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Toxicity to reproduction

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Administrative data

multi-generation reproductive toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
no information
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
reliable with restrictions. Well documented non-guideline publication. Useful for evaluation.

Data source

Reference Type:
Female reproduction and pup survival and growth for mice fed a cadmium-containing purified diet through six consecutive rounds of gestation and lactation
Whelton BD, Bhattacharyya MH, Carnes BA, Moretti ES and Peterson DP
Bibliographic source:
J. Toxicol. Environ. Health 24(3):321-43

Materials and methods

Principles of method if other than guideline:
Method: other, no information
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Reference substance name:
Cadmium chloride
EC Number:
EC Name:
Cadmium chloride
Cas Number:
cadmium dichloride
Details on test material:
-Name of test material: CdCl2

Test animals

Details on test animals or test system and environmental conditions:
- Origin/housing: Virgin female (1175) and virgin male (350, for breeding) CF1 mice (Charles River Breeding Laboratory, Wilmington, Me.) were
maintained from 55 d of age on Wayne Lab BIox diet (Wayne Feeds, Continental Grain, Libertyville, II.) and tap water (pH 2.3) ad libitum until the start ofthe experiment. (Drinking water was acidified with hydrochloric acid to pH 2.3 for control of Pseudomonas. The cadmium concentration in the Wayne Lab BIox diet was 0.25 ppm (Dr. Joel Drews, Wayne Feeds, personal communication) and in the drinking water was 0.1 ppb as determined by fiameless atomic absorption spectrophotometry (IL 555 CTF atomizer, IL 951 AA spectrophotometer, Instrumentation Laboratories, Wilmington, Mass.). The
animal-room lighting schedule was 12 h light (6 a.m. to 6 p.m.) followed by 12 h dark; the relative humidity was 55%, and the room temperature was
22-23°C. Fluorescent lighting was utilized during the breeding portion of the experiment; an ultraviolet-free light source was used after the last
pregnancy/ lactation cycle.
- Age at start of study: At age 67 d, randomly selected females and males weighed 25.2 ± 1.8 g (mean ± SD, n=100) and 29.2 ± 2.2 g (n = 10),
- Weight at study initiation: no data

Administration / exposure

Route of administration:
oral: feed
other: Vehicle: six purified diets were supplemented with Cd at either 0.25, 5.0 or 50 ppm. For each Cd concentration, diets were either sufficient (+) in all dietary constituents or deficient (-) in certain minerals, vitamins and fat. For the environmental leve
Details on exposure:
At age 68 d, female mice [612, pregnant/lactating (PL) group] were selected at random for breeding. Virgin females [540, nonpregnant control (NP)
group] were kept as nonpregnant controls. The PL and NP mice were divided into the cadmium diet groups, and were given stable cadmium in food
and either plain or 109Cd drinking water for the duration of the experiment. At age 70 d, all 8 groups of PL females were bred with males
(two females to one male) for 5 d (Day 0-5 of reproductive round 1) (B), after which the mice were individually housed. (Males were kept on
Wayne Lab Blox diet except when housed with PL females.) On Day 19-24, number of pups per litter at birth was recorded (CP). On Day 24 (median lactation Day 3, median L-3), litters were normalized to 7 pups each (N). (Note that because of the large number of mice bred, litters were normalized on Day 24 of each round, rather than on the specific lactation day for each dam according to her individual delivery date). On Day 38 (median L-17), numbers of pups per litter was again recorded as a final measure of pup survival (CP). For the purpose of determining
reproductive success, a dam bringing a litter of at least one pup to median L-17 was defined as having successfully completed that round of
reproduction. On Day 42 (median L-21), pups were weaned from all dams not scheduled for sacrifice (W), and a 5-d rebreeding period was
started (i.e., on Day 0-5 of the next reproductive round) (B). Re-breeding was continued in this way through 6 consecutive 42-day rounds of pregnancy and lactation. For dams scheduled for sacrifice after a given reproductive round, pups were weaned on true L-21 according to the individual birth date for each dam; numbers of pups and litter weights were determined. At the end of round 5, 119 of 374 remaining PL mice were withheld from
round 6 breeding for future ovariectomy. The remainder were bred with males as previously outlined for the sixth and final time.
Details on mating procedure:
cfr higher under details on exposure
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
Exposure period: continuously during 6 generations
Frequency of treatment:
Details on study schedule:
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
0.25 ppm
Dose / conc.:
5 ppm
Dose / conc.:
50 ppm
No. of animals per sex per dose:
Mouse groups maintained on sufficient or deficient diets at the start of the experiment (a)
Number of mice per group for sufficient (+) and deficient (—) diets
Cd (ppm) (+) ( —) ( + ) ( --)

0.25 67 67 60 60
5 68 68 60 60
109Cd, 5 96 93 75 75
50 77 76 75 75
(a) CF1 mice were maintained on a sufficient or deficient diet containing CdCI2
in one of three concentrations (0.25, 5.0, or 50.0 ppm Cd) and acidified tap water
or acidified tap water containing 109CdCl2 (0.02 µCi/ml, 0.007 ppb 109Cd).
Control animals:


For measurements of fertility, litter size, and pup survival, statistical analyses were conducted based on data for the total population of female miceFor pup growth, analyses were conducted on data from mice that were consecutively pregnant, because data on pup growth were available for the consecutively pregnant population only. Analyses for all end points employed analysis of variance (ANOVA). The fertility analysis was based on proportions using the arc-sine square root transformation (Sokal and Rohlf, 1969). All other analyses were based on means weighted by the inverse
variance. After successively deleting nonsignificant interactions, a single parsimonious ANOVA model with cadmium, diet, and round main effects and a cadmium-by-diet interaction was considered appropriate for interpretation (coefficients of variation ranged from 0.81 to 0.96). Significance of the main effects was determined by the SAS type III sums of squares (SAS Institute Inc., 1979) based on the method of weighted squares of means (Winer, 1971). Significance of specific diet-cadmium levels were based on a posteriori comparisons of least squares means using a Bonferroni
adjustment to the nominal significance level to attain an experiment-wise error rate of 0.05 (Miller, 1966).

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

open allclose all
Dose descriptor:
Effect level:
5 ppm
Based on:
test mat.
Basis for effect level:
other: decreased fertility and litter size
Dose descriptor:
Effect level:
50 ppm
Based on:
test mat.
Basis for effect level:
other: Combined Cd at 50 ppm + deficient diet. Decreased fertility and litter size

Results: F1 generation

Effect levels (F1)

open allclose all
Dose descriptor:
Effect level:
5 ppm
Based on:
test mat.
not specified
Basis for effect level:
body weight and weight gain
Dose descriptor:
Effect level:
50 ppm
Based on:
test mat.
not specified
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Test group 50 mg Cd/kg/day: no effect on fertility and survival, decreased litter size (15%) pup growth (25%)



Parental data:

- Mortality and time to death:Neither cadmium nor deficient diet had a striking effect on survivalof the dams through round 4 of breeding.

- Clinical signs:A measure of sustained reproductive performance can be obtainedby summing percentages of females completing four out of five (4/5)and five out of five (5/5) PL rounds for each dietary regimen. Atthe environmental level of cadmium (0.25 ppm Cd), the sum of femalescompleting 4/5 and 5/5 PL rounds was 42% for PL( + ) females and only7% for PL(-) females, indicating an effect of the deficient diet on consecutive reproduction. At 5 ppm Cd, consecutive pregnancies amongthe PL( + ) females increased over levels at 0.25 ppm Cd (4/5 + 5/5 sumswere 49-60%), while among the PL(1—) females the sums remained thesame (7%). At 50 ppm Cd, the 4/5 + 5/5 sum for PL( + ) females was 43%,essentially the same as at 0.25 ppm Cd (42%). For PL(-) females, how­ever, pregnancies showed a striking decrease at 50 ppm Cd such thatno PL females successfully reproduced 4 times and most (58%) werelimited to 2 nonconsecutive experiences in 5 attempts.

Inspection of both the data and the results of the statistical analysesindicates that, for mice in a given experimental diet group, there were no cumulative, round-by-round decreases from the early to late reproductive rounds in fertility, litter size, pup survival, or pup growth. Consequently, differences in reproductive measures because ofeffects of dietary deficiencies and/or cadmium were essentially repeated in each round and not increased in magnitude with successiverounds.Surprisingly, however, thecadmium-induced decrease in pup growth (2-4 g/pup) was not magnified with increased exposure of the dam to cadmium through successive rounds of breeding.

Because of the absence of cumulative effects with successiverounds, measures of reproductive success are presented not only asmean values for each individual round of gestation and lactation, butalso as grand means for all rounds to allow comparisons of experimental groups independent of reproductive round.

- Fertility:

At 0.25 ppm dietary cadmium, the grand mean for fertility for miceon the deficient diet (57%) was not significantly lower than that for mice on the sufficient diet (65%),indicating that dietary deficienciesplayed little role in determining the fertility of the total population ofmice at environmental levels of cadmium (0.25 ppm).

For groups maintained on the sufficient diet, neither an increase ofdietary cadmium to 50 ppm nor exposure to low doses of109Cd in drinking water decreased fertility; grand mean values for all reproductiverounds ranged from 65 to 77% independent of Cd or109Cd level. Incontrast, for mice on the deficient diet, fertility decreased from a grandmean of 57% at 0.25 ppm Cd to 49-53% at 5 ppm Cd and 36% at 50 ppmCd, indicating that combined exposure to cadmium and di­etary deficiencies had a synergistic effect on fertility that was statisti­cally significant at 50 ppm. Again, the presence of109Cd did not influence fertility.

Results show values for the fertility of PL mice that experienced consecutive rounds of gestation and lactation mice in whichmaximum effects of cadmium or dietary deficiencies had been anticipated. Although these data were not subjected to statistical analysisbecause of data imbalances, the results support the conclusions fromthe total mouse population that exposure to cadmium did notdecrease fertility for mice on the sufficient diet, while combined exposure to 50 ppm Cd and dietary deficiencies had a synergistic effect onfertility. In addition, whereas groups on the sufficient diets successfullyexperienced 6 consecutive PL rounds, groups on the deficient dietswere limited to 3 consecutive reproductive experiences at 0.25 and 5ppm dietary cadmium and to 2 at 50 ppm Cd, for similar numbers ofmice initially bred. Thus, although the fertility of the total population of mice on the deficient diet did not steadily decrease withsuccessive rounds of breeding, dietary deficiencies clearly decreased the number of times that a given female could become consecutively pregnant

- Litter Size at Birth

Litter sizes at birth at 0.25 ppm dietary Cd were significantly smallerby 30% for mice on the deficient diet (7.8 pups/litter) than for mice on the sufficient diet (11.2 pups/litter). Similar, significant,deficiency-dependent decreases in litter size also occurred at 5 and 50ppm Cd for all rounds of breeding.

At 5 ppm Cd, the effect of cadmium on litter size at birth was dependent on the type of diet. For mice on the sufficient diet, litter sizewas smaller by 10% at 5 ppm Cd than at 0.25 ppm Cd. However, formice on the deficient diet, litter size was greater by 10% at 5 ppm Cdthan at 0.25 ppm Cd, suggesting that cadmium at 5 ppm in some waycounteracted the effects of the vitamin, mineral, and fat deficiencies ofthe maternal diet. At 50 ppm Cd, litters were significantly smaller (by15-18%) than at 0.25 ppm Cd for both the sufficient and deficient di­etary groups. The effect of 50 ppm Cd alone on littersize at birth [15% decrease from 0.25(+) to 50{+)] was therefore aboutone-half as great as the effect of dietary deficiencies alone [30% decrease from 0.25(+) to 0.25(-)].

Litters were almost identical in size for the consecutively pregnant/lactating females and for the total population of micefor all diet groups. This was true even for mice on the deficientdiet, where numbers of females that became consecutively pregnantwere strikingly decreased.

- Pup Survival during Lactation

At both 0.25 and 50 ppm Cd, the change of diet from (+) to (-)resulted in a significant (18-20%) decrease in pup survival during lactation. Quite surprisingly, for both the (+) and (-) diet groups,the 200-fold increase in cadmium from 0.25 to 50 ppm caused no significant decrease in pup survival; thus survival to L-17 hinged onmaternal dietary deficiencies and not on cadmium at the concentra­tions used in this experiment. As was seen for litter size at birth, pupsurvival was slightly greater at 5 ppm Cd than at 0.25 ppm Cd for miceon the deficient diet, again suggesting the presence of some protectiveeffect of a moderate concentration of cadmium.

Data for the consecutive PL groups, although not analyzedstatistically, were consistent with conclusions drawn from the data forthe whole population of mice i.e., cadmium did not decreasepup survival, while the deficient maternal diet did.

- Pup Growth during Lactation

Pup weights on the day of weaning, L-21, were measured for the litters of dams that experienced consecutive rounds of pregnancy andlactation. Of all the reproductive parameters measured, pupgrowth was most affected by both dietary deficiencies and exposure to cadmium. At 0.25 ppm Cd, pups weighed 42% less for mice on the deficient diet than for those on the sufficient diet. The overalleffect of dietary deficiencies on pup weight at weaning was highly significant. Pup weights atweaning were essentially the same at 5 ppm Cd as at 0.25 ppm Cd forboth (+) and (-) diet groups. At 50 ppm Cd, however, cadmium de­creased pup weights at weaning by 25% on the (+diet and 41% on the(-) diet compared to the respective weights at 0.25 ppm Cd, with theoverall effect of cadmium being highly significant.Only for the 50(-) group was maternal diet consumption notablylower (31%) than for all other PL groups. At L-21, pups from the 50 ppmCd(-) dams differed from those of the other PL groups, appearing anemic, uncoordinated, listless, and having poor coat condition in additionto being very small.


Summary of the Effects of 50 ppm Cadmium and Dietary Deficiencies on ReproductiveSuccess

Percentage decrease in grand mean valuea

Exposure                    Fertility    Litter size Pup survival     Pup growth

50 ppm Cadmiumb



0     .


Deficient dietc





50 ppm Cadmium +





deficient dietd





aGrand mean values for fertility, litter size, and pup survival and thosefor pup growth

bPercentage decreases are from the 0.25 ppm Cd(+) group to the 50 ppm Cd(+) group.cPercentage decreases are from the 0.25 ppm Cd(+) group to the 0.25 ppm Cd(-) group.dPercentage decreases are from the 0.25 ppm Cd(+) group to the 50 ppm Cd(-) group.

Applicant's summary and conclusion

Litter size and pup growth were most affected by the separate exposures, with decreases of 30-42% for exposure to dietary deficiencies alone, and decreases of 15-25% for exposure to 50 ppm Cd alone. Combined effects of 50 ppm Cd and dietary deficiencies were additive for litter size, pup
survival, and pup growth. In contrast, combined effects on fertility were synergistic. Of these four measures of reproductive success, pup growth was most adversely affected by the exposure conditions studied.

Females in our study were bred through multiple, successive, 42-d rounds of pregnancy and lactation. We therefore had the unique opportunity to look for cumulative effects on reproductive performance with time and with successive pregnancies.

One unexpected finding was the clear synergism between cadmium and the deficient diet in reducing fertility. Possibly the low calcium content of
the deficient diet allowed cadmium to interfere with calcium pathways important to maintaining fertility. A second finding of particular interest was theinvariance in the magnitude of responses to cadmium and dietary deficiencies with successive rounds of breeding.

These results imply that the quality of maternal diet during a given breeding period had a much greater effect on reproductive parameters than does prior dietary exposure.
Executive summary:

Female mice were bred for 6 consecutive 42-day rounds of gestation-lactation. Diet contained either 0.25-5.0 or 50 ppm Cd (as CdCl2) and were either sufficient or deficient in certain vitamins, minerals, and fat. The dose of 5 ppm cadmium combined with a deficient diet

was designed to simulate conditions implicated in the aetiology of Itai-Itai disease.

Exposure to Cd did not decrease fertility for mice on sufficient diets. Combined exposure to cadmium and nutritional deficiencies had a synergistic effect on fertility and litter size that was statistically significant at 50 ppm.