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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:
20 November 1987 to 29 January 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals (1981) No. 474 "Genetic Toxicology: Micronucleus Test"
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
mouse
Strain:
other: albino CFLP
Details on species / strain selection:
Thirty-seven male and thirty-seven female albino CFLP strain mice were supplied by Interfauna (UK) Limited, Wyton, Huntingdon, Cambridgeshire.
The results of the test are believed to be of value in predicting the mutagenic potential of the test material to man. The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is recommended in the test method .
Sex:
male/female
Details on test animals and environmental conditions:
Thirty-seven male and thirty-seven female albino CFLP strain mice were supplied by Interfauna (UK) Limited, Wyton, Huntingdon, Cambridgeshire. At the start of the main study the males weighed 26 – 29 g, and the females 25 – 29 g, and were approximately five to eight weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a unique number within the study by ear punching and a number written on a colour coded cage card.
The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding. With the exception of a 3-4 hour fast immediately before dosing and for approximately two hours after dosing, free access to mains drinking water and food (Rat and Mouse Expanded Diet No. 1, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
The animal room was maintained at a temperature of 20 – 22 °C and relative humidity of 45 - 60%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
For the purpose of this study the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil B.P.

The vehicle was supplied by C.P. Pharmaceuticals Limited, as follows:
Description: clear, straw-coloured oily liquid
Container: plastic screw-top bottle
Supplier's identification: Oil Arachis B.P.
Batch number: 8611797
Storage conditions: room temperature
The identification and stability of the vehicle control were not determined.
Details on exposure:
For the purpose of this study the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil B.P.
Duration of treatment / exposure:
Single treatment/ 24, 48 & 72 hours
Frequency of treatment:
One treatment
Post exposure period:
Animals were killed 24, 48 or 72 hours after dosing.
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (nominal)
Remarks:
maximum practical dose
No. of animals per sex per dose:
30 animals (10 in each test group [5 male/5 female])
30 animals (10 in each vehicle control group [5 male/5 female])
10 animals (positive control [5 male/5 female])
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
The positive control material was supplied by Sigma Chemical Company, as follows:
Description: white powder
Container: brown glass screw-top bottle
Supplier's identification: Cyclophosphamide Monohydrate
Batch number: 85F-0054
Date of arrival: 26 June 1987
Storage conditions: + 4 °C in the dark
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.
The identification and stability of the control material and the preparation was not determined.

Route of administration: Oral: gavage
Dose: 50 mg/kg bw
Concentration: 5 mg/mL
Dose volume: 10 mL/kg

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes were scored for the presence of micronuclei.
Details of tissue and slide preparation:
Immediately following sacrifice (i.e. 24, 48 or 72 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serum (Flow Laboratories Ltd) and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, defatted in xylene and stained in May-Gruenwald/Giemsa.
Evaluation of Slides
Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes (blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (pink stained mature cells) associated with 1000 polychromatic erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48 or 72 hour kill times.
If the above criteria is not demonstrated, the test material is considered to be non-mutagenic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the treatment group mean normochromatic to polychromatic ratio is twice the vehicle control value or when a treatment related increase is shown to be statistically significant.
Statistics:
If necessary, and where possible, all data were statistically analysed using the Kruskal-Wallis one-way analysis of variance by ranks (Kruskal W.H. and Wallis W.A. 1952 J. Am. Statist. Soc. 47 583).

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:
RANGE-FINDING TOXICITY STUDY
Mice treated with Dispersionsblau F-60 768 at 5000 mg/kg, showed no signs of toxicity and therefore 5000 mg/kg was selected as the “maximum practical dose”. No deaths or abnormal clinical observations were recorded.

MICRONUCLEUS STUDY
Mortality Data and Clinical Observations
No deaths abnormal clinical observations were recorded.

Evaluation of Bone Marrow Slides
There were no significant differences between the test material treatment groups and their corresponding vehicle control groups, with regard to the number of micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the known mutagenic activity of cyclophosphamide monohydrate under the conditions of the test.
The test material did not induce an increase in micronucleated PCE’s that was consistent with a mutagenic effect. Dispersions-blau F-60 768 was, therefore, considered to be non-mutagenic under the conditions of the test in which no bone-marrow toxicity was observed.

Any other information on results incl. tables

RANGE-FINDING TOXICITY STUDY

The mortality data are summarised as follows:

DOSE LEVEL

mg/kg

SEX

DEATHS ON DAY

TOTAL DEATHS

0

1

2

3

5000

MALE

0

0

0

0

0/4

FEMALE

0

0

0

0

 

MICRONUCLEUS STUDY

 

SUMMARY OF GROUP MEAN DATA (MALES AND FEMALES COMBINED)

TREATMENT GROUP

NUMBER OF PCE WITH MICRONUCLEI

NUMBER OF NCE WITH MICRONUCLEIa

NCE RATIO

PCE

PER 1000 PCE

PER 1000 NCE

GROUP MEAN

SD

GROUP MEAN

SD

GROUP MEAN

SD

1. DISPERSIONSBLAU

5000 mg/kg

24-hour sampling time

2.2

1.0

1.9

1.3

0.81

0.14

2. DISPERSIONSBALU

5000 mg/kg

49-hour sampling time

2.0

1.2

0.7

1.0

0.75

0.15

3. DISPERSIONBALU

5000 mg/kg

72-hour sampling time

2.5

1.9

1.9

1.3

0.57

0.13

4. VEHICLE CONTROL

24-hour sampling time

2.4

1.3

1.3

1.1

0.78

0.31

5. VEHICLE CONTROL

48-hour sampling time

1.9

1.2

0.9

0.7

0.93

0.22

6. VEHICLE CONTROL

72-hour sampling time

1.7

1.2

0.9

0.8

0.71

0.18

7. POSITIVE CONTROL

24-hour sampling time

44.9

6.2

3.3

0.9

1.39

0.38

Key:

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

SD = standard deviation

a= calculated data

 

MICRONUCLEUS STUDY – INDIVIDUAL AND GROUP MEAN DATA

TREATMENT GROUP

ANIMAL NUMBER & SEX

TOTAL CELLS SCORED

(PCE+NCE)

POLYCHROMATIC ERYTHROCYTES

(PCE)

NORMOCHROMATIC ERYTHROCYTES

(NCE)

NCE

PCE

RATIO

NUMBER SCORED

PCE+MN

NUMBER SCORED

NCE+MN

NCE+MN PER 103NCE

DISPERSIONSBLAU

5000 mg/kg

24-hour sampling time

1 M

2 M

3 M

4 M

5 M

1699

1754

1745

1610

1857

1000

1000

1000

1000

1000

3.0

2.0

3.0

1.0

3.0

699

754

745

610

857

1.0

2.0

2.0

1.0

3.0

1.43

2.65

2.68

1.64

3.50

0.70

0.75

0.75

0.61

0.86

MALE MEANS

S.D.

1733

90

1000

0

2.4

0.9

733

90

1.8

0.8

2.38

0.85

0.73

0.09

6 F

7 F

8 F

9 F

10 F

1888

1797

2119

1835

1752

1000

1000

1000

1000

1000

2.0

4.0

1.0

1.0

2.0

888

797

1119

836

752

0.0

3.0

0.0

2.0

1.0

0.00

3.76

0.00

2.40

1.33

0.89

0.80

1.12

0.84

0.75

FEMALE MEANS

S.D.

1878

144

1000

0

2.0

1.2

878

144

1.2

1.3

1.50

1.62

0.88

0.14

GROUP MEANS

S.D.

1806

136

1000

0

2.2

1.0

806

136

1.5

1.1

1.94

1.30

0.81

0.14

S.D. = standard deviation

TREATMENT GROUP

ANIMAL NUMBER & SEX

TOTAL CELLS SCORED

(PCE+NCE)

POLYCHROMATIC ERYTHROCYTES

(PCE)

NORMOCHROMATIC ERYTHROCYTES

(NCE)

NCE

PCE

RATIO

NUMBER SCORED

PCE+MN

NUMBER SCORED

NCE+MN

NCE+MN PER 103NCE

DISPERSIONSBLAU

5000 mg/kg

48-hour sampling time

11 M

12 M

13 M

14 M

15 M

1720

1772

1620

1841

1531

1000

1000

1000

1000

1000

0.0

2.0

4.0

2.0

3.0

720

772

620

841

531

1.0

2.0

0.0

0.0

0.0

1.39

2.59

0.00

0.00

0.00

0.72

0.77

0.62

0.84

0.53

MALE MEANS

S.D.

1697

123

1000

0

2.2

1.5

697

123

0.6

0.9

0.80

1.17

0.70

0.12

16 F

17 F

18 F

19 F

20 F

1634

1897

2037

1644

1843

1000

1000

1000

1000

1000

2.0

2.0

1.0

3.0

1.0

634

897

1037

644

843

1.0

0.0

0.0

1.0

0.0

1.58

0.00

0.00

1.55

0.00

0.63

0.90

1.04

0.64

0.84

FEMALE MEANS

S.D.

1811

172

1000

0

1.8

0.8

811

172

0.4

0.5

0.63

0.86

0.81

0.17

GROUP MEANS

S.D.

1754

153

1000

0

2.0

1.2

754

153

0.5

0.7

0.71

0.97

0.75

0.15

S.D. = standard deviation

TREATMENT GROUP

ANIMAL NUMBER & SEX

TOTAL CELLS SCORED

(PCE+NCE)

POLYCHROMATIC ERYTHROCYTES

(PCE)

NORMOCHROMATIC ERYTHROCYTES

(NCE)

NCE

PCE

RATIO

NUMBER SCORED

PCE+MN

NUMBER SCORED

NCE+MN

NCE+MN PER 103NCE

DISPERSIONSBLAU

5000 mg/kg

72-hour sampling time

21 M

22 M

23 M

24 M

25 M

1529

1336

1595

1820

1614

1000

1000

1000

1000

1000

4.0

6.0

4.0

2.0

4.0

529

336

595

820

614

1.0

1.0

2.0

3.0

1.0

1.89

2.98

3.36

3.66

1.63

0.53

0.34

0.60

0.82

0.61

MALE MEANS

S.D.

1579

174

1000

0

4.0

1.4

579

174

1.6

0.9

2.70

0.90

0.58

0.17

26 F

27 F

28 F

29 F

30 F

1672

1488

1432

1603

1602

1000

1000

1000

1000

1000

0.0

1.0

1.0

1.0

2.0

672

488

432

603

602

1.0

0.0

1.0

1.0

0.0

1.49

0.00

2.31

1.66

0.00

0.67

0.49

0.43

0.60

0.60

FEMALE MEANS

S.D.

1559

97

1000

0

1.0

0.7

559

97

0.6

0.5

1.09

1.04

0.56

0.10

GROUP MEANS

S.D.

1569

133

1000

0

2.5

1.9

569

133

1.1

0.9

1.90

1.25

0.57

0.13

S.D. = standard deviation

TREATMENT GROUP

ANIMAL NUMBER & SEX

TOTAL CELLS SCORED

(PCE+NCE)

POLYCHROMATIC ERYTHROCYTES

(PCE)

NORMOCHROMATIC ERYTHROCYTES

(NCE)

NCE

PCE

RATIO

NUMBER SCORED

PCE+MN

NUMBER SCORED

NCE+MN

NCE+MN PER 103NCE

VEHICLE CONTROL

24-hour sampling time

31 M

32 M

33 M

34 M

35 M

1459

1839

1600

1485

1593

1000

1000

1000

1000

1000

4.0

5.0

3.0

2.0

2.0

459

839

600

485

593

0.0

2.0

2.0

1.0

1.0

0.00

2.38

3.33

20.6

1.69

0.46

0.84

0.60

0.49

0.59

MALE MEANS

S.D.

1595

150

1000

0

3.2

1.3

594

150

1.2

0.8

1.89

1.22

0.60

0.15

36 F

37 F

38 F

39 F

40 F

1907

1623

1703

2246

2381

1000

1000

1000

1000

1000

2.0

2.0

1.0

1.0

2.0

907

623

703

1246

1381

1.0

0.0

0.0

2.0

1.0

1.10

0.00

0.00

1.61

0.72

0.91

0.62

0.70

1.25

1.38

FEMALE MEANS

S.D.

1972

332

1000

0

1.6

0.5

972

332

0.8

0.8

0.69

0.70

0.97

0.33

GROUP MEANS

S.D.

1784

314

1000

0

2.4

1.3

784

314

1.0

0.8

1.29

1.13

0.78

0.31

S.D. = standard deviation

TREATMENT GROUP

ANIMAL NUMBER & SEX

TOTAL CELLS SCORED

(PCE+NCE)

POLYCHROMATIC ERYTHROCYTES

(PCE)

NORMOCHROMATIC ERYTHROCYTES

(NCE)

NCE

PCE

RATIO

NUMBER SCORED

PCE+MN

NUMBER SCORED

NCE+MN

NCE+MN PER 103NCE

VEHICLE CONTROL

48-hour sampling time

41 M

42 M

43 M

44 M

45 M

1963

1789

1915

1799

2053

1000

1000

1000

1000

1000

0.0

1.0

1.0

3.0

2.0

963

789

915

799

1053

0.0

0.0

1.0

1.0

1.0

0.00

0.00

1.09

1.25

0.95

0.96

0.79

0.92

0.80

1.05

MALE MEANS

S.D.

1904

112

1000

0

1.4

1.1

904

112

0.6

0.5

0.66

0.61

0.90

0.11

46 F

47 F

48 F

49 F

50 F

1507

2019

2336

2050

1878

1000

1000

1000

1000

1000

4.0

1.0

3.0

2.0

2.0

507

1019

1336

1050

878

1.0

1.0

2.0

1.0

0.0

1.97

0.98

1.50

0.95

0.00

0.51

1.02

1.34

1.05

0.88

FEMALE MEANS

S.D.

1958

302

1000

0

2.4

1.1

958

302

1.0

0.7

1.08

0.74

0.96

0.30

GROUP MEANS

S.D.

1931

217

1000

0

1.9

1.2

931

217

0.8

0.6

0.87

0.67

0.93

0.22

S.D. = standard deviation

TREATMENT GROUP

ANIMAL NUMBER & SEX

TOTAL CELLS SCORED

(PCE+NCE)

POLYCHROMATIC ERYTHROCYTES

(PCE)

NORMOCHROMATIC ERYTHROCYTES

(NCE)

NCE

PCE

RATIO

NUMBER SCORED

PCE+MN

NUMBER SCORED

NCE+MN

NCE+MN PER 103NCE

VEHICLE CONTROL

72-hour sampling time

51 M

52 M

53 M

54 M

55 M

1598

1783

1608

1544

1861

1000

1000

1000

1000

1000

2.0

1.0

4.0

3.0

1.0

598

783

608

544

861

1.0

1.0

1.0

0.0

0.0

1.67

1.28

1.64

0.00

0.00

0.60

0.78

0.61

0.54

0.86

MALE MEANS

S.D.

1679

136

1000

0

2.2

1.3

679

136

0.6

0.5

0.92

0.85

0.68

0.14

56 F

57 F

58 F

59 F

60 F

1548

1895

2047

1545

1631

1000

1000

1000

1000

1000

2.0

1.0

0.0

2.0

1.0

548

895

1047

545

631

0.0

1.0

0.0

1.0

1.0

0.00

1.12

0.00

1.83

1.58

0.55

0.90

1.05

0.55

0.63

FEMALE MEANS

S.D.

1733

226

1000

0

1.2

0.8

733

226

0.6

0.5

0.91

0.87

0.73

0.23

GROUP MEANS

S.D.

1706

178

1000

0

1.7

1.2

706

178

0.6

0.5

0.91

0.81

0.71

0.18

S.D. = standard deviation

TREATMENT GROUP

ANIMAL NUMBER & SEX

TOTAL CELLS SCORED

(PCE+NCE)

POLYCHROMATIC ERYTHROCYTES

(PCE)

NORMOCHROMATIC ERYTHROCYTES

(NCE)

NCE

PCE

RATIO

NUMBER SCORED

PCE+MN

NUMBER SCORED

NCE+MN

NCE+MN PER 103NCE

POSITIVE CONTROL

50 mg/kg

24-hour sampling time

61 M

62 M

63 M

64 M

65 M

2508

2366

1938

2817

2906

1000

1000

1000

1000

1000

43.0

44.0

42.0

41.0

54.0

1508

1366

938

1817

1906

5.0

4.0

4.0

2.0

7.0

3.32

2.93

4.26

1.10

3.67

1.51

1.37

0.94

1.82

1.91

MALE MEANS

S.D.

2507

387

1000

0

44.8

5.3

1507

387

4.4

1.8

3.06

1.20

1.51

0.39

66 F

67 F

68 F

69 F

70 F

2404

2226

2840

1963

1905

1000

1000

1000

1000

1000

47.0

36.0

56.0

46.0

40.0

1404

1226

1840

963

905

6.0

4.0

6.0

3.0

3.0

4.27

3.26

3.26

3.12

3.31

1.40

1.23

1.84

0.96

0.91

FEMALE MEANS

S.D.

2268

378

1000

0

45.0

7.6

1268

378

4.4

1.5

3.45

0.47

1.27

0.38

GROUP MEANS

S.D.

2387

383

1000

0

44.9

6.2

1387

382

4.4

1.6

3.25

0.88

1.39

0.38

S.D. = standard deviation

Applicant's summary and conclusion

Conclusions:
The test material, Dispersionsblau F 60 768, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test.
The test substance is not classifiable according to CLP critera.
Executive summary:

A study was performed to assess the potential of Dispersionsblau F60 768 to produce damage to chromosomes or the mitotic apparatus of mice when administered by the oral route. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 474 “Genetic Toxicology: Micronucleus Test”.

 

Following a preliminary range-finding study to confirm the oral toxicity of the test material, the micronucleus study was conducted using Dispersionsblau F-60 768 at the maximum practical dose level (MPD).

 

In the micronucleus study, groups of ten mice (five males and five females) were given a single oral dose of Dispersionsblau F-60 768 at the maximum practical dose (5000 mg/kg). Animals were killed 24, 48 or 72 hours later. Polychromatic erythrocytes were scored for the presence of micronuclei.

 

Further groups of ten mice were treated with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls respectively.

 

There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes in mice treated with Dispersionsblau F-60 768 when compared to the vehicle control groups. No statistical analysis was necessary.

 

The positive control material produced a very marked increase in the number of micronucleated polychromatic erythrocytes.

 

The test material, Dispersionsblau F-60 768, was considered to be non-mutagenic under the conditions of the test.

The test substance is not classified according to CLP criteria.