Dichromium nitride

Endpoint summary

Description of key information

No repeated dose toxicity study with dichromium nitride is available. However, based on the information available from two chronic 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 health hazard to either sex. No adverse effects 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-07-29 to 2004-08-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Deviations from OECD guideline 452 (2009): detailed clinical observations, opthalmological examinations, clinical biochemistry determinations, urinalysis determinations, organ weight determinations were missing; measure of blood clotting time/potential was missing; histopathological examinations of aorta, cervix, coagulating gland, lacrimal gland, peripheral nerve, spinal cord and vagina were missing; individual data missing; historical control data was missing; justification of species selection was 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 50 male and 50 female B6C3F1 mice in feed at concentrations of 0, 2000, 10000 and 50000 ppm (actually ingested: males: approx. 0, 250, 1200 and 6565 mg/kg bw/day; females: approx. 0, 240, 1200 and 6100 mg/kg bw/day) for up to 105 weeks. The following parameters were investigated/recorded: clinical signs, mortality, body weight, food consumption, compound intake, gross pathology, and histopathology.

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 lot OGJ01 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 2-year study using HPLC-UV, and no degradation of the bulk chemical was detected.

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

not specified

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: approx. 5 to 6 weeks
- Weight at study initiation (study day 1; mean):
0 ppm group: 20.3 g (males); 16.6 g (females)
2000 ppm group: 20.5 g (males); 16.7 g (females)
10000 ppm group: 20.4 g (males); 16.6 g (females)
50000 ppm group: 20.5 g (males); 16.5 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) or 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

Five male and five female mice were randomly selected for parasite evaluation and gross observation of disease.

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

Details on route of administration:

Dietary exposure was chosen because humans ingest chromium picolinate in supplements.

Vehicle:

unchanged (no vehicle)

Details on oral 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 (procedure until June 16, 2003; later dose formulations were mixed for only 15 minutes with the intensifier bar).
- Rate of preparation of diet (frequency): approx. monthly
- Storage temperature of food: stored in sealed double-thick plastic bags, protected from light at 5 °C.
- Storage time: 42 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS.
Periodic analyses of the dose formulations of chromium picolinate monohydrate were conducted by the study laboratory using HPLC-UV. During the 2-year studies, the dose formulations were analyzed approximately every 12 weeks. Of the dose formulations analyzed, all 99 were within 10% of the target concentrations;

In addition, samples of dosed feed taken from the animal rooms were analysed periodically during the study. Nine animal room samples of 15 were within 10% of the target concentrations (six animal room samples were between -11 and - 27 %). Low results in the mouse samples were attributed to contamination by urine, faeces, and bedding during the study period when animals were small enough to get into the feeders.

NOTE: homogeneity and stability were only measured for the lot no. OGJ01, which was mixed with another lot in order to provide a combined lot for the 2 year study.

Homogeneity studies of 82 and 50000 ppm dose formulations of lot OGJ01 and stability studies of the 82 ppm dose formulation of lot OGJ01 were performed using ICP-AES. Additional homogeneity studies of 80 and 50,000 ppm dose formulations of this lot 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:

105 weeks

Frequency of treatment:

ad libitum

Dose / conc.:

0 ppm

Remarks:

actually ingested: males and females: 0 mg/kg bw/day

Dose / conc.:

2 000 ppm

Remarks:

actually ingested: males: approx. 250 mg/kg bw/day; females: approx. 240 mg/kg bw/day

Dose / conc.:

10 000 ppm

Remarks:

actually ingested: males: approx. 1200 mg/kg bw/day; females: approx. 1200 mg/kg bw/day

Dose / conc.:

50 000 ppm

Remarks:

actually ingested: males: approx. 6565 mg/kg bw/day; females: approx. 6100 mg/kg bw/day

No. of animals per sex per dose:

50 males / 50 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the dose selection for the 2 year repeated dose oral toxicity study with chromium picolinate monohydrate was based on a 3-month repeated dose oral toxicity study conducted with male and female B6C3F1 mice. Chromium picolinate monohydrate was administered ad libitum at dose levels of 0, 80, 240, 2000, 10000 or 50000 ppm in feed during an exposuer period of 14 weeks. The following parameters were measured/recorded: clinical signs, mortality, body weight, feed consumption, gross pathology, organ weights, histopathology, sperm motility and vaginal cytology.

Results:
Because chromium picolinate monohydrate produced no biologically significant changes in any of the parameters examined in male or female mice, exposure concentrations of 2000, 10000, and 50000 ppm were selected for the 2-year study in mice.

Positive control:

not applicable

Observations and examinations performed and frequency:

NOTE: the parameter haematology was not measured during the 2-year repeated dose oral toxicity study. Method and results were taken from a 3-month repeated dose oral toxicity study conducted prior to the 2-year study.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (clinical findings were recorded monthly)
- Cage side observations checked: mortality and clinical findings

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly for the first 13 weeks, monthly therefater, 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 measured initially, weekly for the first 13 weeks of the study and approximately monthly thereafter.
- Compound intake calculated: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified

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: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
At necropsy, all organs and tissues were examined for grossly visible lesions.

HISTOPATHOLOGY: Yes, all animals
All major tissues were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. The following tissues were examined microscopically: 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.

Statistics:

Survival analyses: Kaplan-Meier surivival curves, means, life table trend test, & life table pairwise comparisons. P values were two-sided.
Neoplasm & nonneoplastic lesions: Poly-k test, continuity-corrected Poly-3 test. P values were one-sided.
Body weight: parametric multiple comparison procedures of Dunnett (1955) & Williams (1971, 1972).
Jonckheere’s test was used to assess the significance of the dose-related trends & 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)*.
Extreme values identified by the outlier test of Dixon and Massey (1957).

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

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:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

NOTE: the parameter haematology was not measured during the 2-year repeated dose oral toxicity study. Method and results were taken from a 3-month repeated dose oral toxicity study conducted prior to the 2-year study.

CLINICAL SIGNS
- 2000, 10000 and 50000 ppm groups: no clinical findings were attributed to chromium picolinate monohydrate exposure.

MORTALITY
Males:
- 2000, 10000 and 50000 ppm groups: survival of exposed groups of males was similar to that of the control groups (Percent probability of survival at end of study: control group: 92 %; 2000 ppm group: 86 %; 10000 ppm group: 76 %; 50000 ppm group: 90 %).
Females:
- 2000, 10000 and 50000 ppm groups: survival of exposed groups of females was similar to that of the control groups (Percent probability of survival at end of study: control group: 90 %; 2000 ppm group: 90 %; 10000 ppm group: 88 %; 50000 ppm group: 78 %).

BODY WEIGHT AND WEIGHT CHANGES
Males:
- 2000, 10000 and 50000 ppm groups: mean body weights of exposed groups of males were generally similar to those of the controls throughout the study.
Females:
- 2000, 10000 and 50000 ppm groups: mean body weights of exposed groups of females were generally decreased (-1 % to -9 %) during the middle of the study but recovered to control values by the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE
Males and females:
- 2000, 10000 and 50000 ppm groups: feed consumption by exposed groups of males and females was similar to that by the controls throughout the study. Dietary concentrations of 2000, 10000, and 50000 ppm resulted in average daily doses of approximately 250, 1200, and 6565 mg chromium picolinate
monohydrate/kg body weight to males (equivalent to 29.8, 143.1, and 783.0 mg Cr/kg/day) and 240, 1200, and 6100 mg/kg to females (equivalent to 28.6, 143.1, and 727.5 mg Cr/kg/day).

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

HISTOPATHOLOGICAL FINDINGS: NEOPLASTIC
1) Liver
Males:
- 2000, 10000 and 50000 ppm groups: in males, the incidences of hepatoblastoma occurred with a positive trend (P ≤ 0.05) (0 ppm, 0/50; 2000 ppm, 1/50; 10000 ppm, 0/50; 50000 ppm, 3/50). Hepatoblastoma is considered a variant of hepatocellular carcinoma. The incidences of hepatocellular adenoma (21/50, 22/50, 21/50, 22/50), hepatocellular carcinoma (15/50, 18/50, 20/50, 16/50), and hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma (combined) (32/50, 32/50, 33/50, 33/50) were similar between control and exposed males. Because of the lack of an increase in the incidences of all these neoplasms combined and the low incidences of hepatoblastoma in exposed animals, the incidences of hepatoblastoma were not considered to be related to chromium picolinate monohydrate exposure.

Females:
- 2000, 10000 and 50000 ppm groups: there were no treatment-related effects in females.

2) Lungs
Males:
- 2000, 10000 and 50000 ppm groups: in males, the incidences of alveolar/bronchiolar carcinoma occurred with a positive trend (P ≤ 0.05) (3/50, 2/50, 5/50, 8/50). The incidences of alveolar/bronchiolar adenoma (13/50, 10/50, 7/50, 8/50) and alveolar/bronchiolar adenoma or carcinoma (combined) (16/50, 12/50, 12/50, 12/50) were decreased in exposed males. Because of the decreases in alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined), the increased incidences of alveolar/bronchiolar carcinoma were not considered related to chromium picolinate monohydrate exposure.

Females:
- 2000, 10000 and 50000 ppm groups: there were no treatment-related effects in females.

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actually ingested: males: approx. 6565 mg/kg bw/day; females:approx. 6100 mg/kg bw/day

Dose descriptor:

NOEL

Effect level:

ca. 783 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

Chromium

Sex:

male

Remarks on result:

other: calculated as Cr3+

Dose descriptor:

NOEL

Effect level:

ca. 727.5 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

Chromium

Sex:

female

Remarks on result:

other: calculated as Cr3+

Critical effects observed:

not specified

Conclusions:

Continuous exposure to chromium picolinate monohydrate in feed (2000, 10000 and 50000 ppm; actually ingested: males: approx. 250, 1200 and 6565 mg/kg bw/day; females: approx. 240, 1200 and 6100 mg/kg bw/day) of male and female B6C3F1 mice for a 2-year period caused no treatment-related effects on clinical signs, mortality, body weights, food consumption and histopathology (neoplastic/non-neoplastic).

Furthermore, no treatment-related effects on haematology were expected based on findings reported for a 3 month repeated dose oral toxicity during which the same concentrations in feed were administered to male and female B6C3F1 mice. The 3 month study was conducted prior to the current study.

Based on the findings from this study and the 3-month study, the NOEL (No-Observed-Effect-Level) of chromium picolinate monohydrate in male and female B6C3F1 mice is considered to be 50000 ppm (actually ingested: males: approx. 6565 mg/kg bw/day (equivalent to approx. 783.0 mg Cr/kg bw/day); females: approx. 6100 mg/kg bw/day (equivalent to approx. 727.5 mg Cr/kg bw/day)) due to the absence of any relevant toxicological effects.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-08-12 to 2004-08-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Deviations from OECD guideline 452 (2009): detailed clinical observations, opthalmological examinations, urinalysis determinations and organ weight determinations were missing; measure of blood clotting time/potential was missing; incomplete clinical chemistry (sodium, potassium, calcium, glucose, and total cholesterol were missing); histopathological examinations of aorta, cervix, coagulating gland, lacrimal gland, peripheral nerve, spinal cord and vagina were missing; individual data missing; historical control data was missing; justification of species selection wasa 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 50 male and 50 female F344/N rats in feed at concentrations of 0, 2000, 10000 and 50000 ppm (actually ingested: males: approx. 0, 90, 460 and 2400 mg/kg bw/day; females: approx. 0, 100, 510 and 2630 mg/kg bw/day) for up to 105 weeks. The following parameters were investigated/recorded: clinical signs, mortality, body weight, food consumption, compound intake, gross pathology, and histopathology.

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 lot OGJ01 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 2-year 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 specified

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: approx. 5 to 6 weeks
- Weight at study initiation (study day 1; mean):
0 ppm group: 114 g (males); 95 g (females)
2000 ppm group: 114 g (males); 95 g (females)
10000 ppm group: 113 g (males); 95 g (females)
50000 ppm group: 113 g (males); 96 g (females)
- Housing: 3 males or 5 females 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: 12 days

Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease.

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

Details on route of administration:

Dietary exposure was chosen because humans ingest chromium picolinate in supplements.

Vehicle:

unchanged (no vehicle)

Details on oral 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 (procedure until June 16, 2003; later dose formulations were mixed for only 15 minutes with the intensifier bar).
- Rate of preparation of diet (frequency): approx. monthly
- Storage temperature of food: stored in sealed double-thick plastic bags, protected from light at 5 °C.
- Storage time: 42 days

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 by the study laboratory using HPLC-UV. During the 2-year studies, the dose formulations were analyzed approximately every 12 weeks. Of the dose formulations analyzed, all 167 were within 10% of the target concentrations.

In addition, samples of dosed feed taken from the animal rooms were analysed periodically during the study. All 12 animal room samples were within 10% of the target concentrations.

NOTE: homogeneity and stability were only measured for the lot no. OGJ01, which was mixed with another lot in order to provide a combined lot for the 2 year study.

Homogeneity studies of 82 and 50000 ppm dose formulations of lot OGJ01 and stability studies of the 82 ppm dose formulation of lot OGJ01 were performed using ICP-AES. Additional homogeneity studies of 80 and 50,000 ppm dose formulations of this lot 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:

105 weeks

Frequency of treatment:

ad libitum

Dose / conc.:

0 ppm

Remarks:

actually ingested: males and females: 0 mg/kg bw/day

Dose / conc.:

2 000 ppm

Remarks:

actually ingested: males: approx. 90 mg/kg bw/day; females: approx. 100 mg/kg bw/day

Dose / conc.:

10 000 ppm

Remarks:

actually ingested: males: approx. 460 mg/kg bw/day; females: approx. 510 mg/kg bw/day

Dose / conc.:

50 000 ppm

Remarks:

actually ingested: males: approx. 2400 mg/kg bw/day; females: approx. 2630 mg/kg bw/day

No. of animals per sex per dose:

50 males / 50 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the dose selection for the 2 year repeated dose oral toxicity study with chromium picolinate monohydrate was based on a 3-month repeated dose oral toxicity study conducted with male and female F344/N rats. Chromium picolinate monohydrate was administered ad libitum at dose levels of 0, 80, 240, 2000, 10000 or 50000 ppm in feed during an exposuer period of 14 weeks. The following parameters were measured/recorded: clinical signs, mortality, body weight, feed consumption, gross pathology, organ weights, haematology, clinical chemistry, histopathology, sperm motility and vaginal cytology.

Results:
Because chromium picolinate monohydrate produced no biologically significant changes in any of the parameters examined in male or female rats, exposure concentrations of 2000, 10000, and 50000 ppm were selected for the 2-year study in rats.

Positive control:

not applicable

Observations and examinations performed and frequency:

NOTE: the parameters haematology and clinical chemistry were not measured during the 2-year repeated dose oral toxicity study. Method and results were taken from a 3-month repeated dose oral toxicity study conducted prior to the 2-year study.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (clinical findings were recorded monthly)
- Cage side observations checked: mortality and clinical findings

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly for the first 13 weeks, monthly thereafter, 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 measured initially, weekly for the first 13 weeks of the study and approximately monthly thereafter.
- Compound intake calculated: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes (method and results taken from a 3 month repeated dose oral toxicity study conducted prior to the current study)
- Time schedule for collection of blood:
clinical pathology study (exposure: 3 weeks): days 3 and 21
core study (exposure: 14 weeks): 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 (method and results taken from a 3 month repeated dose oral toxicity study conducted prior to the current study)
- Time schedule for collection of blood:
clinical pathology study (exposure: 3 weeks): days 3 and 21
core study (exposure: 14 weeks): 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: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
At necropsy, all organs and tissues were examined for grossly visible lesions.

HISTOPATHOLOGY: Yes, all animals
All major tissues were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. The following tissues were examined microscopically: 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.

Statistics:

Survival analyses: Kaplan-Meier surivival curves, means, life table trend test, & life table pairwise comparisons. P values were two-sided.
Neoplasm & nonneoplastic lesions: Poly-k test, continuity-corrected Poly-3 test. P values were one-sided.
Body weight: parametric multiple comparison procedures of Dunnett (1955) & Williams (1971, 1972).
Jonckheere’s test was used to assess the significance of the dose-related trends & 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)*.
Extreme values identified by the outlier test of Dixon and Massey (1957).

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

NOTE: the parameters haematology and clinical chemistry were not measured during the 2-year repeated dose oral toxicity study. Method and results were taken from a 3-month repeated dose oral toxicity study conducted prior to the 2-year study.

CLINICAL SIGNS
Males and females:
- 2000, 10000 and 50000 ppm groups: No clinical findings were attributed to chromium picolinate monohydrate exposure.

MORTALITY
Males:
- 2000, 10000 and 50000 ppm groups: although there was a significant trend for decreases in survival in male rats, the decreases were not significant at any exposure concentration and were not considered related to chromium picolinate monohydrate exposure (Percent probability of survival at end of study: control group: 74 %; 2000 ppm group: 72 %; 10000 ppm group: 70 %; 50000 ppm group: 56 %)..
Females:
- 2000, 10000 and 50000 ppm groups: survival of exposed groups of females was similar to that of the control groups (Percent probability of survival at end of study: control group: 72 %; 2000 ppm group: 70 %; 10000 ppm group: 72 %; 50000 ppm group: 80 %)..

BODY WEIGHT AND WEIGHT CHANGES
Males and females:
- 2000, 10000 and 50000 ppm groups: mean body weights of exposed groups of males and females were similar to those of the controls throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE
Males:
- 2000, 10000 and 50000 ppm groups: feed consumption by exposed groups of males was generally similar to that of the controls throughout the study. Dietary concentrations of 2000, 10000, and 50000 ppm resulted in average daily doses of approx. 90, 460, and 2400 mg chromium picolinate monohydrate/kg body weight to males (equivalent to 10.7, 54.9, and 286.2 mg Cr/kg/day).
Females:
- 2000, 10000 and 50000 ppm groups: feed consumption by exposed groups of females was generally similar to that of the controls throughout the study. Dietary concentrations of 2000, 10000, and 50000 ppm resulted in average daily doses of approx. 100, 510, and 2630 mg chromium picolinate monohydrate/kg body weight to females (equivalent to 11.9, 60.8, and 313.7 mg Cr/kg/day).

HAEMATOLOGICAL FINDINGS
Males and females:
- 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:
- 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.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
Males:
- 10000 ppm group: there were no differences in the incidences of preputial gland hyperplasia between exposed and control groups of rats.
Preputial gland hyperplasia was focal, characterized either by an increase in stratified squamous epithelium of the ducts or by increased numbers of sebaceous cells and possibly basal cells.
Females:
- 2000 ppm group: there were no differences in the incidences of clitoral gland hyperplasia between exposed and control groups of rats.

HISTOPATHOLOGICAL FINDINGS: NEOPLASTIC
Males:
- 10000 ppm group: the incidence of preputial gland adenoma was significantly increased compared to that in the control group and exceeded the historical control ranges for feed studies and for all routes combined. There was no incidence of preputial gland carcinoma.
Preputial gland adenomas were well-circumscribed masses that grew by expansion with compression of the surrounding parenchyma. The neoplastic glands retained some resemblance of acinar structure, although there was some fusion of the acini to form solid clusters of cells (Copeland-Haines and Eustis, 1990)*.
Females:
- 2000 ppm group: the incidence of clitoral gland adenoma was significantly decreased. There was no incidence of clitoral gland carcinoma.

Proliferative lesions of the preputial and clitoral gland comprise a morphological continuum, and separation of these into categories of hyperplasia, adenoma, and carcinoma is based largely on cytological features and degree of altered growth pattern. Lesions classified as hyperplasia are considered to be preneoplastic (Copeland-Haines and Eustis, 1990)*.

*Reference:
- Copeland-Haines, D., and Eustis, S.L. (1990). Specialized sebaceous glands. In Pathology of the Fischer Rat. Reference and Atlas (G.A. Boorman, S.L.
Eustis, M.R. Elwell, C.A. Montgomery, Jr., and W.F. MacKenzie, Eds.), pp. 279–293. Academic Press, Inc., San Diego.

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actually ingested: males: approx. 2400 mg/kg bw/day; females: approx. 2630 mg/kg bw/day

Dose descriptor:

NOEL

Effect level:

ca. 286.2 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

Chromium

Sex:

male

Remarks on result:

other: calculated as Cr3+

Dose descriptor:

NOEL

Effect level:

313.7 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

chromium

Sex:

female

Remarks on result:

other: calculated as Cr3+

Critical effects observed:

not specified

Conclusions:

Continuous exposure to chromium picolinate monohydrate in feed (2000, 10000 and 50000 ppm; actually ingested: males: approx. 90, 460 and 2400 mg/kg bw/day; females: approx. 100, 510 and 2630 mg/kg bw/day) of male and female F344/N rats for a 2-year period caused no treatment-related effects on clinical signs, mortality, body weights, food consumption and histopathology (neoplastic/non-neoplastic).

Furthermore, no treatment-related effects on haematology and clinical chemistry were expected based on findings reported for a 3 month repeated dose oral toxicity during which the same concentrations in feed were administered to male and female F344/N rats. The 3 month study was conducted prior to the current study.

Based on the findings from this study and the 3-month study, the NOEL (No-Observed-Effect-Level) of chromium picolinate monohydrate in male and female F344/N rats is considered to be 50000 ppm (actually ingested: males: approx. 2400 mg/kg bw/day (equivalent to approx. 286.2 mg Cr/kg bw/day); females: approx. 2630 mg/kg bw/day (equivalent to approx. 313.7 mg Cr/kg bw/day)) due to the absence of any relevant toxicological effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

chronic

Species:

rat

Quality of whole database:

key studies available

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No repeated dose toxicity study with dichromium nitride is available. However, based on the information available from two chronic 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 health hazard to either sex. No adverse effects 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 2-year repeated dose oral toxicity study using rats. The substance was administered ad libitum to groups of 50 male and 50 female F344/N rats in feed at concentrations of 0, 2000, 10000 and 50000 ppm (actually ingested: males: approx. 0, 90, 460 and 2400 mg/kg bw/day; females: approx. 0, 100, 510 and 2630 mg/kg bw/day) for up to 105 weeks. According to the authors, chromium picolinate monohydrate did not caused treatment-related effects on clinical signs, mortality, body weights, food consumption and histopathology (neoplastic/non-neoplastic).

Furthermore, no treatment-related effects on haematology and clinical chemistry were expected based on findings reported for a 3 month repeated dose oral toxicity (NTP, 2010; please refer to Section 7.8.1. Toxicity to reproduction: k_NTP_2010_3 months_rats) during which the same concentrations in feed were administered to male and female F344/N rats as in the chronic study. The 3 month study was conducted prior to 2 -year study and was used as a dose range finder for the chronic study.

Based on the findings from the 2 -year and 3-month studies, the NOEL (No-Observed-Effect-Level) of chromium picolinate monohydrate in male and female F344/N rats is considered to be 50000 ppm (actually ingested: males: 2400 mg/kg bw/day (equivalent to 288 mg Cr/kg bw/day); females: 2630 mg/kg bw/day (equivalent to 315.6 mg Cr/kg bw/day)) due to the absence of any relevant toxicological effects.

In addition, the National Toxicology Program (NTP, 2010) evaluated chromium picolinate monohydrate in a 2-year repeated dose oral toxicity study using mice. The substance was administered ad libitum to groups of 50 male and 50 female B6C3F1 mice in feed at concentrations of 0, 2000, 10000 and 50000 ppm (actually ingested: males: approx. 0, 250, 1200 and 6565 mg/kg bw/day; females: approx. 0, 240, 1200 and 6100 mg/kg bw/day) for up to 105 weeks. According to the authors, chromium picolinate monohydrate did not cause treatment-related effects on clinical signs, mortality, body weights, food consumption and histopathology (neoplastic/non-neoplastic).

Furthermore, no treatment-related effects on haematology were expected based on findings reported for a 3 month repeated dose oral toxicity (NTP, 2010; please refer to Section 7.8.1. Toxicity to reproduction: k_NTP_2010_3 months_mice) during which the same concentrations in feed were administered to male and female B6C3F1 mice as in the chronic study. The 3 month study was conducted prior to 2 -year study and was used as a dose range finder for the chronic study.

Based on the findings from the 2 -year and 3-month studies, the NOEL (No-Observed-Effect-Level) of chromium picolinate monohydrate in male and female B6C3F1 mice is considered to be 50000 ppm (actually ingested: males: approx. 6565 mg/kg bw/day (equivalent to approx. 787.8 mg Cr/kg bw/day); females: approx. 6100 mg/kg bw/day (equivalent to approx. 732 mg Cr/kg bw/day)) due to the absence of any relevant toxicological effects.

Dichromium nitride

Since no repeated dose toxicity study 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: 2400 mg/kg bw/day (equivalent to 288 mg Cr/kg bw/day) in the repeated dose toxicity study will be used.

Justification for classification or non-classification

Based on the absence of adverse effects in a chronic repeated dose oral toxicity study in rats, classification for specific target organ toxicity-repeated exposure (oral) is not warranted according to regulation (EC) No. 1272/2008.