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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
OECD repeated sampling micronucleus assay:
Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing :Part I (February, 1983), Ed :Brian J. Dean p 134
(The United Kingdom Environmental Mutagen Society).
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Purity 99.7%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Bodyweight range : 30-35 g (males) and 25-30 g (female) at collection
Age : 9 weeks at collection
Normally in the range: 22+/- 3°C. Humidity was measured daily.
Light cycle: A 12/12 hour light/dark cycle will be maintained, switching an at 0600 h.
Air change: The ventilation system provides 15 air changes per hour in the vented cabinets housing the animals.
Diet: Labsure Animal Diet (CRM Nuts) and tap water will be available at all times.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
sterile double distilled water
Details on exposure:
The mice received single oral application of the test substance by gavage.
Bronopol was tested at following doses: 80 mg/kg bw (12 animals per sex) and
160 mg/kg bw (24 animals/sex), the latter dose being the maximum dose tolerated by the mice.
8 males and 8 females per dose group per sampling time (high dose), 4 males and 4 females per dose group per sampling time (low dose, positive and negative controls).
Frequency of treatment:
single application
Post exposure period:
24, 48, 72 hours after treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
80, 160 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
80 mg/kg bw (12 animals per sex)
160 mg/kg bw (24 animals/sex)
Positive control group (Cyclophosphamide, 75 mg/kg bw): 12 males and 12 females
Negative control group (purified water B.P.): 12 males and 12 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (75 mg/kg bw)

Examinations

Tissues and cell types examined:
After 24, 48 and 72 hours following treatment, 4 animals/sex and 8 animals/sex, respectively from the 80 and the 160 mg/kg bw group were sacrificed, and the femoral bone marrow was extracted, prepared and examined.
Details of tissue and slide preparation:
The polychromatic/normochromatic erythrocytes ratio and the number of micronuclei in 1000 polychromatic erythrocytes per animal.
Statistics:
The statistical assessment of the findings was based on the Analysis of variance, the Freeman-Tukey transformation and the use of F-distribution tables.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Additional information on results:
The main findings can be summarized as follows:
Overt signs of toxicity:
In the 160 mg/kg bw group, 4 males and 4 females died within 48 hours whereas in the 80 mg/kg bw group, one female died within 72 hours.

Ratio of polychromatic to normochromatic erythrocytes in the femoral bone marrow of bronopol treated CD1-mice:
After 72 hours, the polychromatic/normochromatic erythrocytes ratio for the males of the 160 and the 80 mg/kg bw groups as well as for the females of the 80 mg/kg bw group were reduced compared to the normal ration of ca. 1:1; this effect indicated a decrease in haemopoiesis.

Incidence of micronuclei in polychromatic erythrocytes:
The incidence of micronuclei in polychromatic erythrocytes of bronopol-treated mice was within the range of negative control. In contrast, in the cyclophosphamide-treated mice (positive control), statistically significant increases in the incidence of micronuclei in polychromatic erythrocytes were reported after 24 and 48 hours; after 72 hours, the incidence of micronuclei in polychromatic erythrocytes was increased in the males but withoutbeing statistically significant.

Any other information on results incl. tables

Table 1: Ratio of polychromatic to normochromatic erythrocytes in the femoral bone marrow of bronopol treated CD1-mice:

Test group

Dose  (mg/kg bw)

Ratio of polychromatic/normochromatic erythrocytes

24 h

48 h

72 h

M

F

M

F

M

F

Neg. Cont.*

-

1:0.92

1:1.02

1:0.67

1:0.79

1: 0.95

1: 1.04

BN

80

1:1.05

1:0.87

1:1.72

1:1.40

1:6.57

1:4.06

160

1:0.82

1:0.85

1:0.99

1:0.73

1:3.52

1:0.81

Pos. Cont.**

75

1:2.31

1:0.87

1:7.65

1:2.18

1:15.77

1:2.37

BN, Bronopol; *, purified water; **, Cyclophosphamide

Table 2: Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1-mice at time point 24 h.

Test group

Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1

Time point 24 hours

Males

Females

MN/1000 PCE/mouse

Mean (%)

MN/1000 PCE/mouse

Mean (%)

Negative control

 (Purified water)

1

2

0.23

3

0.23

2

1

1

3

2

3

4

4

2

Bronopol 80 mg/kg bw

1

2

0.13

0

0.05

2

2

1

3

1

0

4

0

1

Bronopol 160 mg/kg bw

1

- (Mouse died)

0.10

2

0.10

2

0

0

3

0

0

4

0

3

5

1

- (Mouse died)

6

3

0

7

2

1

8

1

1

Positive control (Cyclophosphamide, 75 mg/kg bw)

1

39

2.73**

42

2.25**

2

13

7

3

34

24

4

23

17

**, Significantly different from negative control (p<0.01)

Table 3: Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1-mice at time point 48 h.

Test group

Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1

Time point 48 hours

Males

Females

MN/1000 PCE/mouse

Mean (%)

MN/1000 PCE/mouse

Mean (%)

Negative control

 (Purified water)

1

0

0.13

0

0.15

2

1

0

3

2

2

4

2

4

Bronopol 80 mg/kg bw

1

2

0.23

0

0.20

2

3

5

3

4

1

4

0

2

Bronopol 160 mg/kg bw

1

- (Mouse died)

0.18

2

0.24

2

2

5

3

0

3

4

- (Mouse died)

- (Mouse died)

5

- (Mouse died)

2

6

3

0

7

2

- (Mouse died)

8

2

- (Mouse died)

Positive control (Cyclophosphamide, 75 mg/kg bw)

1

6

0.78**

11

1.25**

2

5

9

3

7

9

4

13

21

**, Significantly different from negative control (p0.01)

Table 4: Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1-mice at time point 72 h.

Test group

Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1

Time point 72 hours

Males

Females

MN/1000 PCE/mouse

Mean (%)

MN/1000 PCE/mouse

Mean (%)

Negative control

 (Purified water)

1

1

0.13

2

0.28

2

0

2

3

0

3

4

4

4

Bronopol 80 mg/kg bw

1

1

0.18

1

0.17

2

1

1

3

3

3

4

2

- (Mouse died)

Bronopol 160 mg/kg bw

1

1

0.26

0

0.33

2

1

2

3

1

3

4

2

3

5

4

3

6

3

8

7

6

4

8

3

3

Positive control (Cyclophosphamide, 75 mg/kg bw)

1

2

0.30

5

0.25

2

3

2

3

1

0

4

6

3

**, Significantly different from negative control (p0.01)

Applicant's summary and conclusion

Conclusions:
Bronopol at doses up to 160 mg/kg bw, which is the maximum dose tolerated by mice, was not clastogenic in the in vivo mouse micronucleus test.