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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF guideline 59 NohSan Np. 4200 (1985)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
Self-certified laboratory
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material: Copper hydroxide
Lot/Batch number: 021121/1
Purity: 61.14 % copper
Description: Free flowing blue product, needles.
Stability: No evidence of instability was observed.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Source: Covance, Denver, Pennsylvania, USA.
Sex: Female.
Age at study initiation: approximately 5 months.
Weight at study initiation: 2988 to 4412 g (on day 0 of gestation).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % aqueous methylcellulose
Details on exposure:
Concentration in vehicle: 0, 6, 9, or 18 mg Cu/mL
Total volume applied: 1mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of dose formulations confirmed stability, homogeneity and verified the accuracy of formulation. 
Details on mating procedure:
Mating period: Not specified.
Duration of treatment / exposure:
Duration of exposure: Day 7–28 of gestation.
No. of animals per sex per dose:
Number of animals per group: 22 females (1 rabbit of the high dose group was removed on day 7 of gestation, prior to dosing, due to a self-inflicted injury).
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Body weight: Yes (daily)
Food consumption: Yes (daily)
Clinical signs: Yes (daily)
Gross pathology and histopathology: Yes, gross external and visceral examination was performed for all animals.
Post-exposure period: Sacrifice on day 29 of gestation.
Ovaries and uterine content:
Examination of uterine content: Gravid uterine weight, number of corpora lutea, number of implantations, number of resorptions.
Fetal examinations:
General: Litter Size, number of live and dead foetuses, foetal weight, sex ratio, external alterations, retarded renal development.
Skeleton: Yes
Soft tissue: Yes
Statistics:
Linear contrast of means (Maternal weight, weight change, food consumption);
Jonckheere’s test (Live and dead foetuses, resorptions, implantations, incidence of foetal alterations);
Cochran-Armitage test (Incidence of pregnancy, clinical observations, maternal mortality, females with total resorptions, abortions/early deliveries);
Linear contrast of least square means (foetal weight, sex ratio).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Three females of the high dose group were found dead during the study. One of them showed diarrhoea, red cageboard-staining, weakness, and irregular respiration prior to death. Gross necropsy of the three animals included stomach haemorrhage and/or ulceration, dark discolouration or mottling of lung tissue, pale liver, gelatinous tan rectal discharge, and brown liquid in chest cavity. In addition, two females of this dose group aborted. Diarrhoea was observed for one of them and the other animal showed red discoloured stomach lining upon necropsy. No mortality occurred at the low and mid dose levels. All animals of the test substance groups showed diarrhoea. Statistically significant adverse effects on maternal body weights, body weight changes and food consumption were observed at 9 and 18 mg Cu/kg bw/day. Marked reduction in food consumption and body weight losses were observed during the 1st week of dosing. The results are presented in Table 1.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day
Based on:
element
Basis for effect level:
other: Maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The number of foetuses, and numbers of early and late embryonic deaths were not adversely affected by maternal treatment. Mean foetal weight was slightly lower at 18 mg/kg bw/day (9 % lower than controls). The difference from control was considered treatment-related, but it was not statistically significant. The results are presented in Table 2.

There was a total of four foetuses with malformations: one control foetus showed fused ribs, one foetus at 6 mg/kg bw/day showed ectopic kidney, and two foetuses (from separate litters) at 18 mg/kg bw/day showed hemivertebra. These malformations were considered spontaneous and unrelated to treatment. There was a slight increase in incidence of foetuses at 18 mg/kg bw/day with retarded ossification of skull and pelvic bones. However, there was no correlation with foetal weight, and the biological significance of such a slight increase is uncertain, as there was no increase in the incidence of retarded sternebral ossification. Retarded sternebral ossification is a more common indicator of foetal immaturity. Rib alterations occurred at a very high incidence across all groups in this study; almost all litters were affected. The biological significance of an increase in incidence of a very common finding is uncertain. The results are presented in Table 3.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day
Based on:
element
Basis for effect level:
other: Developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1. Maternal effects.

Parameter

Control

low dose

mid dose

high dose

dose response

Number of dams examined

22

22

22

21

 

Clinical findings during application of test substance

Alopecia
Diarrhea
Irregular respiration
Sore
Stain cageboard
Stain tail
Weak



2
0
0
1
0
0
0



5
5*
0
0
0
3
0



2
5*
0
0
0
2
0



1
9*
1
0
3*
5*
1



-
+
-
-
-
+
-

Mortality of dams (%)

0

0

0

14 %*

-

Abortions

0

1

0

2

-

Body weight gain (day 7-29 of gestation) [g]

261.5±130.42

208.5±158.50

179.5±160.97

72.6±211.64*

+

Food consumption (day 7-29 of gestation) [g/day]

144.1 ± 10.61

134.8 ± 21.36

120.3 ± 32.62*

100.6±32.02*

+

Pregnancies
(No. of dams pregnant)

21

21

21

21

-

* statistically significant trend ( p < 0.05)

Table 2. Litter response (Caesarean section data).

Parameter

Control

low dose

medium dose

high dose

dose response + / -

No. mated

22

22

22

21

 

No. pregnant

21

21

21

21

-

No. aborted/delivered early

0

1

0

2

-

No. found dead

0

0

0

3*

-

No. accidentally killed

0

0

0

1

-

No. with total resorptions

0

1

0

0

-

Total number of litters

21

19

21

15

-

Means per litter:

 

 

 

 

 

Mean corpora lutea

10.0±2.06

10.2±1.51

9.1±1.96

10.1±2.26

-

Implantation/Nidations

8.8±2.04

9.0±1.33

7.8±2.04

9.0±2.36

-

Resorptions

Total
Early
Late

 

1.0±1.47
0.8±1.47
0.1±0.48

 

1.0±1.45
0.7±1.41
0.3±0.56

 

0.2±0.54
0.2±0.51
0.0±0.22

 

0.6±0.91
0.4±0.63
0.2±0.56

 

-
-
-

No. of dead fetuses

0

0

0

0

-

No. of Live fetuses

Total
Males
Females

 

7.9±2.63
3.9±1.77
4.0±1.73

 

8.0±1.73
4.7±2.10
3.3±1.45

 

7.6±2.04
3.9±1.59
3.7±1.74

 

8.4±2.03
4.3±2.12
4.1±1.88

 

-
-
-

Mean fetal weight

Total
Males
Females

 

42.95±2.98
43.25±3.14
42.67±3.77

 

41.71±4.51
42.63±4.46
40.85±4.86

 

43.93±5.88
43.32±5.47
43.69±6.06

 

38.91±4.82
38.84±4.95
39.16±5.54

 

-
-
-

Fetal sex ratio

0.48±0.16

0.58±0.18

0.50±0.22

0.48 ±0.23

-

* significant trend (p£ 0.05)

 

Table 3. Examination of the fetuses.

Parameter

Control

low dose

medium dose

high dose

dose response

No. examined (no. of litters)

165 (21)

152 (19)

159 (21)

126 (15)

 

No. of fetuses (litters) with:

 

 

 

 

 

External malformations

0

0

0

0

-

External developmental variations

0

0

0

0

-

External variations due to retarded development

0

0

0

0

-

Skeletal malformations

Rib- fused
Vertebra - hemi

 

1 (1)
0

 

0
0

 

0
0

 

0
2* (2)

 

-
-

Skeletal developmental variations

Rib- supernumerary
Sternebra - fused

 

105 (21)
1 (1)

 

102 (19)
2 (2)

 

127 (20)
0

 

110 (15)*
0

 

-
-

Skeletal variations due to retarded development

Mandible- retarded ossification
Pelvis- retarded ossification
Skull- bent
Skull- retarded ossification
Sternebra- retarded ossification
Vertebra- retarded ossification



0
0
0
0
65 (16)
1 (1)



0
1 (1)
2 (2)
0
60 (12)
0



0
1 (1)
0
1 (1)
76 (18)
0



1 (1)
2 (1)
0
5 (2)*
51 (12)
0



-
-
-
+
-
-

Visceral malformations

Kidney- ectopic

 

0

 

1 (1)

 

0

 

0

 

-

Visceral developmental variations

0

0

0

0

-

Visceral variations due to retarded development

Kidney: Small papilla- size 2
Kidney: Papilla- size 3

 


0
2 (2)

 


1 (1)
5 (5)

 


5 (3)
1 (1)

 


1 (1)
1 (1)

 


-
-

Total (%) with retardation

68 (41%)

67 (44%)

80 (50%)

57 (45%)

-

Total (%) with variations

125 (76%)

124 (82%)

143 (90%)

118 (94%)

-

* statistically significant trend (p < 0.05)

Applicant's summary and conclusion

Conclusions:
Under the conditions of the current study, there was no evidence of test substance-related teratogenicity.

LO(A)EL maternal toxic effects: 9 mg Cu/kg bw/day
NO(A)EL maternal toxic effects: 6 mg Cu/kg bw/day
LO(A)EL developmental effects: 9 mg Cu/kg bw/day 
NO(A)EL developmental effects: 6 mg Cu/kg bw/day
Executive summary:

Materials and Methods

Copper hydroxide was administered at concentrations of 0, 6, 9, or 18 mg Cu/kg bw to pregnant rabbits by gavage from day 7 to day 28 of gestation. Potential effects on maternal and developmental parameters were assessed. The study was conducted according to OECD 414 (1981); method B.31 (87/302/EEC), and USEPA OPPTS 870.3700 (1998).

Results and Discussion

Administration of copper to pregnant rabbits at 18 mg/kg bw/day was associated with marked initial bodyweight loss, inappetance, abortion and death.  Pups in litters from surviving dams showed slightly lower mean foetal weight, and slightly increased incidence of a common skeletal variant.  Maternal treatment at 9 mg/kg bw/day was associated with initial bodyweight loss and inappetance; pups also showed slightly increased incidence of a common skeletal variant, but mean foetal weights were not adversely affected.  Maternal administration of copper hydroxide was not associated with increased incidence of foetal malformations, pre-implantation losses, or foetal (embryonic) deaths.  The maternal and foetal no observed effect level was 6 mg/kg bw/day, based on maternal weight loss, inappetance, and an increased incidence of a common skeletal variant in foetuses at 9 mg/kg bw/day.