Registration Dossier

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The endpoint toxicity to reproduction is addressed by a weight of evidence assessment, taking into account substance specific information as well as read-across data from chromium(III) substances with a higher bioaccessibility and bioavailability. The aspects of bioavailability are further discussed below.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Deviations from the OECD 416 (2001) guideline were noted: purity of the test item missing; dose stepping too large (guideline foresees a dose stepping of three-fold, if a feeding study is carried out); starting weight of the animals unknown; duration of the mating period not stated; mating procedure not described; detailed clinical observations not conducted; timing of oestrus cycle determination unclear; unclear of the utri of all promiparous females were investigated (number of implantation sites, corpora lutea; post-implantation loss); vagina was not histologically examined; thyroid weight of adults missing; individual data missing
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
2001-01-22
Deviations:
yes
Remarks:
please refer to the field "Rationale for reliability incl. deficiencies" above
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: INTOX Pvt. Ltd. India
- Age at study initiation: 7-9 weeks old
- Diet (ad libitum): ‘‘Nutrilab” brand extruded rodent powdered feed (manufactured by M/s Vetcare Pvt. Ltd., Bangalore, India)
- Water (ad libitum)
- Acclimation period: at least one week before the initiation of experiments

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Relative humidity: 30 - 70 %
- Air changes: 10-15 air changes/ hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
The test item was mixed with powdered rodent diet to obtain the three concentration levels. Initially, a small volume of diet premix was prepared which was then mixed with remaining portion of diet to obtain desired homogeneity of the test article concentration in diet.
Details on mating procedure:
The male and female rats of P-generation from each dose group were mated and allowed to deliver normally.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
10 weeks before mating, throughout mating, and continued until their termination
Frequency of treatment:
ad libitum
Details on study schedule:
At weaning, one male and one female pup from each litter from control and treatment groups were selected for first filial (P1) generation and the remaining pups were sacrificed on lactation day 21. The selected animals were exposed to the test item and they were mated to produce second generation (F2).
Dose / conc.:
4 ppm (nominal)
Remarks:
equivalent to 0.5 mg/kg bw/day (actual concentration)
Dose / conc.:
15 ppm (nominal)
Remarks:
equivalent to 2 mg/kg bw/day (actual concentration)
Dose / conc.:
60 ppm (nominal)
Remarks:
equivalent to 8 mg/kg bw/day (actual concentration)
No. of animals per sex per dose:
30 animals/dose/sex
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: since the test item is intended to be consumed by human beings up to a maximum dose of 4 mg of test item/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 the test item on body weight, feed consumption, mating behaviour, fertility, gestation or lactation in rat at dose level up to 60 ppm.
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption (g/rat/day): Yes, weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

Oestrous cyclicity (parental animals):
Oestrous cycle was determined in the P-generation and the P1 parental generation (no further information was given)
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
- cauda epididymis sperm count
- testicular spermatid head count
- sperm motility
- sperm morphology (at least 200 sperms)
- testis weight
- epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
- mean litter size
- sex ratio (at birth)
- number of stillbirths at day 0
- number of live births at day 0
- surivival
- clinical signs
- physical developmental landmarks (unfolding of pinna, hair growth, teeth eruption, eye opening and ear opening) at appropriate lacation days
- body weights (lactation days: 0, 4, 7, 14 and 21)
- sexual maturation (balano-preputial separation and vaginal opening; F2 generation only)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: after the mating and following confirmation of pregnancy status, males were euthanized and were subjected to necropsy and evaluation of sperm parameters (as described above in the field "Sperm parameters").
- Maternal animals: all females were killed after weaning.

GROSS NECROPSY / HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights were recorded at necropsy, and weighed organs from 10 randomly selected animals were subjected to histopathological examination. All animals, which died or were terminated during the study, were also subjected to necropsy and histopathological examination. Organ absolute weights were recorded and relative weights (% of
body weights) were derived for both P and P1 parental animals. The organs weighed and examined included reproductive organs: epididymides (single and total), testes, prostate, ovaries, uterus, and seminal vesicles with Cowper’s glands. In addition to the reproductive organs, other organs such as brain, pituitary gland, liver, kidneys, adrenals and spleen were weighed and examined for histological changes.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age and were subjected to necropsy.
- Organ weights of brain, spleen and thymus were recorded.
Statistics:
For statistical analysis, a litter was considered as the basic sampling unit.
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 (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).
Reproductive indices:
- female fertility index
- gestation index
Offspring viability indices:
- live-born index
- survival index
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
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 specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
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:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS/MORTALITY:
- 4, 15 and 60 ppm: compared to the respective control groups, across the different dose levels for the P generation results of survival and clinical observations recorded for the parental male and female rats of the P 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 the test item. All deaths and abnormal clinical signs observed in the rats during P generation, 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 test item feeding.

BODY WEIGHTS:
- 4, 15 and 60 ppm: the average body weight and body weight gains of the parental male and female rats of the P generations 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 test item exposure at any of the doses, when compared to the respective control groups and across the different dose levels for the P generation.
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.

FOOD CONSUMPTION:
- 4, 15 and 60 ppm: data on feed consumption by the parental male and female rats of the P generation 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 and across the different dose levels for the P generation.

Based on feed intake, the resulting dose of the test item during premating period for the highest dose groups P male and female was calculated as 5.88 and 8.24 mg/kg/day, respectively, for P generation.

ORGAN WEIGHTS:
- 4, 15 and 60 ppm: 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 P generation exposed to the test item at levels of 4, 15, and 60 ppm did not reveal any significant differences from the respective control group, which could be ascribed to the test item.

GROSS PATHOLOGY:
- 4, 15 and 60 ppm: necropsy performed on the parents of the P generation, 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 pathological alterations attributable to their exposure to the test item at dose levels of up to 60 ppm. All the gross 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.

HISTOPATHOLOGY:
- 4, 15 and 60 ppm: histological examinations performed on the parents of the P 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 microscopic pathological alterations attributable to their exposure to the test item at dose levels of up to 60 ppm. All the 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.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE
- 4, 15 and 60 ppm: unaltered length and normalization of oestrous cycles was observed in treated females.

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 4, 15 and 60 ppm: for the P generation exposed to the test item 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.

REPRODUCTIVE PERFORMANCE:
- 4, 15 and 60 ppm: exposure of male and female rats, from the P generation, to the test item 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 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.
The values of male fertility indices for treatment groups in the P generation 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 the P generation in control, 4, 15 and 60 ppm groups were 90%, 100%, 80% and 92.2%, respectively.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
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 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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
CLINICAL SIGNS/MORTALITY:
- 4, 15 and 60 ppm: compared to the respective control groups, across the different dose levels for the P1 generation results of survival and clinical observations recorded for the parental male and female rats of the P1 generation 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 the test item. All deaths and abnormal clinical signs observed in the rats during P1 generation, 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 test item feeding.

BODY WEIGHTS:
- 4, 15 and 60 ppm: the average body weight and body weight gains of the parental male and female rats of the P1 generation 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 test item exposure at any of the doses, when compared to the respective control groups, across the different dose levels for the P1 generation.
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.

FOOD CONSUMPTION:
- 4, 15 and 60 ppm: data on feed consumption by the parental male and female rats of the P1 generation 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 and across the different dose levels for the P1 generation.

Based on feed intake, the resulting dose of the test item during premating period for the highest dose groups P1 male and female was calculated as 9.71 and 9.83 mg/kg/day, respectively, for the P1 generation.

ORGAN WEIGHTS:
- 4, 15 and 60 ppm: 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 P1 generation exposed to the test item at levels of 4, 15, and 60 ppm did not reveal any significant differences from the respective control group, which could be ascribed to the test item.

GROSS PATHOLOGY:
- 4, 15 and 60 ppm: necropsy performed on the parents of the P1 generation, 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 pathological alterations attributable to their exposure to the test item at dose levels of up to 60 ppm. All the gross 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.

HISTOPATHOLOGY:
- 4, 15 and 60 ppm: histological examinations performed on the parents of the P1 generation, 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 microscopic pathological alterations attributable to their exposure to the test item at dose levels of up to 60 ppm. All the 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.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
REPRODUCTIVE FUNCTION: OESTROUS CYCLE
- 4, 15 and 60 ppm: unaltered length and normalization of oestrous cycles was observed in treated females.

REPRODUCTIVE FUNCTION: SPERM MEASURES
For the P1 generation exposed to the test item 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 p1 parents compared to the P parents was found to be slightly lower, it was not considered to be related to the test item 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.

REPRODUCTIVE PERFORMANCE:
- 4, 15 and 60 ppm: exposure of male and female rats, from the P1 generation, to the test item 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 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.
The values of male fertility indices for treatment groups in the P1 generation 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 P1 generation in 4, 15 and 60 ppm groups 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.
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
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
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
CLINICAL SIGNS /MORTALITY:
Compared to the respective control groups of pups and across the different dose levels, data on survival and clinical observations recorded for the offspring 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.
The pups born alive were unaffected, as evidenced from their live birth indices and did not reveal any treatment related adverse effects.
Comparison of the offspring data with the control group did not reveal any adverse effect on 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 the test item at dose levels of 4, 15, and 60 ppm.

BODY WEIGHTS:
- 4, 15 and 60 ppm: the data on average values of body weights of offspring 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 the test item at levels up to 60 ppm, when compared to the respective control groups and across the different dose levels.

SEXUAL MATURATION:
- 4, 15 and 60 ppm: exposure of the parental animals to the test item 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 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 control group.

ORGAN WEIGHTS.
- 4, 15 and 60 ppm: compared to the control group, exposure of the parental animals to the test item at dose levels of up to 60 ppm did not affect the organ weights of the offspring. The group mean values of absolute and relative weights (% of body weights and% of brain weights) of brain, spleen and thymus of pups did not significantly alter between the control and treatment groups.

GROSS PATHOLOGY:
- 4, 15 and 60 ppm: necropsy performed on the offspring on day 4 or at end of the lactation period did not reveal any incidence of gross pathological alterations attributable to exposure of their parents to the test item at the dose levels of up to 60 ppm. All the gross 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.

HISTOPATHOLOGY - NON-NEOPLASTIC
- 4, 15 and 60 ppm: histological examination of brain, thymus and spleen of pups euthanized at the end of lactation period did not reveal any incidence of microscopic pathological alterations attributable to exposure of their parents to the test item at the dose levels of up to 60 ppm. All the 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, the test item treatment did not cause any significant histopathological changes in any organ.

LITER SIZE / SEX RATIO:
- 4, 15 and 60 ppm: comparison of the offspring data with the control group did not reveal any adverse effect on their litter sizes and the sex ratios of litters following exposure of parental females to the test item at dose levels of 4, 15, and 60 ppm.
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
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
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
CLINICAL SIGNS / MORTALITY:
Compared to the respective control groups of pups and across the different dose levels, data on survival and clinical observations recorded for the offspring 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.
Comparison of the offspring data with the control group did not reveal any adverse effect on 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 the test item at dose levels of 4, 15, and 60 ppm.

BODY WEIGHTS:
- 4, 15 and 60 ppm: the data on average values of body weights of offspring 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 the test item at levels up to 60 ppm, when compared to the respective control groups and across the different
dose levels.

SEXUAL MATURATION:
- 4, 15 and 60 ppm: exposure to the test item at any of the dose levels did not affect the age of sexual maturity by the offspring. 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.

- 4, 15 and 60 ppm: exposure of the parental animals to the test item 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 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 control group.

ORGAN WEIGHTS:
- 4, 15 and 60 ppm: compared to the control group, exposure of the parental animals to the test item at dose levels of up to 60 ppm did not affect the organ weights of the offspring. The group mean values of absolute and relative weights (% of body weights and% of brain weights) of brain, spleen and thymus of pups did not significantly alter between the control and treatment groups.

GROSS PATHOLOGY:
- 4, 15 and 60 ppm: necropsy performed on the offspring on day 4 or at end of the lactation period did not reveal any incidence of gross pathological alterations attributable to exposure of their parents to the test item at the dose levels of up to 60 ppm. All the gross 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.

HISTOPATHOLOGY - NON-NEOPLASTIC
- 4, 15 and 60 ppm: histological examination of brain, thymus and spleen of pups euthanized at the end of lactation period did not reveal any incidence of microscopic pathological alterations attributable to exposure of their parents to the test item at the dose levels of up to 60 ppm. All the 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, the test item treatment did not cause any significant histopathological changes in any organ.

LITER SIZE / SEX RATIO:
- 4, 15 and 60 ppm: comparison of the offspring data with the control group did not reveal any adverse effect on their litter sizes and the sex ratios of litters following exposure of parental females to the test item at dose levels of 4, 15, and 60 ppm.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Parental generations:

- Food consumption: data on feed consumption by the parental male and female rats of both (P and P1) 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 across the two generations.

- Clinical signs and mortality: when compared across the 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 the test item.

- Body weights: the average body weight and body weight gains of the parental male and female rats of both the generations (P and P1) 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 the test item exposure when compared across the two generations.

Offspring generation:

- Body weight: the data on average values of body weights of offspring of both the generations (F1 and F2) 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 the test item at levels up to 60 ppm, when compared across the two generations.

- Clinical signs and mortality: when compared across two generations, data on survival and clinical observations recorded for the offspring of both the generations (F1 and F2) during lactation period of 21 days did not reveal any remarkable differences.

- Litter observations: comparison of the offspring data across the F1 and F2 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 the test item at dose levels of 4, 15, and 60 ppm.

Conclusions:
Deshmukh et al. (2009) investigated the reproductive toxicity of novel oxygen-coordinated niacin-bound chromium (III) complex (NBC) in male and female Sprague-Dawley rats during a two-generation study. Rats were maintained on feed containing the test item at dose levels of 0, 4, 15 or 60 ppm for 10 weeks prior to mating, during mating, and for females, through gestation and lactation, across two generations. No test-item related effects were observed for the parental generations in regard to clinical signs, mortality, body weight, food consumption,organ weights, gross pathology, histopathology, reproductive function (oestrous cycle and sperm measures) and reproductive performance. Furthermore, no test item-related effects were observed for the offsprings in regard to the clinical signs, mortality, body weight sexual maturation, organ weights, gross pathology and histopathology.
No NOAEL could be identified.
Endpoint:
fertility, other
Remarks:
based on a 3-months repeated dose oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-10-14 to 2002-01-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study had some experimental and reporting deficiencies: detailed clinical observations, ophthalmological examinations, neurobehavioural examinations and clinical chemistry examinations were missing; measure of blood clotting time/potential was missing; incomplete organ weight determinations (adrenals, uterus, ovaries, spleen, and brain were missing); histopathological examinations of the spinal cord, aorta, and peripheral nerve were missing; individual data was missing; rationale for dose selection was missing; base-line data for haematology and clinical chemistry were missing
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium picolinate monohydrate was administered ad libitum to groups of 10 male and 10 female B6C3F1 mice in feed at concentrations of 0, 80, 240, 2000, 10000 and 50000 ppm (actually ingested: males: approx. 0, 17, 50, 450, 2300 and 11900 mg/kg bw/day; females: approx. 0, 14, 40,370, 1775 and 9140 mg/kg bw/day) for up to 14 weeks. The following parameters were investigated/recorded: clinical signs, survival, body weight, food consumption, compound intake, haematology, organ weights, gross pathology, and histopathology. Furthermore, evaluation of sperm motility and vaginal cytology were carried out in the 0, 2000, 10000 and 50000 ppm groups at the end of the study.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (~25 °C), protected from light, in sealed plastic buckets.

Stability studies of the bulk chemical were performed using ICP-AES and HPLC-UV with detection at 265 nm. These studies indicated that chromium picolinate monohydrate was stable as a bulk chemical for at least 2 weeks when stored in sealed amber glass containers at temperatures up to 60 °C. To ensure stability, the bulk chemical was stored at room temperature (~25° C), protected from light, in sealed plastic buckets. Periodic reanalyses of the bulk chemical were performed during the 3-month study using HPLC-UV, and no degradation of the bulk chemical was detected.
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
not applicable
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation:
0 ppm group: 19.6 ± 0.2 g (males); 16.9 ± 0.2 g (females)
80 ppm group: 19.5 ± 0.3 g (males); 16.7 ± 0.4 g (females)
240 ppm group: 19.5 ± 0.3 g (males); 16.8 ± 0.2 g (females)
2000 ppm group: 19.4 ± 0.3 g (males); 16.7 ± 0.3 g (females)
10000 ppm group: 19.4 ± 0.3 g (males); 16.8 ± 0.3 g (females)
50000 ppm group: 19.6 ± 0.3 g (males); 16.9 ± 0.4 g (females)
- Housing: 1 male or 5 females per polycarbonate cage (Lab Products, Inc., Maywood, NJ; changed weekly (males) or twice weekly (females)); bedding: irradiated, heat-treated hardwood bedding chips (P.J. Murphy Forest Products, Inc., Montville, NJ; changed weekly (males) twice weekly (females)); Rack filters: Reemay® spun-bonded polyester (Andico, Birmingham, AL; changed once every 2 weeks); Racks: stainless steel (Lab Products, Maywood, NJ; changed once every 2 weeks)
- Diet (ad libitum): irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc.,Gardners, PA)
- Water (ad libitum): tap water
- Acclimation period: 12 days (males) or 11 days (females)

Before the studies began, five male and five female mice were randomly selected for parasite evaluation and gross observation of disease. At the end of the studies, serologic analyses were performed on five male and five female control mice.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 to 23.9 °C
- Relative humidity: 50 % ± 15 %
- Air changes: 10/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
A premix of feed and chromium picolinate monohydrate was prepared, then layered into the remaining feed and blended in a Patterson-Kelly twin-shell blender for 30 minutes using an intensifier bar.
- Rate of preparation of diet (frequency): four times
- Storage temperature of food: stored in sealed double-thick plastic bags, protected from light at 5 °C.
- Storage time: 42 days
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of chromium picolinate monohydrate were conducted using HPLC-UV. During the 3-month studies, the dose formulations were analyzed at the beginning, midpoint, and end of the studies; all 35 dose formulations analyzed for mice were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; eight of 10 for mice were within 10% of the target concentrations (the remaining two samples were -11 and -13 % of the target concentration).

Homogeneity studies of 82 and 50000 ppm dose formulations and stability studies of the 82 ppm dose formulation were performed using ICP-AES. Additional homogeneity studies of 80 and 50,000 ppm dose formulations were performed using HPLC-UV. Homogeneity was confirmed, and stability was confirmed for at least 42 days for dose formulations stored in double-thick sealed plastic bags, protected from light at room temperature and for at least 8 days under simulated animal room conditions; to ensure stability, storage at 5° C was recommended.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
ad libitum
Details on study schedule:
not applicable
Dose / conc.:
0 ppm (nominal)
Remarks:
actually ingested: males and females: 0 mg/kg bw/day
Dose / conc.:
80 ppm (nominal)
Remarks:
actually ingested: males: approx. 17 mg/kg bw/day; females: approx. 14 mg/kg bw/day
Dose / conc.:
240 ppm (nominal)
Remarks:
actually ingested: males: approx. 50 mg/kg bw/day; females: approx. 40 mg/kg bw/day
Dose / conc.:
2 000 ppm (nominal)
Remarks:
actually ingested: males: approx. 450 mg/kg bw/day; females: approx. 370 mg/kg bw/day
Dose / conc.:
10 000 ppm (nominal)
Remarks:
actually ingested: males: approx. 2300 mg/kg bw/day; females: approx. 1775 mg/kg bw/day
Dose / conc.:
50 000 ppm (nominal)
Remarks:
actually ingested: males: approx. 11900 mg/kg bw/day; females: approx. 9140 mg/kg bw/day
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, plain diet
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: clinical findings and survival

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly, and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Feed consumption was recorded weekly by cage.
- Compound intake calculate: Yes

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data
OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of study
- Anaesthetic used for blood collection: Yes, carbon dioxide
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: hematocrit, hemoglobin, erythrocyte, reticulocyte, platelet counts, nucleated erythrocytes, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration and leukocyte count and differentials (segmented neutrophils, lymphocytes, activated lymphocytes, monocytes, basophils and eosinophils)

CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data
IMMUNOLOGY: No data
Oestrous cyclicity (parental animals):
For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females of the core study exposed to 0, 2,000, 10000, or 50000 ppm were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus).
Sperm parameters (parental animals):
Parameters examined in all males of the core study of the 0, 2000, 10000 or 50000 ppm groups at the end of the study:
- spermatid heads per testis and per gram testis
- epididymal spermatozoal motility and concentrations
- left cauda, left epididymis, and left testis were weighed

Modified Tyrode's buffer was applied to slides, and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65 °C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10 % dimethylsulfoxide. Homogenization-resistant spermatid nuclei were counted with a hemacytometer.
Litter observations:
not applicable
Postmortem examinations (parental animals):
SACRIFICE
- on the day of last test item exposure the males and females were sacrificed.

GROSS NECROPSY
- necropsies were performed on all animals.

ORGAN WEIGHTS
- heart, right kidney, liver, lung, right testis, and thymus of all animals were weighed.

HISTOPATHOLOGY
Tissues for microscopic examination of all animals were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Complete histopathologic examinations were performed on control and 50,000 ppm rats. The following tissues and organs were examined: gross lesions and tissue masses, adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, gallbladder, harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
Postmortem examinations (offspring):
not applicable
Statistics:
Survival analyses: Kaplan-Meier surivival curves, means, life table trend test, & life table pairwise comparisons. P values were two-sided.
Nonneoplastic lesions: Poly-k test, continuity-corrected Poly-3 test. P values were one-sided.
Organ and body weight data: parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data: nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
Jonckheere’s test was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined, and implausible values were eliminated from the analysis. Because vaginal cytology data are proportions, an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. Proportions of regular cycling females in each exposed group were compared to the control group using the Fisher exact test. Tests for extended periods of estrus and diestrus were constructed based on a Markov chain model proposed by Girard and Sager (1987). For each exposure group, a transition probability matrix was estimated for transitions among the proestrus, estrus, metestrus, and diestrus stages, with provision for extended stays within estrus and diestrus. Equality of transition matrices among exposure groups and between the control group and each exposed group was tested using chi-square statistics.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
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:
no effects observed
Clinical biochemistry 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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not specified
CLINICAL SIGNS
Males and females: there were no clinical findings related to exposure to chromium picolinate monohydrate; reddish-colored faeces of 50000 ppm animals wer believed to be due to excretion of the test article and were not considered a sign of toxicity.

MORTALITY
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: all mice survived to the end of the study.

BODY WEIGHT AND WEIGHT CHANGES
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: final mean body weights and body weight gains of all exposed groups were similar to those of the control groups (no statistical significance).

FOOD CONSUMPTION AND COMPOUND INTAKE
Males:
- 80, 240, 2000, 10000 and 50000 ppm groups: feed consumption by exposed groups of males was similar to that by the controls throughout the study. Dietary concentrations of 80, 240, 2000, 10000, and 50000 ppm resulted in average daily doses of approx. 17, 50, 450, 2300, and 11900 mg chromium picolinate monohydrate/kg body weight to males.

Females:
- 80, 240, 2000, 10000 and 50000 ppm groups: feed consumption by exposed groups of males was similar to that by the controls throughout the study. Dietary concentrations of 80, 240, 2000, 10000, and 50000 ppm resulted in average daily doses of approx. 14, 40, 370, 1775, and 9140 mg chromium picolinate monohydrate/kg body weight to males.

HAEMATOLOGICAL FINDINGS
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: there were no hematological effects in mice administered chromium picolinate monohydrate.

ORGAN WEIGHT FINDINGS
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: there were no biologically significant differences in organ weights between exposed and control groups of mice.

GROSS PATHOLOGICAL FINDINGS
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: no exposure-related lesions occurred in male or female mice.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: no exposure-related lesions occurred in male or female mice.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
- 10000 ppm group: female mice had statistically significant longer estrous cycles (P ≤ 0.05) 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.
- 2000 and 50000 ppm groups: there were no statistically significant changes in estrous cyclicity in female rats.

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 2000, 10000 and 50000 ppm groups: there were no statistically significant changes in sperm parameters in male mice.
There were no significant changes in reproductive organ weights in male mice at any dose.
Dose descriptor:
NOAEL
Remarks:
general toxicity
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Generation:
F1
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified
Conclusions:
Continuous exposure to chromium picolinate monohydrate in feed (80, 240, 2000, 10000 and 50000 ppm; actual ingested: males: approx. 17, 50, 450, 2300 and 11900 mg/kg bw/day; females: approx. 14, 40,370, 1775 and 9140 mg/kg bw/day)) of male and female B6C3F1 mice for an exposure duration of 14 weeks caused no treatment-related effects on clinical signs, survival, body weights, food consumption, haematology, organ weights, gross pathology and histopathology. Furthermore, concentrations of 2000, 10000 and 50000 ppm did not cause treatment-related effects on reproductive functions (sperm measures and estrous cycle) of male and female mice.

Based on the findings from this study, the NOEL (No-Observed-Effect-Level) for systemic toxicity was considered to be 50000 ppm (actually ingested: males: approx. 11900 mg/kg bw/day (equivalent to approx. 1428 mg Cr/kg bw/day); females: approx. 9140 mg/kg bw/day (equivalent to approx. 1096.8 mg Cr/kg bw/day))for male and female mice based on the absence of any relevant toxicological effects. Furthermore, the NOEL for reproductive toxicity was also considered to be 50000 ppm for male and female mice based on the absence of any relevant toxicological effects.
Endpoint:
fertility, other
Remarks:
based on a 3-months repeated dose oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-10-16 to 2002-01-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study had some experimental and reporting deficiencies: detailed clinical observations, ophthalmological examinations, and neurobehavioural examinations were missing; measure of blood clotting time/potential was missing; incomplete clinical chemistry (sodium, potassium, glucose, total cholesterol, urea were missing); incomplete organ weight determinations (adrenals, uterus, ovaries, spleen, and brain were missing); histopathological examinations of the spinal cord, aorta, and peripheral nerve were missing; individual data was missing; rationale for dose selection was missing; base-line data for haematology and clinical chemistry were missing
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium picolinate monohydrate was administered ad libitum to groups of 10 male and 10 female F344/N rats in feed at concentrations of 0, 80, 240, 2000, 10000 and 50000 ppm (actually ingested: males: approx. 0, 7, 20, 160, 800 and 4240 mg/kg bw/day; females: approx. 0, 6, 20,160, 780 and 4250 mg/kg bw/day) for up to 14 weeks. The following parameters were investigated/recorded: clinical signs, survival, body weight, food consumption, compound intake, haematology, clinical chemistry, organ weights, gross pathology, and histopathology. Furthermore, evaluation of sperm motility and vaginal cytology were carried out in the 0, 2000, 10000 and 50000 ppm groups at the end of the study. Lastly, additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 3 weeks. The additional groups were investigaed for the following parameters: haematology and clinical chemistry on days 3 and 21.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (~25 °C), protected from light, in sealed plastic buckets.

Stability studies of the bulk chemical were performed using ICP-AES and HPLC-UV with detection at 265 nm. These studies indicated that chromium picolinate monohydrate was stable as a bulk chemical for at least 2 weeks when stored in sealed amber glass containers at temperatures up to 60 °C. To ensure stability, the bulk chemical was stored at room temperature (~25° C), protected from light, in sealed plastic buckets. Periodic reanalyses of the bulk chemical were performed during the 3-month study using HPLC-UV, and no degradation of the bulk chemical was detected.
Species:
rat
Strain:
other: F344/N
Details on species / strain selection:
not applicable
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation:
0 ppm group: 92 ± 1 g (males); 93 ± 1 g (females)
80 ppm group: 91 ± 2 g (males); 93 ± 1 g (females)
240 ppm group: 91 ± 2 g (males); 93 ± 1 g (females)
2000 ppm group: 92 ± 1 g (males); 93 ± 2 g (females)
10000 ppm group: 92 ± 1 g (males); 94 ± 1 g (females)
50000 ppm group: 92 ± 1 g (males); 93 ± 1 g (females)
- Housing: 5 animals per polycarbonate cage (Lab Products, Inc., Maywood, NJ; changed twice weekly); bedding: irradiated, heat-treated hardwood bedding chips (P.J. Murphy Forest Products, Inc., Montville, NJ; changed twice weekly); Rack filters: Reemay® spun-bonded polyester (Andico, Birmingham, AL; changed once every 2 weeks); Racks: stainless steel (Lab Products, Maywood, NJ; changed once every 2 weeks)
- Diet (ad libitum): irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc.,Gardners, PA)
- Water (ad libitum): tap water
- Acclimation period: 13 days (males) or 14 days (females)

Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation of disease. At the end of the studies, serologic analyses were performed on five male and five female control rats.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 to 23.9 °C
- Relative humidity: 50 % ± 15 %
- Air changes: 10/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
A premix of feed and chromium picolinate monohydrate was prepared, then layered into the remaining feed and blended in a Patterson-Kelly twin-shell blender for 30 minutes using an intensifier bar.
- Rate of preparation of diet (frequency): four times
- Storage temperature of food: stored in sealed double-thick plastic bags, protected from light at 5 °C.
- Storage time: 42 days
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of chromium picolinate monohydrate were conducted using HPLC-UV. During the 3-month studies, the dose formulations were analyzed at the beginning, midpoint, and end of the studies; all 35 dose formulations analyzed for rats were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; all 10 animal room samples for rats were within 10% of the target concentrations.

Homogeneity studies of 82 and 50000 ppm dose formulations and stability studies of the 82 ppm dose formulation were performed using ICP-AES. Additional homogeneity studies of 80 and 50,000 ppm dose formulations were performed using HPLC-UV. Homogeneity was confirmed, and stability was confirmed for at least 42 days for dose formulations stored in double-thick sealed plastic bags, protected from light at room temperature and for at least 8 days under simulated animal room conditions; to ensure stability, storage at 5° C was recommended.
Duration of treatment / exposure:
14 weeks (exception: clinical pathology study: 3 weeks)
Frequency of treatment:
ad libitum
Details on study schedule:
not applicable
Dose / conc.:
0 ppm (nominal)
Remarks:
actually ingested: males and females: 0 mg/kg bw/day
Dose / conc.:
80 ppm (nominal)
Remarks:
actually ingested: males: approx. 7 mg/kg bw/day; females: approx. 6 mg/kg bw/day
Dose / conc.:
240 ppm (nominal)
Remarks:
actually ingested: males: approx. 20 mg/kg bw/day; females: approx. 20 mg/kg bw/day
Dose / conc.:
2 000 ppm (nominal)
Remarks:
actually ingested: males: approx. 160 mg/kg bw/day; females: approx. 160 mg/kg bw/day
Dose / conc.:
10 000 ppm (nominal)
Remarks:
actually ingested: males: approx. 800 mg/kg bw/day; females: approx. 780 mg/kg bw/day
Dose / conc.:
50 000 ppm (nominal)
Remarks:
actually ingested: males: approx. 4240 mg/kg bw/day; females: approx. 4250 mg/kg bw/day
No. of animals per sex per dose:
10 males / 10 females (core and clinical pathology study)
Control animals:
yes, plain diet
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (core study)
- Time schedule: twice daily
- Cage side observations checked: clinical findings and survival

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes (core study)
- Time schedule for examinations: initially, weekly, and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (core study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Feed consumption was recorded weekly by cage.
- Compound intake calculate: Yes

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data
OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes (clinical pathology study and core study)
- Time schedule for collection of blood:
clinical pathology study: days 3 and 21
core study: at the end of study
- Anaesthetic used for blood collection: Yes, carbon dioxide
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: hematocrit, hemoglobin, erythrocyte, reticulocyte, platelet counts, nucleated erythrocytes, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration and leukocyte count and differentials (segmented neutrophils, lymphocytes, activated lymphocytes, monocytes, basophils and eosinophils)

CLINICAL CHEMISTRY: Yes (clinical pathology study and core study)
- Time schedule for collection of blood:
clinical pathology study: days 3 and 21
core study: at the end of study
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data
IMMUNOLOGY: No data
Oestrous cyclicity (parental animals):
For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females of the core study exposed to 0, 2,000, 10000, or 50000 ppm were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus).
Sperm parameters (parental animals):
Parameters examined in all males of the core study of the 0, 2000, 10000 or 50000 ppm groups at the end of the study:
- spermatid heads per testis and per gram testis
- epididymal spermatozoal motility and concentrations
- left cauda, left epididymis, and left testis were weighed

Test yolk was applied to slides, and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65 °C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10 % dimethylsulfoxide. Homogenization-resistant spermatid nuclei were counted with a hemacytometer.
Litter observations:
not applicable
Postmortem examinations (parental animals):
SACRIFICE
- on the day of last test item exposure the males and females were sacrificed.

GROSS NECROPSY
- necropsies were performed on all core study animals.

ORGAN WEIGHTS
- heart, right kidney, liver, lung, right testis, and thymus of all core study animals were weighed.

HISTOPATHOLOGY
Tissues for microscopic examination of all core study animals were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Complete histopathologic examinations were performed on control and 50,000 ppm rats. The following tissues and organs were examined: gross lesions and tissue masses, adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
Postmortem examinations (offspring):
not applicable
Statistics:
Survival analyses: Kaplan-Meier surivival curves, means, life table trend test, & life table pairwise comparisons. P values were two-sided.
Nonneoplastic lesions: Poly-k test, continuity-corrected Poly-3 test. P values were one-sided.
Organ and body weight data: parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data: nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
Jonckheere’s test was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined, and implausible values were eliminated from the analysis. Because vaginal cytology data are proportions, an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. Proportions of regular cycling females in each exposed group were compared to the control group using the Fisher exact test. Tests for extended periods of estrus and diestrus were constructed based on a Markov chain model proposed by Girard and Sager (1987). For each exposure group, a transition probability matrix was estimated for transitions among the proestrus, estrus, metestrus, and diestrus stages, with provision for extended stays within estrus and diestrus. Equality of transition matrices among exposure groups and between the control group and each exposed group was tested using chi-square statistics.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
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:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not specified
CLINICAL SIGNS
Males and females: there were no clinical findings related to exposure to chromium picolinate monohydrate; reddish-colored faeces of 50000 ppm animals were believed to be due to excretion of the test article and were not considered a sign of toxicity.

MORTALITY
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: all rats survived to the end of the study.

BODY WEIGHT AND WEIGHT CHANGES
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: final mean body weights and body weight gains of all exposed groups were similar to those of the control groups (no statistical significance).

FOOD CONSUMPTION AND COMPOUND INTAKE
Males:
- 80, 240, 2000, 10000 and 50000 ppm groups: feed consumption by exposed groups of males was similar to that by the controls throughout the study. Dietary concentrations of 80, 240, 2000, 10000, and 50000 ppm resulted in average daily doses of approx. 7, 20, 160, 800, and 4240 mg chromium picolinate monohydrate/kg body weight to males.

Females:
- 80, 240, 2000, 10000 and 50000 ppm groups: feed consumption by exposed groups of females was similar to that by the controls throughout the study. Dietary concentrations of 80, 240, 2000, 10000, and 50000 ppm resulted in average daily doses of approx. 6, 20, 160, 780, and 4250 mg chromium picolinate monohydrate/kg body weight to females.

HAEMATOLOGICAL FINDINGS
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: all changes were within physiological normal levels, none demonstrated an exposure relationship, and none were considered biologically important or toxicologically relevant.

CLINICAL BIOCHEMISTRY FINDINGS
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: all changes were within physiological normal levels, none demonstrated an exposure relationship, and none were considered biologically important or toxicologically relevant.

ORGAN WEIGHT FINDINGS
Males:
50000 ppm group: relative heart weight was statistically significantly lower (P ≤ 0.05) than the control group.

Females:
- 80, 240, 2000, 10000 and 50000 ppm groups: absolute and relative kidney weights of all exposed groups were significantly (P ≤ 0.05 or P ≤ 0.01) greater than those of the controls, and relative liver weights of exposed groups (80, 240, 2000 and 10000 ppm groups) were significantly (P ≤ 0.05 or P ≤ 0.01) greater than that of the controls. Since there were no significant histologic or clinical chemistry effects in the liver or kidney or dose-related trends in kidney weights, the increases in the weights of these organs were not considered to be biologically significant.

GROSS PATHOLOGICAL FINDINGS
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: no exposure-related lesions occurred in male or female rats.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
Males and females:
- 80, 240, 2000, 10000 and 50000 ppm groups: no exposure-related lesions occurred in male or female rats.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
- 2000, 10000 and 50000 ppm groups: there were no statistically significant changes in estrous cyclicity in female rats at any dose

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 2000, 10000 and 50000 ppm groups: there were no statistically significant changes in sperm parameters at any dose
There were no significant changes in reproductive organ weights in male rats at any dose.
Dose descriptor:
NOAEL
Remarks:
general toxicity
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Generation:
F1
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified
Conclusions:
Continuous exposure to chromium picolinate monohydrate in feed (80, 240, 2000, 10000 and 50000 ppm; actually ingested: males: approx. 7, 20, 160, 800 and 4240 mg/kg bw/day; females: approx. 6, 20,160, 780 and 4250 mg/kg bw/day) of male and female F344/N rats for an exposure duration of 14 weeks caused no treatment-related effects on clinical signs, survival, body weights, food consumption, haematology, clinical chemistry, organ weights, gross pathology and histopathology. Furthermore, concentrations of 2000, 10000 and 50000 ppm did not cause treatment-related effects on reproductive functions (sperm measures and estrous cycle) of male and female rats.

Based on the findings from this study, the NOEL (No-Observed-Effect-Level) for systemic toxicity was considered to be 50000 ppm (actually ingested: males: approx. 4240 mg/kg bw/day (equivalent to approx. 508.8 mg Cr/kg bw/day); females: approx. 4250 mg/kg bw/day (equivalent to approx. 510 mg Cr/kg bw/day)) for male and female rats based on the absence of any relevant toxicological effects. Furthermore, the NOEL for reproductive toxicity was also considered to be 50000 ppm for male and female rats based on the absence of any relevant toxicological effects.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Mode of action

As discussed in the read-across document attached to section 13 of IUCLID, the toxicity of metals and their compounds largely depends on their bioavailability, i.e. the mechanisms of uptake through membranes, intracellular distribution and binding to cellular macromolecules. Data available on the toxicity of trivalent chromium compounds via the oral route suggest that these materials are much less toxic than are the hexavalent compounds, and that the toxicity varies with water solubility. Chromium(III) is absorbed via passive diffusion and phagocytosis, resulting in low total uptake into cells. In general, chromium(III) is very poorly absorbed, with only 0.5 to 2.8% of chromium absorbed via the gastrointestinal tract of humans (ATSDR 2000).

 

As the bioavailability of trivalent chromium is regarded to be of major importance for the respective toxicity, the bioavailability of the insoluble chromium(III) oxide is considered in comparison with more water-soluble inorganic chromium(III) salts and inorganic chromium complexes with organic ligand.

Absorption and excretion of the inorganic chromium salts chromium(III) oxide, chromium chloride hexahydrate and chromium acetate hydroxide were evaluated in male Sprague Dawley rats (Juturu 2003). Rats were orally administered once with 1000 mg/kg bw chromium chloride hexahydrate, chromium acetate hydroxide or chromium oxide. 24-hours urine collection and tissue samples were analyzed for chromium content. Trace levels of chromium were detected in urine samples from rats administered chromium oxide (group mean <0.2 mg/l) while urinary chromium levels for the rats given chromium chloride and chromium acetate were 174 mg/l and 93 mg/l, respectively. Elevated levels of chromium were observed in liver, kidney, heart and pancreas samples collected from rats administered chromium chloride and chromium acetate, compared to chromium levels detected in the chromium oxide and control groups. The tissue chromium concentrations from the chromium oxide group were within or even below control group levels. Evaluation of chromium levels in urine and tissues show that chromium(III) oxide was poorly absorbed (0.0002%) compared to chromium chloride hexahydrate (0.92%) and chromium acetate hydroxide (0.75%). These findings confirm that the bioavailability depends on the water solubility of the chromium(III) compounds.

The very low oral bioavailability of insoluble chromium(III) oxide is reflected by the findings from the acute oral toxicity studies with LD50 values above 5000 mg/kg bw (Loeser 1972, Bomhard 1988). In addition, no biologically relevant systemic effects were indicated in the subchronic and chronic feeding studies with chromium(III) oxide pigment (Ivankovic 1975).

Beside the water solubility, chromium(III) bioavailability is also highly dependent on the nature of the Cr(III) ligand complex ingested or formed in vivo. The absorption/ retention of three trivalent chromium compounds (chromium chloride, chromium picolinate (CrPic), and CrNic (oxygen-coordinated niacin-bound chromium(III) complex, also known as NBC) were evaluated over a 12-hour period in male CD rats (Olin 1994). The rats were gavaged with radioactive-labelled chromium as CrCl3 x 6H20, CrNic or CrPic in a 25% egg white slurry. Rats were killed at 1, 3, 6, and 12 hours post-gavage. Cardiac blood was collected and liver, kidneys, pancreas, testes and gastrocnemius were removed weighed and assayed for 51Cr. The amount of 51Cr in these tissues along with that in urine (collected from the 6 and 12 hour groups), was used to calculate 51Cr absorbed/retained. Chromium(III) in all forms was very poorly absorbed with less than 1% absorption at all time points. For the majority of the time points and tissues, the average percentage of retained 51Cr was higher in CrNic-gavaged rats than in chromium chloride- or CrPic-gavaged rats. Tissues collected 1 hour post-gavage from CrNic rats had retention percentages that were 3.2 to 8.4-fold higher than the CrPic or chromium chloride groups. Three hour post-gavage, CrNic treated rats had blood, muscle, and pancreatic 51Cr retentions that were 2.4 to 8 times higher than CrPic-gavaged rats. By 6 and 12 h post-gavage, the 51Cr retention in tissues were 1.8 to 3.8 times higher in CrNic than in CrPic treated rats. The percentage of the 51Cr dose retained in body tissues and fluids with respect to the administered dose was less than 1 % (0.29% to 0.75%) for all chromium sources at all time points. The authors concluded that there can be significant differences in the bioavailability of different chromium compounds in the rat. However, overall chromium in all forms was very poorly absorbed (less than 1%) at all time points.

This finding is supported by another study with rats, the evaluation of inorganic chromium salt (chromium chloride) and inorganic chromium complexes (chromium nicotinate, chromium picolinate and chromium dinicotinic acid glycine cystein glutamic acid) also revealed a low bioavailability (less than 1%) of chromium from various sources (Polansky 1993 cited in EFSA 2008).

In conjunction with the chronic oral toxicity studies in rats and mice the NTP investigated the stability of the inorganic chromium(III) complex upon oral administration to determine its fate (NTP 2010). The aim of this investigation was whether the complex dissociates in vivo, liberating the chromium(III) cation and the nicotinate anion or whether the stability of the complex is sufficiently high so that it is excreted unchanged. Excretion of picolinate-derived radioactivity in rats is about equally divided between urine and feces. Excretion of chromium, however, is nearly 100% in feces. This implies that much of the picolinate is absorbed without the chromium attached. The major urinary metabolite is a glycine conjugate of picolinic acid. The excretion pattern is similar in mice. The ratio of excretion in urine versus feces may be affected more by formulation in mice. Picolinic acid is cleared rapidly in both rats and mice with elimination being primarily by conjugation with glycine. There is little evidence for accumulation of picolinate-derived material in tissues.

Concluding the oral absorption rates and retention rates of different inorganic chromium salts and organic chromium complexes, the bioavailability of the insoluble chromium oxide is extremely low compared to the other trivalent chromium compounds discussed (bioavailability: chromium nicotinate ≈ chromium picolinate > CrCl3 >> Cr2O3).

Absorption from the respiratory system varies depending on the chemical, solubility, and particle size characteristics of the chromium compound involved. Regarding insoluble chromium(III) particles, after removal to the gastrointestinal tract by mucociliary clearance, uptake is a very slow process presumably mediated by gradual dissolution in phagocytic cells of the lungs and removal via the lymphatic system (ICDA 2006). A very low uptake of insoluble chromium oxide is assumed because of the very low toxicity observed after acute and repeated exposure via the inhalation route. The results of a modern, GLP and guideline compliant study (Gaunt 2009) reports an LC50 value of >5.41 mg/L. Exposure to the test compound was well tolerated and no mortalities occurred. Clinical signs were limited to increased breathing rate during exposure. The principle effects observed in a reliable 13-week inhalation study (Derelanko 1999) were primarily to the respiratory tract, no systemic effects were indicated. The authors concluded that the changes seen in the respiratory tract appeared to be directly associated with the presence of insoluble particles, observed both macroscopically and microscopically in the affected tissues; a non-specific response to the physical presence of test material deposits is discussed. Similar effects have been reported to occur with other inert, respirable particles of low toxicity.

Parallel to the chromic oxide dust basic chromium sulfate was investigated in the 13-week inhalation study. The results of this study demonstrated that there are significant differences in the toxicity of trivalent chromium compounds, chromic oxide and basic chromium sulfate, to the respiratory tract following a 3-month subchronic inhalation exposure in rats. While for both materials the principle effects observed after 90 days exposure were primarily noted in the respiratory tract, the type and location of effects observed were different between the two trivalent chromium compounds despite the animals being exposed to equivalent concentrations of trivalent chromium. The effects on the respiratory tract following 13 weeks of exposure to basic chromium sulfate were more severe nature and more widespread (including the nasal cavity and larynx in addition to the lungs and mediastinal lymph node) than observed for chromium(III) oxide. These effects are most likely related to the acidity of the basic chrome sulfate, which readily forms acidic solutions (ca. pH 2.8), presumably with sulfate group.

Comparing the bioavailability of both chromium salts, beside the localized effects on the respiratory tract, no evidence of systemic toxicity was observed with exposure to chromium(III) oxide. Evidence of systemic toxicity observed with basic chromium sulfate was primarily limited to reductions in body weight not related to reduced food consumption during the 13 weeks of exposure (Derelanko 1999).

In conclusion, the results of this study indicate significant differences in toxicity to the respiratory tract between trivalent chromium compounds chromic oxide and basic chromium sulfate. These are likely related to differences in acidity and water solubility, rather than chromium concentration per se. This conclusion is substantiated by the lack of effect on other internal organs.

 

 

Fertility impairment

The toxicity of trivalent chromium oxide was evaluated in male and female Fischer 344 rats in a 13-week nose-only inhalation study that included a 13-week recovery period (Derelanko 1999). Nose-only exposure to insoluble chromium(III) oxide dust at 4.4, 15 or 44 mg/m³ was carried out for 6 h/day, 5 days per week. Fifteen male and fifteen females Fischer 344 rats were treated per dose group. One group (group 1) served as negative control (0 mg/m³). At the end of the exposures, 10 males and 10 females from each group were sacrificed. The remaining 5 males and females from each group were maintained for an additional 13-week recovery period during which time they received no additional exposures. Each animal was observed immediately prior to and following each exposure for overt toxicity and mortality. Body weights and clinical signs were recorded weekly for each rat. Opthalmoscopic examinations, haematology, clinical biochemistry and gross and microscopic evaluations were performed. No treatment-related death was indicated. In addition, none of the exposure groups, for either sex, exhibited a statistically significant difference from the control group for any haematological, serum biochemical, or urinalysis parameters. No systemic effects of chromium oxide were indicated after inhalation exposure in Fischer 344 rats (see discussion above). The principle effects observed were primarily to the respiratory tract.

At necropsy sperm samples from the left caudal epididymis of 10 males per group were used for evaluation of sperm motility, count and morphology. No substance-related adverse effects on the evaluated sperm parameter were observed in any of the treated males. In addition, no effects on testis and ovaries weights were noted in any of the treated animals compared to control group animals. The macroscopic and microscopic evaluation of the reproduction organs and associated tissues (testis, epididymis, prostate gland, ovary, uterus, cervix, vagina) of high dose animals revealed no treatment-related adverse effects compared to the corresponding negative control.

In summary, nose-only exposure to insoluble chromic dust up to 44 mg/m³ did not induce systemic effects and histological changes or organ weight effects on the reproduction organs of exposed male and female Fischer 344 rats. In addition, no substance-related adverse effects on sperm parameters were noted in any of the treated males. The very low bioavailability of chromic oxide via the inhalation route indicated is in line with lack of systemic effects and absence of effects at the reproduction organs.

 

An early feeding study is considered for supporting reasons. In this early subchronic feeding study (Ivankovic 1975) inbred BD rats were feed for 90 days with chromium(III) oxide. No systemic effects of the test compound were noted in any of the treated rats. Beside the general evaluation done in an early subchronic feeding study, nine females were paired with males from the same dose group following treatment for 60 days (no additional data given). All females became pregnant, gestation periods were normal (23 days) and the resulting offspring did not show any malformations or other adverse effects. Litter sizes were unaffected by treatment (no quantitative data given). In summary, based on the findings from this early subchronic feeding study no indications of adverse effects on fertility or offspring are given. However, the study design and documentation is limited concerning the endpoint toxicity to reproduction. The findings from this study are used only for supporting reasons in a weight of evidence approach.

 

In a read across study data from more water soluble inorganic chromium salts and inorganic chromium complex with organic ligand were considered to assess the reproduction toxicity.

The continuous exposure of rats and mice to chromium picolinate monohydrate in feed at doses of 80, 240, 2000, 10000 and 50000 ppm for an exposure duration of 14 weeks caused no treatment-related effects on clinical signs, survival, body weights, food consumption, haematology, organ weights, gross pathology and histopathology. Furthermore, none of the doses did cause treatment-related effects on reproductive functions (sperm measures and estrous cycle) of male and female rats and mice. On the basis of a complete absence of adverse effects in reproductive organs, the NOAEL for fertility impairment is above 50000 ppm (NTP 2010).

 

More recent and reliable study data are available for an inorganic chromium complex with niacin ligands. The safety of chromium(III) nicotinate (NBC or ChromeMate, niacin-bound chromium) was evaluated in a two-generation reproduction study (Deshumukh 2009). According to the authors (Deshumukh 2009) 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.

Sprague-Dawley rats were maintained on feed containing chromium(III) nicotinate at dose levels of 0, 4, 15 or 60 ppm for 10 week prior to mating, during mating, and for females through gestation and lactation, across two generations. For the parents (F0 and F1) and the offspring (F1 and F2a) reproductive parameters such as fertility and mating, gestation, parturition, litters, lactation, sexual maturity and development of offspring were assessed. Results from this study indicated that dietary exposure to chromium(III) nicotinate to parental male and female rats of both (F0 and F1) the generations during pre-mating and mating periods, for both sexes, and during gestation and lactation in case of female rats, did not cause any significant incidence of mortality or abnormal clinical signs. The findings of this study demonstrate that exposure to dietary dose levels of 4, 15, and 60 ppm (corresponding to approx. 0.5, 2 and 8 mg chromium(III) nicotinate/kg/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. Chromium(III) nicotinate, up to 60 ppm in the diet, did not induce any systemic toxicity in the parental rats and their offspring. In summary, no adverse effects on reproduction as evaluated by sexual maturity, fertility and mating, gestation, parturition, litter properties, lactation and development of the offspring were observed up to 60 ppm chromium(III) nicotinate treatment, 100 times the maximum recommended human dose of NBC.

  

Affects on reproduction and/or reproduction organs were noted in rodents treated with high dosages of the water soluble trivalent chromium compounds chromium sulfate or chromium chloride (Zahid 1990, Bataineh 1997, Elbetiha 1997). As discussed by ICDA (2006) all of these studies suffer from serious deficiencies in study design and performance and therefore no definitive conclusions of the reproductive toxicity of water soluble(III) compounds can be made based on these studies. Because of the deficiencies in study design and performance these studies are disregarded for hazard assessment:

Male mice exposed for 35 days to 15, 30 or 60 mg chromium(III)kg/day (100, 200 or 400 ppm) as chromium sulfate in the diet had reduced sperm count, increased number of morphologically abnormal sperms and degeneration of the outer cellular layer of the seminiferous tubules (Zahid 1990). However, this study is of limited value due to methodological deficiencies and insufficient documentation.

Male Sprague-Dawley rats exposed for 12 weeks to 1000 mg/l chromium chloride in the drinking water (ca. 24 mg Cr(III) kg/day) were mated to unexposed females (1:2) (Bataineh 1997). Male fertility indices (assessed by impregnation, number of implantations, and number of viable foetuses) did not appear to be adversely affected by exposure to chromium chloride, although the untreated females mated to treated males exhibited an increase in the total number of resorptions. Body weight and absolute testes, seminal vesicles and preputial gland weights were significant decreased in Cr(III) treated males. Because of the significant decrease in body weights noted (ca. 30%) at 1000 mg/l a secondary effect might be more likely than a primary effect on fertility.

Male Swiss mice were exposed to 2000 and 5000 mg/l chromium chloride in drinking water (ca. 82 or 204 mg Cr(III)/kg/day) for 12 weeks (Elbetieha 1997). The treated males were mated to unexposed females. The body weights and relative weights of the preputial gland were statistically significant reduced in treated males, whereas the testis weights were significantly increased. The fertility of males of the high dose group (5000 mg/l) was significant decreased. Because of the significant decrease in body weights seen in both male treatment groups a secondary effect might be possible. In a second experiment female Swiss mice were exposed to 2000 and 5000 mg/l chromium chloride in drinking water (ca. 85 or 212 mg Cr(III)/kg/day) for 12 weeks. The treated females were mated to untreated males. No effects on body weight were noted in treated females, the relative ovary and uterus weights of high dose females were statistically significant decreased; however only body weights and organ weights of the control group and 5000 mg/l group are documented, no data given for the 2000 mg/l group (no rational given). Impaired fertility (decreased number of implantations and viable foetuses) was observed in treated females in both dose groups (no. of implantations: control 8.18 ± 1.59, 2000 mg Cr/l: 7.47 ± 2.50, 5000 mg Cr/l: 6.87 ± 2.23; no. of viable foetuses: control 8.18 ± 1.59, 2000 mg Cr/l: 7.33 ± 2.91, 5000 mg Cr/l: 6.00 ± 3.31, Elbetieha 1997). A significant decrease in pregnancy was noted at 5000 mg/l (44%), whereas the pregnancy rate at 2000 mg/l (90%) was comparable to the control group (82.5%). The findings of these studies are limited, because of deficiencies in documentation. Moreover, secondary toxicity effects are likely because of the high dosages used and toxicity observed in treated males (significant reduced body weights in both treatment group). The findings for the females are unclear because auf the limited documentation.

 

Inhalation route:

According to a reliable 13-week inhalation study in rats no compound-related effects on sperm parameters, testicular or ovarian weights were seen after exposure of rats to basic chromium sulfate 6 hours per day at the 3, 10 or 30 mgCr/m3 (corresponding to the inhaled doses of 0.7 to 6.6 mg Cr/kg day). This study was done in parallel with exposures to insoluble chromic oxide dust (see discussion above) (Derelanko 1999).

 

Conclusion

As bioavailability of chromium oxide is regarded to be of major importance for the respective toxicity this aspect was considered in the assessment. The oral absorption of the insoluble chromium oxide is extremely low compared to other inorganic chromium salts and organic chromium complexes. The lack of systemic effects was seen in subchronic and chronic feeding studies in rats (Ivankovic 1975) and 13-week inhalation study (Derelanko 1999). The lack of reproductive effects of trivalent chromium compounds of low bioavailability is in line with the lack of effects at the reproductive organs in the 13 week inhalation study with chromium oxide. As confirmed by literature (Mangelsdorf et al. 2003, Ulbrich & Palmer 1995, Janer et al. 2007 and Dent 2007, Sanbuissho et al. 2009) histopathological examinations in repeated dose toxicity are of high value and high sensitivity for the evaluation of effects on fertility. A read across with data of a two-generation study with chromium(III) nicotinate, an inorganic chromium complex with niacin ligand with a higher absorption rate compared to chromium oxide, did not reveal systemic effects, adverse effects on reproduction as evaluated by sexual maturity, fertility and mating, gestation, parturition, litter properties, lactation and development of the offspring. The absence of reproduction toxicity was in line with the lack of systemic effects noted up to 60 ppm chromium(III) nicotinatein the diet, 100 times the maximum recommended human dose of chromium(III) nicotinate. Thus, even a chromium(III) compound (chromium(III) nicotinate) with a higher bioavailability (chromium(III) nicotinate up to 5000 times increased vs. chromium oxide) did not induced averse effects on reproduction.

Taken together, although a screening reproduction toxicity study or a two or multi-generation reproduction toxicity study were not available for chromium oxide further testing is considered to be of low priority, as the value of additional animal testing should be balanced with animal welfare considerations. In accordance to REACH Annex XI, 1.2, there is sufficient weight of evidence to conclude that chromium oxide is not a reproduction toxicant. Therefore further testing on vertebrate animals for that endpoint is not proposed.

Effects on developmental toxicity

Description of key information

There are no valid developmental toxicity study data available for chromium oxide. Data from an early feeding study gives no indications of malformation or any other adverse effects on offspring (Ivankovic 1975). A read across was performed with study data from inorganic and organic trivalent chromium salts (chromium chloride, chromium nicotinate (NBC), chromium picolinate and the cation [Cr3O(O2CCH2CH3)6(H2O)3]+). No chromium-dependent adverse effects on dams and their offspring were indicated in mice and rats. The bioavailability of chromium oxide is extremely low compared to the assessed inorganic and organic chromium salts (5000 to 300000 times decreased); thus reflecting rather a worst case assumption than the real in vivo situation of the insoluble chromium oxide. Beside the very low bioavailability of chromium oxide, study data indicate that trivalent inorganic and organic chromium salts are poorly transferred to the offspring.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
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

There are no valid developmental toxicity study data available for chromium oxide. Only study data from a limited early 90-day feeding study indicates no malformations or any other adverse effects on offspring (Ivankovic 1975). A read across is performed with recent study data from inorganic and organic chromium salts. The bioavailability and the placenta transfer of trivalent chromium compounds are also regarded because of the decisive importance for the respective developmental toxicity.

 

The developmental toxicity of the dietary chromium compounds chromium(III) picolinate (Cr(pic)3) and the cation [Cr3O(O2CCH2CH3)6(H2O)3]+ (Cr3) were evaluated in CD1- female mice (Bailey 2008). Mated female mice were administered Cr(III) (equivalent to 25 mg/kg bw/d Cr(pic)3, 3.3 mg/kg bw/d Cr3 or 26 mg/kg bw/d Cr3 in the diet from Days 6 -17 of gestation. The current negative control was administered with plaint diet. Food consumption was measured for the intervals gestation days (GD) 6-9, GD 9-13 and GD 13-17; clinical signs were recorded daily. All dams were sacrificed on GD 17. The uteri of all dams were exposed and the numbers of resorptions and dead or live foetuses were recorded. Live foetuses were weighed individually and examined for gross malformations. Maternal body weights were determined minus the gravid uterine weight. Fixed and stained foetuses were subsequently examined for skeletal anomalies. No signs of maternal toxicity were observed in any treatment group. No adverse effects on body weight gain and food consumption were noted in treated dams compared to the control group. 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 (15 mg/kg/day) was lower than the other treated groups (Cr3 120 mg and Cr(pic)3), however this was 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. In a previous study, offspring of Cr(pig)3-treated dams were shown to have a significantly increased incidence of cervical arch defects (Bailey 2006). These findings were not replicated in the current study. The incidence noted for Cr(pic)3 (6.26 ± 1.63) were within the range of the corresponding negative control (4.65 ± 1.14).

In conclusion, maternal exposure to either Cr(pic)3 (equivalent to 25 mgCr/kg/day) or Cr3 dosages (equivalent to 3.3 or 26 mg Cr/kg/day) did not indicate adverse effects on exposed dams and their offspring.

 

In a previous study from the same working group the developmental toxicity of chromium picolinate, chromium chloride and picolinic acid were evaluated in CD-1 mice (Bailey 2006). Pregnant mice were feed from gestation days (GD) 6 to 17 with 200 mg/kg Cr(pic)3 (equivalent to 25 mg/kg), 200 mg/kg CrCl3 (equivalent to 39 mg Cr/kg)or 174 mg/kg picolinic acid in the diet. Dams were sacrificed on GD 17, and their litters were examined for adverse effects. No maternal toxicity was indicated in any of the treatment groups. No adverse effects on foetal weights, implantations, resorbed or dead foetuses were noted. However a significant increase in incidences of bifurcated cervical arches were seen in foetuses from the Cr(pic)3 group compared to the control group (mean litter percentage: 5.79 vs. 2.09%). Foetuses in the picolinic acid-treatment group had an incidence double that of the control group, however the increase was not statistically significant (mean litter percentage: 4.24 vs. 2.09%). Foetuses in the CrCl3 group did not differ from the controls in any variability examined.

 

The developmental toxicity potential of the inorganic chromium(III) niacin complex NBC (also known as chromium nicotinate CrNic) was evaluated in Sprague-Dawley rats (Deshmukh 2009). This developmental study was undertaken as part of a multi-generation reproductive investigation. The animals in this study were selected randomly after weaning from each F2b litter of the F1 generation from the two-generation reproductive toxicity study (Deshmukh 2009) To start the developmental toxicity study, pups (ca. 30/sex/group) from the F2b generation were allowed to grow up to 10 to 12 weeks of age before mating. The rats were exposed directly to NBC through feed. The dietary exposure levels were the same as those chosen for the two-generation reproductive toxicity study (0, 4, 15 or 60 ppm). Following mating at maturity, the pregnant rats were observed daily for clinical signs, and body weight and feed consumption were recorded. The pregnant females were sacrificed on gestation day 20. Necropsy and caesaren section were performed to examine the uterus, ovaries and foetuses. No treatment-related indications of maternal toxicity and no adverse effects on the gravid uteri were noted in treated dams. In addition, no treatment related external abnormalities, no soft tissue abnormalities and skeletal abnormalities were seen in evaluated foetuses.

In conclusion, based on the findings of this developmental toxicity study NBC was found to be non-teratogenic in Sprague-Dawley rats, at dietary exposure levels up to 60 ppm, 100 times the maximum recommended human dose of NBC.

 

 

The developmental toxicity potential of cation [Cr3O(O2CCH2CH3)6(H2O)3]+ (known as CrProp or Cr3) and the mineral status of chromium(III) administered pregnant Wistar rats and their offspring were evaluated (Staniek 2009). Pregnant Wistar rats were feed with CrProp (equivalent 100 mg Cr/ kg diet, equal to 7.2 mg Cr/kg bw/day) in the diet from gestation day 0 to gestation day 21. Control dams received a diet with 0.020 mg Cr/kg bw/day. The dams were sacrificed at gestation day 21. Feed intake, body weight gain, organ weights and haematology were evaluated. In addition, maternal mineral (Cr, Cu, Zn, Fe) indices in liver, kidney, serum and femoral bones were measured. The number of resorptions, dead and live foetuses were also determined. In offspring foetuses body weights, gender, gross malformations and weights of foetal internal organs (liver, kidneys, heart, lungs) were evaluated. No adverse effects on body weight gain, organ weights and haematology were noted in CrProp treated dams. Whereas significant changes were observed in maternal mineral indices in treated animals compared to the control group. In treated dams tissue minerals levels of chromium and copper were increased (liver Cr level 177%, kidney Cr level: 455%); whereas the copper and zinc levels were decreased in the liver (Cu 9% and Zn 12%). No effects on pregnancy and pregnancy outcome were observed. In offspring of treated dams no adverse effects on body weights and foetal organs weight were noted. No abnormalities in gross organ morphology were seen. Concerning mineral levels, supplemented CrProp given to pregnant rats did not effect foetal chromium and iron levels; whereas foetal liver zinc level was increased (181%) and kidney cupper level was decreased (34%) in comparison to the control group (Staniek 2009).

In conclusion, administration of 100 mg CrProp/kg diet (7.2 mg Cr/kg bw/day) to pregnant Wistar rats did not induce adverse effects on pregnancy outcome and offspring; however changes in maternal and foetal tissue zinc and copper levels were noted in treated rats.

 

Cr(III)-transfer to the embryo/foetus

In some early studies the chromium transfer to the embryo/foetus in pregnant rats was evaluated (Mertz 1969). Pregnant rats treated once with 51CrCl3 failed to transmit any radioactivity into the litter. Chromium acetate (2 ppm) given in the drinking water did not supplement chromium-deficient diet. When pregnant rats were given 51Cr3+ (extracted from brewers’ yeast) the radioactivity in litters ranged from 23% to 30% of maternal level. The author suggested that orally administered chromium(III) salt are poorly transferred to the offspring whereas chromium in the form of a natural complex is transported across placenta.

Staniek et al. (2009) showed that CrProp supplemented Wistar rats had significant increased (p<0.001) maternal liver and kidney chromium levels, 1.77 and 4.55-fold compared to control dams. Such an increase in chromium content was not observed in fetal liver and kidneys; the authors suggested that Cr(III) ions are not easily transported cross the placenta barrier and that chromium(III) has got low cumulative potential in rodents.

In conclusion, study data indicates that inorganic and organic chromium salts are poorly transferred to the offspring, whereas chromium in the form of a natural complex is transported more easily across placenta.

 

Conclusion

There are no valid developmental toxicity study data available for chromium oxide. Data from an early feeding study gives no indications of malformation or any other adverse effects on offspring (Ivankovic 1975). A read across was performed with study data from other inorganic and organic trivalent chromium salts. No chromium-dependent adverse effects on dams and their offspring were indicated in mice and rats. The skeletal changes seen inoffspring of CD1 mice (Bailey 2006) were considered to be dependent on picolinic acid and/or by a chemical combination of picolinic acid and chromium but not dependent on chromium(III) alone. The bioavailability of the insoluble chromium oxide is extremely low (0.0002%) compared to the chromium salts evaluated (ca. 1 % chromium chloride and chromium picolinate and 40 to 60% cation [Cr3O(O2CCH2CH3)6(H2O)3]+); thus reflecting rather a worst case assumption than the real in vivo situation of the insoluble chromium oxide. Beside the very low bioavailability of chromium oxide, study data indicate that trivalent inorganic and organic chromium salts are poorly transferred to the offspring.

Taken together, although a developmental toxicity study was not available for chromium oxide further testing is considered to be of low priority. In accordance to REACH Annex XI, 1.2., there is sufficient weight of evidence to conclude that chromium oxide is not a developmental toxicant, and further testing on vertebrate animals for that endpoint shall be omitted.

Justification for classification or non-classification

Taken together, all available data of chromium oxide and additional data from other trivalent inorganic and organic chromium salts, used in a read across, there is sufficient weight of evidence to conclude that chromium oxide is neither a reproduction toxicant nor a developmental toxicant. No classification is required in accordance with regulation (EC) 1272/2008.