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

Endpoint summary

Description of key information

oral, mice, 90 day, feeding, NOAEL = 1090 mg Cr/kg bw/d

oral, rat, 90 day, feeding, NOAEL = 506 mg Cr/kg bw/d

inhalation, rat, 90 day, LOAEC = 17 mg chromium hydroxide sulphate/m³ based on local effects

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

Reference 2

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

506 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-comparable study published in a peer-reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: British Chrome Chemicals (Urlay Nook, Eaglescliff, Cleveland, U.K.)
- Purity, including information on contaminants, isomers, etc.: 25 % Cr(III) as Cr2O3 and less than 0.0003 % Cr(VI) (non-detectable)

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

CDF (Fischer 344)/Crl BR VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 5 weeks, 7 weeks at strat of exposures
- Housing: Following three days of group housing, animals were individually housed in stainless steel, suspended wiremesh cages.
- Diet (e.g. ad libitum): ad libitum during the non-exposure periods
- Water (e.g. ad libitum): ad libitum during the non-exposure periods
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
Purina Certified Chow No. 5002, tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 °C
- Humidity (%): 43 +/- 11%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 4.2 - <= 4.5 µm

Geometric standard deviation (GSD):

2.45

Remarks on MMAD:

MMAD / GSD: Mean particle size distribution in microns (GSD) for three test groups over 13 weeks was 4.2 (2.48), 4.2 (2.37), and 4.5 (2.50) for the low-, mid-, and high-exposure groups, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and acrylic nose-only inhalation chambers
- Method of holding animals in test chamber: restraint tubes
- System of generating particulates/aerosols: Generation of particles was accomplished using an auger dust feeder and an air micronizer.
- Air change rate: 12 chamber air changes per hour

TEST ATMOSPHERE
- Brief description of analytical method used: standard gravimetric methods with periodic analysis for Cr(III) and Cr(VI)
- Samples taken from breathing zone: yes, once per hour

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber samples were determined once per hour by standard gravimetric methods with periodic analysis for Cr(III) and Cr(VI).

Duration of treatment / exposure:

Main study: 13 weeks plus an additional 13-week recovery period for some animals.
Bronchoalveolar lavage evaluation: 5 consecutive days

Frequency of treatment:

Main study: 6 hours per day, 5 days per week for 65 exposures

Dose / conc.:

17 mg/m³ air (nominal)

Dose / conc.:

54 mg/m³ air (nominal)

Dose / conc.:

168 mg/m³ air (nominal)

Dose / conc.:

3 mg/m³ air

Remarks:

chromium (III) equivalents

Dose / conc.:

10 mg/m³ air

Remarks:

chromium (III) equivalents

Dose / conc.:

30 mg/m³ air

Remarks:

chromium (III) equivalents

No. of animals per sex per dose:

15 animals/sex/dose in the main study
5 animals/sex/dose for the bronchoalveolar lavage evaluation

Control animals:

yes

Details on study design:

- Dose selection rationale: The desired exposure levels were selected to be multiples of the threshold limit value (TLV) for trivalent chromium and set at chromium equivalents of 3, 10, and 30 mg/m3.
- Rationale for animal assignment (if not random): Animals were randomly assigned to groups based on body weight.
- Fasting period before blood sampling for clinical biochemistry: fasted overnight
- Rationale for selecting satellite groups: 5 males and 5 females
- Post-exposure recovery period in satellite groups: 13-week recovery period without exposures

Positive control:

No

Observations and examinations performed and frequency:

Individual body weights were recorded weekly during the exposure and recovery periods. All animals received an indirect ophthalmoscopic examination during the acclimation period and prior to terminal necropsy. Standard haematology, clinical biochemistry, and urinalysis determinations were conducted on animals (10/sex/group) at the end of exposures.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (prior to and following each exposure)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (prior to and following each exposure)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimation and prior to necropsy
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of exposures
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes
- How many animals: 10/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of exposures
- Animals fasted: Yes
- How many animals: 10/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: end of exposures
- Metabolism cages used for collection of urine: Yes

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: after 5 exposures on 5 consecutive days
- Dose groups that were examined: all
- Number of animals: 5/sex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At necropsy the heart, lungs, liver, spleen, kidneys, brain, adrenal glands, thyroid/parathyroid glands, testes, and ovaries were weighed. Microscopic evaluation was conducted on all haematoxylin and eosin-stained tissues from the control and high-exposure level groups. The kidneys, liver, nasal tissue, trachea, lungs, larynx, mediastinal and mandibular lymph nodes, and gross lesions from all animals in the low- and mid-exposure level groups were also examined.

Other examinations:

Main study: Sperm motility, count and morphology of 10 males/group
Bronchoalveolar lavage evaluation: Nucleated cell counts and cell differential counts were performed on pooled BALF. Chemical analyses for lactate dehydrogenase, total protein, beta-glucuronidase and glutathione reductase were performed.

Statistics:

One-way analysis of variance on body weights, clinical pathology laboratory tests, BALF data and organ weights. If the result was significant, Bartlett's test for homogeneity of variance was performed. If Bartlett's test was non-significant, Dunnett's t-test was used for pairwise comparisons. If Barlett's test was significant, the Welch t-test with Bonferroni correction was used for pairwise comparisons. The Kruskal-Wallis analysis of variance, followed where appropriate by Mann-Whitney test was used for those parameters where parametric analysis was inappropriate. The level for statistical significance was set at p

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Sporadic laboured breathing in females of the high exposure group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Six animals died on exposure day 1 as a direct result of the restraint tubes. They were replaced. One male of the high exposure group died on day 4. This was not considered to be compound-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant exposure-related reduced mean body weights were observed in the males of the mid- and high exposure groups and the females of the high-exposure group. At the recovery sacrifice the males from the same exposure groups continued to exhibit mean body weights that were significantly lower than the control group, but body weight gains were similar between the exposure groups and the control group. The female mean body weights in all treatment groups were comparable to the control group females at the recovery sacrifice

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No observable decreases in food consumption were noted during the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increased leukocytes associated with increased number of neutrophils, some statistically significant, were noted in the mid- and high-exposure groups for males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phosphatase was statistically elevated in high-exposure group females and serum cholesterol was statistically decreased in mid- and high-exposure group females.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At terminal sacrifice, males and females in all treatment groups demonstrated compound-related, statistically significant increases in mean absolute and relative lung/trachea weights. Statistically significant changes in absolute and/or relative weights of some other organs are listed below (See Table 1); changes in the weights of some organs are secondary to bodyweight effects and, with the exception of the lungs, effects occurred in the absence of microscopic correlates.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gray lung discoloration was commonly observed in animals of the mid- and high-exposure levels. The degree of discoloration increased with exposure level and was present both at the terminal and recovery sacrifices. Similar discoloration was observed in the mediastinal lymph nodes of exposed animals (recovery sacrifice only) while mediastinal lymph node enlargement was seen at all exposure levels. Tan focus/foci were observed in the lungs at the recovery sacrifice of a high percentage of males exposed to the high level.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Chronic inflammation was observed involving the alveoli of all exposure-level groups consisting of alveolar spaces filled with macrophages, neutrophils, lymphocytes, and cellular debris. Multifocal areas of granulomatous inflammation, characterized by infiltration of macrophages and multinucleated giant cells, was observed at all exposure levels and was closely associated with foreign material seen in the lung and presumed to the test article. These changes corresponded to the increased lung weights observed in all exposure groups. Green refractile foreign material was present in the larynx of animals in all treatment groups. Histiocytosis and lymphoid hyperplasia correlated with lymph node enlargement observed at necropsy. Changes in nasal tissues considered to be test article-related, were observed in males and females, and included acute inflammation, suppurative exudate, and mucoid exudate.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No apparent compound-related effects were noted for sperm motility or morphology for any concentration.

At recovery sacrifice, foreign material persisted in the lungs of some animals in all exposure groups, but with decreased incidence in most groups. Granulomatous inflammation of the lung decreased in incidence except in the males and females of the high-exposure group where the incidence was approximately equal to that of the terminal sacrifice animals of this group.

Bronchoalveolar lavage evaluation
Males and females at all exposure levels showed statistically reduced total nucleated cell counts. Segmented neutrophils increased while mononuclear cells decreased, although not to a statistically significant degree, at all concentration levels. Non-statistical increases in protein and lactic dehydrogenase were also observed. Increased amounts of cell debris and lysed cells were noted at all exposure levels.

Details on results:

Main study

Six animals died on exposure day 1 as a direct result of the restraint tubes and they were replaced. One male from the basic chromium sulphate high-exposure (168 mg/m3) group died on exposure day 4 and was not replaced. Although the specific cause of death was not identified, this death was not considered related to exposure to basic chromium sulphate, since there were no significant signs of toxicity observed in any other animals from this group.

Clinical signs of toxicity were limited to sporadic labored breathing, noted during two of the weekly observations, in females exposed to the high concentration of basic chromium sulphate.

Statistically significant, exposure-related reduced mean body weights were observed in the males of the mid- and high-exposure groups and the females of the high-exposure group during the 13-week exposure period. At the recovery sacrifice, males from the same exposure groups continued to exhibit mean body weights that were significantly lower than the control group but body weight gains between treated and control groups were similar.

Most haematology, serum biochemistry, and urinalysis values were similar to the control group. Increased leukocytes associated with increased number of neutrophils, some statistically significant, were noted in mid-and high-exposure groups for males and females. Alkaline phosphatase was statistically elevated in high-exposure group females and serum cholesterol was statistically decreased in mid- and high- exposure group females.

At terminal sacrifice, males and females in all treatment groups demonstrated compound-related, statistically significant increases in mean absolute and relative lung/trachea weights. Statistically significant changes in absolute and/or relative weights of some other organs are listed below (See Table 1); changes in the weights of some organs are secondary to bodyweight effects and, with the exception of the lungs, effects occurred in the absence of microscopic correlates.

Gray lung discoloration was commonly observed in animals exposed to basic chromium sulphate at the mid- and high-exposure levels. The degree of discoloration increased with exposure level and was present both at the terminal and recovery sacrifices. Similar discoloration was observed in the mediastinal lymph nodes of animals exposed to basic chromium sulphate (recovery sacrifice only) while mediastinal lymph node enlargement was seen at all exposure levels. Tan focus/foci were observed in the lungs at the recovery sacrifice of a high percentage of males exposed to the high level of basic chromium sulphate.

Chronic inflammation was observed involving the alveoli of all exposure-level groups consisting of alveolar spaces filled with macrophages, neutrophils, lymphocytes, and cellular debris. Multifocal areas of granulomatous inflammation, characterized by infiltration of macrophages and multinucleated giant cells, was observed at all exposure levels and was closely associated with foreign material seen in the lung and presumed to the test article. These changes corresponded to the increased lung weights observed in all exposure groups. Green refractile foreign material was present in the larynx of animals in all treatment groups. Histiocytosis and lymphoid hyperplasia correlated with lymph node enlargement observed at necropsy. Changes in nasal tissues considered to be test article-related, were observed in males and females, and included acute inflammation, suppurative exudate, and mucoid exudate.

No apparent compound-related effects were noted for sperm motility or morphology for any concentration of basic chromium sulphate.

At recovery sacrifice, foreign material persisted in the lungs of some animals in all exposure groups, but with decreased incidence in most groups. Granulomatous inflammation of the lung decreased in incidence except in the males and females of the high-exposure group where the incidence was approximately equal to that of the terminal sacrifice animals of this group.

Bronchoalveolar lavage evaluation

Males and females at all exposure levels showed statistically reduced total nucleated cell counts. Segmented neutrophils increased while mononuclear cells decreased, although not to a statistically significant degree, at all concentration levels. Non-statistical increases in protein and lactic dehydrogenase were also observed. Increased amounts of cell debris and lysed cells were noted at all exposure levels.

Dose descriptor:

LOAEC

Remarks:

[chromium hydroxide sulphate]

Effect level:

17 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: pathological findings in the respiratory tract

Remarks on result:

other: A NOAEC was not established in this study.

Dose descriptor:

LOAEC

Remarks:

[Cr3+ equivalents]

Effect level:

3 mg/m³ air

Based on:

element

Sex:

male/female

Basis for effect level:

other: pathological findings in the respiratory tract

Remarks on result:

other: A NOAEC was not established in this study.

Critical effects observed:

not specified

Table 1: Selected organ weight changes at terminal and recovery sacrifices of rats exposed to chromium hydroxide sulphate

		Control		17 mg/m^3	54 mg/m^3	168 mg/m^3
Males Lung/trachea
wt (g)		0.99 + 0.70(1.32 + 0.113)		1.26 + 0.071**(1.52 + 0.132)	1.51 + 0.088**(1.95 + 0.068**)	1.86 + 0.89**(2.67 + 0.144**)
wt/bw (% x 10)		4.42 + 0.187(3.89 + 0.214)		5.60 + 0.271**(4.66 + 0.373**)	7.15 + 0.252**(6.37 + 0.298**)	10.69 + 0.688**(8.77 + 0.274**)
Brain
wt (g)		1.79 + 0.087		1.82 + 0.055	1.76 + 0.061	1.71 + 0.069*
wt/bw (% x 10)		8.02 + 0.380		8.12 + 0.374	8.38 + 0.473	9.83 + 0.518**
Kidney
wt (g)		1.54 + 0.106		1.35 + 0.049**	1.62 + 0.085	1.64 + 0.082
wt/bw (% x 10)		7.62 + 0.300		7.28 + 0.207	7.30 + 0.283	7.78 + 0.350**
Liver
wt (g)		5.48 + 0.367		5.63 + 0.271	5.17 + 0.459	4.39 + 0.146**
wt/bw (% x 10)		2.45 + 0.070		2.50 + 0.050	2.45 + 0.091	2.53 + 0.120
Thyroid/parathyroid
wt (mg)		14 + 2.5		15 + 2.9	14 + 1.8	15 + 3.5
wt/bw (% x 10)		6.21 + 1.052		6.64 + 1.475	6.74 + 1.021	8.76 + 2.074*
Spleen
wt (g)		0.45 + 0.038		0.48 + 0.036	0.40 + 0.040*	0.32 + 0.035**
wt/bw (% x 10)		1.99 + 0.149		1.91 + 0.132	1.89 + 0.125	1.84 + 0.151
Testes
wt (g)		2.36 + 0.356		2.39 + 0.261	2.22 + 0.286	2.18 + 0.215
wt/bw (% x 10)		10.54 + 1.315		10.65 + 1.098	10.52 + 1.049	12.53 + 1.238**
Females Lung/trachea
wt (g)		0.81 + 0.081(0.93 + 0.079)		0.98 + 0.094**(1.08 + 0.120)	1.29 + 0.164**(1.59 + 0.120**)	1.66 + 0.084**(2.45 + 0.120**)
wt/bw (% x 10)		5.65 + 0.418(4.74 + 0.384)		6.99 + 0.619**(5.75 + 0.315*)	9.24 + 1.036**(8.02 + 0.750**)	12.89 + 1.134**(13.34 + 0.614**)
Thyroid/parathyroid
wt (mg)		12 + 1.9		11 + 1.3	12 + 1.8	14 + 2.1*
wt/bw (% x 10)		8.26 + 1.493		7.96 + 1.154	8.63 + 1.265	10.77 + 1.522**
Spleen
wt (g)		0.33 + 0.037		0.31 + 0.033	0.30 + 0.033	0.28 + 0.033**
wt/bw (% x 10)		2.32 + 0.268		2.19 + 0.212	2.17 + 0.162	2.19 + 0.273
Note: Organ weight changes, values given as mean + SD: bw = body weight. Non-bracketed values = terminal sacrifice, bracketed values = recovery sacrifice. * p < 0.05; ** p < 0.01

Conclusions:

No systemic toxicity was observed in rats following a 13-week nose-only inhalation study of chromium hydroxide sulphate. Pathological findings were observed in the respiratory tract and are associated with the deposition of inhaled particulate material.

Executive summary:

Chromium hydroxide sulphate was tested in a guideline-compliant 3 -month subchronic inhalation study in rats at doses of 17 mg/m³, 54 mg/m³ and 168 mg/m³ The principle effects observed were noted in the respiratory tract. Exposed rats developed changes in the mediastinal lymphatic tissue, lung, larynx and nasal cavity. Foreign material was absent or decreased after the recovery period in the lungs and was no longer found in the larynx of low- and mid-exposure level rats. This suggests that basic chromium sulphate is most likely rapidly cleared from the respiratory tract due to its high water solubility. Evidence of systemic toxicity was primarily limited to reductions in body weight not related to reduced food consumption. A NOAEC was not established in this study based on the pathological findings in the respiratory tract. The LOAEC for this study was 17 mg/m³ chromium hydroxide sulphate, equivalent to 3 mg/m³ Cr³⁺.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

17 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

oral mice (NTP 2010)

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.

oral rat (NTP 2010)

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.

inhalation rat (Derelanko et al. 1999)

Chromium hydroxide sulphate was tested in a guideline-compliant 3 -month subchronic inhalation study in rats at doses of 17 mg/m³, 54 mg/m³ and 168 mg/m³ The principle effects observed were noted in the respiratory tract. Exposed rats developed changes in the mediastinal lymphatic tissue, lung, larynx and nasal cavity. Foreign material was absent or decreased after the recovery period in the lungs and was no longer found in the larynx of low- and mid-exposure level rats. This suggests that basic chromium sulphate is most likely rapidly cleared from the respiratory tract due to its high water solubility. Evidence of systemic toxicity was primarily limited to reductions in body weight not related to reduced food consumption. A NOAEC was not established in this study based on the pathological findings in the respiratory tract. The LOAEC for this study was 17 mg/m³ chromium hydroxide sulphate, equivalent to 3 mg/m³ Cr³⁺.

Justification for classification or non-classification

Based on the available data no classification is proposed according to Regulation (EC) No 1272/2008.