Calcium acetylide

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive 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 Type:

publication

Title:

The effects of additions of calcium carbonate to the diet of breeding mice. 1. Effects on reproduction and on the heart and thymus weights of the weanlings

Author:

Richards, M.B., Greig, W.A.

Year:

1952

Bibliographic source:

Br J Nutr 6: 265–280

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The test item was applied to 4-8 swiss mice at concentrations of 0, 5000, 10 000, and 20 000 mg/kg food (approx 250, 700, 1400 mg Calcium/kg bw/d based on an estimated food consumption of 17.5% of the body weight). Animals were mated continuously for 80 days. The experiment was stopped after the first litter (8 dams) and the third litter (4 dams), respectively. Liver, heart, thymus, spleen were examined in offspring after 21 days.

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:

CH2O3.Ca

IUPAC Name:

calcium carbonate

Details on test material:

- Name of test material (as cited in study report): CaCO3

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Institute's stock colony
- Diet (e.g. ad libitum): stock diet consisting of Rowett Institute stock cubes with daily supplement of fresh whole mild given ad libitum. Were then given experimental diet a week before being mated

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

Experiment 1:
16 separate diets were created for exposure, based on a basal diet plus varying amounts of CaCO3. The first diet was ground whole wheat (66 parts), dried whole milk (33 parts), and NaCL (1 part) supplemented with either 0, 0.5, 1.0, or 2.0 g CaCO3/ 100g basal diet. The second diet was ground whole wheat (60 parts), maize starch (6 parts), dried whole milk (33 parts), and NaCL (1 part) supplemented with either 0, 0.5, 1.0, or 2.0 g CaCO3/ 100g basal diet. The third diet was ground whole wheat (60 parts), casein (6 parts), dried whole milk (33 parts), and NaCL (1 part) supplemented with either 0, 0.5, 1.0, or 2.0 g CaCO3/100g basal diet.The fourth diet was ground whole wheat (66 parts), casein (5 parts), dried whole milk (33 parts), and NaCL (1 part) supplemented with either 0, 0.5, 1.0, or 2.0 g CaCO3/105g basal diet.

Experiment 2:
The basal diet was diet 78 from Experiment 1 (ground whole wheat (66 parts), casein (5 parts), dried whole milk (33 parts), and NaCL (1 part)). Mice were split into eight groups of eight mice each and arragned into 4 pairs within their groups. One group of each pair was supplemented with 0.5g CaCO3, the other group of pairs were supplemented with 2.0g CaCO3. In addition, one group of each CaCO3 supplements received 10ppm iron, one group of each CaCO3 supplements received 2.87 g sodium dihydrogen phosphate, and one group of each CaCO3 supplements received iron plus sodium dihydrogen phosphate.

Details on mating procedure:

Experiment 1: Four female mice were allotted per group with randomization of litter mates. Males were removed from breeding care after 80 days and females were maintained on diets long enough to rear any conceived litters.
Experiment 2: Eight female mice were allotted per group with randomization of litter mates. Sufficient time was allowed for rearing of two litters.

Each breeding female was reared on the diet to 7 weeks old
Males were reared the same as females.
Mating of litter-mate siblings were avoided.
Continuous monogamous mating was used.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

80 d

Frequency of treatment:

daily

Details on study schedule:

Observations ended by killing litters on weaning at 21 days of age

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 5000, 10 000, and 20 000 mg/kg food
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250, 700, 1400 mg Calcium/kg bw/d
Basis:
nominal in diet

No. of animals per sex per dose:

4 - 8

Details on study design:

Experiment 1:
Maize starch was added to reduce vitamin B content.
Casein was added to reduce vitamin B and increase protein

Experiment 2:
Basal diet 78 was chosen because it produced the best results in Experiment 1.
0.5 and 2.0 g CaCO3 were chosen because they produced the best and worst results, respectively, in Experiment 1.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE: No

Other: organ weights

Oestrous cyclicity (parental animals):

None

Sperm parameters (parental animals):

None

Litter observations:

PARAMETERS EXAMINED
Experiment 1:
First litters - numbers of young weened, total weights weened, weight per litter weened, number and proportion of deaths.
Second and subsequent litters - total weight of animals weened, total nmber of animals weened, average numer of deaths, porportion of deaths.
Effects on organ weights - heart and thymus, liver, spleen, kidneys

Experiment 2:
number born, number and weight weened
Effects on organ weights - heart and thymus

Postmortem examinations (parental animals):

organ weights

Postmortem examinations (offspring):

organ weights

Statistics:

statistical significance, correlation coefficient, regression equations

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

At the highest level of calcium (2.0g/100g diet), there was a significant decrease in the total number weaned (p<0001) and the total weight weaned (p<0.001).
Although the differences in the numbers born at the different CaCO, levels were not statistically significant, it may be noted that there was some decrease at the two higher levels (1.0 and 2.0g/100g diet)
A few animals suffered from vaginal prolapse and failed to produce any litters after the first, which should be attributed to genetic causes.

Details on results (P0)

Experiment 1:
Weight of heart, liver, spleen, and kidneys were not affected in mothers.
Higest level of Ca showed significant decrease in total number weened.
Few animals had vaginal prolapse and failed to produce litters after the first, may be attritubed to genetics.

Experiment 2:
Addition of Fe, P, or higher level of CaCO3 has no significant effects on the number of young born in first or second litter.
The number and weight weaned were affected by the number of failures to rear first litters (6 failures in higher Ca groups, 1 failure in lower Ca group). Mice that failed rearing first litters all succeeded in rearing some young in subsequent litters, therefore the number of total deaths did not differ greatly.
Organ weight - higher levels of CaCO3 increased absolute and relative heart weight and decreased percentage of heart dry matter.

Effect levels (P0)

Key result

Dose descriptor:

LOAEL

Effect level:

20 000 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

First litters: At the highest level of calcium (2.0g/100g diet), there was a significant increase in both the number and proportion of deaths (p<0.01)
Second and subsequent litters showed greater viability as indicated in a lower proportion of deaths. The average number of deaths on diets with the highest level of CaCO3, was significantly higher than on diets with lower levels of CaCO3 (P=0.01) and the highest proportion of deaths also occurred with the highest CaCO3 level (p<0.001).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Considering the weight per litter weaned, thus eliminating the effect of litters dying before weaning, a gradation is apparent in the results, the highest weight being found at the low dose level (0.5g/100g diet), with a marked falling off at the two highest dose levels (1.0g and 2.0g/100g diet). When calculating the mean weight per litter weaned, this effect was most pronounced at the highest dose level (0/0.5/1.0/2.0g/100g diet: 40.5g/53.3g/39.4g/23.3g)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

As the investigation of organ weights was not begun from the start of the experiment, the results are incomplete and are therefore not recorded in detail, but sufficient evidence is available to show that there was a significant effect of CaCO3 in increasing the heart weights of the weanlings. The heart weights of the mothers were not significantly affected. Although the effect of CaCO3 on the thymus weights of the weanlings was not found to be statistically significant when all the results were analysed, there was a tendency for thymus weights to decrease as heart weights increase with increasing levels of CaCO3.
Analysis of the weights (mg/100g body-weight) of liver, spleen and kidneys of the young mice and their mothers did not reveal any significant effects of calcium or other dietary factors on these organs.
In experiment 2, the percentage of dry matter in the heart was significantly lowered by the higher level of Ca (P<0.001). Thymus weights of second litters were significantly affected both by the Ca additions, but in the opposite direction from the heart weights. Thus the absolute weight of the thymus and its weight relative to body-weight were both diminished by the higher level of Ca (P<0.001).

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Experiment 1:
Highest level of Ca had significant decrease in total weight weened and total number weaned and significantly increased number and proportion of deaths. There were non-significant differences in the numbers born at different CaCO3 levels, but some decrease at the two higher levels. Best results were from 0.5g/100g CaCO3.
Second and subsequent litters showed greater viability. Results showed a significant decrease in total weight of weaned animals and total number of weaned animals in the highest level CaCO3. Average number of deaths and highest proportion of deaths occurred at the highest level of CaCO3. Critical periods in rearing young mice were the first and third weeks of life.
Organ weights - frequent occurence of pale speckled livers, enlarged hearts, and small thymus glands, which were more pronounced in groups wiht higher levels of CaCO3. CaCO3 had a significant effect on increasing heart weights of weanlings, but no significant effects on thymus weight. There were no significant effects on weights of liver, spleen, and kidneys in young mice.

Experiment 2:
Total weight weaned was lower in higher CaCO3 group due to litter failures, for groups without litter failures, weigh weaned was similar. Adding Fe and P inproved diet and decreased the effects of higher levels of CaCO3. Weaning weights per litter increased in subsequent groups, amplified by addition of Fe and P, except one group, which showed no difference.
Organ weights - higher levels of CaCO3 were seen to increase absolute heart weight and relative heart weight in weanlings. Fe supplementation significantly decreased relative and absolute heart weight in higher and lower CaCO3 groups. P had no significant effect on relative or absolute heart weight. Percentage of heart dry matter was significantly decreased by higher CaCO3 levels and significantly increased by Fe; P had no significant effect. A significant correlation exists between heart enlargement and degree of anaemia. Thymus weight of second litters were significantly decreased by higher levels of CaCO3 and increased by Fe; P had no significant effect.

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

20 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

In a breeding test on mice, using the method of continuous monogamous mating, three levels of calcium carbonate were added to different diets. On all the diets the highest addition of calcium carbonate, which gave a Ca intake of 1.1% and a Ca:P ratio of approximately 2,3, there was a significantly lowered number and total weight of young weaned and an increased number and proportion of deaths. There was also some decrease in the number of births. Both absolutely and relatively to body-weight the heart weights of the weanlings were significantly increased by Ca and decreased by Fe, whereas thymus weights were decreased by Ca and increased by Fe. The high Ca level also caused a significant increase in the absolute and relative heart weights of the mothers. Although the percentage of dry matter in the hearts of the weanlings was significantly lowered by the higher level of Ca, there was a marked increase in the absolute weight of heart dry matter at this level, indicating the presence of a cardiac hypertrophy. A highly significant relationship was found between heart weight and blood haemoglobin in the weanling mice, an increase in heart weight accompanying a decrease in Hb. The relationship between log heart weight, expressed in mg food/g body-weight, and log Hb was linear.

Applicant's summary and conclusion

Conclusions:

In a breeding test on mice, the highest dose tested (1400 mg Calcium/kg bw/d) significantly lowered the number and total weight of young weaned and increased the number and proportion of deaths. There was also some decrease in the number of births. The study design does not fulfill requirements of an OECD 416 study. Results cannot be used to derive a NOAEL.

Executive summary:

In a multi-generation reproduction study, CaCO3 was administered to 4-8 Swiss mice/sex/dose in diet at dose levels of 0, 5000, 10 000, and 20 000 mg/kg food and 250, 700, 1400 mg Calcium/kg bw/d. The first litter and subsequent litters up to 2 litters were observed. The highest dose tested significantly lowered the number and total weight of young weaned and increased the number and proportion of deaths. There was also a decrease in the number of births. Results cannot be used to derive a NOAEL. This study does not fulfill the requirement for a multi-generational reproductive study (OECD 416) in rodents due to lack of experimental information on animals and test substance, and lack of observational, reproductive, and terminal data; as well as lack of reported data on the previously listed parameters.