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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
three test strains only; solvent used was not defined; tubes were not aerated during pre-incubation by using a shaker; individual data missing; historical control data missing
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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.
Target gene:
TA100: his G 46
TA98: his D 3052
E. coli WP2 uvrA/pKM101: trpE
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (containing 10 % rat S9)
Test concentrations with justification for top dose:
100, 500, 1000, 5000 and 10000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: yes (it was stated that a solvent was used, but the solvent was not further specified)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylenediamine (without metabolic activation; strain TA98) & 2-aminoanthracene (with metabolic activation; all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation (as described in Haworth et al. (1983)*

EXPERIMENTAL PROTOCOL:
- test chemical (0.05 mL), overnight culture of Salmonella (0.05 mL), and S-9 mix or buffer (0.50 mL), were incubated at 37°C, without shaking, for 20 minutes.
- top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium.
- histidine-independent (his+) colonies arising on the plates were counted following two days incubation at 37°C.
- plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
- at the discretion of the investigators, plates with low numbers of colonies, containing precipitated test chemical, or having excessively reduced contrast because of chemical color, were counted by hand.

Two or three trials were conducted for each concentration with and without metabolic activation.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity assay was performed using TA100 or the system developed by Waleh et al. (1982)*. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

* Reference:
- Haworth, S., Lawlor T., Mortelsmans, K., Speck, W., Zeiger E., (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 7 [Suppl 1]: 3 - 142.
- Waleh NS, Rapport SJ, Mortelmans K (1982): Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutat Res 97:247-256.
Rationale for test conditions:
The high dose was set at 10000 μg/plate by experimental design, because no toxicity was observed.
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
mean ± standard error
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was observed in S. typhimurium strains TA100 or TA98 or E. coli strain WP2 uvrA/pKM101 when chromium picolinate monohydrate was tested with or without exogenous metabolic activation (S9).
Precipitation was observed in one trial without metabolic activation and one trial with metabolic activation at the highest concentration (10000 µg/plate) tested with S. typhimurium strain TA98.

Please also refer for results to the field "Attached background material" below.
Conclusions:
Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was observed in S. typhimurium strains TA100 or TA98 or E. coli strain WP2 uvrA/pKM101 with chromium picolinate monohydrate with or without exogenous metabolic activation.
Reason / purpose:
reference to same study
Reference
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:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium picolinate monohydrate was administered ad libitum to groups of 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 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.
Reason / purpose:
reference to same study
Reference
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:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium picolinate monohydrate was administered ad libitum to groups of 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 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.
Reason / purpose:
reference to same study
Reference
Endpoint:
fertility, other
Remarks:
based on a 3-months repeated dose oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-10-14 to 2002-01-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study had some experimental and reporting deficiencies: detailed clinical observations, ophthalmological examinations, neurobehavioural examinations and clinical chemistry examinations were missing; measure of blood clotting time/potential was missing; incomplete organ weight determinations (adrenals, uterus, ovaries, spleen, and brain were missing); histopathological examinations of the spinal cord, aorta, and peripheral nerve were missing; individual data was missing; rationale for dose selection was missing; base-line data for haematology and clinical chemistry were missing
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium picolinate monohydrate was administered ad libitum to groups of 10 male and 10 female B6C3F1 mice in feed at concentrations of 0, 80, 240, 2000, 10000 and 50000 ppm (actually ingested: males: approx. 0, 17, 50, 450, 2300 and 11900 mg/kg bw/day; females: approx. 0, 14, 40,370, 1775 and 9140 mg/kg bw/day) for up to 14 weeks. The following parameters were investigated/recorded: clinical signs, survival, body weight, food consumption, compound intake, haematology, organ weights, gross pathology, and histopathology. Furthermore, evaluation of sperm motility and vaginal cytology were carried out in the 0, 2000, 10000 and 50000 ppm groups at the end of the study.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (~25 °C), protected from light, in sealed plastic buckets.

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

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

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

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

DETAILED CLINICAL OBSERVATIONS: No data

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

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

FOOD EFFICIENCY: No data

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

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

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

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

GROSS NECROPSY
- necropsies were performed on all animals.

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

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

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

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

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

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

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

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

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

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

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
- 10000 ppm group: female mice had statistically significant longer estrous cycles (P ≤ 0.05) than the controls; however, this was likely the result of sampling bias because only three females had regular cycles, and therefore, was not considered biologically significant.
- 2000 and 50000 ppm groups: there were no statistically significant changes in estrous cyclicity in female rats.

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

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

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

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

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

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

DETAILED CLINICAL OBSERVATIONS: No data

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

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

FOOD EFFICIENCY: No data

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Based on the findings from this study, the NOEL (No-Observed-Effect-Level) for systemic toxicity was considered to be 50000 ppm (actually ingested: males: approx. 4240 mg/kg bw/day (equivalent to approx. 508.8 mg Cr/kg bw/day); females: approx. 4250 mg/kg bw/day (equivalent to approx. 510 mg Cr/kg bw/day)) for male and female rats based on the absence of any relevant toxicological effects. Furthermore, the NOEL for reproductive toxicity was also considered to be 50000 ppm for male and female rats based on the absence of any relevant toxicological effects.
Reason / purpose:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
no positive control used; 1000 polychromatic cells counted only; rationale for dose selection/route of administration missing; individual data missing; historical control data missing
GLP compliance:
yes
Type of assay:
other: micronucleus assay
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 specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation:
0 ppm group: 19.6 ± 0.2 g (males); 16.9 ± 0.2 g (females)
80 ppm group: 19.5 ± 0.3 g (males); 16.7 ± 0.4 g (females)
240 ppm group: 19.5 ± 0.3 g (males); 16.8 ± 0.2 g (females)
2000 ppm group: 19.4 ± 0.3 g (males); 16.7 ± 0.3 g (females)
10000 ppm group: 19.4 ± 0.3 g (males); 16.8 ± 0.3 g (females)
50000 ppm group: 19.6 ± 0.3 g (males); 16.9 ± 0.4 g (females)
- Housing: 1 male or 5 females per polycarbonate cage (Lab Products, Inc., Maywood, NJ; changed weekly (males) or twice weekly (females)); bedding: irradiated, heat-treated hardwood bedding chips (P.J. Murphy Forest Products, Inc., Montville, NJ; changed weekly (males) twice weekly (females)); Rack filters: Reemay® spun-bonded polyester (Andico, Birmingham, AL; changed once every 2 weeks); Racks: stainless steel (Lab Products, Maywood, NJ; changed once every 2 weeks)
- Diet (ad libitum): irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc.,Gardners, PA)
- Water (ad libitum): tap water
- Acclimation period: 12 days (males) or 11 days (females)

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

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 to 23.9 °C
- Relative humidity: 50 % ± 15 %
- Air changes: 10/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
- Vehicle(s)/solvent(s) used: none
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
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
ad libitum
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(s):
not specified
Tissues and cell types examined:
The frequency of micronucleated cells in 1000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group was determined. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined per animal as a measure of bone marrow toxicity.
Details of tissue and slide preparation:
SAMPLING TIMES / DETAILS OF SLIDE PREPARATION:
At the end of the 3-month feed study with chromium picolinate monohydrate, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronucleated cells in
1,000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1,000 erythrocytes was determined per animal as a measure of bone marrow toxicity.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials. Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects. Results of the 3-month study were accepted without repeat tests because additional test data could not be obtained.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among normochromatic erythrocytes was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Sex:
female
Genotoxicity:
ambiguous
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No increase in the frequency of micronucleated normochromatic erythrocytes was observed in male B6C3F1 mice administered chromium picolinate monohydrate (80 to 50,000 ppm) in feed for 3 months, indicating no potential for chromium picolinate monohydrate to induce chromosomal alterations in dividing cell populations in this test system. In female mice, however, the small increase in micronucleated normochromatic erythrocytes noted in the highest exposure concentration group (50,000 ppm) was not significant at P=0.0396, but it resulted in a significant trend test (P=0.005).
No significant alterations in the percentage of PCEs among total erythrocytes was observed in exposed mice, indicating that these exposure concentrations of chromium picolinate monohydrate did not induce bone marrow toxicity.

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. All mice survived to the end of the study.

Please also refer to the field "Attached background material" below.
Conclusions:
No increase in the frequency of micronucleated normochromatic erythrocytes was observed in male B6C3F1 mice administered chromium picolinate monohydrate (0, 80, 240, 2000, 10000 and 50000 ppm) in feed for 3 months. A small increase in micronucleated normochromatic erythrocytes was seen in female mice at the highest exposure concentration tested (50000 ppm), and the results in female mice were considered equivocal.
Reason / purpose:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
purity/physical nature of test item missing; housing/feeding conditions missing; body weights missing; individual data & historical control data missing; rationale for vehicle/dose selection/route of administration missing
GLP compliance:
yes
Type of assay:
other: micronucleus assay
Species:
rat
Strain:
other: F344/N
Details on species / strain selection:
not specified
Sex:
male
Details on test animals and environmental conditions:
not specified
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Chromium picolinate was dissolved in corn oil.
Duration of treatment / exposure:
72 hours
Frequency of treatment:
24 hour intervals (total of 3 administrations)
Dose / conc.:
156 other: mg/kg
Dose / conc.:
312 other: mg/kg
Dose / conc.:
625 other: mg/kg
Dose / conc.:
1 250 other: mg/kg
Dose / conc.:
2 500 other: mg/kg
No. of animals per sex per dose:
5 male rats
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: single injection
- Doses / concentrations: 8 mg/kg
Tissues and cell types examined:
2000 polychromatic erythrocytes were scored for the frequency of micronucleated cells in each of five rats per dose group. In addition, the percentage of polychromatic erythrocytes among the total erythrocyte population in the bone marrow was determined per animal as a measure of toxicity.
Details of tissue and slide preparation:
SAMPLING TIMES:
The rats were killed 24 hours after the final treatment, and blood smears were prepared from bone marrow cells obtained from the femurs. The smears were air-dried, fixed, and stained.
Evaluation criteria:
not specified
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among polychromatic erythrocytes was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No induction of micronucleated polychromatic erythrocytes was observed in bone marrow of male F344/N rats treated with chromium picolinate (156 to 2,500 mg/kg) by oral gavage three times at 24-hour intervals, and no significant alterations in the percentage of polychromatic erythrocytes among total erythrocytes was observed in dosed rats, indicating that these doses of chromium picolinate did not induce bone marrow toxicity.
Conclusions:
No induction of micronucleated polychromatic erythrocytes was observed in bone marrow of male F344/N rats treated with chromium picolinate (156, 312, 625, 1250 and 2500 mg/kg) by oral gavage three times at 24-hour intervals.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2010
Reference Type:
publication
Title:
Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals
Author:
Zeiger, E. et al.
Year:
1992
Bibliographic source:
Environmental and Molecular Mutagenesis 19 (21): 2 - 141.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
purity/physical description of the test item missing; solvent used was not defined; tubes were not aerated during pre-incubation by using a shaker; individual data missing; historical control data missing
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
not specified
Specific details on test material used for the study:
not specified

Method

Target gene:
TA98: his D 3052
TA100 & TA1535: his G 46
TA97: hisD6610 hisO1242
TA102 & TA104: hisG428
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium, other: TA104
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix (containing S9 obtained from male Sprague-Dawley rats or male Syrian hamster livers; both types of S9 were used in the study; both types of S9 mix contained either 10 % or 30 % of S9)
Test concentrations with justification for top dose:
Experiment 1:
- 33 (TA98 only),100, 333, 667 (TA97 only), 1000, 2000 (TA97 only) 3333 and 10000 µg/plate (with and without metabolic activation; strains TA97, TA98, TA100, TA102, TA104 &TA1535)

Experiment 2:
- 100, 333, 666 (TA102 only), 1000, 1666, 3333, 6666 and 10000 µg/plate (with and without metabolic activation; strains TA100, TA102 and TA1535)
- 100, 333, 1000, 3333 and 10000 µg/plate (with and without metabolic activation; strains TA97, TA98 and TA104)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: yes (it was stated that a solvent was used, but the solvent was not further specified)
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
mitomycin C
other: 4-nitro-o-phenylenediamine (without metabolic activation) & 2-aminoanthracene (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation (as described in Haworth et al. (1983)*

EXPERIMENTAL PROTOCOL:
- test chemical (0.05 mL), overnight culture of Salmonella (0.05 mL), and S-9 mix or buffer (0.50 mL), were incubated at 37°C, without shaking, for 20 minutes.
- top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium.
- histidine-independent (his+) colonies arising on the plates were counted following two days incubation at 37°C.
- plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
- at the discretion of the investigators, plates with low numbers of colonies, containing precipitated test chemical, or having excessively reduced contrast because of chemical color, were counted by hand.

Two or three trials were conducted for each concentration with and without metabolic activation.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity assay was performed using TA100 or the system developed by Waleh et al. (1982)*. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

* Reference:
- Haworth, S., Lawlor T., Mortelsmans, K., Speck, W., Zeiger E., (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 7 [Suppl 1]: 3 - 142.
- Waleh NS, Rapport SJ, Mortelmans K (1982): Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutat Res 97:247-256.
Rationale for test conditions:
The high dose was set at 10000 μg/plate by experimental design, because no toxicity was observed.
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
mean ± standard error

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA97, TA98, TA100, TA102, TA104 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No induction of gene mutations was observed in two independent studies conducted with chromium picolinate (up to 10000 μg/plate) in several strains of S. typhimurium with and without hamster or rat liver S9.

Precipitation was observed as follows:
TA97:
- 10000 µg/plate (with 10 and 30 % hamster S9)
TA100 & TA1535:
- 3333 µg/plate (without S9 as well as with 10 and 30 % hamster and rat S9)

Please also refer for results to the field "Attached background material" below.

Applicant's summary and conclusion

Conclusions:
No induction of gene mutations was observed in two independent studies conducted with chromium picolinate (up to 10000 μg/plate) in several strains of S. typhimurium with and without hamster or rat liver S9.