Calcium acetylide

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Foetal development in rats fed AIN-76A diets supplemented with excess calcium

Author:

Shackelford ME, Collins TF X, Welsh JJ, Black TN, Ames MJ, Chi RK and O'Donnell M W

Year:

1993

Bibliographic source:

Fd Chem. Toxic. Vol. 31, No. 12, pp. 953-961

Reference 2

Reference Type:

publication

Title:

Mineral interactions in rats fed AIN-76A diets with excess calcium

Author:

Shackelford ME, Collins TF X, Black TN, Ames MJ, Dolan S, Sheikh NS, Chi RK and O'Donnell M W

Year:

1994

Bibliographic source:

Fd Chem. Toxic. Vol. 32, No. 3, pp. 255-263. 1994

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The main objective of the study (Shackelford et al., 1993) was to examine potential effects of high dietary calcium in rats on pregnancy and development of the foetus. Female rats were fed a diet containing various levels of calcium, 6 weeks before mating, throughout mating and 20 days during gestation. Non pregnant animals and pregnant animals fed with normal calcium levels were used as controls. On gestation day 20, the animals were killed and caesarian sections were performed. The uterine contents were examined and the foetuses were evaluated for soft tissue and skeletal changes. In a further publication (Shackelford et al., 1994), the effect of high calcium intake on other mineral tissue levels was analyzed. Some data of this publication are also added in this endpoint summary, cause they provide a better insight on the experimental procedures of the study.

GLP compliance:

not specified

Remarks:

not applicable for publications

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Calcium carbonate

EC Number:

207-439-9

EC Name:

Calcium carbonate

Cas Number:

471-34-1

Molecular formula:

CH2O3.Ca

IUPAC Name:

calcium carbonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

US- Pharmacopeia-grade calcium carbonate (Pfizer)
-Purity: 98.62%

Test animals

Species:

rat

Strain:

other: CD/VAF Plus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc.
- Age at study initiation: 52 days
- Housing: stainless steel
- Diet: modified AIN-76A
The animals were kept on a 6 day quarantine before the initiation of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-27
- Humidity (%): 25-72
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The detailed composition of the diet is provided in Table 1 of the attached document (see below).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Yes, method of the Association of Official Analytical Chemists.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: from 4.30 pm till morning
- Further matings after two unsuccessful attempts: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

6 weeks before mating, throughout mating and 20 days during gestation (only female animals were exposed)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 000 mg/kg diet

Doses / concentrations 2

Dose / conc.:

7 500 mg/kg diet

Doses / concentrations 3

Dose / conc.:

10 000 mg/kg diet

Doses / concentrations 4

Dose / conc.:

12 500 mg/kg diet

No. of animals per sex per dose:

291 animals participated in the study in total. 15 rats were selected randomly for evaluation of the tissue mineral status and histopathology (baseline animals).

Control animals:

yes

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes
- Food consumption for each animal was determined 3 days per week during the 6 wk pre-mating period

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
All organs/tissues were examined grossly. Histopathological examination was performed for the heart, liver, kidneys and bones
-Organ weights measured: liver, kidney, heart

Ovaries and uterine content:

The 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:

Uterine position, sex, weight and length were recorded for each foetus.
- Visceral examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

The incidence of clinical observations: Fisher's exact test. Feed consumption: (ANOVA) and protected least-significant difference (LSD) test (two-tailed). Weight gain: (ANOCOVA), after adjusting for the initial body weight, and an LSD test (two-tailed). Trend analysis was done on feed consumption. It was not done on body weight data, because we analysed these data by covariance. No of corpora lutea, average No of implantations, average No of viable foetuses/litter, average No male or female foetuses/ litter : ANOVA and an LSD test (one-tailed). Implantation efficiency, early deaths, late deaths and total resorptions (early and late deaths): Freeman-Tukey arcsine transformation for binomial proportions (Freeman-Tukey, 1950). Transformed data for each litter by ANOVA and an LSD test (one-tailed), comparing the control group with experimental groups. Data on litters runts and resorbed litters comparing control to treated: Fisher's exact test. Foetal body weights and crown-rump lengths: nested ANOVA (Sokal and Rohlf, 1981) & LSD test (one-tailed). Specific litter incidence of sternebral, skeletal andvisceral variations, control with treatment groups: Fisher's exact test. Proportions of litters with foetuses showing external variations: Fisher's exact test. Average No of foetuses with variations/ litter: Freeman-Tukey arcsine transformation, ANOVA and 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 on the litter data for external variations, using the Cox exact one-tailed test for unadjusted positive trend. A trend is significant if the P value is below 0.05. Trend analysis was not done on data that were analysed by using the Freeman-Tukey arcsine transformation for binomial proportions.

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: alopecia, exudate around the eyes, exudate around the nose, bent teeth, lesion, lump in the left flank or leg and mammary lump. However, no dose response association. Three females died during the study (non-treatment related). The treatment showed some variations on the food consumption; however, not all differences were statistically significant. No body weight gain effects were detected. Relative organ weights to body weights of the treated groups were not significantly different from the controls. Implantations, resorptions and litter sizes were comparable to controls and did not appear influenced by the high calcium consumption.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

227.5 other: mg calcium/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

288.75 other: mg calcium/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No adverse effects were detected on the foetuses. Foetal body weights and lengths were within the normal control values. No effect related to the treatment was seen on the number of litters.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1.25 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

More details on the results are presented in the attached pdf document.

Applicant's summary and conclusion

Conclusions:

There was no adverse developmental/teratogenic effects related to high dietary calcium intake, i.e. 1.25% of the daily diet, of animals, 6 weeks before mating, throughout mating and 20 days during pregnancy. Additionally, such levels did not affect the reproductive performance parameters examined in this study.

Executive summary:

In the present study, the potential effects of high dietary calcium on pregnancy and development of the foetus were studied in rats. Female Charles River CD/VAF Plus rats were fed a diet containing various levels of calcium, i.e. 0.5 (control), 0.75, 1.00, or 1.25% as calcium carbonate, 6 weeks before mating, throughout mating and 20 days during gestation. On gestation day 20, the animals were killed and caesarian sections were performed. The uterine contents were examined and the foetuses were evaluated for soft tissue and skeletal changes. The treatment showed some variations on the food consumption of the females. Nonetheless, not all differences were statistically significant, and they could not be attributed to the treatment. No body weight gain effects were detected. Relative organ weights to body weights of the treated groups were not significantly different from the controls. Implantations, resorptions and litter sizes were comparable to controls and did not appear influenced by the high calcium consumption. It can be concluded, that calcium up to 1.25% in the diet, is not foetotoxic or teratogenic.