Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published peer reviewed study, conducted by the NTP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Species:

other: Rat and Mouse

Strain:

other: F344/N rats and B6C3F1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Farms. They were quarantined for 12 days, and were approximately 5-6 weeks old at study initiation. Animals were distributed randomly into groups of approximately equal initial mean body weights and idenitified by tail tattoo. Male mice were housed singly, male rats were housed in groups of 3, female rats and mice were housed in groups of 5. The animal room was maintained at a temperature of 72±3°F, a relative humidity of 50±15%, a 12 hour light-dark cycle, and 10 air changes per hour. Irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc.) was available ad libitum and changed weekly. Tap water was available ad libitum via automatic watering system.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

CPM was fed in the diet. The dose formulations were prepared monthly by mixing CPM with the feed. The formulations were stored at room temperature in double-thick sealed plastic bags, protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with UV detection was used to analyse the diet preparations. Homogeneity of the high dose formulation was confirmed. The stability of this formulation was confirmed for at least 42 days at room temperature when stored in double-thick sealed plastic bags, protected from light. Periodic analysis confirmed that all 167 dose formulations for rats and all 99 for mice were within 10% of the target concentrations.

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

Daily

Post exposure period:

No post exposure period

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

2 000 ppm (nominal)

Dose / conc.:

10 000 ppm (nominal)

Dose / conc.:

50 000 ppm (nominal)

No. of animals per sex per dose:

50 male and 50 female rats and mice per group.

Control animals:

yes, concurrent no treatment

Details on study design:

Groups of 50 male and 50 female rats and mice were fed diets containing 0, 2000, 10000 or 50000 ppm CPM for 105 weeks. Animals were randomly distributed into groups of approximately equal initial mean body weights. Dose levels were selected based on previous 3 month subchronic toxicity studies. Dosing in feed was chosen as this is the primary route for human exposure (in the diet and in supplements).

Positive control:

Not examined.

Observations and examinations performed and frequency:

All animals were observed twice daily. Animals were weighed initially, once a week for the first 13 weeks, once a month thereafter, and at the end of the studies. Feed consumption was measured weekly for the first 13 weeks of the study and monthly thereafter. Clinical findings were recorded monthly.

Sacrifice and pathology:

Complete necropsies and microscopic examinations were performed on all rats and mice at study termination. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin (eyes were initially fixed in Davidson’s solution), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4–6 lm, 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.

Other examinations:

No further information available.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and non-neoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure that modifies the Cochran–Armitage linear trend test to take survival differences into account. Unless otherwise specified, a value of k = 3 was used in the
analysis of site-specific lesions. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided.

Clinical signs:

no effects observed

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Rat study: In males, there was a significant trend (P = 0.041) for decreased survival (0 ppm, 37/50; 2000 ppm, 36/50; 10,000 ppm, 35/50; 50,000 ppm, 28/50); however, because survival was not significantly different from the control group at any exposure concentration the decreases was not considered to be related to exposure. Survival in exposed females (0 ppm, 36/50; 2000 ppm, 35/50; 10,000 ppm, 36/50; 50,000 ppm, 40/50) was similar to that of the control group. Mean body weights of exposed groups of males and females were similar to those of the controls throughout the study. Feed consumption by exposed groups of males and females was generally similar to that of the controls throughout the study. Body weight and feed consumption data were used to calculate average daily doses of CPM resulting from each concentration (Table 1). Average daily doses were also calculated for Cr(III) and picolinic acid, the components of the CPM complex. Based on the body weight and feed consumption data, the increases in calculated ingested dose were proportional to the increases in exposure concentration. No clinical findings or non-neoplastic lesions were attributed to exposure. The incidence of preputial gland adenoma was significantly increased in males at 10,000 ppm compared to the control group (Table 2). This increase exceeded the historical control ranges for feed studies and for all routes of exposure. The incidence of preputial gland hyperplasia was not increased at any exposure concentration. Preputial gland carcinoma was not observed in control or exposed males. 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. 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). The female counterpart of the preputial gland is the clitoral gland. There were no increases in the incidences of clitoral gland adenoma or hyperplasia over the control group (Table 2). Carcinomas of the clitoral gland were not observed in control or treated females. Proliferative lesions of the preputial and clitoral glands constitute 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 (Copeland-Haines and Eustis, 1990). Lesions classified as hyperplasia are considered preneoplastic.

Mouse study: Survival of exposed groups of males (0 ppm, 46/50; 2000 ppm, 43/50; 10,000 ppm, 38/50; 50,000 ppm, 45/50) and females (0 ppm, 45/50; 2000 ppm, 44/50; 10,000 ppm, 44/50; 50,000 ppm, 39/50) was similar to that of the control groups. Mean body weights of exposed groups of males were generally similar to those of the controls throughout the study. In females, decreases in mean body weights of up to 10% compared to controls were observed during the middle of the study in exposed animals; however, mean body weights recovered to control values by the end of the study. Feed consumption by exposed groups of males and females was similar to that by the controls throughout the study. Body weight and feed consumption data were used to calculate average daily doses of CPM resulting from each concentration (Table 1). Average daily doses were also calculated for Cr(III) and picolinic acid, the components of the CPM complex. Based on the body weight and feed consumption data, the increases in calculated
ingested dose were proportional to the increases in exposure concentration. No clinical findings or neoplastic or non-neoplastic lesions were attributed to CPM exposure.

As part of the 2-year studies, total chromium content in excreta and selected tissues was determined in additional groups of male rats and female mice following 4, 11 or 180 days of exposure and a two day washout. These data will be reported in detail elsewhere; however, the primary findings of the tissue concentration studies aid in the interpretation of the bioassay results and will be discussed briefly here. Accumulation of total chromium with exposure concentration and duration was observed in the liver and kidney of rats and mice, suggesting that Cr(III) is taken up by these tissues; this pattern was less apparent in erythrocytes, forestomach, and glandular stomach. In both rats and mice, chromium tissue concentrations were generally not proportional to exposure concentration. As a result, tissue chromium concentrations in animals exposed to 50,000 ppm CPM were similar to those in animals exposed to lower concentrations. These data suggest that the maximum achievable tissue chromium concentrations were reached in these studies and may offer a partial explanation for the lack of a higher preputial gland neoplasm incidence in male rats exposed to 50,000 ppm than was observed at 10,000 ppm.

Relevance of carcinogenic effects / potential:

The increased incidence of preputial gland adenomas in male rats at 10000ppm was considered equivocal. CPM was not carcinogenic to female rats or male and female mice. The concentrations administered were very high relative to human exposures.

Dose descriptor:

NOEL

Effect level:

50 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: B6C3F1 mice

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOEL

Effect level:

50 000 ppm (nominal)

Sex:

female

Basis for effect level:

other: F344/N female rats

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOEL

Effect level:

2 000 ppm (nominal)

Sex:

male

Basis for effect level:

other: F344/N male rats. An increase in the incidence of preputial gland adenomas was observed at the 10000 ppm dose but the finding was considered equivocal as a similar increase was not seen in the 50000 ppm group.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Table 1. Conversion of CPM exposure concentrations in feed to average daily doses (mg/kg/day) of CPM, Cr(III) and picolinic acid in rats and mice exposed for 2 years.

	Rats			Mice
CPM (ppm)	CPM^a(mg/kg/day)	Cr(III)^b(mg/kg/day)	Picolinic acid^c(mg/kg/day)	CPM^a(mg/kg/day)	Cr(III)^b(mg/kg/day)	Picolinic acid^c(mg/kg/day)
Males
0	-	-	-	-	-	-
2000	90	10.7	79.3	250	29.8	220.2
10000	460	54.9	405.1	1200	143.1	1056.9
50000	2400	286.2	2113.8	6565	783.0	5782.0
Females
0	-	-	-	-	-	-
2000	100	11.9	88.1	240	28.6	211.4
10000	510	60.8	449.2	1200	143.1	1056.9
50000	2630	313.7	2316.3	6100	727.5	5372.5

aCalculated using body weight and feed consumption data.

bCalculated using the average daily dose of CPM and the percent mass of Cr(III) in CPM.

cCalculated using the average daily dose of CPM and the percent mass of picolinic acid in CPM.

Table 2. Accessory sex gland neoplasms in male and female rats exposed to CPM for 2 years.

	Exposure concentration (ppm)
	0	2000	10000	50000
Males
Preputial gland
No. necropsied	50	50	50	50
Hyperplasia	3^a(2.7)^b	1(4.0)	0	2 (2.5)
Adenoma^c	1(2.2)^d	1(2.3)	7(14.9)^*	4 (9.3)
Females
Clitoral gland
No. necropsied	50	50	50	50
Hyperplasia	8^a(2.4)^b	10(2.6)	11 (2.6)	4(2.3)
Adenoma^c	10(21.9)^d	2(4.4)^*	8(17.7)	11(23.4)

aNumber of animals with lesion.

bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild,

3 = moderate, 4 = marked.

cHistorical incidence for 2-year feed studies with controls given NTP-2000 diet

(mean ± standard deviation): 8/250 (3.2% ± 4.2%), range 0–10%; all routes: 43/1193

(3.6% ± 3.5%), range 0–10%.

dSurvival adjusted incidence (%).

eHistorical incidence: 26/200 (13.0% ± 6.2%), range 6–20%; all routes: 104/1096

Conclusions:

There was very little evidence of adverse effect following dietary exposure of rats and mice to CPM for 2 years.

Executive summary:

Chromium picolinate monohydrate (CPM) was fed to male and female F344/N rats and B6C3F1 mice in the diet for 2 years, in order to investigate the potential for CPM to induce chronic toxicity and carcinogenicity. Concentrations of 0, 2000, 10000 or 50000 ppm were fed to groups of 50 male and female mice and rats for 2 years. Exposure to CPM did not induce biologically significant changes in survival, body weight, feed consumption, or non-neoplastic lesions in rats or mice. In male rats, a statistically significant increase in the incidence of preputial gland adenoma at 10,000 ppm was considered an equivocal finding. CPM was not carcinogenic to female rats or to male or female mice.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Groups of 10 male and 10 female rats and mice were fed diets contain ing 0, 80, 240, 2,000, 10,000, or 50,000 ppm chromium picolinate monohydrate for 14 weeks. Chromium picolinate monohydrate (equivalent to average daily doses of approximately 17, 50, 450, 2,300, and 11,900 mg chromium picolinate monohydrate/kg body weight to males and 14, 40, 370, 1,775, and 9,140 mg/kg to females.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: male: 19.5 ± 0.4, female: 16.8 ± 0,5
- Housing: 1 male, 5 female per casge. Cage was Polycarbonate (Lab Products, Inc., Maywood, NJ); changed twice weekly(female mice), bedding was Irradiated, heat-treated hardwood bedding chips (P.J. Murphy Forest Products, Inc., Montville, NJ); changed weekly (male mice) or changed twice weekly, Racks were Stainless steel (Lab Products, Maywood, NJ); changed once every 2 weeks and rack filters were Reemay® spun-bonded polyester (Andico, Birmingham, AL); changed once every 2 weeks
- Diet (e.g. ad libitum): Irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water (Birmingham, AL, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: quarantined for 12 (males) or 11 (females) days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 °C
- Humidity (%): 50% ± 15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): 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. The dose formulations were prepared four times.
- Storage temperature of food: Stored in sealed double-thick plastic bags, protected from light at 5° C.

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. For 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 and mice were within 10% of the target concentrations

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

In feed, available ad libitum.

Dose / conc.:

0 ppm

Remarks:

In feed, available ad libitum

Dose / conc.:

80 ppm

Remarks:

male: 17mg/kg. female: 14mg/kg. Average daily doses of approximately, chromium picolinate monohydrate/ body weight

Dose / conc.:

240 ppm

Remarks:

male: 50mg/kg, female: 40mg/kg

Dose / conc.:

2 000 ppm

Remarks:

male: 450mg/kg female:370mg/kg

Dose / conc.:

10 000 ppm

Remarks:

male: 2300mg/kg female:1775mg/kg

Dose / conc.:

50 000 ppm

Remarks:

male: 11900mg/kg female: 9140mg/kg

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded weekly for core study animals. Clinical findings were recorded monthly.

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Feed consumption by exposed groups of males and females was generally similar to that of the controls throughout the study. Dietary concentrations of 80, 240, 2,000, 10,000, and 50,000 ppm resulted in average daily doses of approximately 17, 50, 450, 2,300, and 11,900 mg chromium picolinate monohydrate/kg body weight to males and 14, 40, 370, 1,775, and 9,140 mg/kg to females.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 3 and 21
- Anaesthetic used for blood collecti: Yes, animals were anesthetized with carbon dioxide
- Animals fasted: Not specified
- How many animals: 10
- Parameters were examined.: hematocrit; hemoglobin; erythrocyte, reticulocyte, and platelet counts; nucleated erythrocytes; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: days 3 and 21
- Animals fasted: Not specified
- How many animals: 8-10

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross lesions and tissue masses.

Optional endpoint(s):

Optional endpoints: Not specified

Statistics:

Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the control group are not significant by Dunnett’s test (body weight), Dunn’s test (estrous cycle length), or Fisher’s exact test (proportion of regular cycling females). By multivariate analysis of variance, exposed females do not differ significantly from the control females in the relative length of time spent in the estrous stages.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical findings related to exposure to chromium picolinate monohydrate; reddish- colored feces of 50,000 ppm animals were believed to be due to excretion of the test article and were not considered a sign of toxicity.

Mortality:

no mortality observed

Description (incidence):

All mice survived to the end of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Final mean body weights and body weight gains of all exposed groups were similar to those of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption by exposed groups of males and females was generally similar to that by the controls throughout the study.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no hematological effects in mice administered chromium picolinate monohydrate.

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no biologically significant differences in organ weights between exposed and control groups of mice.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No exposure-related lesions occurred in male or female mice.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no significant changes in reproductive organ weights in male or female mice or in sperm parameters in male mice. Female mice exposed to 10,000 ppm had significantly longer estrous cycles 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.

Key result

Dose descriptor:

NOAEL

Effect level:

9 140 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects at highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

1 090 mg/kg bw/day (nominal)

Based on:

element

Sex:

female

Basis for effect level:

other: no adverse effects at highest dose tested

Remarks on result:

other: converted to elemental chromium based on the MW (conversion factor: 0.119)

Dose descriptor:

NOAEL

Effect level:

11 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects at highest dose tested

Dose descriptor:

NOAEL

Effect level:

1 419 mg/kg bw/day (nominal)

Based on:

element

Sex:

male

Basis for effect level:

other: no adverse effects at highest dose tested

Remarks on result:

other: converted to elemental chromium based on the MW (conversion factor: 0.119)

Critical effects observed:

no

Conclusions:

There were no clinical findings related to exposure to chromium picolinate monohydrate; reddish-colored feces of 50,000 ppm animals was believed to be due to excretion of the test article and was not considered a sign of toxicity. There were no hematological effects in mice administered chromium picolinate monohydrate. There were no biologically significant differences in organ weights between exposed and control groups of mice. There were no significant changes in reproductive organ weights in male or female mice or in sperm parameters in male mice. Female mice exposed to 10,000 ppm had significantly longer estrous cycles 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. No exposure-related lesions occurred in male or female mice.

Executive summary:

In the subchronic studies groups of 10 male and 10 female mice were fed diets containing 0, 80, 240, 2000, 10000, or 50000 ppm chromium picolinate monohydrate (equivalent to average daily doses of approximately 17, 50, 450, 2300, and 11900 mg chromium picolinate monohydrate/kg body weight to males and 14, 40, 370, 1775, and 9140 mg/kg to females) for 14 weeks. All mice survived to the end of the study. Mean body weights and feed consumption of all exposed groups of males and females were similar to those of the control groups throughout the study. No exposure-related lesions occurred in males or females. Thus, the NOAEL was the highest dose tested: 11900 mg/kg bw/d (1419 mg Cr/kg bw/d) for males and 9140 mg/kg bw/d (1090 mg Cr/kg bw/d) for females.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

- Short description of test conditions: Groups of 10 male and 10 female rats were fed diets containing 0, 80, 240, 2,000, 10,000, or 50,000 ppm chromium picolinate monohydrate (equivalent to average daily doses of approximately 7, 20, 160, 800, or 4,240 mg chromium picolinate monohydrate/kg body weight to males and 6, 20, 160, 780, or 4,250 mg/kg to females) for 14 weeks.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: male: 91 ± 2 g, female: 93 ± 2 g
- Housing: 5 per cage. Cage was Polycarbonate (Lab Products, Inc., Maywood, NJ); changed twice weekly, bedding was irradiated, heat-treated hardwood bedding chips (P.J. Murphy Forest Products, Inc., Montville, NJ); changed twice weekly, Racks were Stainless steel (Lab Products, Maywood, NJ); changed once every 2 weeks and rack filters were Reemay® spun-bonded polyester (Andico, Birmingham, AL); changed once every 2 weeks
- Diet (e.g. ad libitum): Irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water (Birmingham, AL, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: quarantined for 13 (males) or 14 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 °C
- Humidity (%): 50% ± 15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): 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. The dose formulations were prepared four times.
- Storage temperature of food: Stored in sealed double-thick plastic bags, protected from light at 5° C.

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. For 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 and mice were within 10% of the target concentrations

Duration of treatment / exposure:

Core studies: 14 weeks
Clinical pathology study: 3 weeks

Frequency of treatment:

In feed, available ad libitum.

Dose / conc.:

0 ppm

Remarks:

in feed, available ad libitum

Dose / conc.:

80 ppm

Remarks:

in feed, available ad libitum

Dose / conc.:

240 ppm

Remarks:

in feed, available ad libitum

Dose / conc.:

2 000 ppm

Remarks:

in feed, available ad libitum

Dose / conc.:

10 000 ppm

Remarks:

in feed, available ad libitum

Dose / conc.:

50 000 ppm

Remarks:

in feed, available ad libitum

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded weekly for core study animals. Clinical findings were recorded weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Core study animals were weighed initially, weekly, and at the end of the studies

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Feed consumption by exposed groups of males and females was generally similar to that of the controls throughout the study. Dietary concentrations of 80, 240, 2,000, 10,000, and 50,000 ppm resulted in average daily doses of approximately 7, 20, 160, 800, and 4,240 mg chromium picolinate monohydrate/kg body weight to males and 6, 20, 160, 780, and 4,250 mg/kg to females.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 3 and 21
- Anaesthetic used for blood collection: Yes, animals were anesthetized with carbon dioxide
- Animals fasted: Not specified
- How many animals: 8-10
- Parameters were examined: hematocrit; hemoglobin; erythrocyte, reticulocyte, and platelet counts; nucleated erythrocytes; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 3 and 21
- Animals fasted: Not specified
- How many animals: 8-10

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross lesions and tissue masses.

HISTOPATHOLOGY: Yes
The following tissues were examined: 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.

Optional endpoint(s):

Optional endpoints: Not specified

Statistics:

Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the control group are not significant by Dunnett’s test (body weight), Dunn’s test (estrous cycle length), or Fisher’s exact test (proportion of regular cycling females). By multivariate analysis of variance, exposed females do not differ significantly from the control females in the relative length of time spent in the estrous stages.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical findings related to exposure to chromium picolinate monohydrate; reddish-colored feces of 50,000 ppm animals was believed to be due to excretion of the test article and was not considered a sign of toxicity.

Mortality:

no mortality observed

Description (incidence):

All rats survived to the end of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Final mean body weights and body weight gains of all exposed groups were similar to those of the control groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption by exposed groups of males and females was generally similar to that by the controls throughout the study. Dietary concentrations of 80, 240, 2,000, 10,000, and 50,000 ppm resulted in average daily doses of approximately 7, 20, 160, 800, and 4,240 mg chromium picolinate monohydrate/kg body weight to males and 6, 20, 160, 780, and 4,250 mg/kg to females.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were minor sporadic changes in the hematology and clinical chemistry variables in rats. All changes were within physiological normal levels, none demonstrated an exposure relationship, and none were considered biologically important or toxicologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were minor sporadic changes in the hematology and clinical chemistry variables in rats. All changes were within physiological normal levels, none demonstrated an exposure relationship, and none were considered biologically important or toxicologically relevant.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and relative kidney weights of all exposed groups of females were significantly greater than those of the controls, and relative liver weights of exposed groups of females were generally 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. There were no significant changes in reproductive organ weights in male or female rats.

Gross pathological findings:

not specified

Description (incidence and severity):

No exposure-related lesions occurred in male or female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no significant changes in reproductive organ weights in male or female rats, in sperm parameters in male rats, or in estrous cyclicity in female rats at any dose.

Key result

Dose descriptor:

NOAEL

Effect level:

4 240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects in highest dose group

Key result

Dose descriptor:

NOAEL

Effect level:

506 mg/kg bw/day (nominal)

Based on:

element

Sex:

male

Basis for effect level:

other: no adverse effects in highest dose group

Remarks on result:

other: converted to elemental chromium based on the MW (conversion factor: 0.119)

Dose descriptor:

NOAEL

Effect level:

4 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects in highest dose group

Dose descriptor:

NOAEL

Effect level:

507 mg/kg bw/day (nominal)

Based on:

element

Sex:

female

Basis for effect level:

other: no adverse effect in highest dose group

Remarks on result:

other: converted to elemental chromium based on the MW (conversion factor: 0.119)

Critical effects observed:

no

Conclusions:

There were no clinical findings related to exposure to chromium picolinate monohydrate; reddish-colored feces of 50,000 ppm animals was believed to be due to excretion of the test article and was not considered a sign of toxicity. 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. There were no significant changes in reproductive organ weights in male or female rats, in sperm parameters in male rats, or in estrous cyclicity in female rats at any dose. No exposure-related lesions occurred in male or female rats.

Executive summary:

Groups of 10 male and 10 female rats were tested under similar experimental conditions. They were fed diets containing 0, 80, 240, 2,000, 10000, or 50000 ppm chromium picolinate monohydrate (equivalent to average daily doses of approximately 7, 20, 160, 800, or 4240 mg chromium picolinate monohydrate/kg body weight to males and 6, 20, 160, 780, or 4250 mg/kg to females) for 14 weeks. All rats survived to the end of the study. Mean body weights and feed consumption of all exposed groups of males and females were similar to those of the control groups throughout the study. No exposure-related lesions occurred in males or females. Thus, the NOAEL was the highest dose tested: 4240 mg/kg bw/d (506 mg Cr/kg bw/d) for males and 4250 mg/kg bw/d (507 mg Cr/kg bw/d) for females.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only Salmonella typhimurium strains TA98, TA100 and Escherichia coli strain WP2 uvrA/pKM101 were tested

GLP compliance:

no

Remarks:

data from apublication

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

chromium(3+);pyridine-2-carboxylate;hydrate

Cas Number:

27882-76-4

Molecular formula:

C18H12CrN3O6 H2O

IUPAC Name:

chromium(3+);pyridine-2-carboxylate;hydrate

Test material form:

solid: crystalline

Details on test material:

CPM - chromium picolinate monohydrate, CAS no. 27882-76-4. CPM, a reddish-purple crystalline powder, was obtained from TCI America and Sigma-Aldrich. It was identified as CPM by a variety of techniques. Purity was determined to be > 95%. CPM was stable for at least 2 weeks, it was stored at room temperature in sealed plastic buckets protected from light. No degredation was detected during the 2-year studies.

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver of Aroclor 1254-induced male Sprague-Dawley rats
- concentration or volume of S9 mix and S9 in the final culture medium: 10 %
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): positive control for metabolic activation used (2-aminoanthracene)

Test concentrations with justification for top dose:

0, 100, 500, 1000, 5000, 10000 µg/plate
The high dose was set at 10,000 μg/plate by experimental design, because no toxicity was observed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine (TA98, -S9), 2-aminoanthracene (all strains, +S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : 2 (S. typhimurium strain TA 100, E. coli WP2 uvrA), 3 (S. typhimurium strain TA 98)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in buffer (preincubation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Exposure duration/duration of treatment: 2 d

Test articles were incubated with the bacterial tester strains either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rats) for 20 minutes at 37° C. Top agar supplemented with L-histidine and d-biotin was added, and the
contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.

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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Remarks:

not measured/tested

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Test results 5

Species / strain:

S. typhimurium TA 1537

Remarks:

not measured/tested

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: precipitation observed in strain TA 98 in highest dose group with and without metabolic activation

STUDY RESULTS
- Concurrent vehicle negative and positive control data : please refer to table attached

Ames test:
- Mean number of revertant colonies per plate and standard deviation : please refer to table attached

Applicant's summary and conclusion

Conclusions:

In the standard bacterial reverse mutation screening assays conducted by the NTP, chromium picolinate monohydrate showed no clear evidence of genotoxicity. Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was observed in S. typhimurium strains TA100 and TA98 and E. coli strain WP2 uvrA/pKM101 when chromium picolinate monohydrate was tested with or without exogenous metabolic activation (S9).

Executive summary:

Chromium picolinate monohydrate was tested in a bacterial reverse mutation test in in Salmonella typhimurium strains TA98, TA100 and Escherichia coli strain WP2 uvrA/pKM101 at a concentration range of 100 to 10,000 μg/plate with and without exogenous metabolic activation (S9). The substance was negative in all tested strains with and without metabolic activation.