Dichromium nitride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No toxicity data on fertility impairment with dichromium nitride are available. However, based on the information available from two 3 -month repeated dose oral toxicity studies conducted with chromium picolinate monohydrate using male and female F344/N rats and B6C3F1 mice, it was concluded using a read-across concept that dichromium nitride does not present a reproductive toxicity hazard to any sex. No adverse effects on reproductive function could be observed after the administration of chromium picolinate monohydrate. The approach for read-across is described in detail in the document attached in IUCLID section 13.

Link to relevant study records

Reference

Reference 1

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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.

Reference 2

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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

Dose descriptor:

NOAEL

Study duration:

subchronic

Species:

rat

Quality of whole database:

key studies available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

No toxicity data on fertility impairment with dichromium nitride is available. However, based on the information available from two 3 -month repeated dose oral toxicity studies conducted with chromium picolinate monohydrate using male and female F344/N rats and B6C3F1 mice, it was concluded using a read-across concept that dichromium nitride does not present a reproductive toxicity hazard to either sex. No adverse effects on reproductive function could be observed after the administration of chromium picolinate monohydrate. The approach for read-across is described in detail in the document attached in IUCLID section 13.

Chromium picolinate monohydrate

The National Toxicology Program (NTP, 2010) evaluated chromium picolinate monohydrate in a 3 -month repeated dose oral toxicity study using rats. The substance

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. According to the authors, no treatment-related effects on clinical signs, survival, body weights, food consumption, haematology, clinical chemistry, organ weights, gross pathology and histopathology were observed during the study. Furthermore, they stated that 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.

In addition, the National Toxicology Program (NTP, 2010) evaluated chromium picolinate monohydrate in a 3 -month repeated dose oral toxicity study using mice.

The substance 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. According to the authors, no treatment-related effects on clinical signs, survival, body weights, food consumption, haematology, organ weights, gross pathology and histopathology were found. 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.

Dichromium nitride

Since no toxicity data on fertility impairment is available specifically for dichromium nitride, information on chromium picolinate monohydrate will be used for the hazard assessment. For the purpose of hazard assessment of dichromium nitride, the most sensitive species and sex will be used for the DNEL derivation. In this case the male F344/N rats treated with chromium picolinate monohydrate were the most sensitive species and sex and the NOEL of 50000 ppm(actually ingested: males: 4240 mg/kg bw/day (equivalent to 508.8 mg Cr/kg bw/day) will be used.

Justification for classification or non-classification

Due to an absence of adverse effects on reproductive function seen in a subchronic repeated dose oral toxicity study with chromium picolinate monohydrate, as representative of dichromium nitride, a classification for reproductive toxicity in accordance with regulation (EC) 1272/2008 is not warranted.