Registration Dossier

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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Data waiving: In accordance with column 1 of REACH Annex IX, a two-generation reproductive toxicity study does not need to be conducted since the repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with column 1 of REACH Annex IX, an extended on-generation reproductive toxicity study does not need to be conducted since the repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

No data on developmental toxicity is available for the substance. Since the substance is used in cosmetic products, tests on animals are banned. Therefore, a weight of evidence approach is proposed based on information on the anions and cations present in this molecule.

Based on the available oral developmental toxicity data on pyrophosphates, no adverse effects are observed in the tested species (rats, mice, rabbits and hamsters).

Based on the available oral developmental toxicity data on ammonium, no adverse effects are observed in the tested species (rabbits).

Based on the available oral developmental toxicity data on manganese, no adverse effects are observed in the tested species (rats).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Uterine weights and corpora lutea were not determined. One third used for visceral examination instead of 50% as recommended in the guideline. Administration only during periods of organogenesis, not until day before pregnancy)
Principles of method if other than guideline:
Adult female albino CD-1 mice were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 150 mg/kg) or test article in a water suspension (10 mL/kg bw) at 3.35, 15.6, 72.3 and 335.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 17 of gestation all dams underwent Caesarean section. Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
CD-1
Remarks:
Albino
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Weight at study initiation: 26.3 - 29.3 g
- Housing: Gang housing in disposable plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 10 mL/kg bodyweight
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
10 days (Day 6 to Day 15 of gestation)
Frequency of treatment:
Daily
Duration of test:
17 days
Dose / conc.:
3.35 mg/kg bw/day (actual dose received)
Dose / conc.:
15.6 mg/kg bw/day (actual dose received)
Dose / conc.:
72.3 mg/kg bw/day (actual dose received)
Dose / conc.:
335 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 25 25
Aspirin 150.0 24 21
FDA 71-61 3.35 25 23
15.6 25 23
72.3 24 23
335.0 25 22
Control animals:
yes, sham-exposed
other: positive control: 150 mg/kg aspirin
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 11, 15 and 17.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: uterus and urogenital tract
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: one third per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: Yes: two thirds per litter
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 335 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
dead fetuses
food consumption and compound intake
histopathology: non-neoplastic
maternal abnormalities
number of abortions
total litter losses by resorption
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not specified
Visceral malformations:
no effects observed
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 335 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
external malformations
visceral malformations
Remarks on result:
other: developmental toxicity
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin**

3.35

15.6

72.3

335.0

Pregnancies

 

 

 

 

 

 

Total No.

25

21

23

23

23

22

Died or aborted (before Day 17)

0

0

0

0

0

0

To term (on Day 17)

25

21

23

23

23

22

Live litters

 

 

 

 

 

 

Total No.*

25

20

23

23

23

21

Implant Sites

 

 

 

 

 

 

Total No.

286

254

280

279

265

256

Average/dam*

11.4

12.1

12.2

12.1

11.5

11.6

Resorptions

 

 

 

 

 

 

Total No*

17

37

8

3

10

24

Dams with 1 or more sites resorbed

10

13

6

2

9

9

Dams with all sites resorbed

0

1

0

0

0

1

Per cent partial resorptions

40.0

61.9

26.1

8.70

39.1

40.9

Per cent complete resorptions

--

4.76

--

--

--

4.55

Live foetuses

 

 

 

 

 

 

Total No

264

217

271

274

255

229

Average/dam*

10.6

10.3

11.8

11.9

11.5

10.4

Sex ratio (M/F)

0.96

0.75

0.75

0.73

0.82

0.84

Dead Foetuses

 

 

 

 

 

 

Total No.*

5

0

1

2

0

3

Dams with 1 or more dead

4

--

1

2

--

3

Dams with all dead

0

--

0

0

--

0

Per cent partial dead

16.0

--

4.35

8.70

--

13.6

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

0.88

0.84

0.84

0.89

0.86

0.85

* Includes only those dams examined at term

** Positive control: 150 mg/kg

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

Aspirin**

3.35

15.6

72.3

335.0

Live foetuses examined (at term)

183/25

154/20

188/23

190/23

176/23

159/21

Sternebrae

 

 

 

 

 

 

Incomplete oss.

23/10

34/13

19/11

45/13

33/13

29/9

Scrambled

 

 

 

 

 

 

Bipartite

5/4

6/5

9/7

10/9

5/3

9/6

Fused

 

 

 

 

 

 

Extra

 

1/1

 

 

 

1/1

Missing

35/14

26/11

27/11

39/8

30/10

14/6

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

 

 

3/1

 

2/1

 

Fused/split

 

1/1

 

 

 

 

Wavy

 

 

 

 

 

 

Less than 12

 

 

 

 

 

 

More than 13

12/7

37/15

34/15

32/14

38/11

31/15

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

4/3

9/5

12/4

8/3

4/2

4/2

Scrambled

 

 

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

 

 

 

 

 

Tail defects

 

 

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

 

 

1/1

 

 

 

Missing

 

 

1/1

 

 

 

Craniostosis

 

 

 

 

 

 

Other; facial bones, inc

 

 

 

1/1

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

4/3

6/4

12/6

7/3

7/3

4/2

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

29/12

35/15

39/15

52/16

46/15

33/13

Hyoid; reduced

17/13

23/13

24/12

18/10

17/13

23/10

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 150 mg/kg

 

Table 4 Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam

Number of pups

Description

Aspirin

150.0

A 3911

1

Microblepharia

Gastroschisis

FDA 71-61

3.35

M 3010

1

Umbilical hernia

FDA 71-61

15.6

M3033

1

Umbilical hernia

 

 

M 3053

1

Hydrocephalus

FDA 71-61

72.3

M3078

1

Hydrocephalus

Conclusions:
Under the conditions of the study, the test material administered to pregnant mice for 10 days up to a dose level of 335 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 335 mg/kg bw.
Executive summary:

Adult female albino CD-1 mice were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 150 mg/kg) or test article in a water suspension (10 mL/kg bw) at 3.35, 15.6, 72.3 and 335.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 17 of gestation all dams underwent Caesarean section. Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects. Under the conditions of the study, the test material administered to pregnant mice for 10 days up to a dose level of 335 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 335 mg/kg bw.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Uterine weights were not determined. Administration only during periods of organogenesis, not until day before pregnancy)
Principles of method if other than guideline:
Adult female Dutch-belted rabbits were dosed on Day 0 with a single injection of 0.4 mL human chorionic gonadotropin (400 IU) via the marginal ear vein. After three hours each doe was artificially inseminated with 0.3 mL diluted semen from a donor buck using 20 x 10E06 motile sperm. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose) or test article in a water suspension at 1.28, 5.95, 27.6 and 128.0 mg/kg was carried out daily on Days 6 to 18 of gestation. The positive control 6-aminonicotinamide at 2.5 mg/kg was dosed on Day 9. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 29 all does underwent Caesarean section. Number of corpora lutea, implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. Live foetuses of each litter were then placed in an incubator for 24 hours for evaluation of neonatal survival. All pups underwent detailed visceral examination. All foetuses were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
Dutch
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Weight at study initiation: 2.17 - 2.51 kg
- Housing: Individually housed in mesh-bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 1 mL/kg bw at doses equal to or below 250 mg/kg bw and 2 mL/kg at doses up to 500 mg/kg bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: artificial insemination
- Proof of pregnancy: No data
Duration of treatment / exposure:
13 days (Day 6 to Day 18 of gestation)
Frequency of treatment:
Daily
Duration of test:
29 days
Dose / conc.:
1.28 mg/kg bw/day (actual dose received)
Dose / conc.:
5.95 mg/kg bw/day (actual dose received)
Dose / conc.:
27.6 mg/kg bw/day (actual dose received)
Dose / conc.:
128 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 17 11
6-AN 2.5 15 10
FDA 71-61 1.28 15 12
5.95 16 12
27.6 20 9
128.0 15 11
Control animals:
yes, sham-exposed
other: positive control: 2.5 mg/kg 6-aminonicotinamide (6-AN)
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 12, 18 and 29.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus and urogenital tract
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 128 mg/kg bw/day
Basis for effect level:
behaviour (functional findings)
clinical signs
dead fetuses
early or late resorptions
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
number of abortions
pre and post implantation loss
total litter losses by resorption
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No dose related response observed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 128 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

Table 2 Reproduction data

Dose (mg/kg)

Sham

6-AN

1.28

5.95

27.6

128.0

Pregnancies

 

 

 

 

 

 

Total No.

11

10

12

12

9

11

Died or aborted (before Day 29)

1

0

0

2

0

0

To term (on Day 29)

10

10

12

10

9

11

Corpora Lutea

 

 

 

 

 

 

Total no.

175

132

146

191

140

159

Average/dam mated

14.6

12.0

12.2

13.6

9.33

12.2

Live litters

 

 

 

 

 

 

Total No.*

10

6

12

9

9

11

Implant Sites

 

 

 

 

 

 

Total No.

53

42

70

64

44

80

Average/dam*

5.30

4.20

5.83

6.40

4.89

7.27

Resorptions

 

 

 

 

 

 

Total No*

--

18

3

9

1

7

Dams with 1 or more sites resorbed

--

6

2

3

1

5

Dams with all sites resorbed

--

4

--

1

--

--

Per cent partial resorptions

--

60.0

16.7

30.0

11.1

45.5

Per cent complete resorptions

--

40.0

--

10.0

--

--

Live foetuses

 

 

 

 

 

 

Total No

52

19

67

55

43

73

Average/dam*

5.20

1.90

5.58

5.50

4.78

6.64

Sex ratio (M/F)

0.73

5.00

0.63

1.04

1.26

0.92

Dead Foetuses

 

 

 

 

 

 

Total No.*

1

5

--

--

--

73

Dams with 1 or more dead

1

2

--

--

--

--

Dams with all dead

--

--

-

--

--

--

Per cent partial dead

10.0

20.0

--

--

--

--

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

42.2

29.7

37.0

36.0

40.0

35.8

* Includes only those dams examined at term

** Positive control: 2.5 mg/kg 6-AN dosed on Day 9

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

6-AN

1.28

5.95

27.6

128.0

Live foetuses examined (at term)

52/10

19/6

67/12

55/9

43/9

72/11a

Sternebrae

 

 

 

 

 

 

Incomplete oss.

2/2

1/1

 

1/1

2/2

 

Scrambled

 

 

 

 

 

 

Bipartite

 

1/1

 

1/1

 

1/1

Fused

 

3/2

 

 

 

 

Extra

 

1/1

2/1

 

 

6/4

Missing

 

2/2

 

 

 

 

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Fused/split

 

5/2

 

 

3/1

 

Wavy

 

 

 

 

 

 

Less than 12

 

 

 

 

 

 

More than 13

 

 

 

 

 

 

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Scrambled

 

4/2

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

4/3

 

 

 

 

Tail defects

 

13/4

 

 

1/1

1/1

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

 

1/1

 

 

 

 

Missing

 

 

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other; facial bones, inc

 

1/1

 

 

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 2.5 mg/kg 6-AN dosed on Day 9

a One pup lost in processing

Table 4 Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam

Number of pups

Description

6-AN

2.5

Z 6479

1

Medial rotation of hind limbs

 

 

Z 6488

1

Medial rotation of hind limbs

 

 

Z 6489

1

Siamese pups (combined stomach and head)

 

 

Z 6490

3

Anopia, Medial rotation of hind limbs

 

 

 

2

Anopia

FDA 71-61

27.6

M 6051

2

Anopia

Conclusions:
Under the conditions of the study, the test material administered to pregnant rabbits for 13 days up to a dose level of 128 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 128 mg/kg bw.
Executive summary:

Adult female Dutch-belted rabbits were dosed on Day 0 with a single injection of 0.4 mL human chorionic gonadotropin (400 IU) via the marginal ear vein. After three hours each doe was artificially inseminated with 0.3 mL diluted semen from a donor buck using 20 x 10E06 motile sperm. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose) or test article in a water suspension at 1.28, 5.95, 27.6 and 128.0 mg/kg was carried out daily on Days 6 to 18 of gestation. The positive control 6-aminonicotinamide at 2.5 mg/kg was dosed on Day 9. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 29 all does underwent Caesarean section. Number of corpora lutea, implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. Live foetuses of each litter were then placed in an incubator for 24 hours for evaluation of neonatal survival. All pups underwent detailed visceral examination. All foetuses were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects. Under the conditions of the study, the test material administered to pregnant rabbits for 13 days up to a dose level of 128 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 128 mg/kg bw.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Uterine weights and corpora lutea were not determined. One third used for visceral examination instead of 50% as recommended in the guideline. Administration only during periods of organogenesis, not until day before pregnancy)
Principles of method if other than guideline:
Adult female golden hamsters were mated with mature males (1:1). Observation of motile sperm in the vaginal smear was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 250 mg/kg) or test article in a water suspension at 1.66, 7.71, 35.8 and 166.0 mg/kg was carried out daily on Days 6 to 10 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 14 of gestation all dams underwent Caesarean section. Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Genital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
no
Limit test:
no
Species:
hamster
Strain:
other: Golden
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Weight at study initiation: 122.2 - 133.4 g
- Housing: Individually housed in mesh-bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 1 mL/kg bw at doses equal to or below 250 mg/kg bw and 2 mL/kg at doses up to 500 mg/kg bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: 1:1 mating
- Proof of pregnancy: appearence of motile sperm in vaginal smear
Duration of treatment / exposure:
5 days (Day 6 to Day 10 of gestation)
Frequency of treatment:
Daily
Duration of test:
14 days
Dose / conc.:
1.66 mg/kg bw/day (actual dose received)
Dose / conc.:
7.71 mg/kg bw/day (actual dose received)
Dose / conc.:
35.8 mg/kg bw/day (actual dose received)
Dose / conc.:
166 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 22 20
Aspirin 250 22 21
FDA 71-81 1.66 22 20
7.71 24 20
35.8 22 21
166.0 22 22
Control animals:
yes, sham-exposed
other: positive control: 250 mg/kg aspirin
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 8, 10 and 14.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 14
- Organs examined: uterus and genital tract
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 166 mg/kg bw/day
Basis for effect level:
body weight and weight gain
clinical signs
dead fetuses
early or late resorptions
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
number of abortions
pre and post implantation loss
total litter losses by resorption
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 166 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified
Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin

1.66

7.71

35.8

166.0

Pregnancies

 

 

 

 

 

 

Total No.

20

21

20

20

21

22

Died or aborted (before Day 14)

0

0

0

0

1

1

To term (on Day 14)

20

21

20

20

20

21

Live litters

 

 

 

 

 

 

Total No.*

20

21

20

20

20

21

Implant Sites

 

 

 

 

 

 

Total No.

298

311

292

289

290

300

Average/dam*

14.9

14.8

14.6

14.5

14.5

14.3

Resorptions

 

 

 

 

 

 

Total No*

1

8

1

1

1

8

Dams with 1 or more sites resorbed

1

6

1

1

1

5

Dams with all sites resorbed

--

--

--

--

--

--

Per cent partial resorptions

5.00

28.6

5.00

5.00

5.00

23.8

Per cent complete resorptions

--

--

--

--

--

--

Live foetuses

 

 

 

 

 

 

Total No

295

303

291

288

289

292

Average/dam*

14.8

14.4

14.6

14.4

14.5

13.9

Sex ratio (M/F)

0.89

0.94

0.81

0.78

0.75

0.75

Dead Foetuses

 

 

 

 

 

 

Total No.*

2

--

--

--

--

--

Dams with 1 or more dead

2

--

--

--

--

--

Dams with all dead

--

--

--

--

--

--

Per cent partial dead

10.0

--

--

--

--

--

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

1.70

1.72

1.72

1.82

1.82

1.75

* Includes only those dams examined at term

** Positive control: 250 mg/kg

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

Aspirin

1.66

7.71

35.8

166.0

Live foetuses examined (at term)

204/20

209/21

200/20

199/20

202/20

202/21

Sternebrae

 

 

 

 

 

 

Incomplete oss.

131/20

85/20

124/20

69/17

88/18

80/20

Scrambled

 

 

 

 

 

 

Bipartite

18/9

16/11

22/14

20/11

15/9

25/13

Fused

 

 

 

 

 

 

Extra

8/2

 

1/1

6/3

5/3

 

Missing

53/14

48/13

49/12

39/10

33/10

51/14

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Fused/split

 

 

 

 

 

2/1

Wavy

 

 

 

 

 

 

Less than 12

 

 

 

 

 

 

More than 13

62/17

56/13

46/16

69/17

38/13

60/16

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

2/2

15/5

 

1/1

 

8/2

Scrambled

 

 

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

 

 

 

 

 

Tail defects

 

 

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

 

 

 

 

 

 

Missing

 

 

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other

 

 

 

 

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

4/4

17/6

 

6/4

 

13/3

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

6/6

14/6

5/4

4/2

2/1

13/3

Hyoid; reduced

7/7

6/5

9/7

7/3

6/5

8/6

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 250 mg/kg

Table 4 Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam

Number of pups

Description

Sham

0.0

S 5908

1

Meningoencephalocele

 

 

S 5914

1

Hydrocephalus

Aspirin

250.0

A 5912

1

Hepatomegaly

FDA 71-61

1.66

M 5013

1

Hydrocephalus

FDA 71-61

166.0

M 5092

4

Hydrocephalus

 

 

 

2

Hydromyelia

Conclusions:
Under the conditions of the study, the test material administered to pregnant hamsters for 5 days up to a dose level of 166 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 166 mg/kg bw.
Executive summary:

Adult female golden hamsters were mated with mature males (1:1). Observation of motile sperm in the vaginal smear was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 250 mg/kg) or test article in a water suspension at 1.66, 7.71, 35.8 and 166.0 mg/kg was carried out daily on Days 6 to 10 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 14 of gestation all dams underwent Caesarean section. Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Genital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects. Under the conditions of the study, the test material administered to pregnant hamsters for 5 days up to a dose level of 166 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 166 mg/kg bw.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Uterine weights and corpora lutea were not determined. One third used for visceral examination instead of 50% as recommended in the guideline. Administration only during periods of organogenesis, not until day before pregnancy)
Principles of method if other than guideline:
Adult female albino (Wistar derived stock) rats were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 250 mg/kg) or test article in a water suspension at 1.69, 9.24, 42.95 and 169.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. Daily room temperature was recorded. On Day 20 of gestation all dams underwent Caesarean section. Number of implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Weight at study initiation: 214 - 225 g
- Housing: Individual housing in mesh bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 1 mL/kg bw at doses equal to or below 250 mg/kg bw and 2 mL/kg at doses up to 500 mg/kg bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
10 days (Day 6 to Day 15 of gestation)
Frequency of treatment:
Daily
Duration of test:
20 days
Dose / conc.:
1.69 mg/kg bw/day (actual dose received)
Dose / conc.:
9.24 mg/kg bw/day (actual dose received)
Dose / conc.:
42.95 mg/kg bw/day (actual dose received)
Dose / conc.:
169 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 25 23
Aspirin 250.0 24 21
FDA 71-61 1.69 25 24
9.24 25 24
42.95 24 24
169.0 25 21
Control animals:
yes, sham-exposed
other: positive control: 250 mg/kg aspirin
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 11, 15 and 20.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and urogenital tract
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: one third per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: Yes: two thirds per litter
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 169 mg/kg bw/day
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
dead fetuses
early or late resorptions
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
number of abortions
pre and post implantation loss
total litter losses by resorption
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 169 mg/kg bw/day (actual dose received)
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified
Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin

1.69

9.24

42.95

169.0

Pregnancies

 

 

 

 

 

 

Total No.

23

21

24

24

24

21

Died or aborted (before Day 20)

0

1

0

0

0

0

To term (on Day 20)

23

20

24

24

24

21

Live litters

 

 

 

 

 

 

Total No.*

23

18

24

24

24

21

Implant Sites

 

 

293

 

 

 

Total No.

283

234

12.2

284

287

254

Average/dam*

12.3

11.7

 

11.8

12.0

12.1

Resorptions

 

 

 

 

 

 

Total No*

4

35

4

4

4

--

Dams with 1 or more sites resorbed

2

5

4

4

3

--

Dams with all sites resorbed

--

2

--

--

--

--

Per cent partial resorptions

8.70

25.0

16.7

16.7

12.5

--

Per cent complete resorptions

--

10.0

--

--

--

--

Live foetuses

 

 

 

 

 

 

Total No

279

197

288

280

283

254

Average/dam*

12.1

9.85

12.0

11.7

11.8

12.1

Sex ratio (M/F)

1.08

0.80

1.27

1.00

0.98

1.03

Dead Foetuses

 

 

 

 

 

 

Total No.*

--

2

1

--

--

--

Dams with 1 or more dead

--

2

1

--

--

--

Dams with all dead

--

--

--

--

--

--

Per cent partial dead

--

10.0

4.17

--

--

--

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

3.65

2.41

3.71

3.78

3.78

3.75

* Includes only those dams examined at term

** Positive control: 250 mg/kg

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

Aspirin

1.69

9.24

42.95

169.0

Live foetuses examined (at term)

191/23

137/18

198/24

193/24

196/24

176/21

Sternebrae

 

 

 

 

 

 

Incomplete oss.

71/20

86/16

83/19

54/19

61/16

47/17

Scrambled

 

 

 

 

 

 

Bipartite

 

7/5

 

 

2/2

4/3

Fused

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Missing

7/6

117/7

9/7

15/8

1/1

5/4

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

1/1

3/2

3/2

 

 

1/1

Fused/split

 

14/4

 

 

 

 

Wavy

14/8

54/14

34/9

12/7

15/8

13/8

Less than 12

 

 

 

 

 

 

More than 13

1/1

89/15

6/5

3/3

 

6/5

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

6/5

120/17

12/4

21/11

5/4

10/8

Scrambled

 

1/1

 

 

 

 

Fused

 

 

1/1

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

3/3

 

 

 

 

Tail defects

 

1/1

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

23/10

60/13

50/16

31/12

25/13

20/11

Missing

 

9/4

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other

 

1/1

 

 

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

 

4/3

 

 

 

 

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

19/11

69/17

37/15

14/9

26/13

7/6

Hyoid; reduced

7/5

20/8

38/14

27/12

35/16

28/12

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 250 mg/kg

Table 4 Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam

Number of pups

Description

Aspirin

250.0

A 4910

1

Encepalomyelocele

 

 

A 4913

1

Exencephaly; exophthalmos

 

 

A 4917

1

Renal agenesis

 

 

A 4921

1

Encephalomyelocele; gastroschisis

 

 

A 4923

3

Encephalomyelocele

 

 

 

1

Exophthalmos; gastroschisis

 

 

A 4925

2

Encepalomyelocele

FDA 71-61

42.95

M 4076

1

Gastroschisis

Conclusions:
Under the conditions of the study, the test material administered to pregnant rats for 10 days up to a dose level of 169 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 169 mg/kg bw.
Executive summary:

Adult female albino (Wistar derived stock) rats were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 250 mg/kg) or test article in a water suspension at 1.69, 9.24, 42.95 and 169.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. Daily room temperature was recorded. On Day 20 of gestation all dams underwent Caesarean section. Number of implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects. Under the conditions of the study, the test material administered to pregnant rats for 10 days up to a dose level of 169 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 169 mg/kg bw.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Uterine weights were not determined. One third used for visceral examination instead of 50% as recommended in the guideline. Administration only during periods of organogenesis, not until day before pregnancy)
Principles of method if other than guideline:
Adult female albino (Wistar derived stock) rats were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 250 mg/kg) or test article in a water suspension at 1.38, 6.41, 29.7 and 138.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. Daily room temperature was recorded. On Day 20 of gestation all dams underwent Caesarean section. Sex, number of corpora lutea, implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Weight at study initiation: 215 - 221 g
- Housing: Individual housing in mesh bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 26.7
- Humidity (%): 62 - 76

IN-LIFE DATES: 3/9/74 - 2/10/74
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 1 mL/kg bw at doses equal to or below 250 mg/kg bw and 2 mL/kg at doses up to 500 mg/kg bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
10 days (Day 6 to Day 15 of gestation)
Frequency of treatment:
Daily
Duration of test:
20 days
Dose / conc.:
1.38 mg/kg bw/day (actual dose received)
Dose / conc.:
6.41 mg/kg bw/day (actual dose received)
Dose / conc.:
29.7 mg/kg bw/day (actual dose received)
Dose / conc.:
138 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 25 20
Aspirin 250.0 25 19
FDA 73-1 1.38 23 21
6.41 24 20
29.7 25 20
138.0 25 19
Control animals:
yes, sham-exposed
other: positive control: 250 mg/kg aspirin
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 11, 15 and 20.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and urogenital tract
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: one third per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: Yes: two thirds per litter
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 138 mg/kg bw/day
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
dead fetuses
early or late resorptions
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
number of abortions
pre and post implantation loss
total litter losses by resorption
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 138 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified
Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin

1.38

6.41

29.7

138.0

Pregnancies

 

 

 

 

 

 

Total No.

20

19

21

20

20

19

Died or aborted (before Day 20)

0

0

0

0

0

0

To term (on Day 20)

20

19

21

20

20

19

Corpora Lutea

 

 

 

 

 

 

Total no.

244

258

267

263

249

248

Average/dam mated

9.76

10.3

11.6

11.0

9.96

9.92

Live litters

 

 

 

 

 

 

Total No.*

20

19

21

20

20

19

Implant Sites

 

 

 

 

 

 

Total No.

228

225

240

249

237

232

Average/dam*

11.4

11.8

11.4

12.5

11.9

12.2

Resorptions

 

 

 

 

 

 

Total No*

4

17

1

5

8

1

Dams with 1 or more sites resorbed

2

8

1

4

6

1

Dams with all sites resorbed

--

--

--

--

--

--

Per cent partial resorptions

10.0

42.1

4.76

20.0

30.0

5.26

Per cent complete resorptions

--

--

--

--

--

--

Live foetuses

 

 

 

 

 

 

Total No

224

208

239

244

229

231

Average/dam*

11.2

11.0

11.4

12.2

11.5

12.2

Sex ratio (M/F)

0.96

1.04

0.98

0.95

1.14

0.94

Dead Foetuses

 

 

 

 

 

 

Total No.*

--

--

--

--

--

--

Dams with 1 or more dead

--

--

--

--

--

--

Dams with all dead

--

--

--

--

--

--

Per cent partial dead

--

--

--

--

--

--

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

3.90

2.97

4.01

4.01

3.95

4.09

* Includes only those dams examined at term

** Positive control: 250 mg/kg

 

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

Aspirin

4.1

19.0

88.3

410.0

Live foetuses examined (at term)

156/20

145/19

165/21

169/20

163/20

160/19

Sternebrae

 

 

 

 

 

 

Incomplete oss.

20/12

86/18

36/14

19/11

46/15

7/5

Scrambled

 

 

 

 

 

 

Bipartite

 

3/2

1/1

 

 

 

Fused

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Missing

2/2

117/19

9/7

4/3

15/9

 

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

3/2

 

 

2/1

 

 

Fused/split

 

1/1

 

 

 

 

Wavy

21/9

55/16

25/11

23/8

24/13

13/9

Less than 12

1/1

 

 

 

 

 

More than 13

 

110/17

5/1

 

1/1

1/1

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

6/4

64/17

4/2

3/2

2/2

 

Scrambled

 

 

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

 

 

 

 

 

Tail defects

 

 

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

32/12

48/14

19/12

27/11

17/7

18/9

Missing

 

 

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other

 

 

 

 

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

1/1

4/3

 

 

 

 

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

16/8

45/15

10/8

17/9

17/7

10/9

Hyoid; reduced

29/14

9/6

19/10

19/9

13/7

28/10

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 250 mg/kg

 

Table 4 Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam

Number of pups

Description

Aspirin

250.0

43634

1

Encephalomeningocele;

encephalomyelocele

 

 

43639

1

Gastroschisis

 

 

43645

1

Hydrocephalus

 

 

43654

4

Encephalomyelocele

 

 

43655

1

Encephalomyelocele

FDA 73-1

6.41

43707

1

Hydrocephalus; cleft palate

Conclusions:
Under the conditions of the study, the test material administered to pregnant rats for 10 days up to a dose level of 138 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 138 mg/kg bw.
Executive summary:

Adult female albino (Wistar derived stock) rats were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 250 mg/kg) or test article in a water suspension at 1.38, 6.41, 29.7 and 138.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. Daily room temperature was recorded. On Day 20 of gestation all dams underwent Caesarean section. Sex, number of corpora lutea, implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects. Under the conditions of the study, the test material administered to pregnant rats for 10 days up to a dose level of 138 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 138 mg/kg bw.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Uterine weights were not determined. One third used for visceral examination instead of 50% as recommended in the guideline. Administration only during periods of organogenesis, not until day before pregnancy)
Principles of method if other than guideline:
Adult female albino CD-1 mice were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 150 mg/kg) or test article in a water suspension (10 mL/kg bw) at 1.3, 6.0, 28.0 and 130.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 17 of gestation all dams underwent Caesarean section. Sex, numbers of corporea lutea, Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
CD-1
Remarks:
Albino
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Weight at study initiation: 29 - 31 g
- Housing: Gang housing in disposable plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26.67
- Humidity (%): 62 - 76

IN-LIFE DATES: 1/9/74 - 5/10/74
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 10 mL/kg bodyweight
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
10 days (Day 6 to Day 15 of gestation)
Frequency of treatment:
Daily
Duration of test:
17 days
Dose / conc.:
1.3 mg/kg bw/day (actual dose received)
Dose / conc.:
6 mg/kg bw/day (actual dose received)
Dose / conc.:
28 mg/kg bw/day (actual dose received)
Dose / conc.:
130 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 30 21
Aspirin 150.0 25 20
FDA 71-3 1.3 24 20
6.0 27 20
28.0 26 18
130.0 29 21
Control animals:
yes, sham-exposed
other: positive control: 150 mg/kg aspirin
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 11, 15 and 17.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: uterus and urogenital tract
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: one third per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: Yes: two thirds per litter
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 130 mg/kg bw/day
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
dead fetuses
early or late resorptions
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
number of abortions
pre and post implantation loss
total litter losses by resorption
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 130 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin

1.3

6.0

28.0

130.0

Pregnancies

 

 

 

 

 

 

Total No.

21

20

20

20

18

21

Died or aborted (before Day 17)

0

0

0

0

0

0

To term (on Day 17)

21

20

20

20

18

21

Corpora Lutea

 

 

 

 

 

 

Total no.

277

253

239

254

224

261

Average/dam mated

9.23

10.1

10.4

9.41

8.62

9.32

Live litters

 

 

 

 

 

 

Total No.*

21

20

20

20

18

20

Implant Sites

 

 

 

 

 

 

Total No.

250

220

231

237

209

243

Average/dam*

11.9

11.0

11.6

11.9

11.6

11.6

Resorptions

 

 

 

 

 

 

Total No*

9

11

11

13

17

18

Dams with 1 or more sites resorbed

6

8

7

9

10

9

Dams with all sites resorbed

--

--

--

--

--

1

Per cent partial resorptions

28.6

40.0

35.0

45.0

55.6

42.9

Per cent complete resorptions

--

--

--

--

--

4.76

Live foetuses

 

 

 

 

 

 

Total No

240

209

219

223

191

223

Average/dam*

11.4

10.5

11.0

11.2

10.6

10.6

Sex ratio (M/F)

0.86

0.99

0.92

1.14

1.01

1.05

Dead Foetuses

 

 

 

 

 

 

Total No.*

1

--

1

1

1

2

Dams with 1 or more dead

1

--

1

1

1

2

Dams with all dead

--

--

--

--

--

--

Per cent partial dead

4.76

--

5.00

5.00

5.56

9.52

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

0.90

0.91

0.91

0.90

0.91

0.88

* Includes only those dams examined at term

** Positive control: 150 mg/kg

 

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

Aspirin

1.3

6.0

28.0

130.0

Live foetuses examined (at term)

165/21

146/20

150/20

157/20

134/18

157/20

Sternebrae

 

 

 

 

 

 

Incomplete oss.

28/9

66/18

12/7

61/18

14/10

19/9

Scrambled

 

 

 

 

 

 

Bipartite

1/1

3/3

 

 

 

 

Fused

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Missing

17/7

21/10

9/6

17/8

13/7

10/7

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Fused/split

 

 

 

 

 

 

Wavy

 

 

1/1

 

 

 

Less than 12

 

 

 

 

 

 

More than 13

25/13

32/12

31/16

21/11

29/13

38/13

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

7/6

4/4

4/3

2/1

5/4

6/4

Scrambled

 

 

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

 

 

 

 

 

Tail defects

 

 

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

 

 

 

 

 

2/1

Missing

 

 

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other

 

 

 

 

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

4/2

5/4

2/2

3/2

1/1

5/4

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

32/11

33/14

26/12

40/15

37/12

31/14

Hyoid; reduced

13/8

23/11

11/8

21/11

13/8

20/13

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 150 mg/kg

 

Table 4 Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam

Number of pups

Description

Aspirin

150.0

23629

1

Cleft palate

FD 73-1

6.0

23700

1

Exophthalmos; encephalomeningocele

Conclusions:
Under the conditions of the study, the test material administered to pregnant mice for 10 days up to a dose level of 130 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 130 mg/kg bw.
Executive summary:

Adult female albino CD-1 mice were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 150 mg/kg) or test article in a water suspension (10 mL/kg bw) at 1.3, 6.0, 28.0 and 130.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 17 of gestation all dams underwent Caesarean section. Sex, numbers of corporea lutea, Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects. Under the conditions of the study, the test material administered to pregnant mice for 10 days up to a dose level of 130 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 130 mg/kg bw.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Laboratories, Inc.
- Diet (e.g. ad libitum): Each rabbit was provided with 150 g feed (Certified Rabbit Chow #5322) pre-exposure, and 180 g during the exposure.
- Water (e.g. ad libitum): Rabbits were given ab libitum access to water (untreated or treated).
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test formulations of ammonium perchlorate in deionised water were prepared at least weekly, stored refrigerated, and dosage solutions were brought to room temperature prior to use. Test drinking solutions were adjusted to concentrations that yielded target doses of 0, 0.1, 1.0, 10.0, 30.0 and 100.0 mg/kg/day based on actual weekly water consumption. The ammonium perchlorate was considered 100% active for the purpose of dose calculations. Rabbits were given ad libitum access to the water.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Gestational days (GD) 6 to 28 (23 days)
Frequency of treatment:
Daily - ad libitum access to drinking water
Duration of test:
23 days; does were sacrificed on gestational day 29.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
0.1 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
1 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in water
No. of animals per sex per dose:
25 rabbits per group
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected on the basis of dose-finding study carried out by Argus Laboratories, where thyroid histopathology was evident in New Zealand White rabbits at doses of 20, 50 and 100 mg/kg/day. T3 (triiodothyronine), T4 (thyroxine) and TSH (serum thyroid stimulating hormone) blood levels were reduced at all doses and three malformed foetuses from three litters in the 20 mg/kg/day dose group were observed at gross external examination.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS:
- Time schedule:
All rabbits were observed for viability at least twice daily, and for general appearance at least one during the pre-exposure period. The rabbits were also examined daily during the exposure period and on the day of sacrifice (GD 29) for clinical observations of toxicity, abortions, premature deliveries, and deaths.

BODY WEIGHT:
- Time schedule for examinations: Body weights were recorded daily.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: Feed consumption values were recorded daily.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
- Time schedule for examinations: Water consumption values were recorded daily.

OTHER:
Blood samples were collected under anaesthesia on GD 29 for determination of TSH, T3 and T4 levels. Rabbits were then sacrificed and subjected to gross necropsy of the thoracic, abdominal and pelvic viscera. A section of the trachea containing the thyroids/parathryroids of all rabbits was excised. Following fixation the thyroid/parathyroid tissue samples were carefully trimmed, weighed, and evaluated histologically by a veterinary pathologist.
Ovaries and uterine content:
The number of corpora lutea in each ovary was recorded. The uterus was excised and examined for pregnancy, number and distribution of implantations, early and late resorptions, and live and dead foetuses.
Fetal examinations:
Each caesarean-delivered foetus was weighed and examined for gross external alterations. All foetuses were examined by dissection and the brain was examined in situ for approximately one-half of the foetuses in each litter. The remaining foetuses in each litter were decapitated and the heads were examined using Wilson's sectioning technique. All foetuses were examined for skeletal and cartilaginous alterations.
Statistics:
Variance test for homogeneity of the binomial distribution was used for clinical observations and proportion data. Bartlett's test of homogeneity of variances and ANOVA was used for continuous data, followed by Dunnett's test (or Dunn's method of multiple comparisons where K-W was used). If ANOVA wasn't appropriate, the Kruskal-Wallis test was used when 75% or fewer ties were present, but when more than 75% ties were present Fisher's Exact Test was used.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations included localised alopecia, ungroomed coat, scant soft or liquid faeces, and a red perivaginal, perinasal or perioral substance. The clinical observations recorded were considered unrelated to treatment because the incidences were not dose-dependent; the observation was associated with abortion of a litter, and/or the observations were commonly seen in rabbits in the laboratory environment.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related deaths. There were no treatment-related deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The maternal body weights in the control group were consistently lower than the treated groups over the gestation period. There were however, no statistically significant differences in average maternal body weights, gravid uterine weights, body weight gains, or corrected GD29 body weights (GD29 bodyweight minus the gravid uterine weight) among exposure groups during gestation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum T4 levels appeared to decrease in a treatment-related manner, with the 30 and 100 mg/kg groups showing statistically significant decreases compared to controls. This decrease was considered to be treatment-related by the authors because it corresponded to the hypertrophy of follicular epithelium. There were no statistically signficant changes in serum T3 and TSH in treated rabbits compared to controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The only adverse necropsy observation was a mottled liver that occurred in the 1 mg/kg group doe that aborted.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination of the dams' thyroid glands revealed hypertrophy of the follicular epithelium in the 10, 30 and 100 mg/kg groups, this was considered to be treatment related by the authors. No treatment related microscopic changes were observed in the thyroid gland of any rabbits from the 0.1 or 1.0 mg/kg groups. In the affected thyroids, there was an increased height or enlargement of the follicular epithelium, occasionally resulting in a decrease in the lumen of follicles, which contained pale and occasionally vacuolated colloid.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Two does in the 1 mg/kg group aborted on GD 28 and were sacrificed. Both abortions were considered unrealted to treatment because the incidences were not dose-dependent. One dam in the 100 mg/kg group prematurely delivered on GD 27. Because rabbits normally deliver on GD 31, and the pups appeared to be full term (they had fur and were nursing), it was assumed that the rabbit had been incorrectly identified and shipped by the supplier on the wrong day of gestation.
Details on maternal toxic effects:
Caesarean section observations were based on 22, 24, 23, 24 and 23 pregnant does surviving to GD 29. There were no effects on caesarean sectioning or litter parameters. All values were within laboratory historical ranges. All placentae appeared normal and no dam had a litter consisting of only resorbed conceptuses.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 mg/kg bw/day
Basis for effect level:
histopathology: non-neoplastic
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter averages for corpora lutea, implantations, litter sizes, live and dead foetuses, percent dead or resorbed conceptuses, and foetal body weights were comparable and did not differ significantly between groups.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Foetal evaluations were based on 180, 184, 196, 195, 189 and 206 live foetuses in the six exposure groups, respectively. No foetal alterations were attributable to treatment. Only 6 foetuses had gross external alterations: 3 from the control group and two from the 1 mg/kg group, and 1 from the 100 mg/kg group litters. Foetal soft tissue alterations only occurred in four foetuses of the control group, two in the 0.1 mg/kg group, three in the 1 mg/kg groups, two in the 30 mg/kg group and five in the 100 mg/kg group.
Description (incidence and severity):
A statistically significant difference in incidence of folded retina was observed; the foetal incidence in all the treated groups was lower than the control group. Folded retina of the right and/or left eye occurred in several foetuses, but was only seen in the heads examined with Wilson;s technqieu and was therefore considered to be an artefact of processing. Foetuses in some of the exposure groups showed significant increases in skeletal alterations, however none of the changes were considered to be treatment-related as there was no evidence of a dose-response relationship.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

Actual consumed doses of ammonium perchlorate on GDs 6 to 19 were 0, 0.1, 1.0, 10.8, 33.9 and 114.2 mg/kg/day. During GDs 19 to 29, actual consumed doses of ammonium perchlorate were 0, 0.1, 0.8, 10.0, 26.7 and 90.4 mg/kg/day. Therefore, average actual consumed doses for the entire period of gestation were 0, 0.1, 0.9, 10.4, 30.3 and 102.3 mg/kg/day.

Conclusions:
There was no evidence that ammonium perchlorate is toxic to the developing foetus. An increased incidence of thyroid follicular hypertrophy was observed in does, however the thyroid is a target organ for the perchlorate ion therefore this effect is not attributed to the ammonium ion.
Executive summary:

This developmental toxicity study was conducted to evaluate the embryo-foetal toxicity and teratogenic potential of ammonium perchlorate in New Zealand White [Hra:(NZW)SPF] rabbits. Pregnant rabbits were given continual access to ammonium perchlorate in drinking water at target doses of 0, 0.1, 1.0, 10.0, 30.0, and 100.0 mg/kg/day on gestation days 6 through 28. The actual consumed doses in the study were 0, 0.1, 0.9, 10.4, 30.3, and 102.3 mg/kg/day. The rabbits were sacrificed on gestation day 29, and foetuses were examined for developmental alterations. In addition, blood was collected from does for determination of serum thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) levels and the thyroid was subjected to histopathologic examination. No maternal deaths were attributed to perchlorate exposure. Ammonium perchlorate as high as 100.0 mg/kg-day did not affect caesarean sectioning or litter parameters studied, and all values were found to be within the historical ranges of the laboratory. The litter averages for corpora lutea, implantations, litter sizes, live and dead foetuses, percent dead or resorbed conceptuses, and fetal body weights were comparable and also did not differ significantly in the six dose groups. All placentae appeared normal and no dam had a litter consisting of only resorbed conceptuses. The maternal thyroid was the target organ for ammonium perchlorate in this study. Increased incidence of thyroid follicular hypertrophy was observed in does treated with 10 mg/kg/day and above, and significantly decreased T4 was observed in does treated with 30 mg/kg/day and above. Based on these data, the maternal no-observable-adverse-effect level (NOAEL) for ammonium perchlorate was 1.0 mg/kg-day. The developmental NOAEL for ammonium perchlorate was found to be 100.0 mg/kg-day for rabbits.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Female rats were maintained, from the time of weaning, on diets containing different levels of Mn. The animals were mated and the offspring collected by caesarean section at day 21 of pregnancy. The number of implantation sites, resorptions and foetuses were recorded. The foetuses were weighed and observations made. In addition the manganese, iron, copper and zinc concentrations in the foetal body and in the liver of pregnant and non-pregnant (control) animals were determined.
GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orion Yhtyma Oy, Yla- Mankkaa Farm, Mankkaa, Finland
- body weight: 66 g
- Housing: wire bottomed stainless cages
- Diet : ad libitum
- Water :ad libitum
- Acclimation period: 8 weeks before females were placed with males


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Suitable amounts of test material were added to the salt mixture used to make up the diets.
- Storage temperature of food: -10° C
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
10 animals from groups 1, 4 and 5 and 11 animals from groups 2, 3 and 6 were mated with male rats of the same strain that had been kept on a commercial mouse and rat chow. One male was placed overnight with 3 females.
Duration of treatment / exposure:
8-10 weeks. The test substance was added to diet and administered for eight weeks prior to mating and during pregnancy.
Frequency of treatment:
Daily
Dose / conc.:
24 mg/kg diet
Remarks:
approximately 2 mg/kg bw (nominal in diet)
Dose / conc.:
54 mg/kg diet
Remarks:
approximately 5 mg/kg bw (nominal in diet)
Dose / conc.:
154 mg/kg diet
Remarks:
approximately 13 mg/kg bw (nominal in diet)
Dose / conc.:
504 mg/kg diet
Remarks:
approximately 42 a mg/kg bw (nominal in diet)
Dose / conc.:
1 004 mg/kg diet
Remarks:
approximately 84 mg/kg bw (nominal in diet)
No. of animals per sex per dose:
17 animals in each dose group
Control animals:
yes
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: after 21 days blood sample taken and haemoglobin and packed cell volume assessed.

BODY WEIGHT:
- Time schedule for examinations: animals were weighed at the start of the study, week 8 and on gestational day 0 (day of fertilization) and 21

POST-MORTEM EXAMINATIONS:
Maternal: number of implantation sites, resorptions and foetuses were recorded, liver isolated and determination of concentration of Mn, Fe, Cu and Zn was performed
Fetal examinations:
- External examinations: weighed, examination for gross malformations
- Skeletal examinations: skeletal staining
- other: determination of Mn, Fe, Cu, and Zn concentration in body.
Statistics:
Arithmetic means and standard errors were calculated. The significance of differences between the experimental groups was tested by the Student's t-test.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Early or late resorptions:
effects observed, non-treatment-related
Dead fetuses:
effects observed, non-treatment-related
Other effects:
no effects observed
Description (incidence and severity):
Mn, Fe, Cu, Zn in liver
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No impact on reproductive parameters at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
84 mg/kg bw (total dose)
Based on:
other: in diet
Basis for effect level:
dead fetuses
early or late resorptions
number of abortions
pre and post implantation loss
total litter losses by resorption
other: Mn, Fe, Cu and Zn in liver
Reduction in number of live offspring:
no effects observed
Changes in postnatal survival:
no effects observed
Skeletal malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Mn, Fe, Cu and Zn in liver.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Manganese did not cause gross malformations or bone structure anomalies at the concentrations used in this study. Foetal weight was also not affected.
Key result
Dose descriptor:
NOAEL
Effect level:
84 mg/kg bw (total dose)
Based on:
other: in diet
Basis for effect level:
reduction in number of live offspring
changes in postnatal survival
skeletal malformations
other: Mn, Fe, Cu, Zn in liver
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

No gross malformations or bone structure anomalies could be observed in the foetuses, and the Mn intake of the dams was not found to have influenced essentially the foetal weights and the dry matter and ash contents. The iron content of the livers of both pregnant and non-pregnant female rats fell as the Mn level of the diet increased. The haemoglobin values of dams on the highest levels of dietary Mn were also slightly reduced. The haemoglobin values and packed cell volumes in the non-pregnant rats were not affected by increasing dietary concentrations of Mn.

Table 1 : Body weight gain of female rats during the first eight weeks on diets of different manganese levels, and haemoglobin and packed cell volume values of pregnant (21st) day and non-pregnant rats kept on the different experimental diets (mean ± SE)

Group

Mn mg/kg diet

Body weight, g

Weeks on diet

Hb, g/100 mL

PCV, %

0

8

pregnant

non-pregnant

pregnant

non-pregnant

I

4

66 ± 1

238 ± 5

9.8 ± 0.5

14.1 ± 0.3

30 ± 2

43 ± 1

II

24

66 ± 2

235 ± 7

10.4 ± 0.3

14.3 ± 0.1

34 ± 1

43 ± 1

III

54

66 ± 2

235 ± 7

10.2 ± 0.3

14.3 ± 0.2

31 ± 1

43 ± 2

IV

154

66 ± 1

226 ± 6

9.6 ± 0.6

14.1 ± 0.2

29 ± 2

43 ± 1

V

504

66 ± 1

228 ± 6

9.4 ± 0.6

14.0 ± 0.4

30 ± 1

43 ± 1

VI

1004

66 ± 1

221 ± 5

9.4 ± 0.2

13.6 ± 0.6

30 ± 1

41 ± 2

Table 2: Reproductive performance of female rats fed diets of different manganese levels.

Group

Mn mg/kg diet

No. of dams

Body weight gain during pregnancy, g

Implantations per rat

Resorptions

%

Foetuses

Dead

%

Externally normal per rat

I

4

10

117 ± 7

12.6 ± 0.5

10.3

-

11.3 ± 0.7

II

24

11

111 ± 6

11.8 ± 1.1

3.8

-

11.4 ± 1.0

III

54

11

115 ± 5

12.6 ± 0.8

8.6

0.7

11.5 ± 0.7

IV

154

10

110 ± 5

11.6 ± 0.7

11.2

-

10.3 ± 0.8

V

504

10

 99 ± 5

11.9 ± 0.8

12.6

1.7

10.2 ± 0.8

VI

1004

11

110 ± 6

10.5 ± 0.7

8.7

-

 9.5 ± 0.9

Table 3 : Body weight and body dry matter and ash contents of foetuses from dams fed diets of different manganese levels (mean ± SE)

Group

Mn mg/kg diet

Body weight gain during pregnancy, (g)

Dry matter % of bwt

Ash % of dry wt.

I

4

4.63 ± 0.05

13.3 ± 0.1

13.1 ± 0.1

 

II

24

4.72 ± 0.04

13.2 ± 0.1

13.0 ± 0.1

 

III

54

4.73 ± 0.04

13.3 ± 0.1

13.1 ± 0.1

 

IV

154

4.67 ± 0.04

13.2 ± 0.1

13.2 ± 0.2

 

V

504

4.38 ± 0.05

13.0 ± 0.1

13.5 ± 0.3

 

VI

1004

4.75 ± 0.05

13.0 ± 0.1

13.4 ± 0.2

Conclusions:
There was no impact on foetuses at any of the doses studied. There was also no impact on reproductive parameters at any dose level.
Executive summary:

Manganese sulfate was administered to female rats in the diet at concentrations of 24, 54, 154, 504 or 1004 mg manganese/kg dry diet for 8 weeks. After 8 weeks, the animals were allowed to mate. On gestational day 21, the animals were sacrificed , the uterus removed and the number of foetuses, implantation sites and resorptions were determined. The foetuses were subject to skeletal staining and the concentrations of Mn, Fe, Cu and Zn were determined in both whole foetus and maternal liver. There was no impact on foetuses at any of the doses studied. There was also no impact on reproductive parameters at any dose level. It was also observed that increasing dietary concentrations of manganses caused a decrease in iron concentration in the foetus and in the maternal liver. The effect of manganese in diet on concentration of Cu and Zn in foetus and maternal liver was relatively modest.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Manganese chloride (Mn) was dissolved in the drinking water (0,2, or 10 mg/ml) of dams and their litters from conception until postnatal day (PND) 30.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The pH of the 10 mg/ml solution was 6.7 (the pH of the isotonic saline is 5.5).
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Females were mated. Daily vaginal smears were taken for the detection of sperm, which defined gestation day (GD) 1. At this time, the females were individually housed in polypropylene tubs.
Frequency of treatment:
Daily
Duration of test:
From conception until to postnatal day 30.
Dose / conc.:
0 other: mg/ml drinking water
Dose / conc.:
2 other: mg/ml drinking water
Dose / conc.:
10 other: mg/ml drinking water
No. of animals per sex per dose:
10 animals / dose
Control animals:
yes, concurrent vehicle
Fetal examinations:
At birth, the litters were examined and the pups were counted, sexed and weighed. The litter sizes were adjusted to eight male pups with females used to complete the litter until weaning if necessary. This yielded 50 males per group.

One rat each from eight randomly selected litters from each of the three experimental groups was sacrificed for histology at either PND 32 ± 2 or 90 ± 2.

The brains were postfixed for 24 h at 4°C after which they were washed in the phosphate buffer, dehydrated in graded ethanols, cleared in xylene, and then paraffin embedded. Sections were stained with cresyl violet or for glial fibrillary acidic protein (GFAP). Tyrosine hydrosylase (TOH) immunoreaction also using ABC/PAP methods was carried out in frozen 40 µm sections.

The analysis of cortical thickness from brain sections was carried out at PND 32 with the cresyl violet-stained sections using the computer-based image analysis system.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The 10 pregnant rats of each group had healthy pregnancies and all delivered pups. The average daily consumption of manganese during gestation was 0.35 mg/g premating body weight for the dams in the low dose level and 1.42 mg/g for the dams in the high dose level.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
10 other: mg/ml drinking water
Basis for effect level:
effects on pregnancy duration
maternal abnormalities
Key result
Abnormalities:
not specified
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Male/female ratios were also equivalent.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no differences in litter size between control and treated groups.
External malformations:
no effects observed
Description (incidence and severity):
No pup showed evidence of physical malformation.
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At PND 32, cortical thickness was significantly reduced in several areas in the high dose group. At PND 90, cortical thickness was more profoundly and uniformly reduced in both the low and high dose groups.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects


Key result
Dose descriptor:
NOAEL
Effect level:
10 other: mg/ml drinking water
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

The impact of manganese on behaviour, neurochemistry and histopathology was studied in rats exposed to manganese chloride from conception through to post-natal day 30. Exposure was either from suckling or directly from drinking water. No effect on parturition and no physical abnormalities were observed in the offspring. In animals exposed to the highest dose (10 mg/ml) a reduced weight gain was observed from day 9-24 and hyperactivity on day 17. There was also a significantly elevated level of manganese in the brain in these animals. No effect on neurochemistry was observed at either dose group. In both of the treated groups, thinning of the cerebral cortex was observed. This study includes two different exposure scenarios; exposure from suckling and from drinking water following weaning of animals. Accordingly, the exposure is much higher when directly from drinking water than when received from milk. This complicates establishing the actual dose levels. The study helps in assessing the impact of manganese in offspring following maternal exposure.

Conclusions:
There were no differences in litter size between control and treated groups. Male/female ratios were also equivalent.
No pup showed evidence of physical malformation.
Executive summary:

Manganese chloride (Mn) was dissolved in the drinking water (0, 2 or 10 mg/ml) of dams and their litters from conception until postnatal day (PND) 30. Parturition was uneventful in the Mn-exposed rats and no physical abnormalities were observed. The rats exposed to 10 mg/ml Mn showed a 2.5 -fold increase in cortical Mn levels. Their weight gain was attenuated from PND 9 -24 and they were hyperactive at PND 17. Neither the 2 nor the 10 mg/ml Mn-exposed groups differed from the controls on the elevated plus apparatus of on the Morris water maze and the radial arm maze. Brain monoamine levels and choline acetyltransferase activity were unaffected. Tyrosine hydroxylase immunohistochemistry showed that dopamine cells of the substantia nigra were intact. Glial fibrillary acidic protein immunoreactivity was not increased in cortex, caudate, and hippocampus. However, both the low- and high- dose Mn-exposed groups showing thinning of the cerebral cortex. This could have resulted from perinatal malnutrition of from a direct effect of Mn on cortical development.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Oral manganese chloride (MnCl2) doses (0, 25, or 50 mg /kg body wt /day) were given to neonatal rats throughout lactation (i.e. from postnatal day (PND) 1 through 21) and to adult male rats for 21 consecutive days. The MnCl2 doses administered to neonates were ca. 100-fold higher than those resulting from the consumption of an equivalent volume of rat’s milk. Rats were assessed using similar behavioral and neurochemical evaluations.
GLP compliance:
not specified
Species:
rat
Strain:
other: CD
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: Primiparous pregnant (gestational day 13–15) and sixweek- old male CD rats.
- Housing: All animals were housed in polycarbonate cages containing cellulose-fiber chip bedding.
- Diet (e.g. ad libitum): NIH-07 pelleted diet (Zeigler Brothers, Gardner, PA) with ca. 100 ppm manganese, ad libitum
- Water (e.g. ad libitum): filter-purified tapwater (NANOpure System; Barnstead, Boston, MA) containing <0.02 µg manganese/ml, ad libitum.
- Acclimation period: The adult male rats were acclimatized for ca. 2 weeks prior to dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 1°C
- Humidity (%): 50 ± 10%
- Photoperiod (hrs dark / hrs light): 12-h (07.00/19.00) lightdark photoperiod cycle.
Route of administration:
other: oral: by gavage (adult rats) or by mouth using a micropipette (pups)
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Manganese was dissolved in Nanopure water (1 ml/kg)
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Primiparous pregnant (gestational day 13–15).
The date of parturition was designated as postnatal day 0 (PND 0).
Duration of treatment / exposure:
21 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Adult male: 20 rats per dose
Adult female: 10 litters per dose
Control animals:
yes
Maternal examinations:
The weights of adult rats were determined individually and body weight gain was calculated as the difference from the previous body weight.

Clinical examinations were performed on all animals before each daily manganese administration.

Other: motor activity, passive avoidance, acoustic startle, functional observational battery (FOB), brain catecholamine assay, regional manganese determinations, neuropathology.
Fetal examinations:
On PND 4, litters were randomly reduced to four animals per sex whenever possible. Litters of less than eight pups were not used.

Pups were weighed daily.

Spontaneous motor activity was assessed using an automated photobeam activity system (San Diego Instruments, San Diego CA) during the animals’ daily light cycle.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Adult rats given manganese demonstrated no significant change in body weight gain.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant differences in brain weight were observed in any manganese-exposed rats. Manganese exposure was not associated with any treatment-related clinical signs or deaths.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
body weight and weight gain
clinical signs
mortality
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Daily manganese exposure of neonatal pups from the time of birth through weaning (PND 1–21) was associated with significantly decreased body weight gain in the high-dose manganese group.
Other effects:
no effects observed
Description (incidence and severity):
Brain neuropathology
Central nervous system neuropathology was assessed only in neonatal rats. Fixation and processing of the brain tissues from this age group (PND 21) were
excellent. No gross or microscopic abnormalities were observed in the central nervous system of any neonatal rats exposed to manganese.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
other: behaviour
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

No statistically significant treatment-related effects on overall motor activity were observed during any postexposure motor activity test sessions in PND 13, PND 17 or PND 21 ± 1 neonatal or adult rats. No statistically significant differences in FOB assessments were observed in adult rats following high-dose manganese exposure. Neonatal manganese exposure was not associated with altered cognitive function, as assessed by performance pn a step-through passive avoidance task. Initial (day 1) cross-over latencies from each treatment group were not affected by manganese exposure. Although not statistically significant, the neonatal pups given manganese had a trend toward decreased time before cross-over during testing of the memory component of a two-day passive avoidance paradigm.

Neonatal manganese exposure was associated with a significant increase in the magnitude of the acoustic startle reflex during pulse-elicited trials. When compared to control weanling rats, a significant increase in the overall group mean acoustic startle amplitudes elicited during pulse trials was observed in rats neonatally exposed to manganese and then tested on PND 21. For example, mean acoustic startle amplitude elicited during pulse trials was 118 and 121% of the controls in the low- and high-dose manganese groups, respectively. Manganese exposure did not affect the amplitude of the acoustic startle reflex following prepulse trials in any treatment group. A significantly decreased overall mean acoustic startle amplitude elicited during pulse trials was observed in adult rats from the low-dose manganese exposure group. This depression in adult startle reactivity did not demonstrate any dose–response correlation, so the toxicological significance of this observation is unclear.

Conclusions:
No treatment-related changes were observed in clinical signs, motor activity (assessed in neonates on PND 13, 17, 21 ± 1 and in adults), passive avoidance (assessed in neonates on PND 20 ± 1 and in adults) or neuropathology (assessed in PND 21 neonates only).
Executive summary:

Adult and neonatal rats were administered manganese chloride via the oral route at a dose of either 0, 25 or 50 mg/kg bw. The concentration of manganese delivered to the pups was 100-fold higher than in milk from lactating dams. Reduced bodyweight gain and increased acoustic startle reflex were observed in pups in the highest dose group. No other changes in behavioural parameters or effect on neuropathology were seen. The levels of manganese in most regions of the brain were increased in pups and adults following manganese exposure. No treatment-related changes were observed in clinical signs, motor activity (assessed in neonates on PND 13, 17, 21 ± 1 and in adults), passive avoidance (assessed in neonates on PND 20 ± 1 and in adults) or neuropathology (assessed in PND 21 neonates only).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
See discussion.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified for toxicity to reproduction.

Additional information