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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline comparable, published study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline comparable, published study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Method broadly comparable to OECD 414, performed in the mouse using dietary administration

GLP compliance:

no

Remarks:

: published study

Limit test:

no

Specific details on test material used for the study:

The study was performed using the water soluble complexes of Cr (III) - [Cr3O(O2CCH2CH3)6(H2O)3]+ (Cr3)

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, International (Wilmington, MA)
- Housing: AAALAC-approved animal facility in rooms. Mated females were individually housed in shoe-box-type cages with hardwood bedding
- Diet (e.g. ad libitum): Harlan-Teklad LM-485 rodent ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 40–60%
- Photoperiod (hrs dark / hrs light): 12h/ 12h

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
LM-485 milled rodent diet was purchased from Harlan Teklad (Madison, WI). Either Cr(pic)3 or Cr3 was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day. Extensive stability studies indicate that chromium test compounds are extremely stable and that no degradation in the diet would be expected. Because the purpose of this study was to determine the effects of pharmaceutical levels of chromium, no special measures were taken to prevent exposure of the mice to small amounts of chromium that may be introduced into the diet through methods of feed preparation or from the cage hardware. The diet purchased, Teklad LM-485 (7012), contained added chromium in the form of chromium potassium sulphate (0.48 mg/kg of diet).

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Cr(pic) was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Females were exposed from Gestation Day 6-17

Frequency of treatment:

Continuous (dietary)

Duration of test:

Maternal animals were sacrificed on Gestation Day 17.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control, untreated rodent diet

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

providing 3.3 mg Cr/kg/day

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

providing 26 mg Cr/kg/day

No. of animals per sex per dose:

Not stated, however the numbers of litters in each group range from 24-27.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dosages of Cr3 were thus chosen to either deliver an equivalent dosage of chromium based on bioavailability (15 mg/kg/day) or to deliver approximately the same mass of chromium (120 mg/kg/day) as the 200 mg/kg/day dose of Cr(pic)3.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on GD 17

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes

Blood sampling:

- Plasma: No data
- Serum: No data

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes, for cervical arch defects
- Skeletal examinations: Yes
- Head examinations: No data

Statistics:

The data from each study replicate were calculated independently, tested for homogeneity of variance by the Levene statistic using SPSS (SPSS Inc., Chicago, IL), and then the replicates were pooled and analyzed together to give the reported results. All tabular data are presented as the mean+-SEM, and the mean value for each parameter was calculated as the mean of the litter means. Data were analyzed by one-way analysis of variance (ANOVA), followed by an LSD post-hoc test to determine specific significant differences (P <= 0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of maternal toxicity were observed for dams in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Maternal weight gain was not affected by the administration of Cr3.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was virtually identical among the treatment groups, and average food consumption was approximately 7 g diet/day.

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

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:

not specified

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No difference was found in the number of implantations per litter in treated groups as compared to controls.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

percentage of resorbed fetuses did not differ among treatment groups.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

percentage of dead fetuses did not differ among treatment groups.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 26 mg Cr(III)/kg bw/d

Dose descriptor:

NOAEL

Effect level:

26 mg/kg bw/day (nominal)

Based on:

element

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 26 mg Cr(III)/kg bw/d

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal weight and percentage of resorbed or dead fetuses did not differ among treatment groups

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No gross malformations were observed in any of the fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No increased incidence of skeletal defects was observed in exposed fetuses compared to the controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant increase in the incidence of cervical arch defects. Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Maternal exposure to Cr3 at the dosages employed did not appear to cause deleterious effects to the developing offspring in mice.

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Dose descriptor:

NOAEL

Effect level:

26 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Abnormalities:

no effects observed

Developmental effects observed:

no

No signs of maternal toxicity were observed.

Mean foetal weights, foetal viability and the proportion of resorptions were unaffected by treatment. No gross malformations were observed in any of the foetuses. The number of implantations in the low dose Cr₃ group was lower than the other treated groups, however this is not considered to be an effect of treatment in the absence of a dose-response relationship and because implantation occurred prior to exposure. No effects of treatment were observed on the incidence of skeletal anomalies.

Summary of findings

Parameter		Dose group
		0		Cr3 (low)	Cr3(high)
Litters	(#)	27		26	24
Foetuses	(#)	332		275	342
Litter size	(#)	12.30		10.58	14.25
Foetal weight	(g ± SEM)^b	1.02 ± 0.02		1.08 ± 0.03	1.02 ± 0.03
Implantations	(# ± SEM)^b	12.64 ± 0.50		11.00  ± 0.92^a	13.79 ± 0.55
Dead/resorbed foetuses	(# ± SEM)^b	2.74 ± 1.07		3.48 ± 0.95	1.29 ± 0.64
Cervical arch defects^c	(% ± SEM)^b	4.65 ± 1.14		5.18 ± 1.58	3.98 ± 1.33
Maternal weight gain	(g ± SEM)^b	12.39  ± 0.37		11.43 ± 0.48	13.01 ± 0.42

a Significantly different from Cr(pic)3 (25mg Cr/kg/day), and Cr3 (26mg Cr/kg/day) values.

b The mean value for each parameter was calculated as the mean of the litter means.

c Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Conclusions:

No evidence of foetotoxicity, developmental toxicity or teratogenicity was seen in mice exposed to water-soluble complexes of Cr(III) delivering estimated daily doses of Cr(III) equivalent to 3.3 mg/kg bw/d (low dose Cr3 group) and 26 mg/kg bw/d (high dose Cr3 group).

Executive summary:

Mated female mice were administered Cr(III) in the diet from Days 6 -17 of gestation. Dams were sacrificed on Day 17 and the uterine contents investigated. Foetuses were assessed for external defects and skeletal findings following double staining. No maternal toxicity was observed. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen.

Data source

Reference

Reference Type:

publication

Title:

Comparison of the Potential for Developmental Toxicity of Prenatal Exposure to Two Dietary Chromium Supplements, Chromium Picolinate and [Cr3O(O2CCH2CH3)6(H2O)3]+, in Mice

Author:

Bailey MM, Sturdivant J, Jernigan PL, Townsend MB, Bushman J, Ankareddi I, Rasco JF, Hood RD & Vincent JB

Year:

2008

Bibliographic source:

Birth Defects Research (Part B) 83:27–31

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Method broadly comparable to OECD 414, performed in the mouse using dietary administration

GLP compliance:

no

Remarks:

: published study

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tris(picolinate)chromium(III)

Cas Number:

14639-25-9

Molecular formula:

Cr(C6H4NO2)3

IUPAC Name:

Tris(picolinate)chromium(III)

Specific details on test material used for the study:

The study was performed using the water soluble complexes of Cr (III) chromium picolinate
Chromium(III) picolinate, was synthesised according to the methods of Press et al (1990). Cr3 was synthesised according to the methods of Earnshaw et al (1966). The authenticity of both was established by high resolution electron impact mass spectrometry. Picolinic acid was purchased from Fisher Scientific (Pittsburgh, PA).

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, International (Wilmington, MA)
- Housing: AAALAC-approved animal facility in rooms. Mated females were individually housed in shoe-box-type cages with hardwood bedding
- Diet (e.g. ad libitum): Harlan-Teklad LM-485 rodent ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 40–60%
- Photoperiod (hrs dark / hrs light): 12h/ 12h

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
LM-485 milled rodent diet was purchased from Harlan Teklad (Madison, WI). Either Cr(pic)3 or Cr3 was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day. Extensive stability studies indicate that chromium test compounds are extremely stable and that no degradation in the diet would be expected. Because the purpose of this study was to determine the effects of pharmaceutical levels of chromium, no special measures were taken to prevent exposure of the mice to small amounts of chromium that may be introduced into the diet through methods of feed preparation or from the cage hardware. The diet purchased, Teklad LM-485 (7012), contained added chromium in the form of chromium potassium sulphate (0.48 mg/kg of diet).

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Cr(pic) was added to milled rodent chow in sufficient quantities to achieve the appropriate concentration of the test compound. All calculations were based on data from previous studies, which indicated that pregnant CD-1 mice consume an average of 7 g diet/day.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Females were exposed from Gestation Day 6-17

Frequency of treatment:

Continuous (dietary)

Duration of test:

Maternal animals were sacrificed on Gestation Day 17.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control, untreated rodent diet

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

providing 25 mg Cr/kg/day

No. of animals per sex per dose:

Not stated, however the numbers of litters are 27 in the control and 29 in the Cr(pic)3 group.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dosage of chromium picolinate was based on results from a previous study, in which 25 mg Cr/kg/day as Cr(pic)3 was associated with a significant increase in the incidence of cervical arch defects compared to control animals (Bailey et al., 2006).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on GD 17

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes

Blood sampling:

- Plasma: No data
- Serum: No data

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes, for cervical arch defects
- Skeletal examinations: Yes
- Head examinations: No data

Statistics:

The data from each study replicate were calculated independently, tested for homogeneity of variance by the Levene statistic using SPSS (SPSS Inc., Chicago, IL), and then the replicates were pooled and analyzed together to give the reported results. All tabular data are presented as the mean+-SEM, and the mean value for each parameter was calculated as the mean of the litter means. Data were analyzed by one-way analysis of variance (ANOVA), followed by an LSD post-hoc test to determine specific significant differences (P <= 0.05).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of maternal toxicity were observed for dams in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Maternal weight gain was not affected by the administration of Cr(pic)3.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was virtually identical among the treatment groups, and average food consumption was approximately 7g diet/day.

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

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:

not specified

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No difference was found in the number of implantations per litter in treated groups as compared to controls.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

percentage of resorbed fetuses did not differ among treatment groups.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

percentage of dead fetuses did not differ among treatment groups.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not examined

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

element

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 25 mg Cr(III)/kg bw/d.

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No maternal toxicity was observed in either group given equivalent doses of 25 mg Cr(III)/kg bw/d.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal weight and percentage of resorbed or dead fetuses did not differ among treatment groups.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No gross malformations were observed in any of the fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No increased incidence of skeletal defects was observed in exposed fetuses compared to the controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant increase in the incidence of cervical arch defects. Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Maternal exposure to Cr(pic)3 at the dosages employed did not appear to cause deleterious effects to the developing offspring in mice.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

2

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: no effects observed

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

No signs of maternal toxicity were observed.

Mean foetal weights, foetal viability and the proportion of resorptions were unaffected by treatment. No gross malformations were observed in any of the foetuses. No effects of treatment were observed on the incidence of skeletal anomalies. The authors note that a previous study (Bailey et al, 2006) reported an increased incidence of cervical arch defects in the offspring of mice exposed to chromium picolinate, however the incidence of defects in that study (6.26%) is very similar to the control incidence in this study (5.79% and is therefore not considered to be related to treatment.

Summary of findings

Parameter		Dose group
		0		Cr(pic)3
Litters	(#)	27		29
Foetuses	(#)	332		369
Litter size	(#)	12.30		12.72
Foetal weight	(g± SEM)^b	1.02 ± 0.02		1.05 ± 0.02
Implantations	(#± SEM)^b	12.64 ± 0.50		13.18 ± 0.35
Dead/resorbed foetuses	(#± SEM)^b	2.74 ± 1.07		3.29 ± 0.86
Cervical arch defects^c	(%± SEM)^b	4.65 ± 1.14		6.26 ± 1.63
Maternal weight gain	(g± SEM)^b	12.39  ± 0.37		11.96  ± 0.36

a Significantly different from Cr(pic)3 (25mg Cr/kg/day), and Cr3 (26mg Cr/kg/day) values.

b The mean value for each parameter was calculated as the mean of the litter means.

c Cervical arch defects refer to a distal split in the first or second cervical vertebral arch.

Applicant's summary and conclusion

Conclusions:

No evidence of foetotoxicity, developmental toxicity or teratogenicity was seen in this pre-natal developmental toxicity study in mice exposed to water-soluble complexes of Cr(III) delivering estimated daily doses of Cr(III) equivalent to 25 mg/kg bw/d.

Executive summary:

Mated female mice were administered Cr(III) in the diet from Days 6 -17 of gestation. The dose was 200 mg/kg bw/d (25 mg Cr/kg bw/d). Dams were sacrificed on Day 17 and the uterine contents investigated. Foetuses were assessed for external defects and skeletal findings following double staining. No maternal toxicity was observed. No evidence of teratogenicity, foetotoxicity or developmental toxicity was seen.