Dichromium tris(chromate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

12 November 2001 to 12 February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted for the National Toxicology Program

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

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:

This study was conducted to aid in the design and dose selection for a 2 year carcinogenicity study.

GLP compliance:

yes

Remarks:

FDA

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium dichromate dihydrate

IUPAC Name:

Sodium dichromate dihydrate

Constituent 2

Reference substance name:

Chromic acid (H2Cr2O7), disodium salt, dihydrate

EC Number:

616-541-6

Cas Number:

7789-12-0

Molecular formula:

Cr2H2O7.2H2O.2Na

IUPAC Name:

Chromic acid (H2Cr2O7), disodium salt, dihydrate

Details on test material:

Sodium dichromate dihydrate was obtained from Aldrich Chemical Co (Milwaukee) in two lots (15301BI and 13822LI). The two lots were combined at the analytical chemistry laboratory (Battelle Memorial Institute, Columbus) and assigned a new lot no.: 062001. Identity, purity and stability analyses were conducted by the analytical chemistry laboratory and the Southern Research Institute.
The substance was described as an orange crystalline solid, the identity was confirmed by X-ray diffraction, ICP-AES, and PIXE. The overall purity was experimentally determined to be greater than 99.7%.
The chemical was stored at room temperature, protected from light in amber glass bottles. During the study, the stability of the bulk chemical was monitored by the study laboratory using titration; no degredation of the chemical was detected.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were male and female F344/N rats, obtained from Taconic Farms (NY) aged 4 to 5 weeks old. The rats were quarantined for 11 to 14 days, and were 5 to 7 weeks old at study initiation. Five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected from five male and five female control animals at study termination. The sera were analysed for antibody titers to rodent viruses (all results were negative).
The rats were housed in groups of 5 in solid bottom suspended polycarbonate cages fitted with polyester filters. Individuals were identified by tail tattoo. Irradiated NTP-2000 wafer rodent feed (Zeigler Brothers, Inc.) was available ad libitum. Tap water was provided ad libitum from the municipal supply via amber glass water bottles with Teflon lined caps and stainless steel sipper tubes.
The temperature of the animal room was 22.2±1.6°C, relative humidity was 50±15%, fluorescent lighting was provided on a 12 hour light/dark cycle, and there were approximately 18 air changes/hour.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dosing formulations were prepared four times during the study. Formulations were stored in Nalgene containers at room temperature, protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analysed three times during the study using UV-spectroscopy. All formulations were found to be within 10% of the target concentration. Animal room samples and unused carboy storage samples were also analysed; 14/15 animal room samples were within 10% of the target, and all unused carboy samples were within 10% of the target concentration.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuous - ad libitum in drinking water

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 62.5, 125, 250, 500 or 1000 mg/L
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 5, 10, 17, 32, 60 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 1.7, 3.5, 5.9, 11.2 mg/kg bw/d
Basis:
other: Cr (VI) equivalents

No. of animals per sex per dose:

10 rats/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

Animals were randomly distributed into treatment groups of approximately equal initial mean body weights. An additional 10 rats/sex/group were exposed for 4 weeks, for clinical pathology studies.

Positive control:

Not required for this study

Examinations

Observations and examinations performed and frequency:

Animals were observed twice daily. Clinical findings were recorded weekly.
All animals were weighed initially, core study animals were weighed weekly and at study termination.
Water consumption was recorded weekly (core study animals only).
Animals designated for clinical pathology were placed in metabolism cages on day 16 for collection of 24 urine.

Blood was collected from the retroorbital sinus of animals designated for clinical pathology studies, on days 5 and 23. Blood was collected from core study rats at study termination.
Haematology parameters determined were: automated and manual haematocrit, haemoglobin concentration, erythrocyte count, reticulocyte count, nucleated erythrocyte count, platelet count and estimates, erythrocyte and platelet morphology, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, leukocyte count and differentials.
Clinical chemistry parameters determinedwere: urea nitrogen, creatinine, glucose, sodium, potassium, chloride, calcium, phosphorous, total protein, albumin, cholesterol, triglycerides, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, 5'-nucelotidase, total bile acids.
Urinalysis parameters determined were: creatinine, glucose, protein, alkaline phosphatase, aspartate aminotransferase, N-acetyl-glucosaminidase, volume, specific gravity, pH.

Sacrifice and pathology:

Necropsies were performed on all core animals. Organ weights were recorded for the heart, right kidney, liver, lung, sppleen, right testis and thymus.

Complete histopathology was performed on 0 and 1000 mg/L core rats, and on 6 of 10 randomly selected rats in the other exposed groups. The following organs were examined: adrenal gland, bone, brain, clitoral gland, oesophagus, eye, harderian gland, heart and aorta, large intestine, small intestine, kidney, liver, lung and mainstem bronchi, lymph nodes, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skinn, spleen, stomach, testis with epididymis and vaginal tunics, thymus, thyroid gland, trachea, urinary bladder, uterus and gross lesions and tissues masses.

Other examinations:

No other examinations reported.

Statistics:

Lesion incidence was analysed using the Fisher exact test. Continuous variables were analysed using Dunnet's and Williams' tests, Shirley's test, Jonckheere's test, Dunn's test, Mann-Whitney U test or multivariate ANOVA as appropriate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

small reductions at 1000 mg/L

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

reduced intake at 250, 500 and 1000 mg/L

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

exposure-related anaemia

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increase alanine aminotransferase and sorbito dehydrogenase activity in exposed rats

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

transient changes in calcium and phosphorous

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

reduced liver and spleen weights in 500 and 1000 mg/L males; cinreased spleen and kidney weights in 500 and 1000 mg/L females

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

lesions observed in the glandular stomach at 1000 mg/L

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

lesions detected in the glandular stomach, duodenum and pancreatic lymph nodes of males and females, and in liver and bone marrow of females.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no effect of administration on survival, and no clinical signs of toxicity were reported.

BODY WEIGHT AND WEIGHT GAIN
The final mean body weights of male rats in the 1000 mg/L groups were 89% lower than controls, and final mean body weights in female rats at 1000 mg/L was 94% lower than controls. The final mean body weights and weight gain of the 500 mg/L males were less than controls.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumption in the 250, 500 and 1000 mg/L groups was less than the controls.

HAEMATOLOGY
An exposure-related microcytic, hypochromic, responsive anaemia was observed in exposed rats. The microcytosis (evidenced by decreased mean cell volumes) occurred from day 5 to the end of the study in all exposed groups. In 1000 mg/L males, the severity of the microcytosis increased with time. At week 14 erythrocytes in 1000 mg/L rats were approximately 30% smaller in males and 25% smaller in females. At the lower doses, microcytosis generally ameliorated with time. Anaemia (evidenced by decreases in automated and manual haematocrit values, haemoglobin cocentrations and erythrocyte counts) developed in all exposed group by day 23 until week 14, it was most severe at day 23, ameliorating with time. At week 14, erythrocyte couts were increased and contradictory to the lower haematocrit values and haemoglobin concentrations. The increased reticulocyte and nucleated erythrocyte numbers were thought to be indicative of an erythropoietic response. Increased numbers of reticulocytes and nucleated erythrocytes were considered indicative of an erythropoietic response. Whilte there was an apparent erythropoietic response resulting in increased erythrocyte numbers, the erythrocytes produced were smaller, giving rise to a decreased erythron in the 250 mg/L and above groups. Microscopic evaluation of blood smears demonstrated increased erythrocyte fragments, keratocytes, and blebbing that suggested increased erythrocye injury or turnover. Increased numbers of hypochromic microcytes were observed, indicating that blood loss or altered iron metabolism or haemoglobin production was observed. As there was no evidence for blood loss in the animals, the authors concluded that alterations in iron metabolism or haemoglobin production were responsible. In this study, the instrument derived haematocrit values were noticeably lower than the manual haematocrit values in the 125 mg/L and above groups at day 23 and in the 1000 mg/L males at week 14. Microscopic evaluation of the blood smears demonstrated increased numbers of microcytic erythrocyte fragments, keratocytes and hypochromic microcytes. The authors concluded that the decreased instrument-derived haematocrit values could possibly be explained by the instrument's innability to recognise the small erythrocytes or erythrocyte fragments, or determine mean cell volume. Recalculated mean MCHCs using the mean manual haematocrit values were decreased compared to control MCHC values and suggested a hypochromia.
An increased platelet count was observed on day 23 in exposed males, but this increase was not present on week 14. It was believed that the increased platelet count could be indicative of a general increase in haematopoiesis or possibly an iron deficiency-like process.
Increased neutrophil and monocyte counts were primarily observed at higher dose levels, and were considered to be a result of the inflammatory response to lesions observed histologically. Leukocyte and lymphocyte counts were increased. The increase in lymphocyte counts because consistent between the sexes at week 14.

CLINICAL CHEMISTRY
By week 14, alanine aminotransferase activities were increased in all exposed groups by approx. 2 to 8 fold in males and 3 to 7 fold in females. Sorbitol dehydrogenase activities were increased in all exposed groups by approximately 2 to 6 fold in males and 3 to 5 fold in females. There was no dose-related effect. Increased bile acid concentrations occurred on day 23, by week 14 bile acid concentrations were increased in the 500 and 1000 mg/L males and in most female groups (the increases did not occur in an exposure-related fashion). As there was no increase in serum enzyme markers of cholestasis (alkaline phosphatase and 5'-nucleotidase) it was concluded that the bile acid concentrations increased were related to a hepatocellular effect. Lipid metabolism apppeared to be altered; there were decreases in cholesterol and triglyceride concentrations that affected males more than females. Cholesterol decreases of approximately 8% occurred in all exposed males on day 5 and by week 14 the concentrations were decreased in 250, 500 and 1000 mg/L males and 500 and 1000 mg/L females (no dose-relationship was apparent however). Decreased triglyceride concentrations occurred on day 23 in males, by week 14 concentrations were decreased in 1000 mg/L males and 250, 500 and 1000 mg/L females (the decreases showed an dose-relationship in females). Increased creatine kinase activities occurred on day in 500 and 1000 mg/L males and in 250, 500 and 1000 mg/L females. By week 14 creatine kinase activities were increased in 250, 500 and 1000 mg/L rats: the increases in 1000 mg/L males and females were 75% and 120% respectively.

URINALYSIS
Decreased volume and increased specific gravity were in accordance with the decrease in water intake, the authors thought this was indicative of low palatability. There was a small decrease in urea nitrogen also indicative of decreased water intake and slight dehydration. Slight transient changes in calcium and phosphorous concentrations were observed but the mechanism of the change was unknown.

ORGAN WEIGHTS
Absolute and relative liver weights of 500 and 1000 mg/L males were significantly lower than controls. Absolute spleen weights of 500 and 1000 mg/L males and relative spleen weights of 250 and 500 mg/L males were significantly lower than controls. Relative spleen and kidney weights of 250 and 500 mg/L males were significantly lower than controls. Relative spleen and kidney weights of 500 and 1000 mg/L females were significantly increased.

GROSS PATHOLOGY
Gross lesions were observed in the glandular stomach, and described as deformity, pale foci, pale nodules, or thick, pale mucosa. These lesions were observed in the 1000 mg/L rats. The lesions occurred immediately adjacent to the limiting ridge, the anatomic demarcation between the rodent forestomach and glandular stomach.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was an increased incidence of neoplastic lesions in the glandular stomach, duodenum and pancreatic lymph nodes of exposed males and females, and in the liver and bone marrow of exposed females. The severities of the lesions in the duodenum, glandular stomach and pancreatic lymph nodes were generally greater at the highest exposure concentration. Microscopically, the incidences of glandular stomach lesions (including ulcers, regenerative epithelial hyperlasia, and squamous epithelial metaplasia) were significantly increased in the 1000 mg/L rats. The microscopic lesions were similar in all affected rats and were all found immediately adjacent to the limiting ridge. The incidences of minimal to mild histiocytic cell infiltration in the pancreatic lymph nodes were increased in all exposed males and females; the increase was statistically significant in 1000 mg/L females, and in all exposed males except the 125 mg/L group. The incidences of lymphoid hyperplasia and sinusoidal ectasia were significantly increased in 1000 mg/L rats. The incidences of minimal to mild histiocytic infiltration in the duodenum were significantly increased at 125 mg/L and above. The incidences of minimal histiocytic cellular inflammation in the liver were significantly increased in 125 mg/L or greater females; focal chronic inflammation was significantly increased at 1000 mg/L. In the bone marrow, the incidence of minimal hyperplasia was significantly increased in 1000 mg/L females.

Effect levels

Dose descriptor:

NOAEL

Sex:

male/female

Basis for effect level:

other: Haematology parameters were affected in all exposed groups (lowest tested dose: 3.1 mg Cr(VI)/kg bw/d).

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Exposure concentrations of 62.5, 125, 250, 500 and 1000 mg/L resulted in average daily doses of approximately 5, 9, 17, 32 and 60 mg/kg bw for males, and 5, 10, 18, 33 and 61 mg/kg bw in females. These doses were equivalent to approximately 1.7, 3.5, 5.9, 11.2 and 20.9 mg hexavalent chromium/kg body weight per day.

Incidences of selected nonneoplastic lesions.

	0 mg/L	62.5 mg/L	125 mg/L	250 mg/L	500 mg/L	1000 mg/L
Male
Duodenum^a	10	10	10	10	10	10
Histiocytic Cell Infiltration^b	0	0	7**(1.1)^c	9**(1.2)	8**(1.4)	7**(1.4)
Pancreatic Lymph Node	10	10	10	10	10	10
Ectasia	0	0	0	0	1(1.0)	10**(1.7)
Lymphoid Hyperplasia	0	0	0	3(1.0)	3(1.0)	6**(2.7)
Histiocytic Cell Infiltration	0	5*(1.0)	2(1.0)	4*(1.0)	5*(1.0)	9**(1.8)
Glandular Stomach	10	10	10	10	10	10
Ulcer	0	0	0	0	1(2.0)	8**(3.0)
Epithelial Regenerative Focal Hyperplasia	0	0	0	0	0	10**(2.2)
Epithelial Squamous Focal Metaplasia	0	0	0	0	0	7**(2.6)
Female
Duodenum	10	10	10	10	10	10
Histiocytic Cell Infiltration	0	1(1.0)	5*(1.0)	7**(1.4)	8**(1.6)	10**(1.7)
Liver	10	10	10	10	10	10
Histiocytic Cell Infiltration	0	3(1.3)	6**(1.0)	6**(1.0)	9**(1.2)	8**(1.0)
Focal Chronic Inflammation	3(1.0)	5(1.0)	2(1.0)	7(1.0)	2(1.0)	10**(1.0)
Pancreatic Lymph Node	10	10	10	10	10	10
Ectasia	0	0	0	0	1(1.0)	10**(1.8)
Lymphoid Hyperplasia	0	0	2(1.5)	0	0	10**(2.1)
Histiocytic Cell Infiltration	4(1.0)	8(1.4)	7(1.7)	7(1.3)	7(1.7)	9*(1.9)
Glandular Stomach	10	10	10	10	10	10
Ulcer	0	0	0	0	0	10**(3.5)
Epithelial Regenerative Focal Hyperplasia	0	0	0	0	0	10**(2.0)
Epithelial Squamous Focal Metaplasia	0	0	0	0	0	10**(2.4)
Bone Marrow	10	10	10	10	10	10
Hyperplasia	0	0	0	0	0	4*(1.0)

*Significantly different from control P≤0.05 (Fisher’s exact test)

**P≤0.01

a Number of animals with tissue examined microscopically

b Number of animals with lesion

c Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Applicant's summary and conclusion

Conclusions:

Exposure to sodium dichromate dihydrate in the drinking water resulted in hyperplasia and ulceration of the stomach, anaemia, and lesions of the small intestine. A NOAEL could not be identified due to effects being observed at all exposure concentrations.

Executive summary:

The toxicity of sodium dichromate dihydrate was evaluated in a 3 month drinking water study, as a prelude to a longer term carcinogenicity study. Sodium dichromate dihydrate contains hexavalent chromium (CrVI) and therefore a read across is proposed from this substance.

Groups of 10 male and 10 female F344/N rats were exposed to sodium dichromate dihydrate in their drinking water at concentrations of 0, 62.5, 125, 250, 500 or 1000 mg/L, for 3 months. On a molecular weight basis, the doses were equivalent to approximately 1.7, 3.5, 5.9, 11.2 and 20.9 mg hexavalent chromium/kg body weight per day. Additional groups of 10 rats/sex were exposed to the same test concentrations for 4 weeks, and used for clinical pathology studies.

All rats survived to the end of the study, and no clinical signs of toxicity were recorded. Reduced body weights were observed in the 500 and 1000 mg/L males, and the 1000 mg/L females. Water consumption was reduced in animals exposed to 250 mg/L and above, and decreases in urine volume and increases in urine specific gravity were related to the reduced water consumption. Exposed rats developed a microcytic hypochromic anaemia, considered to be evidence of a toxic response resulting from absorption of Cr VI. Serum cholesterol and triglyceride concentrations were lower in exposed rats compared to controls. Increased bile concentrations were observed in exposed rats; this finding was thought to have been due to altered hepatic function. The incidences of histiocytic cellular infiltration were generally significantly increased in the duodenum of all exposed rats, and the liver of female rats. The incidence of nonneoplastic lesions in the glandular stomach at the limiting ridge (focal ulceration, regenerative epithelial hyperplasia, squamous epithelial hyperplasia) was significantly increased in rats exposed to 1000 mg/L compared to controls. Increased histiocytic infiltration of the pancreatic lymph nodes was observed at concentrations as low as 62.5 mg/L.

It was not possible to identify a NOAEL, due to haematological effects and the increased incidence of nonneoplastic lesions in the pancreatic lymph nodes observed at the lowest concentration tested (62.5 mg/L).