Trichromium dicarbide

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

3 months exposure

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

October 2001 - August 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Justification in section 13 read-across analogue approach

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Materials and methods

Principles of method if other than guideline:

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

Test material

Test material information

Constituent 1

Reference substance name:

Chromium picolinate monohydrate

Cas Number:

27882-76-4

Molecular formula:

C18H14CrN3O7,H2O

IUPAC Name:

Chromium picolinate monohydrate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Chromium picolinate monohydrate was obtained from TCI America (Portland, OR) in one lot (OGJ01) and from Sigma-Aldrich (St. Louis, MO) in one lot (CHESS0204DFCI). Lot OGJ01 was used in the 3-month studies. Identity, purity, and stability analyses were performed by the analytical chemistry laboratory. Reports on analyses performed in support of the chromium picolinate monohydrate studies are on file at the National Institute of Environmental Health Sciences.
Tested lot of the chemical, a reddishpurple crystalline powder, were identified as chromium picolinate monohydrate by infrared and proton nuclear magnetic resonance spectroscopy, X-ray diffraction, and electrospray ionization-mass spectrometry (EI-MS).
The moisture contents of lots were determined using Karl Fischer titration. Purity of the test chemical was determined by elemental analyses, proton-induced X-ray emission (PIXE) spectroscopy), inductively coupled plasma-atomic emission spectroscopy (ICP-AES), high - performance liquid chromatography (HPLC) with diode-array detection (HPLC-DAD), UV detection (HPLC-UV), or ICPmass spectrometric detection (HPLC-ICP-MS).
The results of Karl Fischer titration for water content, elemental analyses for carbon, hydrogen, and nitrogen, and ICP-AES analysis for total chromium were all consistent with the theoretical values for chromium picolinate monohydrate. PIXE analysis indicated the absence of significant metallic impurities and a total chromium content of 117% of the theoretical value.
HPLC-DAD revealed a major component at 96%, one impurity at 2.1%, and five additional impurities at greater than or equal to 0.1% each. HPLC-UV by one system followed by HPLC-ICP-MS indicated that the maximum concentrations of free (uncomplexed) Cr(III) or Cr(VI) were less than 0.025%. The overall purity of the lot was determined to be greater than 96%. In an attempt to identify the impurities indicated by HPLC-DAD in the tested lot, preparations of chromium:picolinate complexes were made and analyzed using HPLC-DAD by a second system and HPLC-EI-MS. The results were inconclusive due to insufficient assay sensitivity and lack of authentic reference standards; however, these analyses provided evidence that the impurities in the test chemical were probably chromium:picolinate complexes, although the exact structures and ratios were uncertain.
Stability studies of the bulk chemical were performed using ICP-AES and HPLC-UV. 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, protected from light, in sealed plastic buckets.
Periodic reanalyses of the bulk chemical were performed during the 3-month studies using HPLC-UV, and no degradation of the bulk chemical was detected.

Test animals

Species:

rat

Strain:

other: F244/N

Details on species / strain selection:

Male and female F344/N rats were obtained from Taconic Farms, Inc. (Germantown, NY) for use in the study. Rats and mice were quarantined for 12 days before the beginning of the studies.
Five male and five female rats and mice were randomly selected for parasite evaluation and gross observation of disease. Rats were approximately 5 to 6 weeks old at the beginning of the studies.
The health of the animals was monitored during the studies according to the protocols of the NTP
Sentinel Animal Program.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed three (males) or five (females) per cage. Feed and water were available ad libitum.
Feed consumption was measured weekly for the first 13 weeks of the study and approximately monthly thereafter. Cages were changed and rotated twice weekly; racks were changed and rotated every 2 weeks.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

Groups of 10 male and 10 female rats were fed diets containing 0, 2,000, 10,000, or 50,000 ppm chromium picolinate monohydrate for 14 weeks. Additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 3 weeks. Feed and water were available ad libitum.

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 by system E. 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 tested animals were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; all 10 animal room samples for rats and eight of 10 for mice were within 10% of the target concentrations.

Duration of treatment / exposure:

Core studies: 14 weeks
Clinical pathology study: 3 weeks

Frequency of treatment:

Observed twice daily; core study animals were weighed initially, weekly, and at the end of the studies; clinical findings were recorded weekly. Feed consumption by core study animals was recorded weekly by cage.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Doses / concentrations 3

Dose / conc.:

10 000 ppm

Doses / concentrations 4

Dose / conc.:

50 000 ppm

No. of animals per sex per dose:

Core studies: 10 males and 10 females
Clinical pathology study: 10 male and 10 female rats

Examinations

Parental animals: Observations and examinations:

Clinical findings were recorded weekly for core study animals. Feed consumption by core study animals was recorded weekly by cage. Core study animals were weighed initially, weekly, and at the end of the studies.

Oestrous cyclicity (parental animals):

At the end of the 3-month studies, samples were collected for sperm motility and vaginal cytology evaluations on core study rats and mice exposed to 0, 2,000, 10,000, or 50,000 ppm. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females 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):

Male animals were evaluated for sperm count and motility at terminal sacrifice. The left testis and left epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk (rats) 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 by two observers. 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.

Statistics:

Survival Analyses
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s method for testing two groups for equality and Tarone’s life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Spermatid & epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley and Dunn. Jonckheere’s test was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose related trend

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

Dermal irritation (if dermal study):

not specified

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):

Dietary concentrations of 2,000, 10,000, and 50,000 ppm resulted in average daily doses of approximately 160, 800, and 4,240 mg chromium picolinate monohydrate/kg body weight to males and 160, 780, and 4,250 mg/kg to females.

Food efficiency:

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.

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There were minor sporadic changes in the hematology 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 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.

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

Description (incidence and severity):

There were no significant changes in reproductive organ weights in male or female rats, in sperm par ameters in male rats, or in estrous cyclicity in female rats at any dose. No exposure-related lesions occurred in male or female rats.

Histopathological findings: neoplastic:

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no significant changes in reproductive organ weights in male or female rats, in estrous cyclicity in female rats at any dose. No exposure-related lesions occurred in male or female rats.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no significant changes in reproductive organ weights in male rats, in sperm parameters in male rats at any dose. No exposure-related lesions occurred in male or female rats.

Reproductive performance:

not specified

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

>= 50 000 ppm

Sex:

male/female

Basis for effect level:

clinical signs

mortality

dermal irritation

body weight and weight gain

food consumption and compound intake

food efficiency

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Any other information on results incl. tables

Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Feed Study of Chromium Picolinate Monohydrate (a)

	0 ppm	2000 ppm	10000 ppm	50000 ppm
n	10	10	10	10
Weights (g)
Necropsy body wt	326 ± 8	329 ± 6	319 ± 5	316 ± 9
L. Cauda epididymis	0.2265 ± 0.0427	0.1862  ± 0.0045	0.1823  ± 0.0062	0.1781  ± 0.0073
L. Epididymid	0.4582  ± 0.0088	0.4404  ± 0.0109	0.4440  ± 0.0129	0.4494  ± 0.0157
L. Testis	1.5000  ± 0.0340	1.4757  ± 0.0216	1.4756  ± 0.0272	1.4635  ± 0.0337
Spermatid Mesasurement
Spermatid heads (10^7/g testis)	109.47 ± 4.91	111.75 ± 3.94	100.41 ± 3.45	120.74 ± 7.93
Spermatid heads (107/testis)	147.13 ± 6.02	151.50 ± 7.63	133.25 ± 4.47	158.63 ± 11.27
Epididymal spermatozoal measurements
Sperm motility (%) 7 0.09 ± 0.91	72.34 ± 2.11	71.22 ± 1.33	70.86 ± 0.82	70.09 ± 0.91
Sperm (10^7/g cauda epididymis)	625.59 ± 62.68	576.71 ± 33.76	570.01 ± 32.04	595.47 ± 26.03
Sperm (10^7/cauda epididymis)	120.41 ± 4.68	106.90 ± 5.78	102.78 ± 5.08	106.03 ± 6.29

a Data are presented as mean ± standard error. Differences from the control group are not significant by Dunnett’s test (body and tissue

weights) or Dunn’s test (spermatid and epididymal spermatozoal measurements).

Estrous Cycle Characterization for Female Rats in the 3-Month Feed Study of Chromium Picolinate Monohydrate (a)

	0 ppm	2000 ppm	10000 ppm	50000 ppm
Number weighed at necropsy	10	10	10	10
Necropsy body wt (g)	189 ± 4	190 ± 4	188 ± 4	190 ± 3
Proportion of regular cycling females (b)	6/8	7/10	8/9	10/10
Estrous Cycle length (days)	4.94 ± 0.47 (c)	4.75 ± 0.21	4.44 ± 0.15 (d)	5.00 ± 0.31
Estrous stages (% of cycle)
Diestrus	51.7	38.3	38.3	44.2
Proestrus	6.7	12.5	14.2	7.5
Estrus	25.8	33.3	30.0	30.0
Metestrus	15.8	15.8	17.5	18.3

a 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.

b Number of females with a regular cycle/number of females cycling

c Estrous cycle was longer than 12 days or unclear in 2 of 10 animals.

d Estrous cycle was longer than 12 days or unclear in 1 of 10 animals.

Applicant's summary and conclusion

Conclusions:

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:

Based on the SMVCE results, the reproductive organ weights, and the histopathology of the reproductive organs, there was no evidence of toxicity to the reproductive system in these 3-month studies in rats and mice. Chromium picolinate is marketed as a dietary supplement primarily for weight loss in humans; however, no effect on body weight was observed in rats in the present studies.