Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

2 generation study, rat, NOAEL ≥ 7.80 mg/kg bw/d

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: protocol approved by the Institutional Animal Ethics Committee (Protocol No. P/4489/RT-DT-R/06) based upon the United States Food and Drug Administration Redbook Guidelines for Reproduction Studies IV.C.9.a and Developmental Toxicity Studies IV.C.9.b.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Niacin bound chromium (NBC) is a unique, patented oxygen-coordinated niacin-bound chromium complex commercially known as ChromeMate CM-100M (powder) and was obtained from InterHealth Nutraceuticals, Benicia, CA, USA. Lot#306013

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: bred and reared at the animal breeding facility of INTOX Pvt. Ltd. India
- Age at study initiation: 7–9 weeks old
- Weight at study initiation: F0: 263 ± 18g , F1: 245 ± 17g
- Diet: ad libitum, ‘‘Nutrilab” brand extruded rodent powdered feed manufactured by M/s Vetcare Pvt. Ltd., Bangalore, India, and tested for nutrients and contaminants, was provided ad libitum to the animals during the study period.
- Water: ad libitum
- Acclimation period: The animals were allowed to acclimatize at least one week before the initiation of experiments

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 and 25 °C
- Humidity (%): 30–70%
- Air changes (per hr): 10–15 air changes per h.
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared once a week.
- Mixing appropriate amounts with (Type of food): NBC was mixed with powdered rodent diet (Nutrilab” brand extruded rodent powdered feed manufactured by M/s Vetcare Pvt. Ltd., Bangalore, India) to obtain the three concentration levels. Initially, a small volume of dietpremix was prepared which was then mixed with remaining portion of diet in a mechanized ribbon blender for about 20 min to obtain desired homogeneity of the test article concentration in diet.

Details on mating procedure:

The male and female rats of F0 generation from each dose group were mated and allowed to deliver normally. No further details on mating procedure was provided in the article.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The treatment groups of the F0 parental generation: Receiving feed containing 4, 15, and 60 ppm NBC for a period of 10 weeks before mating, throughout mating, and continued until their termination. The exposure was continued through the next generations, up to completion of developmental toxicity study. At weaning, one male and one female pup from each litter from control and treatment dose groups were selected for first filial (F1 ) generation. The selected F1 animals were exposed to NBC for 10 weeks before mating and then they were mated to produce second generation (F2a).

Frequency of treatment:

continuously via food

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Age at mating of the mated animals in the study: 10 weeks

Dose / conc.:

60 ppm

Remarks:

nominal in diet

Dose / conc.:

15 ppm

Remarks:

nominal in diet

Dose / conc.:

4 ppm

Remarks:

nominal in diet

Dose / conc.:

0 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

30 males and 30 females per dose group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Since NBC is intended to be consumed by human beings up to a maximum dose of 4 mg of NBC/day, the highest dose level for this study was selected so as not to exceed 100 times the maximum recommended human dose, which has a dietary equivalent concentration of 60 ppm. A dose range study revealed no adverse effects of NBC on body weight, feed consumption, mating behavior, fertility, gestation or lactation in rat at dose level up to 60 ppm.
- Rationale for animal assignment (if not random): Sprague–Dawley rats (30/group/sex) were randomly divided into one control and three treatment groups (low, mid and high).

Positive control:

none required

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined daily for signs of toxicity and mortality during the entire period of the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations recorded for the parental male and female rats of both (F0 and F1) the generations during the premating and mating periods

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight and feed consumption of the animals from each group were recorded weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, feed consumption was calculated as g/rat/day

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

For both F0 and F1 parental animals reproductive parameters such as estrous cycle, female fertility index, gestation index, live-born index, mean litter size, sex ratio (at birth), number of stillbirths at day 0, number of live births at day 0, survival index were assessed.

Sperm parameters (parental animals):

For both F0 and F1 parental animals reproductive parameters such as sperm count (epididymal and homogenization resistant testicular), sperm motility and sperm morphology were assessed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring: clinical observations and mortality during lactation, litter observations, sexual maturation, physical development endpoints(such as unfolding of pinna (UP), hair growth (HG), teeth eruption (TE), eye opening (EYO) and ear opening (EO)), organ weights, necropsy and histopathology changes

GROSS EXAMINATION OF DEAD PUPS: Yes, all pups, not selected for next generation, were sacrificed on lactation day 21, and subjected to necropsy.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: After the mating and following confirmation of pregnancy status, males were euthanized with carbon dioxide and were subjected to necropsy and evaluation of sperm parameters such as sperm motility, cauda epididymis sperm count, testicular spermatid head count and sperm morphology.
- Maternal animals: All females were killed after weaning.

GROSS NECROPSY
- At necropsy after the mating period (male rats) or the lactation period (female rats) the group mean values of absolute and relative weight (% of body weight and % of brain weight) of liver, kidneys, brain, spleen, adrenals, pituitary, testes, seminal vesicles (with cowper’s glands), prostate, epididymides, ovaries and uterus, of male and/or female parental rats of F0 generation and F1 generation exposed to NBC at levels of 4, 15, and 60 ppm did not reveal any significant differences from the respective control group,

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights were recorded at necropsy, and weighed organs from 10 randomly selected animals were subjected to histopathological examination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at : All pups, not selected for next generation, were sacrificed on lactation day 21, and subjected to necropsy.

GROSS NECROPSY
- Gross necropsy consisted of brain, thymus and spleen

HISTOPATHOLOGY / ORGAN WEIGTHS: Histological examination of brain, thymus and spleen of pups euthanized at the end of lactation period

Statistics:

For statistical analysis, a litter was considered as the basic sampling unit. To analyze the various parameters, different and appropriate statistical methods were employed. For data on parental body weight and weight gain, feed intake, and organ weights Bartlett’s test followed by ANOVA and Dunnett’s test was employed with statistical significance at P < 0.05. Day 0 and absolute body weights were analyzed by paired t-test. Group differences in litter size were analyzed by Student t-test. Sex ratio was assessed by Chi-square test (2 x 2 contingency tables). Results of the statistical analysis were described as significantly higher (+)/lower (-) than control values at P < 0.05.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All deaths and abnormal clinical signs observed in the rats during F0 and F1 generations, such as transient/ reversible spells of emaciation, abdominal breathing, respiratory rales, hypoactivity, circling disorder and lacrimation, were considered to be incidental and not due to NBC feeding.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All deaths and abnormal clinical signs observed in the rats during F0 and F1 generations, such as transient/ reversible spells of emaciation, abdominal breathing, respiratory rales, hypoactivity, circling disorder and lacrimation, were considered to be incidental and not due to NBC feeding.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

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:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Similarly, the unaltered length and normalization of estrous cycles in treated females, mating performance as evidenced from unaltered indices of male fertility and female fertility, maintenance of normal gestation was evident from unaltered gestation length and gestation indices.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm evaluation. For both the F0 and F1 generations exposed to NBC at dose level up to 60 ppm, evaluations of sperm parameters of male rats during premating and mating period, and the period thereafter up to their termination, did not reveal any changes that could be attributed to the test article

Reproductive performance:

no effects observed

Description (incidence and severity):

NBC at dose levels up to 60 ppm during premating and mating periods and gestation period in females did not reveal any treatment related adverse effects on reproductive performance in terms of fertility and mating, gestation

1. Parental observations (F0 and F1 )
1.1. Food consumption and test compound dosage determination Data on feed consumption by the parental male and female rats of both (F0 and F1 ) the generations during the premating and mating periods, for both sexes, and during gestation and lactation in case of female rats, did not reveal any significant treatment related changes in the average daily feed intake by the male and female rats compared to the respective control groups, across the different dose levels for each of the F0 and F1 generations and also when compared across these two generations. Based on feed intake, the resulting dose of NBC during premating period for the highest dose groups F0 male and female was calculated as 5.88 and 8.24 mg/kg/day, respectively, for F0 generation, while the same was, respectively, 9.71 and 9.83 mg/kg/day in case of F1 generation. The total daily dose of NBC for all groups is presented in Table 2.
1.2. Clinical signs and mortality
Compared to the respective control groups, across the different dose levels for each of the F0 and F1 generations and also when compared across these two generations, results of survival and clinical observations recorded for the parental male and female rats of both (F0 and F1 ) the generations during the premating and mating periods, for both sexes, and during gestation and lactation in case of female rats, did not reveal any remarkable incidence of mortality and abnormal clinical signs among the male and female rats exposed to NBC. All deaths and abnormal clinical signs observed in the rats during F0 and F1 generations, such as transient/reversible spells of emaciation, abdominal breathing, respiratory rales, hypoactivity, circling disorder and lacrimation, were considered to be incidental and not due to NBC feeding.
1.3. Body weights
The average body weight and body weight gains of the parental male and female rats of both the generations (F0 and F1 ) during the premating and mating periods, and during gestation and lactation of female rats, did not reveal any remarkable alterations which could be attributed to NBC exposure at any of the doses, when compared to the respective control groups, across the different dose levels for each of the F0 and F1 generations and also when compared across these two generations. Although, other occasional instances of group mean values of treated animals differing from those of the respective control groups were noted, these were considered incidental or of no toxicological significance due either their lack of dose relation, their small magnitudes, or other procedural reasons unrelated to the treatment.
1.4. Mating, fertility and reproduction
Exposure of male and female rats, from both the F0 and F1 generations, to NBC at dose levels up to 60 ppm during premating and mating periods and gestation period in females did not reveal any treatment related adverse effects on reproductive performance in terms of fertility and mating, gestation, parturition and the litters born. Similarly, the unaltered length and normalization of estrous cycles in treated females, mating performance as evidenced from unaltered indices of male fertility and female fertility, maintenance of normal gestation was evident from unaltered gestation length and gestation indices. Furthermore the pups born alive were unaffected, as evidenced from their live birth indices and did not reveal any treatment related adverse effects.
The values of male fertility indices for treatment groups in F0 and F1 generations did not differ significantly from those of the controls, and also compared well with the historical control data at the test facility. The values of male fertility indices for treatment groups in F0 generation in control, 4, 15 and 60 ppm groups were 90%, 100%, 80% and 92.2%, respectively. Similarly, these values for treatment groups in F1 generation were 103%, 100% and 100% at the doses of 4, 15, and 60 ppm, respectively, while the value was 96% for the control group, respectively.
1.4.1. Sperm evaluation. For both the F0 and F1 generations exposed to NBC at dose level up to 60 ppm, evaluations of sperm parameters of male rats during premating and mating period, and the period thereafter up to their termination, did not reveal any changes that could be attributed to the test article. This was evident by virtue of the group mean values of motility of sperms in cauda epididymis, counts of sperms in cauda epididymis (absolute count and per gram of cauda weight), counts of homogenization resistant spermatids (absolute count and per gram of testis weight) per testis, and the morphological evaluations of the sperms by microscopy of stained smears. Although the sperm motility of F1 parents compared to the F0 parents was found to be slightly lower, it was not considered to be related to the NBC exposure, as the lowering was also observed in the concurrent control group of rats and the altered values were comparable to the historical control data.
1.5. Parental organ weights, necropsy and histopathology: At necropsy after the mating period (male rats) or the lactation period (female rats) the group mean values of absolute and relative weight (% of body weight and % of brain weight) of liver, kidneys, brain, spleen, adrenals, pituitary, testes, seminal vesicles (with cowper’s glands), prostate, epididymides, ovaries and uterus, of male and/or female parental rats of F0 generation and F1 generation exposed to NBC at levels of 4, 15, and 60 ppm did not reveal any significant differences from the respective control group, which could be ascribed to NBC. Necropsy and histological examinations performed on the parents of the F0 and F1 generations, which died during the study or were terminated at end of the mating period (males) or the lactation period (females), did not reveal any incidence of gross and microscopic pathological alterations attributable to their exposure to NBC at dose levels of up to 60 ppm. All the gross and microscopic findings noted were considered to be incidental as the incidence was found to be comparable among the control group and the treatment groups, without any dose dependent trend.

Dose descriptor:

NOAEL

Effect level:

60 ppm

Based on:

test mat.

Remarks:

The equivalent dose in male and female rats was 7.80 and 8.31 mg/kg/day in male and female rats, respectively.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: The parental and offspring no-observed-adverse-effect level (NOAEL) in this two-generation reproduction toxicity study was found to exceed 60 ppm in diet.

Dose descriptor:

NOAEL

Effect level:

7.8 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOAEL

Effect level:

8.31 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All deaths and abnormal clinical signs observed in the rats during F0 and F1 generations, such as transient/ reversible spells of emaciation, abdominal breathing, respiratory rales, hypoactivity, circling disorder and lacrimation, were considered to be incidental and not due to NBC feeding.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All deaths and abnormal clinical signs observed in the rats during F0 and F1 generations, such as transient/ reversible spells of emaciation, abdominal breathing, respiratory rales, hypoactivity, circling disorder and lacrimation, were considered to be incidental and not due to NBC feeding.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Similarly, the unaltered length and normalization of estrous cycles in treated females, mating performance as evidenced from unaltered indices of male fertility and female fertility, maintenance of normal gestation was evident from unaltered gestation length and gestation indices.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm evaluation. For both the F0 and F1 generations exposed to NBC at dose level up to 60 ppm, evaluations of sperm parameters of male rats during premating and mating period, and the period thereafter up to their termination, did not reveal
any changes that could be attributed to the test article

Reproductive performance:

no effects observed

Description (incidence and severity):

NBC at dose levels up to 60 ppm during premating and mating periods and gestation period in females did not reveal any treatment related adverse effects on reproductive performance in terms of fertility and mating, gestation
The values of male fertility indices for treatment groups in F0 and F1 generations did not differ significantly from those of the controls, and also compared well with the historical control data at the test facility

Dose descriptor:

NOAEL

Effect level:

60 ppm

Based on:

test mat.

Remarks:

The equivalent dose in male and female rats was 7.80 and 8.31 mg/kg/day in male and female rats, respectively.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: The parental and offspring no-observed-adverse-effect level (NOAEL) in this two-generation reproduction toxicity study was found to exceed 60 ppm in diet.

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

2. Offspring observations (F1 and F 2a )
2.1. Offspring body weights: The data on average values of body weights of offspring of both the generations (F1 and F 2a ) recorded on lactation days 0, 4, 7, 14 and 21, did not reveal any alterations which could be attributed to exposure of their dams to NBC at levels up to 60 ppm, when compared to the respective control groups, across the different dose levels for each of the F0 and F1 generations and also when compared across these two generations.
2.2. Offspring clinical observations and mortality during lactation: Compared to the respective control groups of pups, across the different dose levels for each of the F1 and F2a generations and also when compared across two generations, data on survival and clinical observations recorded for the offspring of both the generations (F1 and F2a ) during lactation period of 21 days did not reveal any remarkable differences. The observations included clinical abnormalities in pups, and the incidence of normal pups, pups found dead on lactation day 0 and thereafter, pups cannibalized by the dam on lactation day 0 and thereafter, and pups which were terminated in moribund state.
2.3. Litter observations: Comparison of the offspring data with respective control groups and also across the F1 and F2a generations did not reveal any adverse effect on their litter sizes, the sex ratios of litters, the live birth indices and the viability indices of litters calculated for days 4, 7, 14 and 21 of lactation, following exposure of parental females to NBC at dose levels of 4, 15, and 60 ppm.
2.4. Offspring sexual maturation: The sexual maturation was measured only for F2a generation, in terms of age at which there is balano-preputial separation in males and vaginal opening in females. Exposure to NBC at any of the dose levels did not affect the age of sexual maturity by the offspring belonging to the F2a generation. The group mean age at balano-preputial separation in male pups was 31.4 ± 2.58 days, 30.2 ± 2.76 days, 28.9 ± 1.80 days and 27.7 ± 1.42 days, respectively, for the control group and low, mid and high dose levels.
Historical control value (Mean + SD) for the same was 24.6 ± 2.7. The group mean age at vaginal opening in female pups was 57.4 ± 6.85 days, 54.4 ± 8.64 days, 51.7 ± 9.06 days and 50.8 ± 9.64 days, respectively, for the control group and low, mid and high dose levels. Historical control value (Mean + SD) for the same was 49.7 ± 5.3. Although there was a trend for an inverse relationship between dose and sexual maturation in F2a generation, this trend was not statistically significant.
2.5. Offspring physical development endpoints: Exposure of the parental animals of both the F0 and F1 generation to NBC at 4, 15 and 60 ppm had no significant (P > 0.05) adverse effect on the physical development of their litters during the period of lactation, which was evident by the unaltered period, in days, required by pups of F1 and F2a generation to attain certain landmarks of physical development such as days required for unfolding of ear pinna, hair growth on the body, time (days) for eruption of teeth, opening of eyes and opening of ear, compared to the respective control groups.
2.6. Offspring organ weights: Compared to the respective control group, exposure of the parental animals of both the F0 and F1 generation to NBC at dose levels of up to 60 ppm did not affect the organ weights of their F1 and F2a offspring. The group mean values of absolute and relative weights (% of body weights and% of brain weights) of brain, spleen and thymus of pups of F1 and F2a generation did not significantly alter between the control and treatment groups (Table 8).
2.7. Offspring necropsy and histopathology changes: Necropsy performed on the offspring of the F1 and F2a generations, on day 4 or at end of the lactation period, and histological examination of brain, thymus and spleen of pups euthanized at the end of lactation period did not reveal any incidence of gross or microscopic pathological alterations attributable to exposure of their parents to NBC at the dose levels of up to 60 ppm. All the gross and microscopic pathology findings encountered in this study were considered incidental as the incidence was found to be comparable among the control group and the treatment groups, without any dose dependent trend. Thus, NBC treatment did not cause any significant histopathological changes in any organ.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

60 ppm

Based on:

test mat.

Remarks:

The equivalent dose in male and female rats was 7.80 and 8.31 mg/kg/day in male and female rats, respectively.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: The parental and offspring no-observed-adverse-effect level (NOAEL) in this two-generation reproduction toxicity study was found to exceed 60 ppm in diet.

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

60 ppm

Based on:

test mat.

Remarks:

The equivalent dose in male and female rats was 7.80 and 8.31 mg/kg/day in male and female rats, respectively.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: The parental and offspring no-observed-adverse-effect level (NOAEL) in this two-generation reproduction toxicity study was found to exceed 60 ppm in diet.

Critical effects observed:

no

Reproductive effects observed:

not specified

Conclusions:

In this two generation reproduction toxicity study with Niacin bound chromium (NBC) no adverse or non-adverse effects to rats were noted up to the maximum dose tested (60 ppm in diet, equivalent to 8 mg/kg bw/d) and thus the NOAEL = NOEL can be set to the highest dose tested in this study being 8 mg/kg bw/d. No effects on fertility, developmental toxicity or through lactation were seen in rats.

Executive summary:

The findings of this two-generation reproduction toxicity study demonstrate that exposure of male and female Sprague–Dawley rats to Niacin bound chromium (NBC) at the dietary dose levels of 4, 15, and 60 ppm, (approximately corresponding to 0.5, 2 and 8 mg/kg bw/day, respectively) for over two generations was without any adverse effects on various parameters of reproductive performance such as growth, sexual maturity, fertility and mating, gestation, parturition, litter properties, lactation and development of their offspring. NBC, at these dose levels, did not induce any systemic toxicity in the parental rats and their offspring. The present two-generation study serves as a better model for observing the influences of NBC on germ cell development, spermatogenesis, and sexual maturity. Results from the present study did not reveal any adverse effects of NBC on spermatogenesis at dose levels up to 60 ppm in F0 and F1 generation of male rats, respectively.

Overall, the results of two-generation reproductive toxicity in conjunction with earlier studies suggest that under the conditions of the study NBC is safe in male and female rats. The parental and offspring no-observed-adverse-effect level (NOAEL) in this two-generation reproduction toxicity study was found to exceed 60 ppm in diet. The equivalent dose in male and female rats was 7.80 and 8.31 mg/kg/day in male and female rats, respectively.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7.8 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

2 generation study (Deshmukh et al. 2009)

The findings of this two-generation reproduction toxicity study demonstrate that exposure of male and female Sprague–Dawley rats to Niacin bound chromium (NBC) at the dietary dose levels of 4, 15, and 60 ppm, (approximately corresponding to 0.5, 2 and 8 mg/kg bw/day, respectively) for over two generations was without any adverse effects on various parameters of reproductive performance such as growth, sexual maturity, fertility and mating, gestation, parturition, litter properties, lactation and development of their offspring. NBC, at these dose levels, did not induce any systemic toxicity in the parental rats and their offspring. The present two-generation study serves as a better model for observing the influences of NBC on germ cell development, spermatogenesis, and sexual maturity. Results from the present study did not reveal any adverse effects of NBC on spermatogenesis at dose levels up to 60 ppm in F0 and F1 generation of male rats, respectively.

Overall, the results of two-generation reproductive toxicity in conjunction with earlier studies suggest that under the conditions of the study NBC is safe in male and female rats. The parental and offspring no-observed-adverse-effect level (NOAEL) in this two-generation reproduction toxicity study was found to exceed 60 ppm in diet. The equivalent dose in male and female rats was 7.80 and 8.31 mg/kg/day in male and female rats, respectively.

Screening studies with basic chromium sulphate

A 90-day inhalation study with basic chromium sulphate (Derelanko et al, 1999) showed no effects on sperm parameters at exposure concentrations of up to 168 mg/m3. A number of drinking water studies using chromium chloride have suggested effects on male and/or female reproductive performance at high dose levels, however reviews have identified a number of deficiencies in these studies and the effects on the reproductive system are unclear.

Studies with chromium (III) chloride

Administration of high dose levels of chromium chloride in the drinking water (Elbetieha & Al-Hamood, 1997) to mice indicate adverse effects on male and female fertility. The same authors also report effects on fertility and sexual behaviour in male rats.

Screening studies performed with chromium (III) compounds

In an oral 90 day study there were no significant changes in reproductive organ weights in male or female mice or in sperm parameters in male mice. Female mice exposed to 1090 Cr/kg bw/d had significantly longer estrous cycles than the controls. However, this was likely the result of sampling bias because only three females had regular cycles, and therefore, was not considered biologically significant. No exposure-related lesions occurred in male or female mice. In rats there were no significant changes in reproductive organ weights in male or female rats, in sperm parameters in male rats, or in estrous cyclicity in female rats at any dose up to 506 mg Cr/kg bw/d. No exposure-related lesions occurred in male or female rats (NTP 2010 - please refer to section 7.5.1).

No effects on male fertility, female fertility or embryofoetal development were seen in rats mated following exposure to dietary levels of up to 5% chromium (III) oxide for 60 days (Ivankovic & Preussmann, 1975), equivalent to mean achieved intakes in excess of 2000 mg/kg bw/d. No effects on testes or ovary weights were seen following administration at this dose level for 2 years. No effects on sperm parameters were seen in male rats exposed by inhalation to chromium oxide dust at concentrations of up to 30 mg/m3 in a 90-day study (Derelanko et al, 1999). The available information therefore do not indicate any adverse effect of chromium (III) oxide administration on either the reproductive organs or on reproductive function. Kinetic data show that this Cr(III) is poorly absorbed. Chromium (III) is an essential element and highly water-soluble forms (such as the picolinate complex) are used as dietary supplements; data indicate that chromium (III) supplementation may be of benefit in controlling insulin-dependent diabetes and it has been suggested that chromium (III) supplements may be of benefit in the control of gestational diabetes.

Effects on developmental toxicity

Description of key information

Mice, diet, exposure gd 6 -17 d, NOAEL = 25 mg Cr/kg bw/d

Rat, gavage, exposure gd 1 - 3 and 4 - 6, No effects at 102 mg/kg body weight/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline comparable, published study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Method broadly comparable to OECD 414, performed in the mouse using dietary administration

GLP compliance:

no

Remarks:

: published study

Limit test:

no

Specific details on test material used for the study:

The study was performed using the water soluble complexes of Cr (III) chromium picolinate
Chromium(III) picolinate, was synthesised according to the methods of Press et al (1990). Cr3 was synthesised according to the methods of Earnshaw et al (1966). The authenticity of both was established by high resolution electron impact mass spectrometry. Picolinic acid was purchased from Fisher Scientific (Pittsburgh, PA).

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, International (Wilmington, MA)
- Housing: AAALAC-approved animal facility in rooms. Mated females were individually housed in shoe-box-type cages with hardwood bedding
- Diet (e.g. ad libitum): Harlan-Teklad LM-485 rodent ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 40–60%
- Photoperiod (hrs dark / hrs light): 12h/ 12h

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
LM-485 milled rodent diet was purchased from Harlan Teklad (Madison, WI). Either Cr(pic)3 or Cr3 was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day. Extensive stability studies indicate that chromium test compounds are extremely stable and that no degradation in the diet would be expected. Because the purpose of this study was to determine the effects of pharmaceutical levels of chromium, no special measures were taken to prevent exposure of the mice to small amounts of chromium that may be introduced into the diet through methods of feed preparation or from the cage hardware. The diet purchased, Teklad LM-485 (7012), contained added chromium in the form of chromium potassium sulphate (0.48 mg/kg of diet).

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Cr(pic) was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Females were exposed from Gestation Day 6-17

Frequency of treatment:

Continuous (dietary)

Duration of test:

Maternal animals were sacrificed on Gestation Day 17.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control, untreated rodent diet

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

providing 25 mg Cr/kg/day

No. of animals per sex per dose:

Not stated, however the numbers of litters are 27 in the control and 29 in the Cr(pic)3 group.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dosage of chromium picolinate was based on results from a previous study, in which 25 mg Cr/kg/day as Cr(pic)3 was associated with a significant increase in the incidence of cervical arch defects compared to control animals (Bailey et al., 2006).

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on GD 17

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes

Blood sampling:

- Plasma: No data
- Serum: No data

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes, for cervical arch defects
- Skeletal examinations: Yes
- Head examinations: No data

Statistics:

The data from each study replicate were calculated independently, tested for homogeneity of variance by the Levene statistic using SPSS (SPSS Inc., Chicago, IL), and then the replicates were pooled and analyzed together to give the reported results. All tabular data are presented as the mean+-SEM, and the mean value for each parameter was calculated as the mean of the litter means. Data were analyzed by one-way analysis of variance (ANOVA), followed by an LSD post-hoc test to determine specific significant differences (P <= 0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of maternal toxicity were observed for dams in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Maternal weight gain was not affected by the administration of Cr(pic)3.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was virtually identical among the treatment groups, and average food consumption was approximately 7g diet/day.

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

Endocrine 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 specified

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No difference was found in the number of implantations per litter in treated groups as compared to controls.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

percentage of resorbed fetuses did not differ among treatment groups.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

percentage of dead fetuses did not differ among treatment groups.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

element

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 25 mg Cr(III)/kg bw/d.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 25 mg Cr(III)/kg bw/d.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal weight and percentage of resorbed or dead fetuses did not differ among treatment groups.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No gross malformations were observed in any of the fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No increased incidence of skeletal defects was observed in exposed fetuses compared to the controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant increase in the incidence of cervical arch defects. Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Maternal exposure to Cr(pic)3 at the dosages employed did not appear to cause deleterious effects to the developing offspring in mice.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

No signs of maternal toxicity were observed.

Mean foetal weights, foetal viability and the proportion of resorptions were unaffected by treatment. No gross malformations were observed in any of the foetuses. No effects of treatment were observed on the incidence of skeletal anomalies. The authors note that a previous study (Bailey et al, 2006) reported an increased incidence of cervical arch defects in the offspring of mice exposed to chromium picolinate, however the incidence of defects in that study (6.26%) is very similar to the control incidence in this study (5.79% and is therefore not considered to be related to treatment.

Summary of findings

Parameter		Dose group
		0		Cr(pic)3
Litters	(#)	27		29
Foetuses	(#)	332		369
Litter size	(#)	12.30		12.72
Foetal weight	(g± SEM)^b	1.02 ± 0.02		1.05 ± 0.02
Implantations	(#± SEM)^b	12.64 ± 0.50		13.18 ± 0.35
Dead/resorbed foetuses	(#± SEM)^b	2.74 ± 1.07		3.29 ± 0.86
Cervical arch defects^c	(%± SEM)^b	4.65 ± 1.14		6.26 ± 1.63
Maternal weight gain	(g± SEM)^b	12.39  ± 0.37		11.96  ± 0.36

a Significantly different from Cr(pic)3 (25mg Cr/kg/day), and Cr3 (26mg Cr/kg/day) values.

b The mean value for each parameter was calculated as the mean of the litter means.

c Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Conclusions:

No evidence of foetotoxicity, developmental toxicity or teratogenicity was seen in this pre-natal developmental toxicity study in mice exposed to water-soluble complexes of Cr(III) delivering estimated daily doses of Cr(III) equivalent to 25 mg/kg bw/d.

Executive summary:

Mated female mice were administered Cr(III) in the diet from Days 6 -17 of gestation. The dose was 200 mg/kg bw/d (25 mg Cr/kg bw/d). Dams were sacrificed on Day 17 and the uterine contents investigated. Foetuses were assessed for external defects and skeletal findings following double staining. No maternal toxicity was observed. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline comparable, published study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Method broadly comparable to OECD 414, performed in the mouse using dietary administration

GLP compliance:

no

Remarks:

: published study

Limit test:

no

Specific details on test material used for the study:

The study was performed using the water soluble complexes of Cr (III) - [Cr3O(O2CCH2CH3)6(H2O)3]+ (Cr3)

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, International (Wilmington, MA)
- Housing: AAALAC-approved animal facility in rooms. Mated females were individually housed in shoe-box-type cages with hardwood bedding
- Diet (e.g. ad libitum): Harlan-Teklad LM-485 rodent ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 40–60%
- Photoperiod (hrs dark / hrs light): 12h/ 12h

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
LM-485 milled rodent diet was purchased from Harlan Teklad (Madison, WI). Either Cr(pic)3 or Cr3 was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day. Extensive stability studies indicate that chromium test compounds are extremely stable and that no degradation in the diet would be expected. Because the purpose of this study was to determine the effects of pharmaceutical levels of chromium, no special measures were taken to prevent exposure of the mice to small amounts of chromium that may be introduced into the diet through methods of feed preparation or from the cage hardware. The diet purchased, Teklad LM-485 (7012), contained added chromium in the form of chromium potassium sulphate (0.48 mg/kg of diet).

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Cr(pic) was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Females were exposed from Gestation Day 6-17

Frequency of treatment:

Continuous (dietary)

Duration of test:

Maternal animals were sacrificed on Gestation Day 17.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control, untreated rodent diet

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

providing 3.3 mg Cr/kg/day

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

providing 26 mg Cr/kg/day

No. of animals per sex per dose:

Not stated, however the numbers of litters in each group range from 24-27.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dosages of Cr3 were thus chosen to either deliver an equivalent dosage of chromium based on bioavailability (15 mg/kg/day) or to deliver approximately the same mass of chromium (120 mg/kg/day) as the 200 mg/kg/day dose of Cr(pic)3.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on GD 17

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes

Blood sampling:

- Plasma: No data
- Serum: No data

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes, for cervical arch defects
- Skeletal examinations: Yes
- Head examinations: No data

Statistics:

The data from each study replicate were calculated independently, tested for homogeneity of variance by the Levene statistic using SPSS (SPSS Inc., Chicago, IL), and then the replicates were pooled and analyzed together to give the reported results. All tabular data are presented as the mean+-SEM, and the mean value for each parameter was calculated as the mean of the litter means. Data were analyzed by one-way analysis of variance (ANOVA), followed by an LSD post-hoc test to determine specific significant differences (P <= 0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of maternal toxicity were observed for dams in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Maternal weight gain was not affected by the administration of Cr3.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was virtually identical among the treatment groups, and average food consumption was approximately 7 g diet/day.

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

Endocrine 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 specified

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No difference was found in the number of implantations per litter in treated groups as compared to controls.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

percentage of resorbed fetuses did not differ among treatment groups.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

percentage of dead fetuses did not differ among treatment groups.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 26 mg Cr(III)/kg bw/d

Dose descriptor:

NOAEL

Effect level:

26 mg/kg bw/day (nominal)

Based on:

element

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 26 mg Cr(III)/kg bw/d

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal weight and percentage of resorbed or dead fetuses did not differ among treatment groups

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No gross malformations were observed in any of the fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No increased incidence of skeletal defects was observed in exposed fetuses compared to the controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant increase in the incidence of cervical arch defects. Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Maternal exposure to Cr3 at the dosages employed did not appear to cause deleterious effects to the developing offspring in mice.

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Dose descriptor:

NOAEL

Effect level:

26 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Abnormalities:

no effects observed

Developmental effects observed:

no

No signs of maternal toxicity were observed.

Mean foetal weights, foetal viability and the proportion of resorptions were unaffected by treatment. No gross malformations were observed in any of the foetuses. The number of implantations in the low dose Cr₃ group was lower than the other treated groups, however this is not considered to be an effect of treatment in the absence of a dose-response relationship and because implantation occurred prior to exposure. No effects of treatment were observed on the incidence of skeletal anomalies.

Summary of findings

Parameter		Dose group
		0		Cr3 (low)	Cr3(high)
Litters	(#)	27		26	24
Foetuses	(#)	332		275	342
Litter size	(#)	12.30		10.58	14.25
Foetal weight	(g ± SEM)^b	1.02 ± 0.02		1.08 ± 0.03	1.02 ± 0.03
Implantations	(# ± SEM)^b	12.64 ± 0.50		11.00  ± 0.92^a	13.79 ± 0.55
Dead/resorbed foetuses	(# ± SEM)^b	2.74 ± 1.07		3.48 ± 0.95	1.29 ± 0.64
Cervical arch defects^c	(% ± SEM)^b	4.65 ± 1.14		5.18 ± 1.58	3.98 ± 1.33
Maternal weight gain	(g ± SEM)^b	12.39  ± 0.37		11.43 ± 0.48	13.01 ± 0.42

a Significantly different from Cr(pic)3 (25mg Cr/kg/day), and Cr3 (26mg Cr/kg/day) values.

b The mean value for each parameter was calculated as the mean of the litter means.

c Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Conclusions:

No evidence of foetotoxicity, developmental toxicity or teratogenicity was seen in mice exposed to water-soluble complexes of Cr(III) delivering estimated daily doses of Cr(III) equivalent to 3.3 mg/kg bw/d (low dose Cr3 group) and 26 mg/kg bw/d (high dose Cr3 group).

Executive summary:

Mated female mice were administered Cr(III) in the diet from Days 6 -17 of gestation. Dams were sacrificed on Day 17 and the uterine contents investigated. Foetuses were assessed for external defects and skeletal findings following double staining. No maternal toxicity was observed. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Mated female mice were administered Cr(III) in the diet from Days 6 -17 of gestation. The dose was 200 mg/kg bw/d (25 mg Cr/kg bw/d). Dams were sacrificed on Day 17 and the uterine contents investigated. Foetuses were assessed for external defects and skeletal findings following double staining. No maternal toxicity was observed. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen (Bailey et al. 2008).

Chromium trichloride was administered to rats via gavage at doses of 25 mg/rat (102 mg/kg body weight/day) on days 1 – 3 or 4 – 6 of pregnancy. The pregnancy rate of treated rats, when dosed during day 1 - 3 of pregnancy, was slightly reduced (P < 0.05), numbers of implantations and viable fetuses were not significantly affected. When dosed during days 4 - 6 of pregnancy, neither pregnancy rate, nor number of implantations or fetuses were significantly affected. Thus, chromium trichloride had no significant effect in this study (Betaineh et al. 2007).

The results of an EST assay performed with the water-soluble chromium (III) chloride salt were negative.

Justification for classification or non-classification

Based on the available data no classification is proposed according to Regulation (EC) No 1272/2008.