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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:
1984-09-25 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).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and according to peer reviewed methods.
Qualifier:
according to
Guideline:
other: NTP peer reviewed methods
Principles of method if other than guideline:
Male and Female mice received diets containing varying concentrations of MnSO4 for 103 weeks. Groups of 9 or 10 animals were evaluated after 9 and 15 months of chemical exposure.
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Mice were approximately 41 days old at the beginning of the study.
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 1500, 5000, 15000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
70 male and 70 female per dose group.
Control animals:
yes
Observations and examinations performed and frequency:
All animals were observed twice daily. Clinical findings were recorded weekly for the first 13 weeks and monthly thereafter.


Sacrifice and pathology:
All animals were necropsied. At necropsy all organs were examined for gross lesions and all major tissues were fixed and preserved for histopathological examinations.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified

CLINICAL SIGNS AND MORTALITY: Survival rats of exposed mice were similar to that of the controls. No clinical findings were attributed to the administration of MnSO4.

BODY WEIGHT AND WEIGHT GAIN: The mean bodyweights of exposed male mice were similar to that of the controls. The final mean body weights for the 1500, 5000 and 15000 groups were 6, 9 and 13% lower than the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption by all groups was similar to that of the controls.

HAEMATOLOGY AND CLINICAL CHEMISTRY: No chemical-related differences between exposed and control groups occurred in haematology or clinical chemistry parameters.

TISSUE CONCENTRATIONS OF MANGANESE: Concentrations of manganese were significantly elevated in the livers of mice exposed to 5000 and 15000ppm at the 9 and 15 month evaluations. Hepatic iron levels were significantly lower in exposed females at the 9 month evaluation and in 5000 and 15000 ppm males and all exposed females at the 15 month evaluation.

GROSS PATHOLOGY: Incidences of thyroid follicular dilatation and hyperplasia were significantly greater in 15000 ppm exposed mice than in the controls. Follicular cell adenomas occurred in one 15000ppm male at the 15 month evaluation and in three 15000 ppm males at the end of the study.

Follicular cell adenomas also occurred in two control, one 1500 and five 15000 ppm female mice at the end of the study. It is uncertain if the slightly increased incidence of follicular cell adenoma is related to the ingestion of MnSO4.

OTHER FINDINGS: 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.

Conclusions:
No clinical findings were attributed to MnSO4 treatment.
Reason / purpose:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and according to peer reviewed methods.
Qualifier:
according to
Guideline:
other: NTP peer reviewed methods
Principles of method if other than guideline:
Groups of 10 male and 10 female rats were fed diets containing 0, 1600, 3130, 6250, 12500 or 25000 ppm manganese sulphate. Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses. A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.
GLP compliance:
yes
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Stone Ridge, NY)
- Age at study initiation: 50 days



Route of administration:
oral: feed
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 1600, 3130, 6250, 12500 or 25000 which was equivalent to doses from 110 to 1700 mg/kg in males and 115 to 2000 mg/kg in females
Basis:
nominal in diet
No. of animals per sex per dose:
10 male and 10 female per dose group
Control animals:
yes
Observations and examinations performed and frequency:
Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses.
Sacrifice and pathology:
A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
-BODY WEIGHT AND WEIGHT GAIN: The mean bodyweight gain in males receiving 3130 ppm was marginally lower than that of the controls and was significantly lower in the three highest female dose groups than the controls.
-FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption by exposed rats was similar to that of the controls. Females ingested an average of 20% more manganese sulphate than males in the corresponding exposure groups.
-HAEMATOLOGY: Neutrophil counts were significantly higher in all exposed male groups. Lymphocyte counts were significantly lower in the three highest dose groups.
In females: leukocyte counts were significantly lower in the three highest dose groups.
A significant increase in the percent haematocrit and erythrocyte counts occurred in males exposed to the three highest dose levels.
-ORGAN WEIGHTS: Absolute and relative liver weights of all exposed males and of the female 25000 ppm group were significantly lower than the controls.
Dose descriptor:
NOAEL
Effect level:
1 700 mg/kg bw/day (nominal)
Based on:
other: food intake and concentration in food.
Sex:
male
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
other:
Sex:
female
Critical effects observed:
not specified

No clear relationship between observed differences and the ingestion of manganese sulphate has been defined.

Table 1.Survival, Body Weights, and Feed Consumption of Rats in the Rats in the 13-Week Feed Study of Manganese (II) Sulphate Monohydrate

 

Concentration (ppm)

Survivala

Mean body weight and weight changesbrelative

Final Weight Feed to controls (%)

Consumptionc

Initial

Final

Change

Week 1

Week 13

 

Male

 

 

 

 

 

 

 

0

10/10

136±5

291±4

155±4

 

14.9

13.1

1, 600

10/10

142±4

294±5

152±4

101

14.5

13.5

3,130

10/10

149±3

291±4

141±4

100

14.8

13.6

6, 250

10/10

148±2

294±3

146±3

101

15.0

9.6

12, 500

10/10

150±11

290±6

140±11

99

14.9

14.9

25, 000

10/10

140±4

284±6

144±4

97

14.1

14.4

Female

 

 

 

 

 

 

 

0

10/10

99±1

184±2

84±2

 

10.7

9.2

1, 600

10/10

103±1

181±2

79±2

99

10.8

9.3

3,130

10/10

96±1

175±2*

80±3

95

10.9

9.2

6, 250

10/10

101±1

176±2*

75±1**

96

10.7

14.3

12, 500

10/10

106±1**

178±1*

73±2**

97

10.7

10.5

25, 000

10/10

104±1**

174±3**

70±2**

95

12.1

10.3

* Significantly different (P≤0.05) from the control group by Williams’ or Dunnett’s test

** P≤0.01

a Number of animals surviving at 13 weeks/ number initially in group

b Weight given as mean ± standard error

c Feed consumption is expressed as grams per animal per day

 

Conclusions:
The high level of MnSO4 consumed on a daily basis for 13 weeks in this study, without mortality, supports the lack of acute toxicity. Although some changes in lung weight and certain haematological parameters were significant compared to controls, the lack of clinical and histopathological findings despite the very high daily oral dose over a sub-chronic period, is an indication of the relatively low toxicity of MnSO4, at least for the parameters studied in this report.
Reason / purpose:
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:
1984-09-11 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:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
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 (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
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 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
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
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
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
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
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
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
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).
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
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 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.

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 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
mouse
Strain:
B6C3F1
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals 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

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
Details on study schedule:
not applicable
Doses / concentrationsopen allclose all
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

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:
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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
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

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 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

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)
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

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 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.