Sodium dichromate

Carcinogenicity

Description of key information

An oral carcinogenicity study in rats and mice is available for sodium dichromate. The discussion is included in the endpoint summary of the hexavalent chromium substances category and the read-across approach is described in detail in the attached document in section 13 of the IUCLID.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-10-02 to 2004-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Minimal methodlogical deficiencies: food consumption not measured; blood smears not collected for differential blood count; few histopathological parameters missing (muscular tissue, peripheral nerve, spinal cord, sternum, femur with joint)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Sodium dichromate dihydrate was administered ad libitum to groups of 50 male and 50 female F344/N rats in drinking water at concentrations of 0, 14.3, 57.3, 172, and 516 mg/L(actually ingested: males: approx. 0, 0.6, 2.2, 6, and 17 mg/kg bw/day; females: approx. 0, 0.7, 2.7, 7, and 20 mg/kg bw/day) for up to 105 -106 weeks. The following parameters were investigated/recorded: clinical signs, mortality, body weight, water consumption, compound intake, gross pathology and histopathology.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Aldrich Chemical Company (Milwaukee, WI)
- Lot/batch number of test material: 062001

Species:

rat

Strain:

other: F344/N

Details on species / strain selection:

not specified

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: 6 to 7 weeks
- Weight at study initiation (mean range): males: 117 - 118 g; females: 99 - 100g
- Housing: 3 males or 5 females per cage; solid-bottom polycarbonate cages (Lab Products, Maywood, NJ), changed twice weekly; bedding: heat-treated, irradiated hardwood chips (P.J. Murphy Forest Products, Inc., Montville, NJ), changed twice weekly; Rack filters: Reemay® spun-bonded polyester (Andico, Birmingham, AL), changed every 2 weeks; Racks: stainless steel (Lab Products, Maywood, NJ), changed and rotated every 2 weeks
- Diet (ad libitum): irradiated NTP-2000 wafers (Zeigler Brothers, Inc., Gardners, PA); changed weekly
- Water (ad libitum): tap water (Birmingham municipal supply); changed twice weekly
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 3 °C
- Relative humidity:: 50% ± 15%
- Air changes: 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease.

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared approximately every 2 weeks by mixing sodium dichromate dihydrate with tap water. Formulations were stored in NALGENE® containers at room temperature for up to 42 days.
The sodium dichromate dihydrate dosed water used was slightly acidic. Based on an equilibrium constant of 50, dichromate predominates at the highest exposure concentration and the chromate:dichromate ratio approaches 1 at the lowest exposure concentration. These ratios would be obtained when the starting material was a chromate or dichromate salt.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies of a 41.8 μg/mL dose formulation were performed using ion chromatography. Stability was confirmed for at least 42 days for dose formulations stored in sealed NALGENE® containers, protected from light, at temperatures up to room temperature and for at least 7 days when stored in drinking water bottles under simulated animal room conditions.
Periodic analyses of the dose formulations of sodium dichromate dihydrate were conducted using ultraviolet/visible/near infrared spectroscopy (350 to 390 nm). The dose formulations were analyzed approximately every 10 weeks. Of the dose formulations analyzed, all 44 were within 10 % of the target concentrations. Animal room samples and unused carboy storage samples of these dose formulations were also analyzed; all 16 animal room samples were within 10% of the target concentrations. Fourteen of 16 carboy samples were within 10% of the target concentrations.

Duration of treatment / exposure:

105 to 106 weeks

Frequency of treatment:

ad libitum

Post exposure period:

not specified

Dose / conc.:

14.3 mg/L drinking water

Remarks:

actually ingested: males: approx. 0.6 mg/kg bw/day; females: approx. 0.7 mg/kg bw /day

Dose / conc.:

57.3 mg/L drinking water

Remarks:

actually ingested: males: approx. 2.2 mg/kg bw/day; females: approx. 2.7 mg/kg bw /day

Dose / conc.:

172 mg/L drinking water

Remarks:

actually ingested: males: approx. 6 mg/kg bw/day; females: approx. 7 mg/kg bw /day

Dose / conc.:

516 mg/L drinking water

Remarks:

actually ingested: males: approx. 17 mg/kg bw/day; females: approx. 20 mg/kg bw /day

No. of animals per sex per dose:

50 males / 50 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: the 2-year studies were designed after a NTP public meeting on hexavalent chromium to consider the results of the prechronic studies and recommendations from the panel of scientific experts convened at this meeting (NTP, 2006)*. Exposure concentrations were selected based on these public discussions and review of the data from the 3-month toxicity studies. Based on source water concentrations, an additional low exposure concentration group was added to the 2-year studies to provide a concentration that was closer to concentrations to which humans might be exposed through the drinking water. A wider spacing of the exposure concentrations was also recommended to extend the dose‑response curve. The highest exposure concentration in rats was limited by toxicity observed in the 3-month studies. In the 3-month studies, reductions in body weight and water consumption and increased incidences of ulcers in the glandular stomach were observed in male and female rats exposed to 1000 mg sodium dichromate dihydrate/L. Based on these considerations, the exposure concentrations of total chromium recommended were 0, 5, 20, 60, or 180 mg/L for male and female rats. These exposure concentrations are equivalent to 0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate.

*Reference:
- National Toxicology Program (NTP) (2006). NTP Study of the Hexavalent Chromium Compound Sodium Dichromate Dihydrate. 13859>

Positive control:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; clinical findings were recorded at 4-week intervals beginning at week 5.

DETAILED CLINICAL OBSERVATIONS: No data
DERMAL IRRITATION: No data

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly for the first 13 weeks, at 4-week intervals thereafter, and at the end of the studies

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly for the first 13 weeks and then every 4 weeks for a 7-day period each time.

OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Complete necropsies and microscopic examinations were performed on all rats. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, trimmed and processed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. The following tissues were examined microscopically: gross lesions, tissue masses, adrenal gland, bone (with marrow), brain, clitoral gland, oesophagus, eye, 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, tongue, trachea, urinary bladder, and uterus.

During the audit of the pathology specimens for the rat study, the residual wet tissues of the gastrointestinal tract from male and female rats from all groups were examined to ensure that the entire gastrointestinal tract was adequately opened. Any untrimmed potential lesions were collected for microscopic evaluation.

For the 2-year study, slides of all neoplasms and all potential target organs for all animals were evaluated, which included the glandular stomach, small intestine (duodenum, ileum, jejunum), large intestine (cecum, colon), liver, oral mucosa, and tongue of rats. In addition, slides for all animals were reviewed for specific lesions in the bone marrow, kidney, pancreatic and mesenteric lymph nodes, and salivary glands of rats.

The oral mucosa and tongue are not protocol-required tissues, and as such, only lesions that are observed grossly in these sites are routinely evaluated histologically. Because the gross lesions observed in the oral mucosa were diagnosed as neoplasms, the oral mucosa (present on slides of the three standard sections of the nasal cavity required to be evaluated for each study) from all male and female rats in all exposure concentration groups was evaluated for neoplastic and nonneoplastic lesions. Histologic sections of the tongue were prepared for all male and female rats in all exposure concentration groups and were evaluated for neoplastic and nonneoplastic lesions.

Statistics:

Survival analyses: Kaplan-Meier surivival curves, means, life table trend test, & life table pairwise comparisons. P values were two-sided.
Neoplasm & nonneoplastic lesions: Poly-k test, continuity-corrected Poly-3 test. P values were one-sided.
Body weight: parametric multiple comparison procedures of Dunnett (1955) & Williams (1971, 1972).
Jonckheere’s test was used to assess the significance of the dose-related trends & to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test)*.
Extreme values identified by the outlier test of Dixon and Massey (1957).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Males & females:
172 and 516 mg/L: water consumption was less than that by the controls throughout the study. Decreases were evident from the first week of the study and continued until terminal sacrifice. During the second year of the study, the average water consumption was reduced by 15% and 22% and by 15% and 27% that of the controls in the 172 and 516 mg/L male and female rats, respectively.

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver:
The incidences of minimal to moderate histiocytic cellular infiltration in 516 mg/L males and in females exposed to 57.3 mg/L or greater were significantly greater than those in the controls. The incidences of chronic inflammation in 172 mg/L males and all exposed groups of females were significantly increased. Chronic inflammation was generally of minimal to mild severity in most groups, including the controls, except 516 mg/L females in which there appeared to be a slight increase in the severity (mild to moderate). There were statistically significant, exposure concentration-related increases in the incidences of fatty change in female rats exposed to 57.3 mg/L or greater. The incidences of basophilic focus in males exposed to 57.3 or 172 mg/L were significantly increased. The incidence of clear cell focus in females exposed to 172 mg/L was significantly increased.

Histiocytic infiltrates were characterized by the presence of individual, small clusters and sometimes syncytia of histiocytes (macrophages) that were typically randomly scattered throughout the hepatic parenchyma but often occurred in increased numbers within the portal areas. The histiocytes were large (approximately 20 to 80 microns in diameter) and had abundant pale, lightly eosinophilic, faintly stippled cytoplasm and single, small, peripheral, dark basophilic nuclei. In minimally affected livers, the histiocytes often occurred singly, whereas in more severely affected livers, the histiocytes occurred as small clusters of a few cells. Chronic inflammation consisted of small, randomly scattered aggregates of macrophages, lymphocytes, and neutrophils in variable numbers and in some cases were associated with the larger histiocytic infiltrates. Chronic inflammation is consistent with changes that are considered to be background or spontaneous lesions commonly observed in aged rats and appears to be exacerbated by exposure. Fatty change consisted of scattered hepatocytes that contained one to several variably sized, discrete, clear, cytoplasmic vacuoles (fat droplets).

- Small intestine (duodenum):
The incidences of minimal to mild histiocytic cellular infiltration were significantly increased in males exposed to 57.3 mg/L or greater and in 172 and 516 mg/L females. The infiltrating histiocytes were morphologically similar to those observed in the liver and occurred singly or as clusters of cells within the lamina propria at the tips of the duodenal villi.

- Mesenteric lymph node:
The incidences of histiocytic cellular infiltration in males exposed to 57.3 mg/L or greater and in 172 and 516 mg/L females were significantly increased compared to the controls; there was a slight increase in the severity of infiltrates in 172 and 516 mg/L females with more animals in these groups having mild severity grades relative to the controls and the lower exposure concentration groups. The incidences of minimal lymph node hemorrhage in males exposed to 57.3 mg/L or greater and in 516 mg/L females were significantly increased and may have been caused by the histiocytic infiltrates in the lymph node. The histiocytic infiltrates were morphologically similar to those that occurred in the liver and duodenum. They formed numerous variably sized clusters randomly scattered within the cortex and/or medullary sinuses. Frequently, the clusters coalesced to form sheets that in some cases replaced much of the lymph node parenchyma.

Please also refer to the field "Attached background material" below.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Oral cavity (oral mucose and tongue):
Exposure to sodium dichromate dihydrate resulted in the development of neoplasms of the squamous epithelium that lines the oral mucosa and tongue. The incidences of squamous cell carcinoma in the oral mucosa of 516 mg/L male and female rats were significantly greater than those in the controls. None were seen in concurrent or historical controls for drinking water studies. Squamous cell carcinomas were also observed in the oral mucosa of two 172 mg/L female rats and exceeded the historical control ranges for drinking water studies and for all routes of administration. One squamous cell carcinoma of the tongue occurred in a 14.3 mg/L male and one in a 172 mg/L female. A squamous cell papilloma of the oral mucosa occurred in one 516 mg/L male, and a squamous cell papilloma of the tongue occurred in one 516 mg/L male. One control and one 14.3 mg/L female had squamous cell papillomas of the tongue. The incidences of squamous cell papilloma or squamous cell carcinoma (combined) of the oral mucosa or tongue of 516 mg/L male and female rats were significantly greater than those in the controls.

Microscopically, the squamous cell carcinomas of the oral mucosa were highly invasive, irregular masses. Typically, they appeared to originate in the oral mucosa of the palate adjacent to the upper molar teeth; invaded the tongue, Harderian gland, and the soft tissues surrounding the nose in some animals; and penetrated the maxilla and invaded the brain in one case. Carcinomas consisted of a mixture of variably sized islands and short, irregular cords of relatively well differentiated stratified squamous epithelium, surrounded by moderate amounts of dense fibrous connective tissue stroma. Frequently, the epithelium formed cyst-like structures filled with keratin (keratin pearls). The squamous cell papillomas of the oral mucosa and tongue were exophytic masses that projected from the mucosa and consisted of irregular papillary proliferations of mature squamous epithelium supported by a core of fibrovascular stroma. In the tongue, squamous cell carcinoma was diagnosed when the stroma of these papillary masses had evidence of invasion in the form of irregular foci of dysplastic epithelium.

Please also refer to the field "Attached background material" below.

Other effects:

not specified

Details on results:

CLINICAL SIGNS:
Males and females:
- 14.3, 57.3, 172, and 516 mg/L groups: no clinical findings were attributed to sodium dichromate dihydrate exposure.

MORTALITY
Males:
- 14.3, 57.3, 172, and 516 mg/L groups: survival of exposed groups was similar to that of the control groups (Percent probability of survival at end of study: control group: 56 %; 14.3 mg/L group: 60 %; 57.3 mg/L group: 61 %; 172 mg/L group: 72 %; 516 mg/L group: 59 %).

Please also refer to the field "Attached background material" below.

Females:
- 14.3, 57.3, 172, and 516 mg/L groups: survival of exposed groups was similar to that of the control groups (Percent probability of survival at end of study: control group: 66 %; 14.3 mg/L group: 64 %; 57.3 mg/L group: 64 %; 172 mg/L group: 72 %; 516 mg/L group: 62 %).

BODY WEIGHT AND WEIGHT CHANGES:
Males & females:
- 14.3, 57.3, and 172 mg/L groups: mean body weights of male and female rats were generally similar to those of the control groups throughout the study.
- 516 mg/L: mean body weights of males and females were less than those of controls throughout the study and by the end of the study were 88% and 89% that of the respective controls. The lower body weights were partly attributed to poor palatability of the dosed water and consequent reductions in water consumption rather than direct toxic effects of sodium dichromate dihydrate exposure.

Please also refer to the field "Attached background material" below.

WATER CONSUMPTION:
Males and females:
Drinking water concentrations of 14.3, 57.3, 172, or 516 mg/L resulted in average daily doses of approximately 0.6, 2.2, 6, or 17 mg sodium dichromate dihydrate/kg body weight for male rats and 0.7, 2.7, 7, or 20 mg/kg for female rats.

HISTOPATHOLOGY- NON-NEOPLASTIC:
- Pancreatic lymph node:
The incidences of histiocytic cellular infiltration were increased in the 516 mg/L males and in 57.3 mg/L or greater females with the incidence in 172 mg/L females being significantly greater than that in the controls; the average severity of infiltration in 57.3 mg/L or greater females increased with increasing dose. Histiocytic infiltrates were morphologically similar to those that occurred in the liver, duodenum, and mesenteric lymph nodes.

- Salivary gland:
The incidence of salivary gland atrophy was significantly increased in 172 mg/L females compared to that in the controls; in general, the average severity was minimal in the controls and in the 14.3, 57.3, and 172 mg/L groups, but mild in the 516 mg/L group. Microscopically, atrophy occurred as single, focal lesions in the salivary glands. Minimal lesions consisted of small, poorly defined foci of atrophic acini, whereas mild lesions were larger, well-defined foci. The atrophic foci consisted of shrunken glandular acini lined by low cuboidal epithelial cells devoid of secretory material, accompanied by an increased prominence of the interstitial stroma and ducts, some of which were variably dilated. Atrophy is an age-related spontaneous change commonly observed in rats. The biological significance of the increases in female rats in this study is uncertain.

Please also refer to the field "Attached background material" below.

HISTOPATHOLOGY-NEOPLASTIC:
Exposure to sodium dichromate dihydrate resulted in statistically significant increased incidences of neoplasms in one or more exposed groups. In general, the increased incidences occurred in one sex, one or more exposed groups, and were not exposure concentration related. These lesions included pancreatic acinar adenoma in males (0 mg/L, 1/50; 14.3 mg/L, 2/50; 57.3 mg/L, 6/49; 172 mg/L, 2/50; 516 mg/L, 2/49); benign pheochromocytoma of the adrenal medulla in males (6/49, 13/50, 14/49, 5/50, 4/49); and mononuclear cell leukemia in females (8/50, 18/50, 13/50, 7/50, 11/50). The relationship of these changes to exposure is uncertain.

Please also refer to the field "Attached background material" below.

Relevance of carcinogenic effects / potential:

Clear evidence of site-of-contact carinogenicity was seen under the conditions of this study with sodium dichromate administered in drinking water. There was no evidence of systemic carcinogenicity.

Dose descriptor:

LOAEL

Remarks:

(toxicity)

Effect level:

57.3 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 50.4 mg sodium dichromate anhydrous/L

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

516 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: equivalent to 454.1 mg sodium dichromate anhydrous/L

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

172 mg/L drinking water

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: equivalent to 454.1 mg sodium dichromate anhydrous/L

Conclusions:

Groups of 50 male and 50 female rats were exposed to drinking water containing 0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate (equivalent to 0, 5, 20, 60, or 180 mg/L chromium) for 2 years (equivalent to average daily doses of approximately 0.6, 2.2, 6, or 17 mg sodium dichromate dihydrate/kg body weight for males and 0.7, 2.7, 7, or 20 mg/kg for females). Survival of exposed groups was similar to that of the control groups. Mean body weights of 516 mg/L males and females were less than those of the controls throughout the study. The lower body weights were partly attributed to poor palatability of the dosed water and consequent reductions in water consumption. Water consumption by 172 and 516 mg/L rats was less than that by the controls throughout the study. Exposure to sodium dichromate dihydrate caused a microcytic hypochromic anemia in rats that ameliorated with time.

Exposure to sodium dichromate dihydrate resulted in the development of neoplasms of the squamous epithelium that lines the oral mucosa and tongue. The incidences of squamous cell carcinoma in the oral mucosa of 516 mg/L male and female rats were significantly greater than those in the controls. The incidence in 172 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration. The incidences of squamous cell papilloma or squamous cell carcinoma (combined) of the oral mucosa or tongue of 516 mg/L male and female rats were significantly greater than those in the controls.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-09-04 to 2004-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Minimal methodlogical deficiencies: food consumption not measured; blood smears not collected for differential blood count; few histopathological parameters missing (muscular tissue, peripheral nerve, spinal cord, sternum, femur with joint)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Sodium dichromate dihydrate was administered ad libitum to groups of 50 male and 50 female B6C3F1 mice in drinking water at concentrations of 0, 14.3, 28.6, 85.7, and 257.7 mg/L for males and 14.3, 57.2, 172, 516 mg/L for females (actually ingested: males: approx. 0, 1.1, 2.6, 7, and 17 mg/kg bw/day; females: approx. 0, 1.1, 3.9, 9, and 25 mg/kg bw/day) for up to 105 -106 weeks. The following parameters were investigated/recorded: clinical signs, mortality, body weight, water consumption, compound intake, gross pathology and histopathology.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Aldrich Chemical Company (Milwaukee, WI)
- Lot/batch number of test material: 062001

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

not specified

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: 6 to 7 weeks
- Housing: 1 males or 5 females per cage; solid-bottom polycarbonate cages (Lab Products, Maywood, NJ), changed once (male mice) or twice weekly (female mice); bedding: heat-treated, irradiated hardwood chips (P.J. Murphy Forest Products, Inc., Montville, NJ), changed once (male mice) or twice weekly (female mice); Rack filters: Reemay® spun-bonded polyester (Andico, Birmingham, AL), changed every 2 weeks; Racks: stainless steel (Lab Products, Maywood, NJ), changed and rotated every 2 weeks
- Diet (ad libitum): irradiated NTP-2000 wafers (Zeigler Brothers, Inc., Gardners, PA); changed weekly
- Water (ad libitum): tap water (Birmingham municipal supply); changed twice weekly
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 3 °C
- Relative humidity: 50% ± 15%
- Air changes: 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Five male and five female mice were randomly selected for parasite evaluation and gross observation of disease.

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared approximately every 2 weeks by mixing sodium dichromate dihydrate with tap water. Formulations were stored in NALGENE® containers at room temperature for up to 42 days.
The sodium dichromate dihydrate dosed water used was slightly acidic. Based on an equilibrium constant of 50, dichromate predominates at the highest exposure concentration and the chromate:dichromate ratio approaches 1 at the lowest exposure concentration. These ratios would be obtained when the starting material was a chromate or dichromate salt.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies of a 41.8 μg/mL dose formulation were performed using ion chromatography. Stability was confirmed for at least 42 days for dose formulations stored in sealed NALGENE® containers, protected from light, at temperatures up to room temperature and for at least 7 days when stored in drinking water bottles under simulated animal room conditions.
Periodic analyses of the dose formulations of sodium dichromate dihydrate were conducted using ultraviolet/visible/near infrared spectroscopy (350 to 390 nm). The dose formulations were analyzed approximately every 10 weeks. Of the dose formulations analyzed, all 84 were within 10 % of the target concentrations. Animal room samples and unused carboy storage samples of these dose formulations were also analyzed; 34 of 35 animal room samples were within 10% of the target concentrations. Fourteen of 16 carboy samples were within 10% of the target concentrations.

Duration of treatment / exposure:

105 to 106 weeks

Frequency of treatment:

ad libitum

Post exposure period:

not specified

Dose / conc.:

14.3 mg/L drinking water

Remarks:

male and female; actually ingested: males: 1.1 mg/kg bw/day; females: 1.1 mg/kg bw/day

Dose / conc.:

28.6 mg/L drinking water

Remarks:

male mice only; actually ingested: males: 2.6 mg/kg bw/day

Dose / conc.:

57.2 mg/L drinking water

Remarks:

female mice only, actually ingested: females: 3.9 mg/kg bw/day

Dose / conc.:

85.7 mg/L drinking water

Remarks:

male mice only; actually ingested: males: 7 mg/kg bw/day

Dose / conc.:

172 mg/L drinking water

Remarks:

female mice only; actually ingested: females: 9 mg/kg bw/day

Dose / conc.:

257.4 mg/L drinking water

Remarks:

male mice only; actually ingested: males: 17 mg/kg bw/day

Dose / conc.:

516 mg/L drinking water

Remarks:

female mice only; actually ingested: females: 25 mg/kg bw/day

No. of animals per sex per dose:

50 males / 50 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: the 2-year studies were designed after a NTP public meeting on hexavalent chromium to consider the results of the prechronic studies and recommendations from the panel of scientific experts convened at this meeting (NTP, 2006)*. Exposure concentrations were selected based on these public discussions and review of the data from the 3-month toxicity studies. Based on source water concentrations, an additional low exposure concentration group was added to the 2-year studies to provide a concentration that was closer to concentrations to which humans might be exposed through the drinking water. A wider spacing of the exposure concentrations was also recommended to extend the dose‑response curve. The highest exposure concentration in mice was limited by toxicity observed in the 3-month studies. In mice, significant reductions in body weight and water consumption were observed in males exposed to 500 or 1,000 mg/L and females exposed to 1,000 mg/L. Based on these considerations, the exposure concentrations of total chromium recommended were 0, 5, 10, 30, or 90 mg/L for male mice and 0, 5, 20, 60, or 180 mg/L for female mice. These exposure concentrations are equivalent to 0, 14.3, 28.6, 85.7, or 257.4 mg/L and 0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate for males and females, respectively.

*Reference:
- National Toxicology Program (NTP) (2006). NTP Study of the Hexavalent Chromium Compound Sodium Dichromate Dihydrate. 13859>

Positive control:

not secified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; clinical findings were recorded at 4-week intervals beginning at week 5.

DETAILED CLINICAL OBSERVATIONS: No data
DERMAL IRRITATION: No data

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly for the first 13 weeks, at 4-week intervals thereafter, and at the end of the studies

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly for the first 13 weeks and then every 4 weeks for a 7-day period each time.

OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Complete necropsies and microscopic examinations were performed on all mice. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, trimmed and processed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. The following tissues were examined microscopically: gross lesions, tissue masses, adrenal gland, bone (with marrow), brain, clitoral gland, oesophagus, eye, gallbladder, 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, tongue, trachea, urinary bladder, and uterus.

During the audit of the pathology specimens for the mouse study, the residual wet tissues of the gastrointestinal tract from male and female mice from all groups were examined to ensure that the entire gastrointestinal tract was adequately opened. Any untrimmed potential lesions were collected for microscopic evaluation.

For the 2-year study, slides of all neoplasms and all potential target organs for all animals were evaluated, which included the forestomach, glandular stomach, small intestine (duodenum, ileum, jejunum), and large intestine (cecum, colon) of mice. In addition, slides for all animals were reviewed for specific lesions in the bone marrow, kidney, pancreatic and mesenteric lymph nodes, pancreas, spleen, thymus, and urinary bladder of mice.

The oral mucosa and tongue are not protocol-required tissues, and as such, only lesions that are observed grossly in these sites are routinely evaluated histologically. Because the gross lesions observed in the oral mucosa were diagnosed as neoplasms, the oral mucosa (present on slides of the three standard sections of the nasal cavity required to be evaluated for each study) from all male and female mice in all exposure concentration groups was evaluated for neoplastic and nonneoplastic lesions. Histologic sections of the tongue were prepared for all male and female mice in all exposure concentration groups and were evaluated for neoplastic and nonneoplastic lesions.

Statistics:

Survival analyses: Kaplan-Meier surivival curves, means, life table trend test, & life table pairwise comparisons. P values were two-sided.
Neoplasm & nonneoplastic lesions: Poly-k test, continuity-corrected Poly-3 test. P values were one-sided.
Body weight: parametric multiple comparison procedures of Dunnett (1955) & Williams (1971, 1972).
Jonckheere’s test was used to assess the significance of the dose-related trends & to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test)*.
Extreme values identified by the outlier test of Dixon and Massey (1957).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Males and females:
Water consumption by 85.7 and 257.4 mg/L males and 172 and 516 mg/L females was less than that by the controls throughout the study. During the second year of the study, the average water consumption was reduced by 15% and 35% than that of the controls in 85.7 and 257.4 mg/L males, respectively, and by 25% and 32% than that of the controls in the 172 and 516 mg/L females, respectively.

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Small intestine (duodenum, jejunum, and ileum):
Low incidences of focal epithelial hyperplasia occurred in the duodenum of exposed male and female mice. Although the increased incidences were not exposure concentration-related or statistically significant, this lesion was considered a preneoplastic lesion related to exposure to sodium dichromate dihydrate because of its morphologic similarities to adenoma. Focal epithelial hyperplasias were distinguished from adenomas in that they were smaller, commonly located in the superficial mucosa, and were less discrete with margins that tended to blend with the normal surrounding mucosal epithelium. These hyperplastic lesions occurred as focal disorganized areas of increased cellularity that stained more basophilic than the surrounding histologically normal mucosa. They were composed of crypts and villi that were lined by increased numbers of cuboidal to tall columnar epithelial cells that were morphologically similar to those of the adenomas.

The incidences of diffuse epithelial hyperplasia were significantly increased in the duodenum of all exposed groups of male and female mice. In the jejunum, the incidence of diffuse epithelial hyperplasia was significantly increased in 516 mg/L females. Diffuse epithelial hyperplasia generally involved the entire mucosa. Compared to the controls, the duodenal villi of exposed mice were short, broad, blunt, and lined by densely packed, tall columnar epithelial cells that were more basophilic than the shorter epithelial cells lining the duodenum villi of the controls. The epithelial cells and cell nuclei were often piled up in multiple layers along the long axis of the villi. Intestinal crypts were often elongated and generally appeared to contain increased numbers of epithelial cells with increased numbers of mitotic figures. Collectively, these lesions are considered consistent with regenerative hyperplasia secondary to previous epithelial cell injury.

The incidences of histiocytic cellular infiltration were significantly increased in the duodenum of 85.7 and 257.4 mg/L males and of 172 and 516 mg/L females. In the jejunum, the incidence of histiocytic cellular infiltration was significantly increased in 516 mg/L females compared to the controls. Infiltrates ranged from
small clusters of three to five cells in the lower exposure groups to larger clusters that expanded the tips of the affected villi in the higher exposure groups.

- Liver:
The incidences of histiocytic cellular infiltration were significantly increased in all exposed groups of females.

- Mesenteric lymph node:
The incidences of minimal to moderate histiocytic cellular infiltration in all exposed groups of male and female mice were significantly greater than those in the controls. In general, there was a slight increase in the severity with increasing exposure concentration.

- Pancreatic lymph node:
The incidences of minimal to moderate histiocytic cellular infiltration in 85.7 and 257.4 mg/L males and in 172 and 516 mg/L females were significantly greater than those in the controls.

- Pancreas:
The incidences of cytoplasmic alteration in the pancreatic acini of males exposed to 85.7 or 257.4 mg/L and in all exposed groups of females were significantly increased. Cytoplasmic alteration was characterized by depletion of cytoplasmic zymogen granules from the pancreatic acinar epithelial cells. The biological significance of this change was uncertain.

Please also refer to the field "Attached background material" below.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Small intestine (duodenum, jejunum, and ileum):
In the duodenum, the incidences of adenoma occurred with a positive trend in males and females. The incidences of adenoma in 257.4 mg/L males and 172 and 516 mg/L females were significantly greater that those in the controls. The incidences of multiple adenoma were also significantly increased in 257.4 mg/L males and in 516 mg/L females. Adenomas were observed in one control, one 28.6 mg/L, and five 85.7 mg/L males and in two 57.3 mg/L females; none of these increased incidences were statistically significant. However, the incidences of adenoma in 85.7 mg/L males and 57.3 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration. The incidences of carcinoma of the duodenum were increased in 85.7 and 257.4 mg/L males and in 172 and 516 mg/L females; the incidence in 516 mg/L females was significantly greater than that in the controls. The incidences of carcinoma in 257.4 mg/L males and 516 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration.

In the jejunum, the incidence of adenoma in 516 mg/L females was significantly increased compared to that in the controls. In females, one adenoma occurred in the 14.3 mg/L group and two adenomas occurred in the 172 mg/L group; the incidence in 172 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration. The incidence of adenoma was greater in 257.4 mg/L males but was not significantly increased compared to the controls. However, the incidence exceeded the historical control ranges for drinking water studies and for all routes of administration. In the historical controls for the jejunum, adenoma has not been observed in mice in drinking water studies, and only one adenoma has been observed in males for all routes of administration. Low incidences of carcinoma of the jejunum occurred in exposed male and female mice; in males, the incidences of carcinoma in the 14.3, 85.7, and 257.4 mg/L groups did not exceed the historical control ranges for drinking water studies or for all routes of administration. In females, the incidences of carcinoma of the jejunum in the 57.3 and 172 mg/L groups exceeded the historical control ranges for drinking water studies and for all routes of administration.

For the entire small intestine (duodenum, jejunum, and ileum), the incidences of adenoma and carcinoma in 257.4 mg/L males and the incidences of adenoma or carcinoma (combined) in 85.7 and 257.4 mg/L males were significantly greater than those in the controls. The incidences of adenoma in 85.7 mg/L males exceeded the historical control ranges for drinking water studies and for all routes of administration. For the entire small intestine, the incidence of adenoma in 172 mg/L females, the incidences of adenoma and carcinoma in 516 mg/L females, and the incidences of adenoma or carcinoma (combined) in 172 and 516 mg/L females were significantly increased. The incidences of adenoma, carcinoma, and adenoma or carcinoma (combined) in 57.3 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration. The incidences of carcinoma in 172 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration.

Adenomas were discrete, broad based, focally extensive, plaque-like lesions that thickened the mucosa and protruded into the lumen. They were composed of irregular, elongated, often branching crypts and irregular, glandular structures lined by one to several layers of densely packed, atypical, cuboidal to tall columnar, hyperchromatic epithelial cells with large oval to elongated nuclei. Increased mitotic activity was commonly evident.

Carcinomas were extensive, sessile, plaque-like neoplasms distinguished from adenomas by dysplastic epithelial growth, complete effacement of the mucosa with invasion of the submucosa and/or muscularis mucosa, degree of cellular atypia, and the presence of variable fibroplasia/desmoplasia. They were composed of hyperchromatic, atypical, pleomorphic (cuboidal, round, polygonal, columnar) epithelial cells that had round, oval, or elongated hyperchromatic nuclei. Increased mitotic activity was evident to a greater extent than in adenomas, and many mitoses were atypical.

Please also refer to the field "Attached background material" below.

Other effects:

not specified

Details on results:

CLINICAL SIGNS:
Males and females:
No clinical findings were attributed to sodium dichromate dihydrate exposure.

MORTALITY
Males:
- 14.3, 28.6, 85.7, and 257.4 mg/L groups: survival of exposed groups was similar to that of the control groups (Percent probability of survival at end of study: control group: 66 %; 14.3 mg/L group: 70 %; 28.6 mg/L group: 70 %; 85.7 mg/L group: 76 %; 257.4 mg/L group: 64 %).

Females:
- 14.3, 57.3, 172, and 516 mg/L groups: survival of exposed groups was similar to that of the control groups (Percent probability of survival at end of study: control group: 74 %; 14.3 mg/L group: 78 %; 57.3 mg/L group: 90 %; 172 mg/L group: 84 %; 516 mg/L group: 84 %).
Please also refer to the field "Attached background material" below.

BODY WEIGHT AND WEIGHT CHANGES:
Males and females:
The mean body weights of 257.4 mg/L male mice were less than those of the controls from months 2 through 6 of the study but were only slightly less than the control group by the end of the study; mean body weights of 172 mg/L females were less than those of the controls from months 3 through 12 of the study; and mean body weights of 516 mg/L females were less than those of the controls from month 2 until the end of the study. By the end of the study, the mean body weight of 172 mg/L females was 8% less than that of the controls, and the mean body weight of 516 mg/L females was 15% less than that of the control group. The lower body weights were partly attributed to poor palatability of the dosed water and consequent reductions in water consumption rather than direct toxic effects of sodium dichromate dihydrate exposure.
Please also refer to the field "Attached background material" below.

WATER CONSUMPTION.
Males and females:
Drinking water concentrations of 14.3, 28.6, 85.7, or 257.4 mg/L for males and 14.3, 57.3, 172, or 516 mg/L for females resulted in average daily doses of approximately 1.1, 2.6, 7, or 17 mg sodium dichromate dihydrate/kg body weight for male mice and 1.1, 3.9, 9, or 25 mg/kg for female mice.

HISTOPATHOLOGY-NON-NEOPLASTIC
- Liver:
The incidence of chronic inflammation was significantly increased in 172 mg/L females. The incidence of clear cell focus in 257.4 mg/L males was significantly less than that in the controls. The incidences of eosinophilic focus in 257.4 mg/L males and 172 and 516 mg/L females were significantly less than those in the controls.

Please also refer to the field "Attached background material" below.

HISTOPATHOLOGY-NEOPLASTIC
- Liver:
The incidences of hepatoblastoma in 14.3, 85.7, and 257.4 mg/L males were significantly less than that in the controls (0 mg/L, 17/50; 14.3 mg/L, 8/50; 28.6 mg/L, 11/50; 85.7 mg/L, 2/50; 257.4 mg/L, 0/50). The incidences of hepatocellular adenoma or carcinoma (combined) in 28.6 mg/L males (42/50, 42/50, 33/50, 37/50, 41/50) and hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma (combined) in 28.6 and 85.7 mg/L males (46/50, 42/50, 37/50, 37/50, 41/50) were significantly decreased. The incidences of hepatocellular adenoma in 172 and 516 mg/L females were significantly less than that in the controls (0 mg/L, 14/49; 14.3 mg/L, 21/50; 57.3 mg/L, 13/50; 172 mg/L, 6/50; 516 mg/L, 3/50). The biological significance of these decreases is unknown.

Please also refer to the field "Attached background material" below.

Relevance of carcinogenic effects / potential:

The results of this study indicate a clear carcinogenic effect of sodium dichromate dihaydrate on the mucosa of the small intestine in the mouse.

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

85.7 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: equivalent to 75.4 mg sodium dichromate anhydrous/L

Dose descriptor:

LOAEL

Effect level:

57.3 mg/L drinking water

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: equivalent to 50.4 mg sodium dichromate anhydrous/L

Conclusions:

Groups of 50 male mice were exposed to drinking water containing 0, 14.3, 28.6, 85.7, or 257.4 mg/L sodium dichromate dihydrate (equivalent to 0, 5, 10, 30, or 90 mg/L chromium) for 2 years (equivalent to average daily doses of approximately 1.1, 2.6, 7, or 17 mg sodium dichromate dihydrate/kg body weight). Groups of 50 female mice were exposed to drinking water containing 0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate (equivalent to 0, 5, 20, 60, or 180 mg/L chromium) for 2 years (equivalent to average daily doses of approximately 1.1, 3.9, 9, or 25 mg/kg). Survival of exposed groups was similar to that of the control groups. Mean body weights of 257.4 mg/L males were less than those of controls from months 2 through 6 of the study, but by the end of the study, the mean body weight of 257.4 mg/L males was only slightly less than that of the control group. Mean body weights of 172 mg/L females were less than those of the controls from months 3 through 12 of the study, and mean body weights of 516 mg/L females were less than those of the controls from month 2 until the end of the study. By the end of the study, the mean body weight of 172 mg/L females was 8% less than that of the controls, and the mean body weight of 516 mg/L females was 15% less than that of the controls. The lower body weights were partly attributed to poor palatability of the dosed water and consequent reductions in water consumption. Water consumption by 85.7 and 257.4 mg/L males and 172 and 516 mg/L females was less than that by the controls throughout the study. A treatment‑related microcytosis occurred in exposed mice; the mice were less affected than the rats.

The incidences of neoplasms of the small intestine (duodenum, jejunum, or ileum) were increased in exposed groups of male and female mice. The incidences of adenoma of the duodenum in 257.4 mg/L males and 172 and 516 mg/L females were significantly greater than those in the controls. The incidence of carcinoma of the duodenum was significantly increased in 516 mg/L females. The incidence of adenoma of the jejunum in 516 mg/L females was significantly increased compared to that in the controls. When the incidences of adenoma and carcinoma were combined for all sites of the small intestine, the incidences were significantly increased in 85.7 and 257.4 mg/L males and 172 and 516 mg/L females compared to those in the controls. The incidences in 57.3 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

7 mg/kg bw/day

Study duration:

chronic

Species:

cat

System:

gastrointestinal tract

Organ:

oral cavity

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available