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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
based on chronic oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-09-11 to 1986-09-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Neurological, optical and effects on urinalysis not examined. Food intake measured for a 7-day period every 4 weeks instead of weekly observation during first 13 weeks and then in 3-months intervals. Hematology and clinical chemistry scheduled for 9- and 15 months. Incomplete histopathology (aorta, accessory genital organs, musculature, peripheral nerve, spinal cord, eyes missing). Incomplete clinical chemistry (total protein, albumin, carbohydrate metabolism (i.e. blood glucose) missing).
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-09-11 to 1986-09-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Neurological, optical and effects on urinalysis not examined. Food intake measured for a 7-day period every 4 weeks instead of weekly observation during first 13 weeks and then in 3-months intervals. Hematology and clinical chemistry scheduled for 9- and 15 months. Incomplete histopathology (aorta, accessory genital organs, musculature, peripheral nerve, spinal cord, eyes missing). Incomplete clinical chemistry (total protein, albumin, carbohydrate metabolism (i.e. blood glucose) missing).
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a repeated dose oral toxicity study manganese (II) sulfate monohydrate was administered via feed ad libitum to three groups, each of 70 male and 70 female F344/N rats, for 103 weeks. The test item concentrations used were 1500, 5000, and 15000 ppm (actual dose received: 60, 200, and 615 mg/kg/day for males and 70, 230, and 715 mg/kg/day for females, respectively). A control group was run concurrently. Ten animals/sex/group were evaluated after 9 months and 15 months of test item exposure. The remaining animals were sacrificed at the end of the study. The following parameters were evaluated: clinical signs, survival, body weight, food consumption, haematology (intrim sacrifices only), clinical chemistry (intrim sacrifices only), organ weights (brain, kidney, and liver; intrim sacrifices only), macroscopic examination, and histopathology. Lastly, blood plasma, brain, kidneys, liver, and pancreas were examined for metal concentration of copper, iron, manganese, and zinc during intrim sacrifices.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in the dark at room temperature throughout the study
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: Frederick Cancer Research Facility (Frederick, MD)
- Age at study initiation: approx. 41 days
- Housing: housed 5 animals/cage (polycarbonate (Lab Products, Inc., Garfield, NJ); bedding material: BetaChips, hardwood chips (Northeastern Products, Inc., Warrensburg, NY); cage filters: spun-bonded DuPont 2024 polyester (Snow Filtration Co., Cincinnati, OH): racks: stainless steel (Lab Products, Inc., Garfield, NJ)
- Diet (ad libitum): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA); diet contains 60 g manganous oxide per 2000 lbs feed
- Water (ad libitum): automatic watering system (Edstrom Industries, Waterford, WI)
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 23.9 °C
- Relative humidity: 35 - 65 %
- Air changes: minimum of 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Details on route of administration:
not specified
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): dose formulations were prepared by mixing manganese (II) sulfate monohydrate with feed (NIH-07 open formula meal rat and mouse diet; diet contains 60 g manganous oxide per 2000 lbs feed). A premix of the test item and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes.
Dose formulations were discarded 21 days after the date of preparation.
- Storage temperature of food containing the test item: stored in plastic buckets with lids in the dark at 25 °C.

The level of manganese in the diet received by controls was approx. 92 ppm.
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. Homogeneity was confirmed; 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. A subsequent study confirmed the stability of the dose formulations for 3 weeks. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings support the conclusion that the manganese remained in divalent state.

Periodic analyses of the dose formulations were performed using spectrophotometric methods every two months during the 2-year study. All dose formulations were within the specified 10 % of the target concentrations throughout the studies. Results of periodic referee analyses were also within 10 % of the target concentrations.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
ad libitum
Dose / conc.:
1 500 ppm
Remarks:
actual dose received: 60 mg/kg/day for males and 70 mg/kg/day for females (20 and 23 mg Mn/kg/day, respectively)
Dose / conc.:
5 000 ppm
Remarks:
actual dose received: 200 mg/kg/day for males and 230 mg/kg/day for females (65 and 75 mg Mn/kg/day, respectively)
Dose / conc.:
15 000 ppm
Remarks:
actual dose received: 615 mg/kg/day for males and 715 mg/kg/day for females (200 and 233 mg Mn/kg/day, respectively)
No. of animals per sex per dose:
70 males / 70 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on decreases in body weight gain and the lower absolute and relative liver weights in the 25000 ppm groups in the 13-week study, doses of 0, 1500, 5000 and 15000 ppm were selected.
- Section schedule (intrim sacrifice): 10 rats/sex/group were evaluated after 9-months and 15 months of chemical exposure.
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; clinical findings were recorded weekly for the first 13 weeks and monthly thereafter.
- Cage side observations checked: survival and clinical findings

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly during first 13 weeks of study, monthly thereafter and at interim evaluations

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption measured for a 7-day period once every 4 weeks
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated: Yes

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
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: erythrocytes, hemoglobin, hematocrit, platelets, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, reticulocytes, nucleated erythrocytes, leukocyte count and differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, creatinine

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE:
- intrim sacrifce were performed after 9 months and 15 months of chemical exposure (10 animals/sex/group).
- terminal sacrifice: at the end of study

GROSS PATHOLOGY: Yes
- necropsy was performed on all animals
- at necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 µm, and stained with hematoxylin and eosin for microscopic examination.
- organ weights: brain, kidneys, and liver from animals selected for the 9- and 15-month evaluations were weighed at necropsy.

HISTOPATHOLOGY: Yes
- complete histopathologic examinations were performed on control (0 ppm) and high-dose (15000 ppm) animals at the 9- and 15-month interim evaluations and gross lesions were examined for the low- and mid-dose groups (1500 and 5000 ppm) groups.
- complete histopathologic examinations were performed on all animals surviving until the end of the studies and on those that died or were killed moribund during the studies.
- the following tissues were examined during the histopathology: gross lesions, tissue masses, associated lymph nodes, adrenal gland, bone, bone marrow, brain, cecum, colon, rectum, oesophagus, heart, kidney, liver, lung, mandibular and mesenteric lymph nodes, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, skin, small intestine, spleen, stomach (forestomach and glandular), testes/epididymis, thyroid gland, trachea, thymus, urinary bladder, uterus.

TISSUE METAL CONCENTRATION ANALYSIS (intrim sacrifice after 9 months and 15 months only)
- tissues examined in metal concentration analyses for copper, iron, manganese, and zinc were blood plasma, brain, kidney, liver, and pancreas.
Other examinations:
SCREENING OF HEALTH
- at the beginning of the study, serum samples were collected from 10 female rats for murine virus assay.
- serum samples were collected from 5 male and 5 female at 6, 12 and 18 months into the study and from 5 male and 5 female rats at terminal sacrifice for screening for the following: CARB, Mycoplasma arthridis and pulmonis, PVM, RCV/SDA, Sendai, H-1 and KRV
- results: possible M. arthritidis was found during the 6 month examination (1/10 animals). In addition, CARB was discovered during the 24 months examination (2/10 animals).
- before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- significant lower survival of males in the 15000 ppm dose group occuring after week 93 of the study, due to nephropathy and renal failure
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS
- no clinical findings were chemical-related.

MORTALITY
- survival of 1500 and 5000 ppm males and all exposed groups of females was similar to that of controls.

BODY WEIGHT
- mean body weights of 1500 and 5000 ppm male rats exposed to manganese (II) sulfate monohydrate were similar to those of controls throughout the 2-year study.
- mean body weights of 15000 ppm male rats were within 5% of that of controls until week 89. From week 89, the mean body weights ranged from 8% to 13% lower than that of controls; at the end of the 2-year study, the final mean body weight of 15,000 ppm males was 10% lower than that of controls.
- mean body weights of exposed females were similar to that of controls throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- feed consumption by exposed groups was similar to that by control groups
- rats exposed to 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate received approx. daily doses of 60, 200, or 615 mg/kg body weight (males) or 70, 230, or 715 mg/kg (females).

HAEMATOLOGICAL FINDINGS:
- values for hematology parameters were generally similar among exposed and control groups at the 9- and 15-month interim evaluations

CLINICAL BIOCHEMISTRY FINDINGS
- values for clinical chemistry parameters were generally similar among exposed and control groups at the 9- and 15-month interim evaluations.

ORGAN WEIGHTS FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS:
- at the 9- and 15-month interim evaluations, the absolute kidney weights of exposed rats were similar to those of the controls.

HISTOPATHOLOGCAL FINDINGS - non-neoplastic
- pancreas: hyperplasia of the pancreatic islets occurred in a few males in each of the exposure groups but not in the control group (hyperplasia: control, 0/52; 1,500 ppm, 2/50; 5,000 ppm, 2/51; 15,000 ppm, 3/51).

- kidney: chronic nephropathy occurred in all male rats examined at both interim evaluations and most of the control and exposed males at the end of the study (severity was slightly greater in the high-dose group).
The incidences of several lesions commonly associated with advanced nephropathy and renal failure were significantly increased in 15000 ppm male rats. These lesions included mineralization of blood vessels (4/52, 10/51, 6/51, 17/52), mineralization of the glandular stomach (8/52, 13/51, 9/51, 23/52), fibrous osteodystrophy of the femur (12/52, 14/51, 12/51, 24/52), and parathyroid gland hyperplasia (14/51, 14/46, 12/49, 23/50).

- adrenal gland: In females, medullary hyperplasia occurred with a significant negative trend and a significantly decreased incidence in the 15000 ppm
group (control 12/50, 1,500 ppm 11/50, 5,000 ppm 6/51, and 15,000 ppm 1/48). The incidence of medullary hyperplasia in exposed males was similar to that of the controls.

HISTOPATHOLOGCAL FINDINGS - neoplastic
- pancreas: adenoma of the pancreatic islets occurred in a few males in each of the exposure groups but not in the control group (adenoma: 0/52, 3/50, 4/51, 3/51). In addition, a carcinoma of the pancreatic islets was found in one 15000 ppm male. However, neither the trend test nor pairwise comparisons were significant, and the incidences in each of the dose groups were within the range of historical control groups (adenoma, 0% to 12%; carcinoma, 0% to 6%).
- adrenal gland: benign pheochromocytomas of the adrenal medulla in males occurred with a significant negative trend, but the decreases were not significant by pairwise comparison (14/52, 17/51, 14/51, and 6/52).

TISSUE METAL CONCENTRATION ANALYSES:
- at both the 9- and 15-month interim evaluations, a significantly increased manganese concentrations were observed in livers of the 5000 and 15000 ppm dose groups (males and females), accompanied by depression of hepatic iron
-significantly increased manganese concentrations were observed in brain and kidney (9-month interim evaluation) as well as in brain, kidney and pancreas (15-month interim evaluation) in males of the 15000 ppm dose group
- significantly increased copper concentrations were observed in kidneys of the 15000 ppm males (9-month interim evaluation) and in 15000 females (9- and 15-month interim evaluation)
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
After the oral administration of 1500, 5000, and 15000 ppm of manganese (II) sulfate monohydrate (actual dose received: 60, 200, and 615 mg/kg/day for males and 70, 230, and 715 mg/kg/day for females, respectively) survival of the 1500 and 5000 ppm males and all exposed groups of females was similar to that of controls. A significant lower survival of the 15000 ppm males occurred due to nephropathy and renal failure after week 93 of the study. Furthermore, no test item-related effects were found for clinical signs, body weight, food consumption, haematology (intrim sacrifices), clinical chemistry (intrim sacrifices), organ weights (intrim sacrifices), and histopathology.

At both the 9- and 15-month interim evaluations, a significantly increased manganese concentrations were observed in livers of the 5000 and 15000 ppm dose groups (males and females), accompanied by depression of hepatic iron. Also, a significant increase of manganese concentrations were observed in brain and kidney (9-month interim evaluation) as well as in brain, kidney and pancreas (15-month interim evaluation) in males of the 15000 ppm dose group. Lastly, a significant increased copper concentrations were observed in kidneys of the 15000 ppm males (9-month interim evaluation) and in 15000 females (9- and 15-month interim evaluation).

Based on the absence of any adverse effects related to the test substance, a No Observed Adverse Effect Level (NOAEL) for test item of 15000 ppm (actual dose received: 615 mg/kg/day for males and 715 mg/kg/day for females (200 and 233 mg Mn/kg/day, respectively) was concluded for male and female rats.
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-09-25 to 1986-10-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Neurological, optical and effects on urinalysis not examined. Food intake measured for a 7-day period every 4 weeks instead of weekly observation during first 13 weeks and then in 3-months intervals. Hematology and clinical chemistry scheduled for 9- and 15 months. Incomplete histopathology (aorta, accessory genital organs, musculature, peripheral nerve, spinal cord, eyes missing). Incomplete clinical chemistry (total protein, albumin, carbohydrate metabolism (i.e. blood glucose) missing).
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a repeated dose oral toxicity study manganese (II) sulfate monohydrate was administered via feed ad libitum to three groups, each of 70 male and 70 female B6C3F1 mice, for 103 weeks. The test item concentrations used were 1500, 5000, and 15000 ppm (actual dose received: 160, 540, and 1800 mg/kg/day for males and 200, 700, and 2250 mg/kg/day for females, respectively). A control group was run concurrently. Ten animals/sex/group were evaluated after 9 months and 15 months of test item exposure. The remaining animals were sacrificed at the end of the study. The following parameters were evaluated: clinical signs, survival, body weight, food consumption, haematology (intrim sacrifices only), clinical chemistry (intrim sacrifices only), organ weights (brain, kidney, and liver; intrim sacrifices only), macroscopic examination, and histopathology. Lastly, brain, kidneys, liver, and pancreas were examined for metal concentration of copper, iron, manganese, and zinc during intrim sacrifices.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in the dark at room temperature throughout the study
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: Frederick Cancer Research Facility (Frederick, MD)
- Age at study initiation: approx. 41 days
- Housing: housed 5 animals/cage (polycarbonate (Lab Products, Inc., Garfield, NJ); bedding material: BetaChips, hardwood chips (Northeastern Products, Inc., Warrensburg, NY); cage filters: spun-bonded DuPont 2024 polyester (Snow Filtration Co., Cincinnati, OH): racks: stainless steel (Lab Products, Inc., Garfield, NJ)
- Diet (ad libitum): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA); diet contains 60 g manganous oxide per 2000 lbs feed
- Water (ad libitum): automatic watering system (Edstrom Industries, Waterford, WI)
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 23.9 °C
- Relative humidity: 35 - 65 %
- Air changes: minimum of 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Details on route of administration:
not specified
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): dose formulations were prepared by mixing manganese (II) sulfate monohydrate with feed (NIH-07 open formula meal rat and mouse diet; diet contains 60 g manganous oxide per 2000 lbs feed). A premix of the test item and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes.
Dose formulations were discarded 21 days after the date of preparation.
- Storage temperature of food containing the test item: stored in plastic buckets with lids in the dark at 25 °C.

The level of manganese in the diet received by controls was approx. 92 ppm.
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. Homogeneity was confirmed; 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. A subsequent study confirmed the stability of the dose formulations for 3 weeks. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings support the conclusion that the manganese remained in divalent state.

Periodic analyses of the dose formulations were performed using spectrophotometric methods every two months during the 2-year study. All dose formulations were within the specified 10 % of the target concentrations throughout the studies. Results of periodic referee analyses were also within 10 % of the target concentrations.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
ad libitum
Dose / conc.:
1 500 ppm
Remarks:
actual dose received: 160 mg/kg/day for males and 200 mg/kg/day for females (51.2 and 64 mg Mn/kg/day, respectively)
Dose / conc.:
5 000 ppm
Remarks:
actual dose received: 540 mg/kg/day for males and 700 mg/kg/day for females (172.8 and 224 mg Mn/kg/day, respectively)
Dose / conc.:
15 000 ppm
Remarks:
actual dose received: 1800 mg/kg/day for males and 2250 mg/kg/day for females (576 and 720 mg Mn/kg/day, respectively)
No. of animals per sex per dose:
70 males / 70 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: selected doses for the 2 year study 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 13week study.
- Section schedule (intrim sacrifice): 10 mice/sex/group were evaluated after 9-months and 15 months of chemical exposure.
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; clinical findings were recorded weekly for the first 13 weeks and monthly thereafter.
- Cage side observations checked: survival and clinical findings

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly during first 13 weeks of study, monthly thereafter and at interim evaluations

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption measured for a 7-day period once every 4 weeks
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated: Yes

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
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: erythrocytes, hemoglobin, hematocrit, platelets, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, reticulocytes, nucleated erythrocytes, leukocyte count and differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, and creatinine

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE:
- intrim sacrifces were performed after 9 months and 15 months of chemical exposure (10 animals/sex/group).
- terminal sacrifice: at the end of study

GROSS PATHOLOGY: Yes
- necropsy was performed on all animals
- at necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 µm, and stained with hematoxylin and eosin for microscopic examination.
- organ weights: brain, kidneys, and liver from animals selected for the 9- and 15-month evaluations were weighed at necropsy.

HISTOPATHOLOGY: Yes
- complete histopathologic examinations were performed on control (0 ppm) and high-dose (15000 ppm) animals at the 9- and 15-month interim evaluations and gross lesions were examined for the low- and mid-dose groups (1500 and 5000 ppm) groups.
- complete histopathologic examinations were performed on all animals surviving until the end of the studies and on those that died or were killed moribund during the studies.
- the following tissues were examined during the histopathology: gross lesions, tissue masses, associated lymph nodes, adrenal gland, bone, bone marrow, brain, cecum, colon, rectum, oesophagus, gallbladder, heart, kidney, liver, lung, mandibular and mesenteric lymph nodes, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, skin, small intestine, spleen, stomach (forestomach and glandular), testes/epididymis, thyroid gland, trachea, thymus, urinary bladder, uterus.

TISSUE METAL CONCENTRATION ANALYSIS (intrim sacrifice after 9 months and 15 months only)
- tissues examined in metal concentration analyses for copper, iron, manganese, and zinc were brain, kidney, liver, and pancreas.
Other examinations:
SCREENING OF HEALTH
- serum samples for viral screening were collected from six male and five female mice and from five mice of each sex on two separate occasions prior to the start of the 2-year study.
- an additional collection was taken at 8 months into the study for screening of the mouse hepatitis virus only.
- serum samples were also collected from sentinel animals at 6, 12, and 18 months into the study, and from five male and five female animals in the 15,000 ppm group at the end of the study.
- Sera were processed appropriately and screened for the following: LCM, Mycoplasma arthridis, Mycoplasma pulmonis, MHV, PVM, Sendai, Ectromelia virus, GDVII, mouse adenoma virus, Reovirus 3, papovavirus, MVM, polyoma virus, epizootic siarrhea of infant mice (EDIM)
- results: EDIM was found in mice during the 6 month (5/10 animals), 12 month (4/9 amimals), 18 month (4/10 animals) and 24 month (6/10 animals examinations. In adiition, M. arthritidis was discovered in mice at the 24 month examinations (3/10 animals).
- before the studies began, five male and five female mice were selected for parasite evaluation and gross observation for evidence of disease.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- survival of exposed males and females was similar to that of the control (at study termination: males: 46/50 animals (control group), 44/50 animals (1500 ppm dose group), 46/50 animals (5000 ppm dose group), and 46/50 animals (15000 ppm); females: 42/50 animals (control group), 46/50 animals (1500 ppm dose group), 38/50 animals (5000 ppm dose group), 42/50 animals (15000 ppm dose group).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
females: final mean body weight for the 15000 ppm groups was 13% lower than that of the control group (decrease of equal to or greater than 10% started in week 86 and lasted until the end of the study).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- percent hematocrit, hemoglobin concentrations, and erythrocyte counts in 15000 ppm male mice at the 15-month interim evaluation were greater than those of the controls (significance uncertain; not observed in a 13 week study).
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Neuropathological findings:
not specified
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 15000 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 15000 ppm males and 5000 and 15000 ppm females. A significantly increased incidence of focal hyperplasia of follicular epithelium occurred in 15000 ppm males and in all exposed females.
Follicular dilatation at the 9-month evaluation was characterized by a uniform increase in the follicular diameter throughout the gland. Follicular dilatation in mice at the end of the study differed from that observed in mice at the 9-month interim evaluation in that the dilated follicles were limited to the periphery of the glands. The affected follicles contained pale eosinophilic colloid and were lined by a single layer of flat to slightly cuboidal follicular epithelial cells. Follicular cell hyperplasia and adenoma constitute a morphological continuum. Follicular cell hyperplasia consisted of single or multiple collections of variably sized follicles with irregular hypertrophy and increased cellularity of the follicular epithelium. Minimal to mild follicular cell hyperplasia consisted of one or several follicles lined by columnar epithelium with small and infrequent papillary infoldings. Moderate to marked hyperplasia involved clusters of variably sized follicles with more prominent papillary formations. Follicular cell adenomas were generally more discrete collections of altered follicles compressing the surrounding parenchyma.

- forestomach: a statistically significant increased incidence of focal squamous hyperplasia of the forestomach occurred in the 15000 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:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS:
- no clinical findings were attributed to the administration of manganese (II) sulfate monohydrate.

BODY WEIGHT AND WEIGHT CHANGES:
- 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
- in females, the final mean body weights for the 1500, and 5000 ppm groups were 6% and 9% lower than that of the control group, respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- feed consumption by exposed male and female mice was similar to that of the control groups
- mice exposed to 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate received approx. daily doses of 160, 540, or 1800 mg/kg body weight (males) or 200, 700, or 2,250 mg/kg body weight (females), respectively.

HAEMATOLOGICAL FINDINGS:
- no notable differences were observed in the haematology parameters

CLINICAL BIOCHEMISTRY FINDINGS
- no notable differences were observed in the clinical chemistry parameters.

ORGAN WEIGHTS FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS:
- liver: at the 9-month interim evaluation, absolute liver weights of 15000 ppm males and of 5000 and 15000 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.

HISTOPATHOLOGCAL FINDINGS - non-neoplastic
- liver: 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 foci in exposed females were similar to those of the controls

HISTOPATHOLOGCAL FINDINGS - neoplastic
- thyroid gland: follicular cell adenomas were found in three (6%) 15000 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 15000 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 15000 ppm males and females were not significantly greater than those of the controls.

- liver: one male in the 15000 ppm group and two females in the 5000 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. The incidences of adenoma in exposed females were similar to those of the controls

TISSUE METAL CONCENTRATION ANALYSES:
- at the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of the 5000 and 15000 ppm groups.
- hepatic iron levels were significantly lower in exposed females at the 9- and 15month interim evaluations and in 5000 and 15000 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.
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
histopathology: non-neoplastic
Remarks on result:
other: 5000 ppm is equivalent to the following actual doses received: 540 mg/kg/day for males and 700 mg/kg/day for females (172.8 and 224 mg Mn/kg/day, respectively
Critical effects observed:
not specified
Conclusions:
After the oral administration of 1500, 5000, and 15000 ppm of manganese (II) sulfate monohydrate (actual dose received: 160, 540, and 1800 mg/kg/day for males and 200, 700, and 2250 mg/kg/day for females, respectively) survival of exposed males and females was similar to that of the control and no clinical signs were attributed to the administration of the test substance. Furthermore, mean body weights of exposed males were similar to those of the control group. In female mice, mean body weights of all exposed groups were lower than that of the control after week 37 and the final mean body weights for the 1500, 5000, and 15000 ppm groups were 6 %, 9 %, and 13 % lower than that of the control. Food consumption of exposed male and female mice was similar to that of the control group. In addition, at the 9 month and 15 month intrim evaluation, no treatment-related effects were observed for clinical chemistry and organ weights. The haematological evaluation revealed that the percent hematocrit, hemoglobin concentrations, and erythrocyte counts in 15000 ppm male mice at the 15-month interim evaluation were greater than those of the controls. The significance of this finding is uncertain.

During the histopathology thyroid follicular dilatation and focal hyperplasia as well as focal hyperplasia in the forestomach were found and considered to be treatment-related findings. No significant increase in the incidence of neoplasia in mice could be found.

At the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of the 5000 and 15000 ppm groups. Hepatic iron levels were significantly lower in exposed females at the 9- and 15month interim evaluations and in 5000 and 15000 males at the 15-month interim evaluation. Lastly, 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.

Based on the histopathological findings in the thyroid (follicular dilatation and focal hyperplasia), decreased final body weight in females, and haematological findings in the male mice at the 15000 ppm dose level, a No Observed Adverse Effect Level (NOAEL) for test item of 5000 ppm (actual dose received: 540 mg/kg/day for males and 700 mg/kg/day for females (172.8 and 224 mg Mn/kg/day, respectively)) was concluded for male and female mice.
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
fertility, other
Remarks:
based on chronic oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-09-25 to 1986-10-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Neurological, optical and effects on urinalysis not examined. Food intake measured for a 7-day period every 4 weeks instead of weekly observation during first 13 weeks and then in 3-months intervals. Hematology and clinical chemistry scheduled for 9- and 15 months. Incomplete histopathology (aorta, accessory genital organs, musculature, peripheral nerve, spinal cord, eyes missing). Incomplete clinical chemistry (total protein, albumin, carbohydrate metabolism (i.e. blood glucose) missing).
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a repeated dose oral toxicity study manganese (II) sulfate monohydrate was administered via feed ad libitum to three groups, each of 70 male and 70 female B6C3F1 mice, for 103 weeks. The test item concentrations used were 1500, 5000, and 15000 ppm (actual dose received: 160, 540, and 1800 mg/kg/day for males and 200, 700, and 2250 mg/kg/day for females, respectively). A control group was run concurrently. Ten animals/sex/group were evaluated after 9 months and 15 months of test item exposure. The remaining animals were sacrificed at the end of the study. The following parameters were evaluated: clinical signs, survival, body weight, food consumption, haematology (interim sacrifices only), clinical chemistry (interim sacrifices only), organ weights (brain, kidney, and liver; interim sacrifices only), macroscopic examination, and histopathology. Lastly, brain, kidneys, liver, and pancreas were examined for metal concentration of copper, iron, manganese, and zinc during intrim sacrifices.
GLP compliance:
yes
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in the dark at room temperature throughout the study
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: Frederick Cancer Research Facility (Frederick, MD)
- Age at study initiation: approx. 41 days
- Housing: housed 5 animals/cage (polycarbonate (Lab Products, Inc., Garfield, NJ); bedding material: BetaChips, hardwood chips (Northeastern Products, Inc., Warrensburg, NY); cage filters: spun-bonded DuPont 2024 polyester (Snow Filtration Co., Cincinnati, OH): racks: stainless steel (Lab Products, Inc., Garfield, NJ)
- Diet (ad libitum): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA); diet contains 60 g manganous oxide per 2000 lbs feed
- Water (ad libitum): automatic watering system (Edstrom Industries, Waterford, WI)
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 23.9 °C
- Relative humidity: 35 - 65 %
- Air changes: minimum of 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): dose formulations were prepared by mixing manganese (II) sulfate monohydrate with feed (NIH-07 open formula meal rat and mouse diet; diet contains 60 g manganous oxide per 2000 lbs feed). A premix of the test item and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes.
Dose formulations were discarded 21 days after the date of preparation.
- Storage temperature of food containing the test item: stored in plastic buckets with lids in the dark at 25 °C.

The level of manganese in the diet received by controls was approx. 92 ppm.
Details on mating procedure:
not applicable
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. Homogeneity was confirmed; 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. A subsequent study confirmed the stability of the dose formulations for 3 weeks. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings support the conclusion that the manganese remained in divalent state.

Periodic analyses of the dose formulations were performed using spectrophotometric methods every two months during the 2-year study. All dose formulations were within the specified 10 % of the target concentrations throughout the studies. Results of periodic referee analyses were also within 10 % of the target concentrations.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
ad libitum
Details on study schedule:
not applicable
Dose / conc.:
1 500 ppm
Remarks:
actual dose received: 160 mg/kg/day for males and 200 mg/kg/day for females (51.2 and 64 mg Mn/kg/day, respectively)
Dose / conc.:
5 000 ppm
Remarks:
actual dose received: 540 mg/kg/day for males and 700 mg/kg/day for females (172.8 and 224 mg Mn/kg/day, respectively)
Dose / conc.:
15 000 ppm
Remarks:
actual dose received: 1800 mg/kg/day for males and 2250 mg/kg/day for females (576 and 720 mg Mn/kg/day, respectively)
No. of animals per sex per dose:
70 males / 70 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: selected doses for the 2 year study 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 13week study.
- Section schedule (intrim sacrifice): 10 mice/sex/group were evaluated after 9-months and 15 months of chemical exposure.
Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; clinical findings were recorded weekly for the first 13 weeks and monthly thereafter.
- Cage side observations checked: survival and clinical findings

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly during first 13 weeks of study, monthly thereafter and at interim evaluations

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption measured for a 7-day period once every 4 weeks
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated: Yes

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
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: erythrocytes, hemoglobin, hematocrit, platelets, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, reticulocytes, nucleated erythrocytes, leukocyte count and differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, and creatinine

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
not applicable
Postmortem examinations (parental animals):
SACRIFICE:
- intrim sacrifces were performed after 9 months and 15 months of chemical exposure (10 animals/sex/group).
- terminal sacrifice: at the end of study

GROSS PATHOLOGY: Yes
- necropsy was performed on all animals
- at necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 µm, and stained with hematoxylin and eosin for microscopic examination.
- organ weights: brain, kidneys, and liver from animals selected for the 9- and 15-month evaluations were weighed at necropsy.

HISTOPATHOLOGY: Yes
- complete histopathologic examinations were performed on control (0 ppm) and high-dose (15000 ppm) animals at the 9- and 15-month interim evaluations and gross lesions were examined for the low- and mid-dose groups (1500 and 5000 ppm) groups.
- complete histopathologic examinations were performed on all animals surviving until the end of the studies and on those that died or were killed moribund during the studies.
- the following tissues were examined during the histopathology: gross lesions, tissue masses, associated lymph nodes, adrenal gland, bone, bone marrow, brain, cecum, colon, rectum, oesophagus, gallbladder, heart, kidney, liver, lung, mandibular and mesenteric lymph nodes, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, skin, small intestine, spleen, stomach (forestomach and glandular), testes/epididymis, thyroid gland, trachea, thymus, urinary bladder, uterus.
Postmortem examinations (offspring):
not applicable
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- survival of exposed males and females was similar to that of the control (at study termination: males: 46/50 animals (control group), 44/50 animals (1500 ppm dose group), 46/50 animals (5000 ppm dose group), and 46/50 animals (15000 ppm); females: 42/50 animals (control group), 46/50 animals (1500 ppm dose group), 38/50 animals (5000 ppm dose group), 42/50 animals (15000 ppm dose group).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
females: final mean body weight for the 15000 ppm groups was 13% lower than that of the control group (decrease of equal to or greater than 10% started in week 86 and lasted until the end of the study).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
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 15000 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 15000 ppm males and 5000 and 15000 ppm females. A significantly increased incidence of focal hyperplasia of follicular epithelium occurred in 15000 ppm males and in all exposed females.
Follicular dilatation at the 9-month evaluation was characterized by a uniform increase in the follicular diameter throughout the gland. Follicular dilatation in mice at the end of the study differed from that observed in mice at the 9-month interim evaluation in that the dilated follicles were limited to the periphery of the glands. The affected follicles contained pale eosinophilic colloid and were lined by a single layer of flat to slightly cuboidal follicular epithelial cells. Follicular cell hyperplasia and adenoma constitute a morphological continuum. Follicular cell hyperplasia consisted of single or multiple collections of variably sized follicles with irregular hypertrophy and increased cellularity of the follicular epithelium. Minimal to mild follicular cell hyperplasia consisted of one or several follicles lined by columnar epithelium with small and infrequent papillary infoldings. Moderate to marked hyperplasia involved clusters of variably sized follicles with more prominent papillary formations. Follicular cell adenomas were generally more discrete collections of altered follicles compressing the surrounding parenchyma.

- forestomach: a statistically significant increased incidence of focal squamous hyperplasia of the forestomach occurred in the 15000 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:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
CLINICAL SIGNS:
- no clinical findings were attributed to the administration of manganese (II) sulfate monohydrate.

BODY WEIGHT AND WEIGHT CHANGES:
- 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
- in females, the final mean body weights for the 1500, and 5000 ppm groups were 6% and 9% lower than that of the control group, respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- feed consumption by exposed male and female mice was similar to that of the control groups
- mice exposed to 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate received approx. daily doses of 160, 540, or 1800 mg/kg body weight (males) or 200, 700, or 2,250 mg/kg body weight (females), respectively.

HAEMATOLOGICAL FINDINGS:
- percent hematocrit, hemoglobin concentrations, and erythrocyte counts in 15000 ppm male mice at the 15-month interim evaluation were greater than those of the controls (significance uncertain; not observed in a 13 week study).
- no other notable differences were observed in the haematology parameters

CLINICAL BIOCHEMISTRY FINDINGS
- no notable differences were observed in the clinical chemistry parameters.

ORGAN WEIGHTS FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS:
- liver: at the 9-month interim evaluation, absolute liver weights of 15000 ppm males and of 5000 and 15000 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.

HISTOPATHOLOGCAL FINDINGS - non-neoplastic
- liver: 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 foci in exposed females were similar to those of the controls

HISTOPATHOLOGCAL FINDINGS - neoplastic
- thyroid gland: follicular cell adenomas were found in three (6%) 15000 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 15000 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 15000 ppm males and females were not significantly greater than those of the controls.

- liver: one male in the 15000 ppm group and two females in the 5000 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. The incidences of adenoma in exposed females were similar to those of the controls
Dose descriptor:
NOAEL
Remarks:
(reproduction toxicity)
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
(general toxicity)
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
histopathology: non-neoplastic
Remarks on result:
other: 5000 ppm is equivalent to the following actual doses received: 540 mg/kg/day for males and 700 mg/kg/day for females (172.8 and 224 mg Mn/kg/day, respectively
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
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
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
not applicable
Generation:
F1
Remarks on result:
not measured/tested
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
NOAEL (fertility; male and female mice) > 15000 ppm (actual dose received: 1800 mg/kg/day for males and 2250 mg/kg/day for females (576 and 720 mg Mn/kg/day, respectively))

Histopathological examination performed on mammary gland, ovary, prostate gland, testes/epididymis, and uterus did not reveal any test item-related effects.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a repeated dose oral toxicity study manganese (II) sulfate monohydrate was administered via feed ad libitum to three groups, each of 70 male and 70 female F344/N rats, for 103 weeks. The test item concentrations used were 1500, 5000, and 15000 ppm (actual dose received: 60, 200, and 615 mg/kg/day for males and 70, 230, and 715 mg/kg/day for females, respectively). A control group was run concurrently. Ten animals/sex/group were evaluated after 9 months and 15 months of test item exposure. The remaining animals were sacrificed at the end of the study. The following parameters were evaluated: clinical signs, survival, body weight, food consumption, haematology (interim sacrifices only), clinical chemistry (interim sacrifices only), organ weights (brain, kidney, and liver; intrim sacrifices only), macroscopic examination, and histopathology. Lastly, blood plasma, brain, kidneys, liver, and pancreas were examined for metal concentration of copper, iron, manganese, and zinc during intrim sacrifices.
GLP compliance:
yes
Limit test:
no
Justification for study design:
not applicable

Test material

Constituent 1
Chemical structure
Reference substance name:
manganese(2+);sulfate;hydrate
Cas Number:
10034-96-5
Molecular formula:
MnSO4 H2O
IUPAC Name:
manganese(2+);sulfate;hydrate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Manganese (II) sulfate monohydrate (produced by J.T. Baker Chemical Company, Glen Ellyn, IL)
- Physical state: white, slightly efflorescent crystalline compound
- Analytic purity: 97.7 % ± 0.4 % (Chelometric titration); overall purity > 97 % (elemental analyses, weight loss on drying, chelometric titration, spark source mass spectometry)(no manganate (VI) or permanganate (VII) were identified by infrared and ultraviolet/visible spectroscopy)
- Impurities (identity and concentrations): sodium (640 ppm), potassium (120 ppm), silicon (160 ppm)
- Lot No.: 003261
- Periodic reanalyses of the bulk chemical: no degradation of the bulk chemical was detected
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in the dark at room temperature throughout the study

Test animals

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: Frederick Cancer Research Facility (Frederick, MD)
- Age at study initiation: approx. 41 days
- Housing: housed 5 animals/cage (polycarbonate (Lab Products, Inc., Garfield, NJ); bedding material: BetaChips, hardwood chips (Northeastern Products, Inc., Warrensburg, NY); cage filters: spun-bonded DuPont 2024 polyester (Snow Filtration Co., Cincinnati, OH): racks: stainless steel (Lab Products, Inc., Garfield, NJ)
- Diet (ad libitum): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA); diet contains 60 g manganous oxide per 2000 lbs feed
- Water (ad libitum): automatic watering system (Edstrom Industries, Waterford, WI)
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 23.9 °C
- Relative humidity: 35 - 65 %
- Air changes: minimum of 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): dose formulations were prepared by mixing manganese (II) sulfate monohydrate with feed (NIH-07 open formula meal rat and mouse diet; diet contains 60 g manganous oxide per 2000 lbs feed). A premix of the test item and feed was prepared by blending with a spatula; premix and remainder of feed was layered in a Patterson-Kelley twin-shell blender and mixed for 15 minutes with an intensifier bar on for the first 5 minutes.
Dose formulations were discarded 21 days after the date of preparation.
- Storage temperature of food containing the test item: stored in plastic buckets with lids in the dark at 25 °C.

The level of manganese in the diet received by controls was approx. 92 ppm.
Details on mating procedure:
not applicable
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. Homogeneity was confirmed; 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. A subsequent study confirmed the stability of the dose formulations for 3 weeks. No direct speciation was performed. However, complete recovery from dose formulations was achieved and other likely species are not soluble in dilute acid which was used for extraction. These findings support the conclusion that the manganese remained in divalent state.

Periodic analyses of the dose formulations were performed using spectrophotometric methods every two months during the 2-year study. All dose formulations were within the specified 10 % of the target concentrations throughout the studies. Results of periodic referee analyses were also within 10 % of the target concentrations.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
ad libitum
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 ppm
Remarks:
actual dose received: 60 mg/kg/day for males and 70 mg/kg/day for females (20 and 23 mg Mn/kg/day, respectively)
Dose / conc.:
5 000 ppm
Remarks:
actual dose received: 200 mg/kg/day for males and 230 mg/kg/day for females (65 and 75 mg Mn/kg/day, respectively)
Dose / conc.:
15 000 ppm
Remarks:
actual dose received: 615 mg/kg/day for males and 715 mg/kg/day for females (200 and 233 mg Mn/kg/day, respectively)
No. of animals per sex per dose:
70 males / 70 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on decreases in body weight gain and the lower absolute and relative liver weights in the 25000 ppm groups in the 13-week study, doses of 0, 1500, 5000 and 15000 ppm were selected.
- Section schedule (intrim sacrifice): 10 rats/sex/group were evaluated after 9-months and 15 months of chemical exposure.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; clinical findings were recorded weekly for the first 13 weeks and monthly thereafter.
- Cage side observations checked: survival and clinical findings

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly during first 13 weeks of study, monthly thereafter and at interim evaluations

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption measured for a 7-day period once every 4 weeks
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated: Yes

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
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: erythrocytes, hemoglobin, hematocrit, platelets, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, reticulocytes, nucleated erythrocytes, leukocyte count and differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the 9- and 15-month interim evaluation
- Animals fasted: No
- How many animals: all intrim animals
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, creatinine

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
not applicable
Postmortem examinations (parental animals):
SACRIFICE:
- intrim sacrifce were performed after 9 months and 15 months of chemical exposure (10 animals/sex/group).
- terminal sacrifice: at the end of study

GROSS PATHOLOGY: Yes
- necropsy was performed on all animals
- at necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 µm, and stained with hematoxylin and eosin for microscopic examination.
- organ weights: brain, kidneys, and liver from animals selected for the 9- and 15-month evaluations were weighed at necropsy.

HISTOPATHOLOGY: Yes
- complete histopathologic examinations were performed on control (0 ppm) and high-dose (15000 ppm) animals at the 9- and 15-month interim evaluations and gross lesions were examined for the low- and mid-dose groups (1500 and 5000 ppm) groups.
- complete histopathologic examinations were performed on all animals surviving until the end of the studies and on those that died or were killed moribund during the studies.
- the following tissues were examined during the histopathology: gross lesions, tissue masses, associated lymph nodes, adrenal gland, bone, bone marrow, brain, cecum, colon, rectum, oesophagus, heart, kidney, liver, lung, mandibular and mesenteric lymph nodes, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, skin, small intestine, spleen, stomach (forestomach and glandular), testes/epididymis, thyroid gland, trachea, thymus, urinary bladder, uterus.
Postmortem examinations (offspring):
not applicable
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- significant lower survival of males in the 15000 ppm dose group occuring after week 93 of the study, due to nephropathy and renal failure
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Details on results (P0)

CLINICAL SIGNS
- no clinical findings were chemical-related.

MORTALITY
- survival of 1500 and 5000 ppm males and all exposed groups of females was similar to that of controls.

BODY WEIGHT
- mean body weights of 1500 and 5000 ppm male rats exposed to manganese (II) sulfate monohydrate were similar to those of controls throughout the 2-year study.
- mean body weights of 15000 ppm male rats were within 5% of that of controls until week 89. From week 89, the mean body weights ranged from 8% to 13% lower than that of controls; at the end of the 2-year study, the final mean body weight of 15,000 ppm males was 10% lower than that of controls.
- mean body weights of exposed females were similar to that of controls throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- feed consumption by exposed groups was similar to that by control groups
- rats exposed to 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate received approx. daily doses of 60, 200, or 615 mg/kg body weight (males) or 70, 230, or 715 mg/kg (females).

HAEMATOLOGICAL FINDINGS:
- values for hematology parameters were generally similar among exposed and control groups at the 9- and 15-month interim evaluations

CLINICAL BIOCHEMISTRY FINDINGS
- values for clinical chemistry parameters were generally similar among exposed and control groups at the 9- and 15-month interim evaluations.

ORGAN WEIGHTS FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS:
- at the 9- and 15-month interim evaluations, the absolute kidney weights of exposed rats were similar to those of the controls.

HISTOPATHOLOGCAL FINDINGS - non-neoplastic
- pancreas: hyperplasia of the pancreatic islets occurred in a few males in each of the exposure groups but not in the control group (hyperplasia: control, 0/52; 1,500 ppm, 2/50; 5,000 ppm, 2/51; 15,000 ppm, 3/51).

- kidney: chronic nephropathy occurred in all male rats examined at both interim evaluations and most of the control and exposed males at the end of the study (severity was slightly greater in the high-dose group).
The incidences of several lesions commonly associated with advanced nephropathy and renal failure were significantly increased in 15000 ppm male rats. These lesions included mineralization of blood vessels (4/52, 10/51, 6/51, 17/52), mineralization of the glandular stomach (8/52, 13/51, 9/51, 23/52), fibrous osteodystrophy of the femur (12/52, 14/51, 12/51, 24/52), and parathyroid gland hyperplasia (14/51, 14/46, 12/49, 23/50).

- adrenal gland: In females, medullary hyperplasia occurred with a significant negative trend and a significantly decreased incidence in the 15000 ppm
group (control 12/50, 1,500 ppm 11/50, 5,000 ppm 6/51, and 15,000 ppm 1/48). The incidence of medullary hyperplasia in exposed males was similar to that of the controls.

HISTOPATHOLOGCAL FINDINGS - neoplastic
- pancreas: adenoma of the pancreatic islets occurred in a few males in each of the exposure groups but not in the control group (adenoma: 0/52, 3/50, 4/51, 3/51). In addition, a carcinoma of the pancreatic islets was found in one 15000 ppm male. However, neither the trend test nor pairwise comparisons were significant, and the incidences in each of the dose groups were within the range of historical control groups (adenoma, 0% to 12%; carcinoma, 0% to 6%).
- adrenal gland: benign pheochromocytomas of the adrenal medulla in males occurred with a significant negative trend, but the decreases were not significant by pairwise comparison (14/52, 17/51, 14/51, and 6/52).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(reproduction toxicity)
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
(general toxicity)
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
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
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

not applicable

Effect levels (F1)

Generation:
F1
Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (fertility; male and female rats) > 15000 ppm (actual dose received: 1800 mg/kg/day for males and 2250 mg/kg/day for females (576 and 720 mg Mn/kg/day, respectively))

Histopathological examination performed on mammary gland, ovary, prostate gland, testes/epididymis, and uterus did not reveal any test item-related effects.