours after administration.

 

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

 

The number of polychromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with C. I. Disperse Red 82 and was not less than 20% of the control value.

 

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

 

Under the conditions of the present study the results indicate that C. I. Disperse Red 82 is not mutagenic in the micronucleus test.