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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Foetal Development in Rats Fed AIN-76A Diets Supplemented with Excess Calcium
Author:
Shackelford ME, Collins TFX, Welsh JJ, Black TN, Ames MJ, Chi RK & O'Donnell MW
Year:
1993
Bibliographic source:
Fd. Chem. Toxic., 31(12), 953-961

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Minor deviations from the guideline which do not affect the outcome of the study.
Principles of method if other than guideline:
This study was designed to evaluate the developmental effects of moderate dietary calcium increases in rats fed nutritionally adequate diets. Female Charles River CD/VAF Plus rats were given 0.50 (control), 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate in AIN-76A diets for 6 weeks before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 98.62 %

Test animals

Species:
rat
Strain:
other: CD/VAF Plus Charles River
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Wilmington, MA, USA)
- Age at study initiation: Females - 52 days old; Males - 44 days old
- Weight at study initiation: Females - approximately 217 ± 1 g
- Fasting period before study: None
- Housing: Stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 27 °C
- Humidity (%): 25 - 72 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Once
- Mixing appropriate amounts with (Type of food): AIN-76A Diet
- Storage temperature of food: -1 - -6 °C

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration of calcium analysed using the methods of the Association of Official Analytical Chemists
Details on mating procedure:
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: No data - at least overnight for one night
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Females: 6 weeks prior to mating, through mating to day 20 of gestation
Frequency of treatment:
Rats allowed access to feed ad libitum
Duration of test:
Approximately 62 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0.50 (control), 0.75, 1.00 or 1.25 % calcium
Basis:
nominal in diet
No. of animals per sex per dose:
Females: Pre-mating - 69/dose; Pregnant rats - 45-48/dose
Males: Received test material only during the mating period
Control animals:
other: The group receiving 0.5% calcium were used as the control group

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Every 3 days for the 6 weeks before mating and during gestation, but not during mating (females only)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes (females only)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Major organs (no further details reported)


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
The incidence of clinical parameters was analysed by comparing treatment groups with the control group by using Fisher's exact test.
Data on feed consumption were analysed using analysis of variance (ANOVA) and a protected least significant difference (LSD) test (two-tailed).
Weight gains were analysed using analysis of covariance (ANOCOVA) after adjusting for the initial body weight, and an LSD test (two-tailed).
Trend analysis was performed on feed consumption.
The number of corpora lutea, the average number of implantations, the average number of viable foetuses for each litter and the average number of male or female foetuses for each litter were analysed by ANOVA and a LSD test (one-tailed).
Data on implantation efficiency, early deaths, late deaths, and total resorptions (early and late deaths) were analysed by the Freeman-Tukey arcsine transformation for binomial proportions. The transformed data for each litter were then analysed by ANOVA and a LSD test (one-tailed), comparing the control group with experimental groups.
Fisher's exact test was used to analyse data on litters with runts and litters totally resorbed, comparing the controls group with treatment groups.
Foetal body weights and crown-rump lengths were analysed by a nested ANOVA and a LSD (one-tailed).
Data on the specific litter incidence of sternebral, skeletal and visceral variations were analysed using Fisher's exact test.
Proportions of litters with foetuses showing external variations were analysed using Fisher's exact test.
The average number of foetuses with variations per litter was analysed using the Freeman-Tukey arcsine transformation and then an ANOVA and a protected LSD test (one-tailed).
Litters that had foetuses with one or more sternebral, skeletal and visceral variations were analysed with Fisher's exact test.
Trend analysis was performed on the litter data for external variations using the Cox exact one-tailed test for unadjusted positive trend.
Historical control data:
No historical control data were provided

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical findings included alopecia, exudate around the eyes, exudate around the nose, bent teeth, lesion, lump in the left flank or leg and mammary lump. The number of animals with alopecia was significantly increased in animals fed 1.00 % calcium (8.7 vs 0 % in the control) during the 6 weeks before mating and in the groups fed 0.75 and 1.00 % calcium (10 and 16.3 % respectively vs 0 % in the control) during gestation.
One non-pregnant animal was observed to have diarrhoea on day 4 after assignment to the 1.25 % calcium diet.
One pregnant female fed 1.00 % dietary calcium developed bleeding from the vagina on days 15 and 16 of gestation. The incidence of diarrhoea and bleeding from the vagina was not considered to be related to the dietary calcium.
Three females died during the study, but the deaths were not attributable to excess calcium in the diet.
Values for mean daily feed consumption per female given excess dietary calcium for 6 weeks before mating and during gestation are shown in Table 1. The 0.75, 1.00 and 1.25 % groups ate slightly more than the controls during most of the study, but not all the increases were statistically significant.
Mean values for body weight gain for animals given excess dietary calcium for 6 weeks before mating and gestation are given in Table 2. Although some differences in weight gain between control and treated groups were significant, there was no consistent pattern of increase or decease in the body weight gain.
Maternal reproductive parameters are listed in Table 3. There were no adverse reproductive effects associated with ingestion of excess dietary calcium. Significant increases were observed in implantation efficiency at 1.25 % dietary calcium and in the average number of viable female foetuses at 0.75 and 1.25 % dietary calcium. These increases were not dose related and were not considered to be caused by dietary calcium.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
> 1.25 other: % w/w calcium
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
> 1.25 other: % w/w calcium
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no adverse effects to the foetuses caused by feeding excess dietary calcium to the dams (Table 3). Foetal body weights and crown-rump lengths of male and female animals given excess dietary calcium were similar to those of the control groups. Feeding excess dietary calcium had no effect on the number of litters with male or female runts. The incidences of specific external variations are shown in Table 4. In the control group, one foetus did not possess a mouth (astomia) and a lower jaw (agnathia). At the 1.25 % dietary calcium level, one foetus had multiple anomalies: agnathia, cleft palate, protruding tongue, beak-like snout, malformed head, open eye on the right side, oedema of the trunk, omphalocele, exposed heart and lungs (diaphragmatic cleft), club foot on the left front paw, scoliosis, imperforate anus and kinked tail. The occurrence of this foetus with multiple anomalies was not thought to be related to feeding the rats excess dietary calcium before and during pregnancy. The data in table 4 were analysed by trend analysis and Fisher's exact test. The Cox exact trend test showed no significant positive trends in the litter incidence of individual or combined variations. When analysed by Fisher's exact test, there were no significant differences between treated and control groups in the litter incidences of these same parameters. Under the conditions of the study, there were no significant increases in the litter incidence of specific or combined external variations. The numbers of foetuses with sternebral ossification deficiencies and the litter incidences are summarised in Table 5. A comparison of the proportion of litters with foetuses having sternebral variations showed no significant differences among the groups given excess calcium and the control group. The increase in the number of litters with foetuses with bipartite sternebrae in the 1.25 % group was not significant (p=0.0854). The incidences of sternebral variations are presented in Table 6. The variations counted are based on the classification of the types and not on the individual sternebrae affected. The average number of foetuses per litter with one or more sternebral variations increased in the 0.75, 1.00 and 1.25 % groups, but the differences were not significant. There was no significant increase in the average number of foetuses per litter with at least two or three sternebral variations in the groups fed excess dietary calcium. Statistical analysis of the proportion of litters that had foetuses with variations (at least one, two or three) showed no significant increase between the excess calcium and the control groups. The incidences of specific skeletal variations, excluding sternebrae, were not dose related and there were no significant differences among any of the values (Table 7). The average number of sacral and caudal vertebrae was not affected by dietary calcium (data not shown). The average number of foetuses per litter with at least one skeletal variation was lower in the 0.75, 1.00 and 1.25 % dietary calcium groups than in the control (Table 8). However, the ANOVA of the Freeman-Tukey arcsine-transformed data showed no differences in the average number of foetuses with variations (p>0.10). The proportion of litters that had foetuses with variations (at least one, two or three) showed no dose-related or significant differences between any of the values obtained in this study. There was no dose related effect on the incidence of any other type of soft-tissue variation (Table 9). The proportion of litters with hydrocephalus was greater in the control group than in the treatment groups, but the differences were not significant. The incidence of visceral problems by foetus and by litter is shown in Table 10. The average number of foetuses with at least one, two or three soft-tissue variations was similar in all groups. Although the average number of foetuses with at least one variation was lower in the 1.00 and 1.25 % groups, ANOVA did not indicate a significant decrease compared with controls. Although there was a dose-dependent decrease in the proportion of litters with foetuses with at least one variation. Fisher's exact test (two-tailed) did not show any significant difference. Thus the proportion of litters with foetuses with at least one two or three soft-tissue variations was similar in all groups.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean daily feed consumption (g/animal/day) of rats given excess dietary calcium.

Concentration of Calcium in the Diet (%)

Days

0.5

0.75

1.00

1.25

Non-pregnant rats 6 weeks before mating

(69)

(69)

(69)

(69)

0-6

15.2 ± 0.4

15.7 ± 0.3

15.8 ± 0.3

16.4 ± 0.4

6-12

17.4 ± 0.3

17.7 ± 0.3

17.8 ± 0.3

18.2 ± 0.3

12-18

17.0 ± 0.3

17.6 ± 0.3

18.1 ± 0.3b

18.4 ± 0.3c

18-24

17.9 ± 0.3

18.1 ± 0.3

18.8 ± 0.3a

18.9 ± 0.3b

24-30

17.6 ± 0.3

17.9 ± 0.3

18.8 ± 0.3b

18.6 ± 0.3a

30-36

17.8 ± 0.3

17.7 ± 0.3

18.6 ± 0.3d

18.4 ± 0.2

36-42

17.8 ± 0.3

17.8 ± 0.3*

18.6 ± 0.3a

18.5 ± 0.3d

0-42

17.3 ± 0.2

17.5 ± 0.2*

18.1 ± 0.2a

18.2 ± 0.2b

Pregnant rats from days 0-20

(45)

(47)

(44)

(48)

0-6

19.6 ± 0.4

20.4 ± 0.4

20.8 ± 0.4a

21.0 ± 0.3b

6-12

22.8 ±0.4

23.3 ± 0.3

23.9 ± 0.4d

24.2 ± 0.4a

12-18

23.5 ± 0.4

24.3 ± 0.3

24.1 ± 0.5

24.2 ± 0.3

18-20

23.7 ± 0.5

24.0 ± 0.4

23.7 ± 0.6

23.0 ± 0.5

0-20

22.2 ± 0.4

22.8 ± 0.3

23.0 ± 0.4

23.1 ± 0.3

* Because one animal was autopsied in extremis on day 40 in the 0.75% group the number of rats was reduced to 68 for the data for days 36 -42 and 0 -42.

Number of animals are indicated in parantheses. Values are means ± SEM. superscripts indicate differences between control and test values ( aP ≤0.05; bP≤0.01; cP ≤0.001; d 0.05 P 0.01 )

Table 2: Mean body weight gains and initial body weights of rats given excess dietary calcium

Concentration of Calcium in the Diet (%)

Days

0.5

0.75

1.00

1.25

Body weight  gain (g) of non-pregnant rats 6 weeks before mating

(69)

(69)

(69)

(69)

0-6

8.1 ± 1.4

9.9 ± 1.2

9.1 ± 1.1

7.7 ± 1.4

6-12

22.1 ± 1.1

21.1 ± 0.9

21.7 ± 1.1

20.1 ± 1.1

12-18

12.1 ± 0.8

14.7 ± 0.9 a

16.1 ±1.0 a

16.3 ± 0.7 a

18-24

16.3 ± 0.8

14.0 ± 0.7 a

15.7 ± 0.9

14.1 ± 0.7 a

24-30

9.0 ± 0.6

9.8 ± 0.8

11.9 ± 0.9 a

10.6 ± 0.7

30-36

11.0 ±0.9

9.2 ± 0.8

10.1 ± 1.0

9.6 ± 0.8

36-42

5.3 ± 0.7

5.8 ± 0.9*

4.9 ± 0.8

6.4 ± 0.6

0-42

83.9 ± 2.9

85.1 ± 3.1*

89.5 ± 3.0

84.7 ± 2.6

Initial body weight (g) of non pregnant rats

0

217.7 ± 1.0

217.6 ± 1.0

217.4 ± 1.0

217.7 ± 1.0

Body weight gain (g) of pregnant rats

(45)

(47)

(44)

(48)

0-6

29.8 ±1.2

29.7 ± 1.3

29.8 ± 1.1

29.4 ± 1.0

6-12

30.4 ± 1.0

32.0 ± 0.8

30.2 ± 0.7

31.9 ± 0.9

12-18

45.2 ± 1.8

51.1 ± 1.4a

48.0 ± 2.6

48.9 ± 1.5

18-20

25.8 ± 1.1

27.9 ± 0.9

26.2 ± 1.1

25.1 ± 1.0

0-20

131.2 ± 3.1

140.7 ± 2.6

134.2 ± 3.9

135.3 ± 3.0

Initial body weight (g) of pregnant rats

0

297.6 ± 3.6

300.8 ± 3.7

306.5 ± 4.8

300.0 ± 3.8

* Because one animal was autopsied in extremis on day 40 in the 0.75% group the number of rats was reduced to 68 for days 36-42 and 0 -42

Values marked with superscripts indicate significant differences between control and test values aP ≤0.05; bP≤0.01; cP ≤0.001; d 0.05 P 0.10

Table 3: Maternal and reproductive outcome at autopsy and foetal data from rats given excess dietary calcium

Concentration of Calcium in the Diet (%)

Parameter

0.5

0.75

1.00

1.25

No. of pregnant females

45

47

44

48

No. of corpora lutea/female

16.27 ± 0.4

16.83 ± 0.40

16.52 ± 0.27

15.93 ± 0.32

Implantation frequency

 (% female)*$

82.01 ± 3.73

90.29 ± 1.89

86.51 ± 3.46

93.41 ± 2.28b

No of implants/female*

13.58 ± 0.68

15.26 ± 0.43

14.25 ±  0.60

15.15 ± 0.50

No of viable foetuses/female*

11.82 ± 0.66

13.53 ± 0.46

 12.82 ± 0.65

13.54 ± 0.58

No of viable foetuses/litter*

6.29 ± 0.43

6.49 ± 0.33

6.95 ± 0.49

6.83 ± 0.37

Viable female foetuses/litter*

5.53 ± 0.39

7.04 ± 0.38b

5.86 ± 0.37

6.71 ± 0.43a

Resorptions/female (mean %)*‡

12.50 ± 1.98

11.15 ± 1.73

12.92 ± 3.19

13.81 ± 3.09

No of early deaths/litter$

1.71

1.70

1.41

1.56

No. of late deaths/litter$

0.04

0.02

0.02

0.04

No of early and late deaths/litter$‡

1.76

1.72

1.43

1.60

No of litters totally resorbed

0

0

1

2

Body weight (g)

Males

3.84

3.90

3.83

3.79

Females

3.61

3.69

3.66

3.58

Crown rump length (cm)

Males

4.0

4.0

4.0

4.0

Females

3.9

3.9

3.9

3.9

No of runts

Males

5

5

3

3

Females

4 (4)

4 (1)

1 (1)

3 (2)

* Means ± SEM

$ Statistical analysis was performed on these data after application of the Freman-Tukey arcsine transformation

‡ The number of resorptions includes early deaths and late deaths per litter

§ The number of litters is indicated in parentheses

Values marked with superscripts indicate significant differences between control and test values aP ≤0.05; bP≤0.01; cP ≤0.001; d 0.05 P 0.10

Table 4: Incidence of specific external variations in foetuses from rats given excess dietary calcium

Concentration of Calcium in the Diet (%)

Parameter

0.5

0.75

1.00

1.25

No. foetuses (litters examined)

532 (45)

636 (47)

564 (43)

650 (46)

No of foetuses (litters with variations)

2 (2)

2 (2)

1 (1)

8 (6)

Heamorrhages, external

1 (1)

-

1 (1)

2 (2)

Cleft palate

-

1 91)

-

2 (2)

Agnethia

1 (1)

-

-

1 (1)

Astomia

1 (1)

-

-

-

Thread like tail

-

1 (1)

-

1 (1)

Kinked tail

-

-

-

2 (2)

Oedema

-

-

-

3 (2)

Malformed head

-

-

-

1 (1)

Beaked snout

-

-

-

1 (1)

Protruding tongue

-

-

-

1 (1)

Open eye

-

-

-

1 (1)

Diaphragmatic cleft*

-

-

-

1 (1)

Omphalocele

-

-

-

1 (1)

Club foot

-

-

-

1 (1)

Seollosis

-

-

-

1 (1)

Imperforate anus

-

-

-

2 (2)

The number of litters is indicated parentheses

* This parameter includes exposed heart and lungs

Table 5: Incidence of specific external variations in foetuses from rats given excess dietary calcium

Concentration of Calcium in the Diet (%)

Parameter

0.5

0.75

1.00

1.25

No. foetuses (litters examined)

251 (44)

305 (47)

273 (43)

313 (46)

No of foetuses with specific stenebral variations

Incomplete ossification

74 (31)

84 (35)

88 (36)

95 (35)

Bipartite

4 (4)

3 (3)

4 (3)

10 (10)d

Unossified

50 (27)

49 (27)

40 (18)

54 (31)

Malagnified

8 (7)

2 (2)

10 (9)

11 (11)

Fused

1 (1)

0 (0)

0 (0)

1 (1)

The number of litters is indicated parentheses

Superscript indicates differences between control and test values (d0.05  P 0.10)

Table 6: Incidence of sternebral variations in foetuses of rats given excess dietary calcium

Concentration of Calcium in the Diet (%)

Parameter

0.5

0.75

1.00

1.25

Sternebral variations*

Total no.

137

138

142

171

Mean no./litter

3.11

2.94

3.30

3.72

Foetuses with one or more sternbral variations

Total no.

105

118

112

138

Mean no./litter$

2.39

2.51

2.60

3.00

Foetuses affected (%)

41.83

38.69

41.03

44.09

Litters with foetuses with one or more variations

Total no.

37

42

38

39

Litters affected (%)

84.09

89.36

88.37

84.78

No of foetuses examined

251

305

273

313

No of litters examined

44

47

43

46

* Statistical analysis was not performed on this parameter

$ Statistical analysis was performed on these data after application of the Freeman-Tukey arcsine transformation

Applicant's summary and conclusion

Conclusions:
Both the non-pregnant and pregnant rats in the 0.75, 1.00 and 1.25% groups ate slightly more than did the control group during most of the intervals measured, but not all the increases were statistically significant. There was no consistent pattern of increase or decrease in weight gain. No dose-related changes were found in maternal clinical findings, the average number of implantations, resorptions and viable foetuses, or foetal length or weight. Under the conditions of the study, there were no statistically significant increases as compared with the control group in the litter incidence regarding specific external, visceral or skeletal variations of the foetuses. Dietary calcium was neither foetotoxic nor teratogenic at the concentrations used.

Therefore, as no adverse effects were noted at this dose level (1.25% Ca in diet), the NOAEL for teratogenic and maternal toxic effects in rats is in excess of 1963 - 2188 mg/kg bw/day of calcium carbonate.