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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The whole report summarizes the data of three mutagenic testing approaches, which were conducted in 1974, prior to the implementation of guidelines. Furthermore, no data referring to purity and stability of the tested substance were provided. However, the results obtained can be considered as scientifically acceptable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1974

Materials and methods

Principles of method if other than guideline:
Method: other: according to the method of Bateman AJ (Heredity 12: 213-232, 1958) and Bateman AJ and Epstein SS (in Chemical Mutagens, Vol. 2, ed. Hollaender, Plenum Press, 1971)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
No further data.

Test animals

Species:
mouse
Strain:
other: OLAC
Sex:
male
Details on test animals and environmental conditions:
Animals: OLIC ex-breeding stock males of proven fertility. OLIC virgin females, 12 weeks old when mated.
Temperature range 22-24 °C; daylight supplemented by fluorescent tubes on a controlled li.ght/dark cycle, 16 hours on/8 hours off.
Diet: Oxoid cubes and water ad libitum.
Four hours after completion of the dosing, each male was housed with 3 females for mating, which were replaced at weekly intervals for 4 weeks.

Administration / exposure

Route of administration:
other: gavage and i.p., respectively
Details on exposure:
Application volume: 0.1 ml/10 g bw
Duration of treatment / exposure:
Oral dosage: once daily for 6 consecutive days.
Intraperitoneal dosage: single i.p. injection
Frequency of treatment:
Substance: once daily
Positive control: once (last day of substance dosing)
Post exposure period:
4 hours (i.e. 4 hours after the last dosing, the males were housed together with the females for mating.)
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
oral application
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
oral application
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
intraperitoneal application, (0.9% saline)
No. of animals per sex per dose:
10 for test group and positive control
20 for negative control
Control animals:
other: Twenty 20 males served as negative control. They were treated orally with water.
Positive control(s):
Ten males mice were used for positive control.
Positive controls received a single i.p. injection of 25mg/kg bw tris(2-methyl-1-aziridinyl)-phosphine oxide (METEPA) in 0.1 ml/10 g bw of 0.9% physiological saline.

Examinations

Tissues and cell types examined:
The females were killed 14 days from the midpoint of the mating week, corresponding to gestation days 9 to 16. Numbers of pregnancies, and live and dead implants were recorded.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Positive controls validity:
valid
Additional information on results:
Summary of the main findings:

Oral treatment with 100 and 20 mg/kg bw bronopol:
Four of the 10 males treated with 100 mg/kg bw bronopol died within 2 weeks (Table 1). The implantation rate was significantly reduced in weeks 2 and 3; this effect was further accompanied by a reduction in fertility as indicated by a decreased pregnancy rate observed in this group (Table 2, 3). No such effects were seen at 20 mg/kg bw. Moreover, for both test doses, no increase in the frequency of dead implants were seen. In fact, for 100 mg/kg bw, the number of dead implants/pregnant female ranged was 0.5, 0.4, 0.2 and 0.5 respectively after 1, 2, 3 and 4 weeks; the corresponding control values respectivley were 0.6, 0.5, 1.0 and 0.7. For 20 mg/kg bw, the values also were within control range.

Injection (i.p.) of 10 mg/kg bw bronopol:
One male from the group receiving 10 mg/kg i.p. died during the third week (Table 1). The implantation rate was significantly reduced in week 4; this effect was further accompanied by a reduction in fertility as indicated by a decreased pregnancy rate observed in this group (Table 2, 3). In this group, a decrease in the frequency of dead implants was seen after 4 weeks (0.2 dead implant per pregnant female versus 0.7 for control).

Positive control (METEPA, 25 mg/kg bw, i.p. injection):
In this group, the number of dead implants per pregnant female was significantly increased compared to untreated control. Mean values of 3.5, 3.9 and 3.1 were reported respectively after week 1, 2 and 3, versus 0.6, 0.5 and 1.0 for controls.

For the highest orally applied dose of bronopol (i.e. 100 mg/kg bw, 6 times) and for the single i.p. injection, a conspicuous decrease in pregnancy rate was observed, which was indicative of a reduced fertility related again to the toxicity of the tested doses of bronopol observed for the males in these groups (Table 2).

Any other information on results incl. tables

Table 1: Cumulative mortalities of Bronopol-treated animals.

Dose

Cumulative deaths per week (W)

W1

W2

W3

W4

0 mg/kg bw (negative control; oral)

0

0

0

0

Bronopol, 6 x 100 mg/kg bw(oral)

1

4

4

4

Bronopol, 6 x  20 mg/kg bw(oral)

0

0

0

0

Bronopol, 1 x 10 mg/kg bw(i.p.)

0

0

1

1

Positive control (METEPA 1x 25 mg/kg bw (i.p.)

0

0

0

0

Table 2: Number of pregnant females per week (per cent).

Dose

Pregnant females per week (W)

W1

W2

W3

W4

0 mg/kg bw (negative control; oral)

42 (70%)

34 (57%)

33 (55%)

34 (56%)

Bronopol, 6 x 100 mg/kg bw(oral)

8 (44%)

7 (39%)

9 (50%)

10 (56%)

Bronopol, 6 x  20 mg/kg bw(oral)

20 (67%)

22 (73%)

24 (80%)

23 (76%)

Bronopol, 1 x 10 mg/kg bw(i.p.)

9 (33%)

13 (45%)

14 (48%)

12 (44%)

Positive control (METEPA 1x 25 mg/kg bw (i.p.)

24 (83%)

20 (67%)

21 (70%)

17 (56%)

Table 3: Implants per pregnant female (live, dead, total).

Dose

Live implants per pregnant female and week (W)

W1

W2

W3

W4

0 mg/kg bw (negative control; oral)

9.4

9.6

9.4

10.4

Bronopol, 6 x 100 mg/kg bw(oral)

8.5

6.7

6.0*

9.6

Bronopol, 6 x  20 mg/kg bw(oral)

9.8

8.4

9.3

10.6

Bronopol, 1 x 10 mg/kg bw(i.p.)

9.2

8.1

9.9

8.7*

Positive control (METEPA 1x 25 mg/kg bw (i.p.)

7.4***

4.2***

6.9**

8.7*

Dose

Dead implants per pregnant female and week (W)

W1

W2

W3

W4

0 mg/kg bw (negative control; oral)

0.6

0.5

1.0

0.7

Bronopol, 6 x 100 mg/kg bw(oral)

0.5

0.4

0.2

0.5

Bronopol, 6 x  20 mg/kg bw(oral)

1.0

0.9

0.5

0.7

Bronopol, 1 x 10 mg/kg bw(i.p.)

0.7

1.2

0.3

0.2*

Positive control (METEPA 1x 25 mg/kg bw (i.p.)

3.5***

3.9***

3.1***

1.2

Dose

Total Number of implants per female and week (W)

W1

W2

W3

W4

0 mg/kg bw (negative control; oral)

10.0

10.1

10.4

11.1

Bronopol, 6 x 100 mg/kg bw(oral)

9.0

7.1*

6.2*

10.1

Bronopol, 6 x  20 mg/kg bw(oral)

10.8

9.3

9.8

11.2

Bronopol, 1 x 10 mg/kg bw(i.p.)

9.9

9.3

10.2

8.9*

Positive control (METEPA 1x 25 mg/kg bw (i.p.)

10.9

8.1*

10.0

9.9

*, p=<0.05; **, p=<0.01; ***, p=<0.001

Applicant's summary and conclusion

Conclusions:
CL-Freetext:
Overt signs of toxicity were seen after 6 daily oral doses
of 100 mg/kg Bronopol in male mice, but not after 6 daily oral doses of 20 mg/kg, nor after a single i.p. injection of 10 mg/kg. Reduced fertility was seen in the high dose group, but not in the others. This effect was considered to be related to toxicity rather than dominant lethality. No increase in dead implants was observed after Bronopol treatment. From these results it was concluded that Bronopol lacks cytotoxicity for mouse meiotic and post-meiotic sperm stages.