Calcium 3,5,5-trimethylhexanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented publication. Under physiological conditions the carbonate contained in the test substance calcium carbonate is released as CO2 upon contact with the acid environment in the stomach and is therefore not relevant for consideration of systemic toxicity. The study allows the derivation of a NOAEL value for effects of Ca regarding developmental toxicity.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

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

Author:

Shackelford, M.E.; et al.

Year:

1993

Bibliographic source:

Fd Chem. Toxicol. 31, 953-961

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

no

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:

CaCO3

IUPAC Name:

calcium carbonate

Details on test material:

- Name of test material (as cited in study report): Calcium carbonate
- Purity: 98.62%
- Physical state: solid
No further details are given.

Test animals

Species:

rat

Strain:

other: Charles River CD/VAF Plus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Wilmington, MA, USA)
- Age at study initiation: when received, the females were 52 days old and the males were 44 days old
- Weight at study initiation: 217 g (females)
- Housing: Animals, identified by numbered metal ear tags, were housed in stainless-steel cages.
- Diet: ad libitum; females received powdered AIN-76A diet for 6 weeks before and throughout mating and for 20 days of gestation; male rats were fed pelleted Purina Rodent Chow except during mating, when they also received one of the four experimental diets.
- Water: ad libitum, deionised water
- Acclimation period: 6 days

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

No further details are given.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with laboratory diet: The formulation of powdered AIN-76A diet is recommended as nutritionally adequate for rats by the American Institute of Nutrition. The mineral mix of the AIN-76a diet was slightly modified. The copper content was inceased from 6 to 9 mg/kg diet by seperate addition of cupric carbonate. Dietary calcium was adjusted with calcium carbonate.
- Storage temperature of food: During the duration of the study, the diets were stored in a walk-in refrigerator (-1 to -6°C) to maintain nutrient stability.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The diets were assayed for homogeneity and concentrations of Ca, P, Mg, Mn, Zn, Fe and Cu by the New Jersey Feed Laboratory, Inc., using the methods of the Association of Official Analytical Chemists.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: female rats were mated to males at a 2:1 ratio
- Length of cohabitation: approximately 24 hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

No further details are given.

Duration of treatment / exposure:

Rats were given calcium rations in diet for 6 weeks before mating, during mating and for 20 days of gestation.

Frequency of treatment:

continuously

Duration of test:

until day 20 of gestation

No. of animals per sex per dose:

- 156 male rats
- From the total of 291 female animals, 276 female rats were divided into 4 groups of 69 animals
- 15 female rats were randomly selected for evaluation of tissue mineral status and histopathology before assignment to the 4 dietary groups

Control animals:

yes

Details on study design:

no further details given

Examinations

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Female animals were weighed every 3 days for 6 weeks before mating and during gestation, but not during mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Feed consumption was measured every 3 days for 6 weeks before mating and during gestation, but not during mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data; not applicable

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Laparotomy was performed on each dam, and the major organs were examined. In each case, 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.

OTHER:
- 135 female rats were randomly selected for determination of tissue mineral level at three time points during the study: 15 animals before assignment of diet groups, 15 animals per group (total of 60) after 6wks on the diet, and 15 animals per group (total of 60) at Caesarian section.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: Yes, uterine position, sex, weight and crown-rump lenght of each foetus

Fetal examinations:

- External examinations: Yes: Foetuses were examined individually for gross abnormalities. The uterine position, sex, weight and crown-rump length of each foetus were recorded.
- Soft tissue and skeletal examinations: Yes: Foetuses were assigned to skeletal or visceral analysis. Approximately half of the foetuses were fixed and stained and examined microscopically for skeletal variations. The remaining half were fixed and microscopically examined for internal variations of the soft tissues.
- Head examinations: Yes
- Other: Yes, from 12 litters in each group two foetuses were randomly selected for whole body mineral determinations.

Statistics:

All statistical analyses were performed by the Division of Methematics, US FDA.

Indices:

Implantation efficiency

Historical control data:

no data given

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Both the non-pregnant and pregnant rats in the 0.75, 1.0 and 1.25 % groups ate slightly more than the control group, but this increase was not 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 and resorptions.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 1.25 other: % Ca in diet

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 dose-related changes were found in the average number of 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.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Dietary calcium was neither foetotoxic nor teratogenic at the concentrations used. The highest dietary dose of 1.25 % Ca is considered to be the NOAEL for developmental effects in this study.