Reaction mass of calcium dihydroxide and calcium peroxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

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 Type:

publication

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Qualifier:

no guideline followed

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 wk 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

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

Specific details on test material used for the study:

Source: Pfizer (Adams, MA, USA)
Purity: 98.62%

Test animals

Species:

rat

Strain:

other: CD/VAF

Details on test animals or test system 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 (non-pregnant) 217 g.
- 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
- Humidity (%): 25-72
- Photoperiod (hrs dark / hrs light): 12-hrlight/dark cycle

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION:
The test material was incorporated into a modified AIN-76A. Calcium phosphate and calcium carbonate were added separately to adjust the diets to specified levels of calcium and phosphorus. Calcium phosphate was the primary source of calcium and phosphorus. Dietary calcium was adjusted to 0.50, 0.75, 1.00 and 1.25% with calcium carbonate, and dietary phosphorus was maintained at approximately 0.40%. At the time of preparation, a separate lot was prepared for each of the specified dose levels of calcium. The
diets were assayed for homogeneity and concentrations of calcium, phosphorus, magnesium, manganese, zinc, iron and copper. The diets at 1.0 and 1.25% calcium were slightly higher in iron than the other diets used in this study because of contributions from calcium carbonate. The diets were stored in a walk-in refrigerator (-1 to -6°C) to maintain nutrient stability

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

After 6 wk, the female rats were mated to the males in a 2:1 ratio, with cohabitation beginning at approximately 4.30 pm. The following morning, a vaginal smear was made for each female to determine copulation. The day on which sperm was found in the vaginal smear was considered to be day 0 of gestation.

Duration of treatment / exposure:

6 wk before mating, throughout mating and for 20 days of gestation.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.5 other: % calcium in diet

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

0.75 other: % calcium in diet

Doses / concentrations 3

Dose / conc.:

1 other: % calcium in diet

Doses / concentrations 4

Dose / conc.:

1.25 other: % calciumm in diet

No. of animals per sex per dose:

A total of 156 male and 291 female

Control animals:

yes

Examinations

Maternal examinations:

TISSUE MINERAL LEVELS:
Of the 291 female animals used in the study, 135 were randomly selected for the determination of tissue mineral levels at three times during the study: 15 animals before assignment of the remaining 276 to diet groups; 60 non-pregnant animals (15 per group) after 6wk on the diets; and 60 pregnant animals (15 per group) at the time of caesarean section on day 20 of gestation.

BODY WEIGHT AND FEED CONSUMPTION
Female animals were weighed and feed consumption was measured every 3 days for 6 wk before mating and during gestation, but not during mating.

POST-MORTEM EXAMINATIONS
A laparotomy was then performed on each dam, and the major organs were examined.

Ovaries and uterine content:

The uterus was opened and examined in situ for the presence and position of resorption sites and foetuses (dead or alive) and the number of implantation sites. Deciduomas (brownish implantation sites without placentas) were considered early deaths; implantation sites with placentas and complete but non-viable foetuses that were of subnormal size, showed retarded development or were in a macerated condition were classified as late deaths. The uteri of animals that did not appear to be pregnant were stained with sodium sulfide to enhance resorption sites. Corpora lutea were counted under a lighted magnifying lens.

Fetal examinations:

Foetuses were examined individually for gross abnormalities. The uterine position, sex, weight and crown-rump length of each foetus were recorded.
Any foetus that weighed less than 70% of the average weight of the concurrent male or female controls was considered to be a runt.
Foetuses were assigned to skeletal or visceral analysis.
From 12 litters in each group, two foetuses (one from each uterine horn) were randomly selected and used for whole-body mineral determinations.
Approximately half of the foetuses were examined microscopically for skeletal variations.
The remaining half were examined for internal variations of the soft tissues.

Statistics:

Data on feed consumption were submitted to an analysis of variance (ANOVA) and a protected least-significant difference (LSD) test (two-tailed). Data on weight gain were submitted to an analysis of covariance (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, since analysed by covariance. A trend is present if the linearity P value is below 0.05 (borderline trend if below 0.10) and, at the same time, the departure P value is above 0.10. 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 an LSD test (one-tailed). Data on implantation efficiency, early deaths, late deaths and total resorptions (early and late deaths) were analysed by using the Freeman-Tukey arcsine transformation for binomial proportions. Then transformed data for each litter by ANOVA and an LSD test (one-tailed) was analysed. Data on litters with runts and litters totally resorbed was analysed by Fishcer's exact test. Foetal body weights and crown-rump lengths were analysed by a nested ANOVA and an LSD test (one-tailed). Specific litter incidence of sternebral, skeletal and visceral variations was analysed by using Fisher's exact test. Proportions of litters with foetuses showing external variations were analysed by using Fisher's exact test. The average number of foetuses with variations per litter was analysed by 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 on the litter data for external variations was done, using the Cox exact one-tailed test for unadjusted positive trend (significant if P < 0.05).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical findings included the following: alopecia, exudate around the eyes, exudate around the nose, bent teeth, lesion, lump in the left flank or leg and mammary lump. None of the above findings was dose related. 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three females died during the study, but the deaths were not attributable to excess calcium in the diet.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Initial body weight on day 0 for non-pregnant rats and sperm-positive females was similar for all groups. Although some differences in weight gain between control and treated groups were significant, there was no consistent pattern of increase or decrease in the body weight gain. Weight gain increased during some intervals measured but decreased for other intervals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The 0.75, 1.00 and 1.25% groups ate slightly more than the controls during most of the intervals measured, but not all the increases were statistically significant. The
trend analysis for feed consumption for non-pregnant rats showed a significant (P < 0.05) positive trend during most intervals measured, but the trend was borderline positive (P =0.0514) during days (6-12). There was no linear trend during days 12-18 and 18-20 of gestation, but there was a significant positive trend (P < 0.05) during the other intervals measured.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1.25 other: % calcium in diet

Based on:

test mat.

Basis for effect level:

other: No test item related adverse effects observed at the highest dose tested

Maternal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no adverse effects to the foetuses caused by feeding excess dietary calcium to the dams. 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.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

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 fight 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.

Skeletal malformations:

no effects observed

Description (incidence and severity):

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.

Visceral malformations:

no effects observed

Description (incidence and severity):

There was no dose-related effect on the incidence of any type of soft-tissue variation. The proportion of litters with hydrocephalus was greater in the control group than in the treated groups, but he differences were not significant.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1.25 other: % calcium in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related adverse effects observed at the highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for maternal toxicity, reproduction and developmental toxicity was determined to be 1.25% calcium in diet since no test related adverse effect was observed at the highest dose tested in rats.

Executive summary:

The 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 wk before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed. 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. Based on these results, the NOAEL for maternal toxicity, reproduction and developmental toxicity was determined to be 1.25% calcium in diet since no test related adverse effect was observed at the highest dose tested.