Dimanganese trioxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

- Principle of test: Groups of 70 male and 70 female B6C3F1 mice were fed diets containing 0, 1,500, 5,000, or 15,000 ppm manganese (II) sulfate monohydrate for 103 weeks.
- Parameters analysed / observed: Survival, body weights, feed consumption, hematology, clinical chemistry, tissue metal concentration analyses, histopathological and clinical observations were made.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

manganese(2+);sulfate;hydrate

EC Number:

600-072-9

Cas Number:

10034-96-5

Molecular formula:

MnSO4.H2O

IUPAC Name:

manganese(2+);sulfate;hydrate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Source: J.T. Baker Chemical Company, Glen Ellyn, IL, Batch No.: 003261
- Purity: 97%
- Appearance: white, slightly efflorescent crystalline compound
- Solubility: Soluble in water and insoluble in alcohol

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dose formulations were prepared by mixing manganese (II) sulfate monohydrate with feed

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in feed: Stability of the dose formulations was established for 2 weeks in the dark at room temperature and for 1 week exposed to air and light.

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility, Frederick, MD
- Age at study initiation: approximately 41 days old
- Housing: 5 per cage; Animals were housed in polycarbonate cages with BetaChips, hardwood chips, which was changed twice weekly or more frequently when needed. Cage filters: Spun-bonded, DuPont 2024, changed once every 2 weeks; Racks: Stainless steel, changed once every 2 weeks
- Diet (e.g. ad libitum): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9
- Humidity (%): 35 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared by mixing manganese (II) sulfate monohydrate with feed. Homogeneity was confirmed and stability of the dose formulations was established for 2 weeks in the dark at room temperature and for 1 week exposed to air and light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability analyses of the dose formulations were conducted using a spectrophotometric method.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

daily; ad libitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Doses / concentrations 4

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

70

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses selected for the 2-year study in mice were 0, 1 500, 5 000, and 15 000 ppm. These doses were based on the significantly lower mean body weight gains of all exposed males and 50 000 ppm females and the significantly lower absolute and relative liver weights of 50 000 ppm males in the 13¬-week study.
- 10 mice per group were evaluated after 9 months and 15 months of chemical exposure.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded initially, weekly during the first 13 weeks of study, monthly thereafter ant at interim (9 and 15 months) evaluations.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 9 and 15 months
- Parameters analysed: erythrocytes, hemoglobin, hematocrit, platelets, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, reticulocytes, nucleated erythrocytes, and leukocyte count and differential
- Anaesthetic used for blood collection: Not specified
- Animals fasted: not specified
- How many animals: as many as 10 per group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 9 and 15 months
- Parameters analysed: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, and creatinine
- Animals fasted: not specified
- How many animals: as many as 10 per group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for the first 13 weeks and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: Yes
- Time schedule: measured for a 7-day period once every 4 weeks

OTHER:
- Organ weight: brain, kidneys, and liver from animals selected for the 9- and 15-month evaluations were weighed at necropsy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Complete histopathologic examinations were performed on all 0 and 15 000 ppm animals at the 9- and 15-month interim evaluations and gross lesions examined for the 1 500 and 5 000 ppm groups. Complete histopathologic examinations were performed on all animals at the end of the studies and on all animals that died or were killed moribund during the studies. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, bone, bone marrow, brain, cecum, colon and rectum, esophagus, heart, kidney, liver, lung, mandibular and mesenteric lymph nodes, mammary gland, nose, gallbladder, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, skin, small intestine, spleen, stomach (forestomach and glandular), testes/epididymis, thymus, thyroid gland, trachea, uterus, and urinary bladder.

Other examinations:

- Tissue metal concentration analyses: analyses for copper, iron, manganese, and zinc were brain, kidney, liver and pancreas

Statistics:

- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier
- Organ and body weight data, which have approximately normal distributions, were analyzed using the parametric multiple comparison procedures of Dunnett and Williams
- Jonckheere's test was used to assess the significance of the dose-response trends and 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-response trend (Dunnett's or Dunn's test).
- Average severity values were analyzed for significance using the Mann-Whitney U test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical findings were attributed to the administration of the test item.

Mortality:

no mortality observed

Description (incidence):

Survival of exposed males and females was similar to that of the control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of exposed males were similar to those of the control group.
After week 37, mean body weights of all exposed groups of females were lower than that of the controls. The final mean body weights for the 1 500, 5 000, and 15 000 ppm groups were 6%, 9%, and 13% lower than that of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption by exposed male and female mice was similar to that of the control groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Percent hematocrit, hemoglobin concentrations, and erythrocyte counts in 15 000 ppm male mice at the 15-month interim evaluation were greater than those of the controls but the significance of these slight increases is uncertain.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related effects were observed at the 9-or 15-month interim evaluations.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Liver: At the 9-month interim evaluation, absolute liver weights of 15 000 ppm males and of 5 000 and 15 000 ppm females were significantly lower than those of controls. Since these groups also had lower mean body weights, and relative liver weights were similar to controls, the lower absolute liver weights are not considered chemical related. At the 15-month interim evaluation, absolute and relative liver weights of exposed mice were similar to controls.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Gland: At the 9- and 15-month interim evaluations, thyroid follicle dilatation was present in 15 000 ppm males and females but not in the controls. At the end of the 2-year study, the incidence of follicular dilatation increased significantly in 15 000 ppm males and 5 000 and 15 000 ppm females. A significantly increased incidence of focal hyperplasia of follicular epithelium also occurred in 15 000 ppm males and in all exposed females.

Forestomach: A statistically significant increased incidence of focal squamous hyperplasia of the forestomach occurred in the 15 000 ppm males and females, accompanied by ulceration/erosion and inflammation. Hyperplasia of the squamous epithelium occurred focally at various sites of the forestomach mucosa. The lesion was characterized by broad-based areas of either proliferative epithelial thickening and hyperkeratosis or by polypoid projections of thickened epithelium protruding directly from the mucosa into the lumen of the stomach. Inflammation of the lamina propria and submucosa subjacent to the ulcerative lesions consisted of a mixture of infiltrating neutrophils and mononuclear leukocytes.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thyroid gland: Follicular cell adenomas were found in three (6%) 15 000 ppm males. This rate is marginally higher than the average rate of 2% and just within the range of 0% - 6% for historical control male mice. The incidence of this neoplasm was 10% in 15 000 ppm females, which is slightly above the average of 3% and range of 0%-9% for historical control female mice. The incidences of adenoma in 15 000 ppm males and females were not significantly greater than those of the controls.

Liver: One male in the 15 000 ppm group and two females in the 5 000 ppm group had hepatocellular adenomas at the 15-month interim evaluation. At the end of the 2-year study, hepatocellular adenomas occurred with a statistically significant negative trend in males (30/50, 29/49, 19/51, 20/50) that was also significant by pairwise comparison in the 5 000 and 15 000 ppm groups. Hepatocellular foci did not occur in an exposure-related pattern (foci of any type, males: 4/50, 16/49, 9/51, 1/50). The incidences of adenoma or foci in exposed females were similar to those of the controls.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Tissue metal concentration analyses: At the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of the 5 000 and 15 000 ppm groups. Hepatic iron levels were significantly lower in exposed females at the 9- and 15-month interim evaluations and in 5 000 and 15 000 males at the 15-month interim evaluation. Tissue concentrations of manganese in the brain (except 1 500 and 5 000 ppm females at 15 months), kidney, and pancreas (except 1 500 males at 9 months and 1 500 ppm females at 15 months) of exposed groups were significantly greater those of controls.

Details on results:

The study suggests equivocal evidence of carcinogenicity in male and female mice based on marginally increased incidences of thyroid gland follicular cell adenoma and significantly increased incidences of follicular cell hyperplasia.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

540 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

3

Dose descriptor:

LOAEL

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

4

Dose descriptor:

LOAEL

Effect level:

540 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In this study equivalent to OECD 453, a NOAEL of 5 000 ppm can be considered for males, which is equal to approx. 540 mg/kg body weight. Subsequently, the LOAEL for males is determined to be 15 000 ppm which is equal to 1800 mg/kg body weight. For females, a NOAEL of 1 500 ppm can be considered, which is equal to 200 mg/kg body weight.

Executive summary:

In a chronic toxicity study similar to guideline OECD 453, manganese sulphate monohydrate (97% purity) was administered to young adult B6C3F1 mice (70/sex/dose) in diet at dose levels of 0, 1 500, 5 000 and 15 000 ppm for 103 weeks.

Lower mean body weights were observed in female mice (6% at 1 500 ppm; 9% at 5 000 ppm and 13% at 15 000 ppm. Furthermore, focal squamous hyperplasia of the forestomach in male and female mice at 15 000 ppm was observed. In the thyroid gland, the incidence of follicular dilatation increased significantly in 15 000 ppm males and 5 000 and 15 000 ppm females. There was equivocal evidence of carcinogenic activity of the test item in male and female mice, based on a marginally increased incidence of thyroid gland follicular cell adenoma and a significantly increased incidence of follicular cell hyperplasia.

Based on the results the oral NOAEL was determined with 200 mg/kg body weight for the females and 540 mg/kg body weight for males.