Chromium trichloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Chromium trichloride had no effects on fertility of turkey hens

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: fully reliable study performed under GLP without deviations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Sprague–Dawley rats, 7–9 weeks old, bred and reared at the animal breeding facility of INTOX Pvt. Ltd. India were used. The animals were maintained under controlled conditions in a room ventilated with 100% fresh and filtered air, with 10–15 air changes per h. The room temperature was maintained between 19 and 25 °C with relative humidity 30–70% and a 12 h light/dark cycle. The animals were allowed to acclimatize at least one week before the initiation of experiments with food and water available ad libitum.

Route of administration:

oral: feed

Vehicle:

other: NBC was mixed with powdered rodent diet to obtain the three concentration levels.

Details on exposure:

‘‘Nutrilab” brand extruded rodent powdered feed manufactured by M/s Vetcare Pvt. Ltd., Bangalore, India, and tested for nutrients and contaminants, was provided ad libitum to the animals during the study period. NBC was mixed with powdered rodent diet to obtain the three concentration levels. Initially, a small volume of dietpremix was prepared which was then mixed with remaining portion of diet in a mechanized ribbon blender for about 20 min to obtain desired homogeneity of the test article concentration in diet. The experimental diets were prepared once a week.
Test article, NBC, for the study was provided by InterHealth Nutraceutical Inc. NBC was administered to rats orally, by dietary admixture. Since NBC is intended to be consumed by human beings up to a maximum dose of 4 mg of NBC/day, the highest dose level for this study was selected so as not to exceed 100 times the maximum recommended human dose, which has a dietary equivalent concentration of 60 ppm. A dose range study revealed no adverse effects of NBC on body weight, feed consumption, mating behavior, fertility, gestation or lactation in rat at dose level up to 60 ppm. Sprague–Dawley rats (30/group/sex) were randomly divided into one control and three treatment groups (low, mid and high). The treatment groups of the F0 parental generation received feed containing 4, 15, or 60 ppm NBC for a period of 10 weeks before mating, throughout mating, and continued until their termination. The exposure was continued through the next generations, up to completion of developmental toxicity study. Control group of animals were fed normal diet. The male and female rats of F0 generation from each dose group were mated and allowed to deliver normally. At weaning, one male and one female pup from each litter from control and treatment dose groups were selected for first filial (F1 ) generation. The selected F1 animals were exposed to NBC for 10 weeks before mating and then they were mated to produce second generation (F2a).

Details on mating procedure:

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

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

see frequency of treatment

Frequency of treatment:

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

Remarks:

Doses / Concentrations:
0, 4, 15 and 60 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

30 males and 30 females per dose group

Control animals:

yes, plain diet

Positive control:

none required

Parental animals: Observations and examinations:

Animals were examined daily for signs of toxicity and mortality during the entire period of the study. Body weight and feed consumption of the animals from each group were recorded weekly. Feed consumption was calculated as g/rat/day. For both F0 and F1 parental animals reproductive parameters such as estrous cycle, female fertility index, gestation index, live-born index, mean litter size, sex ratio (at birth), number of stillbirths at day 0, number of live births at day 0, survival index, sperm count (epididymal and homogenization resistant testicular), sperm motility and sperm morphology were assessed. Pups from both generations were examined for survival, clinical signs, and their physical developmental landmarks such as unfolding of pinna (UP), hair growth (HG), teeth eruption (TE), eye opening (EYO) and ear opening (EO) were observed and recorded for appropriate lactation day to monitor postnatal growth. All pups, not selected for next generation, were sacrificed on lactation day 21, and subjected to necropsy.

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

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

Litter observations:

Pups from both generations were examined for survival, clinical signs, and their physical developmental landmarks such as unfolding of pinna (UP), hair growth (HG), teeth eruption (TE), eye opening (EYO) and ear opening (EO) were observed and recorded for appropriate lactation day to monitor postnatal growth. All pups, not selected for next generation, were sacrificed on lactation day 21, and subjected to necropsy.

Postmortem examinations (parental animals):

After the mating and following confirmation of pregnancy status, males were euthanized with carbon dioxide and were subjected to necropsy and evaluation of sperm parameters such as sperm motility, cauda epididymis sperm count, testicular spermatid head count and sperm morphology. Sperm motility was assessed by observing percentile motile sperms and graded byassigning a numerical score, based on the 6 point scale. The cauda epididymal sperm count was done by counting the sperms using a haemocytometer and was expressed as number of sperms per cauda epididymis and number of sperms per gram cauda epididymal weight. The homogenization-resistant testicular spermatid head count was done by counting the spermatids using a hemocytometer and was expressed as number of (homogenization-resistant) spermatids per testis and number of spermatids per gram testis weight.
The same cauda epididymis that was used for sperm motility and sperm count, was utilized for preparation of smears for sperm morphology evaluations which were then examined under 40 x magnification. At least 200 sperms were counted per slide and abnormalities such as misshapen sperm-head, flagella, and sperm-head separation from flagella were recorded.
All females were killed after weaning. 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 F0 and F1 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):

All pups, not selected for next generation, were sacrificed on lactation day 21, and subjected to necropsy.

Statistics:

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

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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

Dose descriptor:

NOEL

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested; equivalent to NOEL of 7.80 mg/kg bw/d for males and 8.31 mg/kg bw/d for females

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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

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

Dose descriptor:

NOEL

Generation:

F1

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested; equivalent to NOEL of 7.80 mg/kg bw/d for males and 8.31 mg/kg bw/d for females

Dose descriptor:

NOEL

Generation:

F2a

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested; equivalent to NOEL of 7.80 mg/kg bw/d for males and 8.31 mg/kg bw/d for females

Reproductive effects observed:

not specified

Conclusions:

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

Executive summary:

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

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

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-standard screening study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Deviations:

not applicable

Principles of method if other than guideline:

Effects on sperm parameters measured during a 90-day inhalation study performed according to OECD 413 (subchronic inhalation study).

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: CDF

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female CDF (Fischer 344)/Crl BR VAF/Plus rats, approximately 5 weeks of age, were obtained from Charles River Laboratories (Raleigh, NC). Following 3 days of group housing, animals were individually housed in stainless steel, suspended wire-mesh cages and given free access to commercial laboratory feed (Purina Certified Rodent Chow No. 5002) and tap water during the non-exposure periods. Animal rooms were maintained on a 12-h light/dark cycle; temperature range was maintained at 21 ±2°C, and the relative humidity range was 43 ±11%

Route of administration:

inhalation: dust

Type of inhalation exposure (if applicable):

nose only

Vehicle:

unchanged (no vehicle)

Details on exposure:

Rats were exposed in stainless steel and acrylic nose-only inhalation chambers operated with at least 12 chamber air changes per h. Basic chromium sulfate particles were generated using an auger dust feeder and an air micronizer. Chamber samples were determined once per h by standard gravimetric methods, with periodic analysis for Cr(III) and Cr(VI). Particle-size measurements were made from each exposure level using a cascade impactor once per day for the first two weeks and weekly thereafter.

Details on mating procedure:

Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Standard gravimetric methods, with periodic analysis for Cr(III) and Cr(VI)

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week

Details on study schedule:

Doses basic chromium sulfate (17, 54, or 168 mg/m 3 ). The desired exposure levels were selected to be multiples of the threshold limit value (TLV) for trivalent chromium and set at chromium equivalents of 3, 10, and 30 mg/m3 for each test article.

Remarks:

Doses / Concentrations:
17, 54, 168 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

Clinical observations: Animals were observed daily prior to and following each exposure for clinical signs of toxicity, and were observed twice daily for morbidity and mortality during the recovery period and on weekends. Individual body weights were recorded weekly during the exposure and recovery periods. All animals received an indirect ophthalmoscopic examination during the acclimation period and prior to terminal necropsy.
Clinical pathology: Standard hematology, clinical biochemistry, and urinalysis determinations were conducted on animals, 10 per sex per group, designated for necropsy at the end of exposures. Animals were fasted overnight prior to blood sampling, with water available. Blood samples were obtained from the orbital sinus plexus. At necropsy, bone marrow smears were prepared and differential cell counts were evaluated. All clinical procedures were performed using automated instrumentation except bone marrow smears, which were examined microscopically. Urinalysis determinations were conducted on samples collected overnight in stainless-steel metabolism cages.
Urinalysis determinations were performed by gross observation, microscopy, and automated clinical analyzer. Following urinalysis testing, aliquots of the remaining urine from 5 animals per sex from the control group, and the high-exposure level groups for both test articles were submitted for Beta 2-microglobulin analysis.
Pathology: Animals found dead or euthanized by design at study termination were necropsied. At necropsy the heart, lungs, liver, spleen, kidneys, brain, adrenal glands, thyroid/parathyroid glands, testes, and ovaries were weighed. Tissues typically harvested for subchronic studies were also removed and preserved. All tissues were placed in 10% neutral buffered formalin, except eye tissue, which was fixed in Davidson’s fixative. Microscopic evaluation was conducted on all hematoxylin and eosin-stained tissues from the control group and high-exposure-level groups of both test articles. The kidneys, livers, nasal tissues, trachea, lungs, larynx, mediastinal and mandibular lymph nodes, and gross lesions from all animals in the low- and mid-exposure level groups for both test articles were also examined. A formal peer review of the histopathologic findings was performed.
Bronchoalveolar lavage evaluation: Bronchoalveolar lavage (BAL) analyses were conducted on 5 animals per sex per group exposed for 5 consecutive days with the main study animals. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The lungs, heart, trachea, larynx, and tongue were removed en-block. Following tracheal cannulation, the airways were washed 3 successive times with warmed, physiological saline (30 µL per gram of body weight) and the resulting BALF was pooled. Nucleated cell counts were performed using a Neubauer hemocytometer, and cell differential counts were performed on Wright-Giemsa-stained smears. Chemical analyses performed spectrophotometrically included for lactate dehydrogenase (LDH), total protein, beta-glucuronidase, and glutathione reductase. LDH determination was based on the reduction of pyruvate to lactate with concurrent oxidation of NADH to NAD, measured by a decrease in absorbance. Total protein was measured by increased absorbance associated with pyrogallol red-molybdate complex with basic amino acids. Beta-glucuronidase activity was determined by enzymatic conversion to alcohol, D-glucuronate, and p-nitrophenol. Glutathione reductase activity was determined from the enzymatic reduction of oxidized glutathione and concurrent oxidation of NADPH to NADH.
Sperm evaluation: At necropsy, sperm samples from the left caudal epididymis of 10 males per group were used for automated evaluation of sperm motility, count, and morphology. The concentration and morphology of the sperm were evaluated using visual methods. Two hundred intact sperm were evaluated from each animal for morphology. Intact sperm were evaluated as normal or abnormal. The number of disarticulated sperm in each field was also assessed.

Sperm parameters (parental animals):

In a 13-week nose-only inhalation study, sperm samples from male rats were collected at necropsy and were used for automated evaluation of sperm motility, count and morphology.
Sperm evaluation: At necropsy, sperm samples from the left caudal epididymis of 10 males per group were used for automated evaluation of sperm motility, count, and morphology. The concentration and morphology of the sperm were evaluated using visual methods. Two hundred intact sperm were evaluated from each animal for morphology. Intact sperm were evaluated as normal or abnormal. The number of disarticulated sperm in each field was also assessed.

Litter observations:

none

Postmortem examinations (parental animals):

Pathology: Animals found dead or euthanized by design at study termination were necropsied. At necropsy the heart, lungs, liver, spleen, kidneys, brain, adrenal glands, thyroid/parathyroid glands, testes, and ovaries were weighed. Tissues typically harvested for subchronic studies were also removed and preserved. All tissues were placed in 10% neutral buffered formalin, except eye tissue, which was fixed in Davidson’s fixative. Microscopic evaluation was conducted on all hematoxylin and eosin-stained tissues from the control group and high-exposure-level groups of both test articles. The kidneys, livers, nasal tissues, trachea, lungs, larynx, mediastinal and mandibular lymph nodes, and gross lesions from all animals in the low- and mid-exposure level groups for both test articles were also examined. A formal peer review of the histopathologic findings was performed.

Postmortem examinations (offspring):

none

Statistics:

Statistical analyses were performed of body weights, clinical pathology laboratory tests, BALF data, and organ weights using one-way analysis of variance. If the result was non-significant, no additional analysis was performed. If the result was significant, Bartlett’s test for homogeneity of variance was performed. If Bartlett’s test was non-significant, Dunnett’s t-test was used for pairwise comparisons. If Bartlett’s test was significant, the Welch t-test with Bonferonni correction was used for pairwise comparisons. The Kruskal-Wallis analysis of variance, followed where appropriate by the Mann-Whitney U test, was used for those parameters where parametric analysis was inappropriate. The level for statistical significance was set at p 0.05.

Reproductive indices:

none

Offspring viability indices:

none

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced mean body weights in mid- and high dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced mean body weights in mid- and high dose group

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Chronic inflammation was observed involving the alveoli of all exposure-level groups, consisting of alveolar spaces filled with macrophages, neutrophils, lymphocytes, and cellular debris.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

In this 90d-inhalation study sperm parameters were additionaly investigated. No exposure-related effects were noted for either test article in the ophthalmologic evaluations or for sperm motility, morphology, or concentration.

Dose descriptor:

NOAEL

Remarks:

for sperm parameters

Effect level:

ca. 30 mg/m³ air (nominal)

Based on:

element

Remarks:

chromium III

Sex:

male/female

Basis for effect level:

other: No effects were seen at the highest dose tested (168 mg/m3 chromium (III) hydroxide sulphate, equivalent to 30 mg/m3 Cr(III)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Offsprings were not investigated in this study

Reproductive effects observed:

not specified

No compound-related effects were noted for sperm motility or morphology in rats with nose-only exposures to chromium hydroxide sulphate dust at 17, 54, or 168 mg/m3 for 6 h/day, 5 days/week for 13 weeks. Ovary and testes weights were also unaffected by treatment.

Conclusions:

Inhalation exposure to chromium oxide (insoluble chromium III compound) and chromium hydroxy sulfate (soluble chromium III compound) dust in this study did not have any adverse effects on sperm parameters.

Executive summary:

Sperm parameters were assessed in male rats exposed by inhalation to chromium (III) oxide and chromium (III) hydroxy sulfate dusts in a 90-day study at concentrations of 0, 17, 54 or 168 mg/m3. No treatment-related effects on fertility parameters (sperm parameters) were apparent.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

see discussion on developmental toxicity.

Short description of key information:
Chromium(III) compounds have no effects on fertility (weight of evidence)

Effects on developmental toxicity

Description of key information

Chromium(III) compounds have no effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline comparable, published study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

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

GLP compliance:

no

Remarks:

: published study

Limit test:

no

Species:

mouse

Strain:

CD-1

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Male and female CD-1 mice, obtained from Charles River Breeding Laboratories, International (Wilmington, MA) were housed in an AAALAC-approved animal facility in rooms maintained at 22721C, with 40–60% humidity and a 12-hr photoperiod. Animals were bred naturally, two females with one male. Observation of a copulation plug was designated GD 0. Mated females were individually housed in shoe-box-type cages with hardwood bedding and were given Harlan-Teklad LM-485 rodent diet and tap water ad libitum.

Duration of treatment / exposure:

Females were exposed from Gestation Day 6-17

Frequency of treatment:

Continuous (dietary)

Duration of test:

Maternal animals were sacrificed on Gestation Day 17.

Remarks:

Doses / Concentrations:
Control
Basis:
other: untreated diet

Remarks:

Doses / Concentrations:
200 mg/kg bw/d chromium picolinate
Basis:
other: based on predicted food consumption

Remarks:

Doses / Concentrations:
15 mg/kg bw/d Cr3
Basis:
other: based on predicted food consumption

Remarks:

Doses / Concentrations:
120 mg/kg bw/d Cr3
Basis:
other: based on predicted food consumption

No. of animals per sex per dose:

Not stated, however the numbers of litters in each group range from 24-29

Control animals:

yes, plain diet

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

26 mg/kg bw/day (nominal)

Based on:

element

Remarks:

chromium III

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

26 mg/kg bw/day (nominal)

Based on:

element

Remarks:

chromium III

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

No signs of maternal toxicity were observed.

Mean foetal weights, foetal viability and the proportion of resorptions were unaffected by treatment. No gross malformations were observed in any of the foetuses. The number of implantations in the low dose Cr3 group was lower than the other treated groups, however this is not considered to be an effect of treatment in the absence of a dose-response relationship and because implantation occurred prior to exposure. No effects of treatment were observed on the incidence of skeletal anomalies. The authors note that a previous study (Bailey et al, 2006) reported an increased incidence of cervical arch defects in the offspring of mice exposed to chromium picolinate, however the incidence of defects in that study (6.26%) is very similar to the control incidence in this study (5.79% and is therefore not considered to be related to treatment.

Summary of findings

Parameter		Dose group
		0	Cr picolinate	Cr3 (low)	Cr3 (high)
Litters	(#)	27	29	26	24
Foetuses	(#)	332	369	275	342
Litter size	(#)	12.30	12.72	10.58	14.25
Foetal weight	(g)	1.02	1.05	1.08	1.02
Implantations	(#)	12.64	13.18	11.00	13.79
Dead/resorbed foetuses	(#)	2.74	3.29	3.48	1.29
Cervical arch defects	(#)	4.65	6.26	5.18	3.98

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

Conclusions:

No evidence of foetotoxicity, developmental toxicity or teratogenicity was seen in mice exposed to water-soluble complexes of Cr(III) delivering estimated daily doses of Cr(III) equivalent to 25 mg/kg bw/d (picolinate group), 3.3 mg/kg bw/d (low dose Cr3 group) and 26 mg/kg bw.d (high dose Cr3 group).

Executive summary:

Mated female mice were administered Cr(III) in the diet from Days 6 -17 of gestation. Dams were sacrificed on Day 17 and the uterine contents investigated. Foetuses were assessed for external defects and skeletal findings following double staining. No maternal toxicity was observed. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen. The results of this study also show that a reported increased incidence of cervical arch defects in a previous study by the same authors was within the background range.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

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

Chromium(III) compounds such as chromium trichloride have been assessed in several studies for effects on fertility and developmental effects.

Elbetieha A & Al-Hamood MH (1997) reported about a non-guideline experiment in which male and female Swiss mice were administered chromium chloride in drinking water (0, 2000 and 5000 mg/L) for 12 weeks prior to mating with untreated animals. Female mice were sacrificed one week following treatment; the numbers of pregnant animals, total implantations, viable foetuses and resorptions were recorded. Satellite groups of animals were also treated for 12 weeks (without mating) and the weights of the reproductive organs recorded.

There were no deaths or signs of toxicity; slightly reduced bodyweights were seen in both groups of treated males but without any relationship to dose. Relative testes weights were significantly increased in both satellite groups, however findings may be secondary to body weight effects. Relative seminal vesicle and preputial gland weights were significantly lower; however the toxicological significance of these findings is unclear in the absence of histopathology. Relative ovary and uterus weight was increased in females at 5000 mg/l. Mean numbers of implantation sites were lower in treated groups.

Due to some shortcomings in study design, these finding should be interpreted with caution and considering the high doses applied should not become overestimated. Also the US Department of Health and Human Services in its Toxicological Profile for Chromium, September 2012, states that the results should be interpreted with caution due to concerns regarding experimental methods, including decreased water consumption in the higher concentration group (resulting in a potential overestimate of exposure and uncertainty regarding daily dose calculations). Also sperm counts were not conducted and no standard mating protocol was used.

In 1998 M.H. Al-Hamood, A. Elbetieha and H. Bataineh published results on fertility of males, exposed through their gestational and lactation phase was not affected significantly. Females showed slight effects with regards to total number of resorptions and number of pregnant females, whereas number of implantations and numbers of viable fetuses were not affected.

This study also should be seen with caution as parental toxicity was not observed/recorded and only one dose was tested, resulting in no possibility to assess dose-response relationship. Also the US EPA (see Toxicological Profile for Chromium by US Department of Health and Human Services, Sept. 2012) has questioned the results of this study due to shortcomings in methodology and documentation and had converted the exposure information to 74 mg/kg bw/d.

Derelanko (1999) investigated fertility effects as part of a 90d-repeated dose toxicity study in which sperm parameters were assessed in male rats exposed by inhalation to chromium (III) oxide and chromium (III) hydroxy sulfate dusts in a 90-day study at concentrations of 0, 17, 54 or 168 mg/m3. No treatment-related effects on fertility parameters (sperm parameters) were apparent.

H. Bataineh, M. H. Al-Hamood, A. Elbetieha and I. Bani Hani (1997) investigated chromium(III) and chromium(VI) with regards to fertility in rats. Only one dose group (1000 ppm in water) was assessed and the purity of starting materials is unclear – therefore, the study was assessed unreliable. However, generally there was no mortality or clinical sign of toxicity in male rats exposed to chromium chloride and potassium dichromate at the concentration used in this study. The results presented in this paper show that neither trivalent chromium compound (chromium chloride) nor hexavalent chromium compound (potassium dichromate) had effects on fertility of male rats. The increase in the number of resorptions observed in this study in female rats impregnated by male rats that were exposed to chromium chloride or potassium dichromate may be attributed to an increase in postimplantation mortality of fertilized ova. Although there were significant decreases in body weight gain in chromium chloride and potassium dichromate-exposed male rats, despite unlimited access to food this effect on body weight did not affect the fertility. It has been found that even 30% decrease in body weight gain had only minimal reproductive effects. The results presented in this work show that the reduction in relative seminal vesicles and preputial weights in chromium chloride - and potassium dichromate - exposed males had no effect on reproduction in the male rat. The reduction in seminal vesicles and preputial gland weights in the present study might suggest an alteration in the pattern of testosterone secretion.

However, it should be noted, that only one dose chromium trichloride has been applied in this study and thus a dose response relationship on organ weight findings could not be seen. Also, a potential influence by Cr(VI) contamination of chromium trichloride cannot be excluded, as the test materials were not assessed for purity (at least this is not reported). From Levis & Majone (1981) it is known that chromium(III) compounds, contaminated with chromium(VI) may show similar effects as pure chromium(VI) compounds. Thus, the results of this study with respect to effects on certain organ weights and number of resorptions should be taken with caution. However, the lack of effects on male fertility can be seen with more confidence, as it was shown that also potassium dichromate had no effects.

N.S. Deshmukh, M. Bagchi, F.C. Lau, D. Bagchi (2009) investigated the influence of chromium(3+) tri(pyridine-3-carboxylate) (NBC) in a two-generation study design. NBC is a complexed chromium(III) compound designed for better absorption of chromium(III) and was dosed via feed in this study.

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

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

Ivankovic (1975) assessed fertility of chromium(III), dosed as chromium(III)oxide as part of a 90 day feeding study in rats as of day 60 altogether nine females (3 females per dose group) were paired with males (3 males per dose group) from the same dosage group (0, 2 and 5% Cr2O3 in diet). All the females became pregnant in due course, the gestation period was normal (23 days) and the young showed no malformations or other adverse effects. Litter sizes were in the normal range, averaging eight pups. Some of the progeny were retained for lifetime observation and (600 days) no tumours have been detected. It was thus shown that no toxic or teratogenic effects were associated with Cr2O3 treatment throughout the gestation period and fertility was not adversely affected.

Bailey MM, Sturdivant J, Jernigan PL, Townsend MB, Bushman J, Ankareddi I, Rasco JF, Hood RD & Vincent JB (2008) studies developmental effects upon exposure to chromium picolinate monohydrate (CPM). Mated female mice were administered Cr(III) in the diet from Days 6 -17 of gestation. Dams were sacrificed on Day 17 and the uterine contents investigated. Foetuses were assessed for external defects and skeletal findings following double staining. No maternal toxicity was observed. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen.

Stummann (2007) reported about a new in vitro screening assay to investigate the embryotoxicity of chromium trichloride using mouse embryonic stem cells. The study indicates that chromium (III) compounds are not embryotoxic; the results are consistent with the available in vivo data. In contrast, chromium VI was tested in parallel and was found to be embryotoxic, proving the test system being valid.

HAMEED N. BATAINEH, ZIAD M. BATAINEH and HAYTHAM DARADKA (2007) reported in a published study that several industrial metal salts have been investigated via intragastric intubation to rats; manganese sulfate, lead acetate, aluminum chloride, ferrous chloride and ferric chloride in doses of 50 mg/rat (204 mg/kg body weight/day) and chromium chloride and potassium dichromate in doses of 25 mg/rat (102 mg/kg body weight/day) on days 1 – 3 or 4 – 6 of pregnancy.

Whereas the pregnancy rate to chromium trichloride, when dosed during day 1 - 3 of pregnancy, was slightly reduced (P < 0.05), numbers of implantations and viable fetuses were not significantly affected. When dosed during days 4 - 6 of pregnancy, neither pregnancy rate nor number of implantations or fetuses were significantly affected. Thus, chromium trichloride had no significant effect in this study, whereas in contrast chromium dichromate (chromium(VI) compound) had significant effects, especially when dosed during days 1 - 3, corresponding to the period of pre-implantation. The same applies to number of resorptions compared to total number of implantations, where chromium trichloride had no significant effect but potassium dichromate had shown very severe effects.

Although the study deviates significantly from the OECD414 test protocol, as it dosed during days 1 - 3 and 4 - 6 of pregnancy, rather than during days 6 - 20 and the study has various shortcomings such as limited level of details reported it shows a distinct difference between chromium(III) and chromium(VI). The aim of this work was to determine the critical period for embryolethality of industrial metal salts using a short duration of exposure in the early stages of pregnancy. Whereas chromium(VI) as potassium dichromate had very significant effects, chromium(III) dosed as chromium trichloride had almost no significant effects. Only the number of pregnant rats when dosed at days 1 - 3 were slightly reduced but given the low number of animals used this should not be overstressed.

In conclusion, the most reliable and valuable study is the 2-generation reproductive toxicity study using chromium(3+) tri(pyridine-3-carboxylate) (NBC) as chromium(III) source. In this study the parental and offspring no-observed-adverse-effect level (NOAEL) in this two-generation reproduction toxicity study was found to exceed 60 ppm in diet (highest dose group in this study). Also, the prenatal study reported by Bailey et al. according to OECD414 study design applying two dietary chromium(III) supplements, chromium picolinate and [Cr3O(O2CCH2CH3)6(H2O)3]+, to mice resulted in no maternal toxicity. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen. In contrast, the studies performed by Bataineh et al and Hamood et al. do not follow any standard protocols and do have some shortcomings in study design, which were also stressed by the US EPA when assessing these studies. However, typically no effects on male fertility (rats and mice) were seen and fertility by females was only slightly affected, mainly due to resorptions. As the purity of chromium trichloride in these studies were not reported and effects seen with chromium(VI), also investigated, were generally much stronger, the results should be seen with caution.

 

No effects on fertility were seen in studies reported by Derelanko (90d-inhalation to dichromium trioxide and chromium hydroxyl sulfate as insoluble and soluble chromium(III) source) and in studies reported by Ivankovic applying up to 5% dichromium trioxide in diet over 90 days to rats.

Additionally, the study reported by Stummann, an in vitro study using mouse embryonic stem cells, did not show any embryotoxic effects towards chromium trichloride whereas potassium chromate (chromium VI) showed clear embryonic effects.

Thus, chromium(III) compounds are considered unlikely for showing reproductive toxicity when tested in rats and mice weighing the evidence seen in existing studies.

Toxicity to reproduction: other studies

Additional information

Frobish (1980) reported on investigations in turkey hens, fed with chromium trichloride and although not a classical reproduction study and only dosed up to 10 ppm chromium in diet, the study showed that there were no effects on fertility of white turkey hens and hatchability of the eggs. However, the number of eggs was significantly reduced, indicative of parental effects. As only an abstract was published, no detailed information is available and the results should be taken with caution, in particular as no rodent species was investigated.

Justification for classification or non-classification

Based on a weight of evidence approach assessing all available information on fertility and developmental toxicity of chromium(III) compounds it was concluded that chromium trichloride is not classifiable for reproductive toxicity according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC).