Sodium permanganate

Carcinogenicity

Description of key information

WoE Read-Across Source MnSO4: NTP (1993) - Rat

There was no evidence of carcinogenic activity in rats fed diets containing up to 15000 ppm managanese (II) sulfate monohydrate for 2 years.

 

WoE Read-Across Source MnSO4: NTP (1993) - Mouse

There was equivocal evidence of carcinogenic activity of manganese (II) sulfate monohydrate in male and female B6C3F1 mice, based on the marginally increased incidences of thyroid gland follicular cell adenoma and the significantly increased incidences of follicular cell hyperplasia.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please see the read across justification.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

other:

Critical effects observed:

not specified

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 September 1984 to 25 September 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted by the National Toxicology Program

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: NTP guideline

Principles of method if other than guideline:

Assessment of carcinogenicity

GLP compliance:

yes

Remarks:

FDA

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were male and female F344/N rats obtained from Frederick Cancer Research Facility (Frederick, MD). They were quarantined for 12 days prior to exposure. Before the beginning of the studies, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease. Serology samples were collected for viral screening. The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program. The rats were approximately 41 days old at the start of the study.
Rats were housed 5 per cage, individuals were identified by toe markp. Feed (NIH-07 open formula meal rat and mouse diet, Ziegler Brothers, Inc.) and water were available ad libitum. The rats were housed in polycarbonate cages (Lab Products, Inc.) on stainless steel racks, with hardwood chips as bedding (BetaChips). Cages were rotated every 2 weeks; racks were rotated every 2 weeks.
The temperature of the animal room was 20.6 to 23.9°C, the relative humidity was 35-65%. Fluorescent lighting was provided for 12 hours/day, and there were a minimum of 10 air changes per hour.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

A premix with manganese (II) sulfate monohydrate 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 prepared once.
Groups of 70 male and 70 female rats were fed diets containing 0, 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate. The level of manganese in the diet received by controls was approximately 92 ppm. The appropriate feed was supplied twice weekly and was available ad libitum.

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 by the analytical chemistry laboratory 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 under the conditions listed above. 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 strongly support the conclusion that the manganese remained in the divalent state. The dose formulations were prepared weekly. Dose formulations were discarded 21 days after the date of preparation.
Periodic analyses of the dose formulations of manganese (II) sulfate monohydrate were conducted at the study laboratory and at the analytical chemistry laboratory using spectrophotometric methods. Dose formulations were analyzed three times during the study. All dose formulations were within the specified 10% of the target concentration.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

Daily - ad libitum in diet

Post exposure period:

There was no post exposure period

Remarks:

Doses / Concentrations:
0, 1500, 5000 and 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

70 rats/sex/dose

Control animals:

yes, plain diet

yes, historical

Details on study design:

Animals were assigned to treatment groups by weight intervals. Animals from each interval were randomized and proportionately assigned to cages, then the cages were assigned to dose groups using an appropriate table of random numbers.
Dose selection was based on the results from a 13 week study. Decreases in body weight gain and the absolute and relative liver weights were seen in the 25000 ppm groups, therefore dose levels selected for the 2 year study were 0, 1500, 5000 and 15000 ppm.
Interim sacrifices were carried out at months 9 and 15.

Positive control:

A positive control was not included.

Observations and examinations performed and frequency:

Rats were observed twice daily. Clinical observations and animal weights were recorded initially, weekly during the first 13 weeks of the study, then monthly thereafter and at interim evaluations. Feed consumption was measured for a 7 day period one every 4 weeks.
Blood was collected at the 9 and 15 month interim evaluations for haematology and clinical chemistry determinations. Terminal blood samples were also collected. Haematology parameters evaluated were: erythrocytes, haemoglobin, haematocrit, platelets, mean erythrocyte volume, mean erythrocyte haemoglobin, mean erythrocyte haemoglobin concentration, reticulocytes, nucleated erythrocytes, and leukocyte count and differential. Clinical chemistry parameters evaluated were: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, and creatinine.

Sacrifice and pathology:

Interim sacrifices were carried out after 9 and 15 months. Organs weighed at the interim evaluations were brain, kidneys and liver. Terminal sacrifice was carried out after 103 weeks exposure. At necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 µm, and stained with
haematoxylin and eosin for microscopic examination.
Complete histopathologic examinations were performed on all 0 and 15000 ppm animals at the 9- and 15-month interim evaluations and gross lesions examined for the 1500 and 5000 ppm groups. Complete histopathologic examinations were performed on all animals at the end of the studies and on all animals that died or were killed moribund during the studies. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, bone, bone marrow, brain, cecum, colon and rectum, 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, thymus, thyroid gland, trachea, uterus, and urinary bladder.

Other examinations:

Samples of blood plasma, kidneys, liver and pancreas were collected 9 and 15 month interim evaluations for tissue metal concentration analyses (manganese, iron, copper, and zinc).

Statistics:

The probability of survival was estimated by the product limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related
effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. Logistic regression analysis was used to assess the incidence of neoplasms. Pairwise comparisons and were used to identify differences between control and treated groups. The significance of pairwise comparisons was determined according to the methods of Dunnett (1955), Williams (1971, 1972), Shirley (1977), Dunn (1964), Jonckheeres test (1954) or the Mann-Whitney U test.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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:

effects observed, treatment-related

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Survival of 15000 ppm male rats was significantly lower than that of the controls; survival of 1500 and 5000 ppm males and all exposed groups of females was similar to that of controls. The significant reduction in survival of 15000 ppm males was attributed to increased severity of nephropathy and renal failure. The decreased survival did not occur until approximately week 93 of the study.
The 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. The 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 15000 ppm males was 10% lower than that of controls. Mean body weights of exposed females were similar to that of controls throughout the study. 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 approximate daily doses of 60, 200, or 615 mg/kg body weight (males) or 70, 230, or 715 mg/kg (females). No clinical findings were chemical related.
Values for haematology and clinical chemistry parameters were generally similar among exposed and control groups at the 9- and 15-month interim evaluations. At both the 9- and 15-month interim evaluations, the manganese levels in the liver of 5000 and 15000 ppm males and females were significantly greater than those in controls. The hepatic iron concentrations for these exposure groups were lower than for controls. The concentrations of manganese in the brain, kidney, and pancreas of exposed and control rats were variable; 15000 ppm males had a significantly higher
concentration of manganese in the brain and kidney at the 9-month interim evaluation and in the brain, kidney, and pancreas at the 15-month interim evaluation. Copper levels in the kidney of 15000 ppm males at 9 months and in 15000 ppm females at 9 and 15 months were significantly greater than those of the controls.
No chemical-related lesions were observed at the 9- or 15-month interim evaluations.
Hyperplasia or adenoma 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; 1500 ppm, 2/50; 5000 ppm, 2/51; 15000 ppm, 3/51; 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 NTP historical control groups (adenoma, 0% to 12%; carcinoma, 0% to 6%).
At the 9- and 15-month interim evaluations, the absolute kidney weights of exposed rats were similar to those of the controls. 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 (Table 1). The average severity of nephropathy was slightly greater in the high-dose group, but the difference was not statistically significant. Because of the subjective nature of the severity grading, an additional evaluation of the kidney of high-dose and control male rats was performed without knowledge of the previous diagnoses. The result of the additional evaluation confirmed the presence of a marginally increased severity of nephropathy in the high-dose group, and the difference was significant (P=0.04) by a two-sided MannWhitney U test. The severity of nephropathy varied from minimal to marked. Minimal nephropathy was characterized by a few sparsely scattered cortical foci of regenerating tubules with increased epithelial cytoplasmic basophilia and slightly thickened glomerular basement membranes. Nephropathy of mild severity had similar morphologic features, but these features occurred with greater frequency. Also present were occasional dilated tubules filled with homogenous hyaline material and lined by flattened epithelial cells. Nephropathy of moderate to marked severity had similar but more severe and extensive tubule lesions. In addition, variable interstitial fibrosis and mineralization with mononuclear leukocyte infiltration, variable tubule loss and atrophy, and degenerative glomerular changes occurred. In the most severe cases, cystic tubules lined by cuboidal or attenuated epithelial cells were present.
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).
In females, adrenal gland medullary hyperplasia occurred with a significant negative trend and a significantly decreased incidence in the 15000 ppm group (control 12/50, 1500 ppm 11/50, 5000 ppm 6/51 and 15000 ppm. 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) the incidence of medullary
hyperplasia in exposed males was similar to that of the controls.

Relevance of carcinogenic effects / potential:

There was no evidence of carcinogenic activity in rats.

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

other: No evidence of carcinogenicity at the highest dose level.

Critical effects observed:

not specified

Incidence and severity of nephropathy

Dose (ppm)	0	1500	5000	15000
Males
9 month interim evaluation
Kidney^a	8	10	10	10
Nephropathy^b	8 (1.3)^c	10 (1.1)	10 (1.1)	10 (1.3)
15 month interim evaluation
Kidney	10	9	9	8
Nephropathy	10 (1.6)	9 (1.9)	9 (1.9)	8 (2.0)
2 year study
Kidney	52	50	51	52
Nephropathy (initial evaluation)	50 (2.9)	49 (3.0)	51 (3.0)	50 (3.2)
Nephropathy (additional evaluation)	52 (2.8)			51 (3.1)*
Females
2 year study
Kidney	50	50	51	48
Nephropathy	48 (1.8)	50 (1.5)	49 (1.7)	48 (1.9)

* Significantly different (P is less than or equal to 0.05) from the control group by two-sided Mann-Whitney U test

a Number of animals with organ examined microscopically

b Number of animals with lesion

c Average severity grade of lesions in all animals (0=normal; 1=minimal; 2=mild; 3=moderate; 4=marked)

Conclusions:

There was no evidence of carcinogenic activity in rats fed diets containing up to 15000 ppm managanese (II) sulfate monohydrate for 2 years.

Executive summary:

Groups of 70 male and 70 female F344/N rats were fed diets containing 0, 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate for 2 years, to determine the carcinogenic potential of the substance. Based on average daily feed consumption, these doses resulted in the daily ingestion of 60, 200, or 615 mg/kg body weight (males) or 70, 230, or 715 mg/kg (females). Intermin sacrifices were carried out on groups of 8 to 10 rats at 9 and 15 months.

Survival of 15000 ppm male rats in the 2-year study was significantly lower than that of the control group. The deaths of males in the control and exposure groups were attributed to a variety of spontaneous neoplastic and nonneoplastic lesions; however, the greater number of deaths in the 15000 ppm group resulted from increased incidences of advanced renal disease related to ingestion of manganese (II) sulfate monohydrate. The decreased survival of the 15000 ppm males did not occur until approximately week 93 of the study; before week 93, survival was similar in all groups. Survival of exposed females was similar to that of the controls. The mean body weight of 15000 ppm male rats was within 5% of the control group until week 89; by week 104, the mean body weight of 15000 ppm males was 10% lower than that of the control group. The mean body weights of 1500 and 5000 ppm male rats and all exposed female groups were similar to those of the controls throughout the study. Feed consumption by all exposure groups was similar to that by the control groups. No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion.

The ingestion of diets containing 15000 ppm manganese (II) sulfate monohydrate was associated with a marginal increase in the average severity of nephropathy in male rats (0 ppm, 2.9; 1500 ppm, 3.0; 5000 ppm, 3.0; 15000 ppm, 3.2). The increased severity of nephropathy in the 15000 ppm male rats was accompanied by significantly increased incidences of mineralization of the blood vessels and glandular stomach, parathyroid gland hyperplasia, and fibrous osteodystrophy of the femur. These lesions are manifestations of renal failure, uremia, and secondary hyperparathyroidism. The increased incidence of advanced renal disease caused reduced survival of the high-dose male rats. No increase in the incidence of neoplasms in male or female rats was attributed to the ingestion of diets containing manganese (II) sulfate monohydrate. Under the conditions of the study, there was no evidence of carcinogenic activity (according to the NTP Levels of Evidence of Carcinogenic Activity) of manganese (II) sulfate monohydrate in male or female rats.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please see the read across justification.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Sex:

male/female

Basis for effect level:

other:

Critical effects observed:

not specified

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

8 October 1984 to 10 October 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted by the National Toxicology Program

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

other: NTP protocol

Principles of method if other than guideline:

Lifetime carcinogenicity assay

GLP compliance:

yes

Remarks:

FDA

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were male and female B6C3F1 mice, obtained from Frederick Cancer Research Facility (MD). Mice were quarantined for 13 days prior to exposure. Before the beginning of the studies, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Serology samples were collected for viral screening. The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program. Mice were approximately 41 days old at the beginnning of the study.
Mice were housed 5 per cage, individuals were identified by toe mark. Feed (NIH-07 open formula meal rat and mouse diet, Ziegler Brothers, Inc.) and water were available ad libitum. The mice were housed in polycarbonate cages (Lab Products, Inc.) on stainless steel racks, with hardwood chips (BetaChips)as bedding. Cages were rotated every 2 weeks; racks were rotated every 2 weeks.
The temperature of the animal room was 20.6 to 23.9°C, the relative humidity was 35-65%. Fluorescent lighting was provided for 12 hours/day, and there were a minimum of 10 air changes per hour.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

A premix with manganese (II) sulfate monohydrate 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 prepared once.
Groups of 70 male and 70 female mice were fed diets containing 0, 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate. The level of manganese in the diet received by controls was approximately 92 ppm. The appropriate feed was supplied twice weekly and was available ad libitum.

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 by the analytical chemistry laboratory 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 under the conditions listed above. 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 strongly support the conclusion that the manganese remained in the divalent state. The dose formulations were prepared weekly. Dose formulations were discarded 21 days after the date of preparation.
Periodic analyses of the dose formulations of manganese (II) sulfate monohydrate were conducted at the study laboratory and at the analytical chemistry laboratory using spectrophotometric methods. Dose formulations were analyzed three times during the study. All dose formulations were within the specified 10% of the target concentration.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

Daily - ad libitum in diet

Post exposure period:

There was no post exposure period

Remarks:

Doses / Concentrations:
0, 1500, 5000 and 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

70 mice/sex/dose

Control animals:

yes, plain diet

yes, historical

Details on study design:

Animals were assigned to treatment groups by weight intervals. Animals from each interval were randomized and proportionately assigned to cages, then the cages were assigned to dose groups using an appropriate table of random numbers.
The doses selected for the 2 year study were 0, 1500, 5000 and 15000 ppm. These doses were based on the significantly lower mean body weight gains of all exposed males and 50000 ppm females and the significantly lower absolute and relative liver weights of 50000 ppm males in the 13 week study.
Interim sacrifices were carried out at months 9 and 15.

Positive control:

A positive control was not included.

Observations and examinations performed and frequency:

Mice were observed twice daily. Clinical observations and animal weights were recorded initially, weekly during the first 13 weeks of the study, then monthly thereafter and at interim evaluations. Feed consumption was measured for a 7 day period one every 4 weeks.
Blood was collected at the 9 and 15 month interim evaluations for haematology and clinical chemistry determinations. Terminal blood samples were also collected. Haematology parameters evaluated were: erythrocytes, haemoglobin, haematocrit, platelets, mean erythrocyte volume, mean erythrocyte haemoglobin, mean erythrocyte haemoglobin concentration, reticulocytes, nucleated erythrocytes, and leukocyte count and differential. Clinical chemistry parameters evaluated were: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, and creatinine.

Sacrifice and pathology:

Interim sacrifices were carried out after 9 and 15 months. Organs weighed at the interim evaluations were brain, kidneys and liver. Terminal sacrifice was carried out after 103 weeks exposure. At necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 µm, and stained with
haematoxylin and eosin for microscopic examination.
Complete histopathologic examinations were performed on all 0 and 15000 ppm animals at the 9- and 15-month interim evaluations and gross lesions examined for the 1500 and 5000 ppm groups. Complete histopathologic examinations were performed on all animals at the end of the studies and on all animals that died or were killed moribund during the studies. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, bone, bone marrow, brain, cecum, colon and rectum, 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, thymus, thyroid gland, trachea, uterus, and urinary bladder.

Other examinations:

The brain, kidney liver and pancreas were examined in metal concentration analyses for copper, iron, magnesium and zinc.

Statistics:

The probability of survival was estimated by the product limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related
effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. Logistic regression analysis was used to assess the incidence of neoplasms. Pairwise comparisons and were used to identify differences between control and treated groups. The significance of pairwise comparisons was determined according to the methods of Dunnett (1955), Williams (1971, 1972), Shirley (1977), Dunn (1964), Jonckheeres test (1954) or the Mann-Whitney U test.

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:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Survival of exposed males and females was similar to that of the control groups. The mean body weights of exposed males were similar to those of the control group. After week 37, mean body weights of all exposed groups of females were lower than that of the controls. The final mean body weights for the 1500, 5000, and 15000 ppm groups were 6%, 9%, and 13% lower than that of the control group. 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 approximate daily doses of 160, 540, or 1800 mg/kg body weight (males) or 200, 700, or 2250 mg/kg body weight (females). No clinical findings were attributed to administration.
Percent haematocrit, haemoglobin concentrations, and erythrocyte counts in 15000 ppm male mice at the 15 month interim evaluation were greater than those of the controls. These slight increases are not consistent with the findings in the 13 week study and their significance was uncertain. No other notable differences were observed in the haematology or clinical chemistry parameters. 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 15 month interim evaluations and in 5000 and 15000 males at the 15 month interim evaluation. Tissue concentrations of manganese in the brain (except 1500 and 5000 ppm females at 15 months), kidney, and pancreas (except 1500 males at 9 months and 1500 ppm females at 15 months) of exposed groups were significantly greater those of controls.
At the 9- and 15-month interim evaluations, thyroid follicle dilatation was present in 15000 ppm males and females but not in the controls (Table 1). 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 also occurred in 15000 ppm males and in all exposed females. 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. 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.
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 (Table 2). 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.
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. 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 5000 and 15000 ppm groups. Hepatocellular foci did not occur in an exposure-related pattern (foci of any type, males: 4/50, 16/49, 9/51, 1/50). The incidences of adenoma or foci in exposed females were similar to those of the controls.

Relevance of carcinogenic effects / potential:

There was equivocal evidence of carcinogenic activity of manganese (II) sulfate monohydrate in male and female B6C3F1 mice, based on the marginally increased incidences of thyroid gland follicular cell adenoma and the significantly increased incidences of follicular cell hyperplasia.

Dose descriptor:

NOAEL

Effect level:

5 000

Sex:

male/female

Basis for effect level:

other: There was equivocal evidence of carcinogenicity at 15000 ppm

Critical effects observed:

not specified

Incidences of selected lesions of the thyroid gland

Dose (ppm)	0	1500	5000	15000
Males
9 month interim evaluation
Thyroid Gland^a	10	0	0	9
Follicular Dilatation^b	0	^c	-	6**(1.0)^d
15 month interim evaluation
Thyroid Gland	10	2	1	10
Follicular Dilatation	0	0	0	9**(1.0)
Follicular Cell Adenoma	0	0	0	1
2 Year Study
Thyroid Gland	50	49	51	50
Follicular Dilatation	2(1.0)	2(1.5)	5(1.0)	23**(1.2)
Follicular Cell, Hyperplasia, Focal	5(1.0)	2(1.5)	8(1.5)	27**(1.9)
Follicular Cell Adenoma^e
Overall rate^f	0/50(0%)	0/49(0%)	0/51(0%)	3/50(6%)
Adjusted rate^g	0.0%	0.0%	0.0%	6.5%
Terminal rate^h	0/46(0%)	0/44(0%)	0/46(0%)	3/46(7%)
First incidence (days)	-	-	-	729(T)
Logistic regression test^i	P=0.015	-	-	P=0.121
Females
9 month interim evaluation
Thyroid Gland	10	0	1	10
Follicular Dilatation	0	-	1(1.0)	7**(1.0)
15 month interim evaluation
Thyroid Gland	9	0	2	9
Follicular Dilatation	0	-	0	5*(1.0)
2 Year Study
Thyroid Gland	50	50	49	51
Follicular Dilatation	1(1.0)	5(1.0)	11**(1.4)	24**(1.2)
Follicular Cell, Hyperplasia, Diffuse	1(1.0)	1(1.0)	0	0
Follicular Cell, Hyperplasia, Focal	3(2.3)	15**(1.5)	27**(1.5)	43**(2.1)
Follicular Cell Adenoma^j
Overall rate	2/50(4%)	1/50(2%)	0/49(0%)	5/51(10%)
Adjusted rate	4.8%	2.2%	0.0%	11.9%
Terminal rate	2/42(5%)	1/46(2.2%)	0/37(0%)	5/42(12%)
First incidence (days)	729(T)	729(T)	-	729(T)
Logistic regression test	P=0.037	P=0.468N	P=0.267N	P=0.216

(T)Terminal Sacrifice

* Significantly different (P≤0.05) from the control group by the Fisher exact test (interim evaluations) or by the logistic regression test (2-year study)

** P≤0.01

a Number of animals with organ examined microscopically

b Number of animals with lesion

c Not applicable; tissue not examined microscopically in this group

d Average severity grade of lesions in affected animals (1=minimal; 2=mild; 3=moderate; 4=marked)

e Historical incidence for 2-year feed studies with untreated control groups (mean ± standard deviation): 19/1,105 (1.7% ± 1.7%); range 0%-4%

f Number of neoplasm-bearing animals/number of animals microscopically examined

g Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

h Observed incidence at terminal kill

i Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards these neoplasms as nonfatal. A lower

incidence in an exposure group is indicated byN.

j Historical incidence: 27/1,099 (2.5% ± 2.9%); range 0%-9%

Incidence of lesions in the forestomach

Dose (ppm)	0	1500	5000	15000
Males
2 Year Study
Forestomach^a	50	49	51	50
Erosion, Focal^b	0	0	0	2(3.0)^c
Squamous Hyperplasia, Focal	2(2.0)	1(2.0)	5(1.2)	14**(2.3)
Inflammation, Chronic Active	0	0	0	5*(2.0)
Ulcer	0	0	0	6*(2.5)
Squamous Cell Papilloma	1	1	0	0
Females
9 month interim evaluation
Forestomach	10	0	0	9
Squamous Hyperplasia, Focal	0	0	0	1(2.0)
15 month interim evaluation
Forestomach	9	0	1	9
Squamous Hyperplasia, Focal	0	0	1(2.0)	1(2.0)
Inflammation, Chronic Active	0	0	0	1(1.0)
2 Year Study
Forestomach	51	50	49	50
Squamous Hyperplasia, Focal	1(2.0)	3(1.7)	3(2.0)	9**(2.4)
Ulcer	2(2.0)	0	0	3(2.7)
Inflammation, Chronic Active	0	1(2.0)	1(2.0)	3(1.7)
Squamous Cell Papilloma	1	0	0	0

* Significantly different (P≤0.05)

from the control group by the logistic regression test

** (P≤0.01)

a Number of animals with organ examined microscopically

b Number of animals with lesion

c Average severity grade of lesions in affected animals (1=minimal; 2=mild; 3=moderate; 4=marked)

Conclusions:

There was equivocal evidence of carcinogenic activity of manganese (II) sulfate monohydrate in male and female B6C3F1 mice, based on the marginally increased incidences of thyroid gland follicular cell adenoma and the significantly increased incidences of follicular cell hyperplasia.

Executive summary:

Groups of 70 male and 70 female B6C3F₁ mice received diets containing 0, 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate for 2 years, to determine the carcinogenic potential of the substance. These levels resulted in an average daily ingestion of 160, 540, or 1800 mg/kg body weight (males) or 200, 700, or 2250 mg/kg (females). Interim sacrifices were carried out on groups of 9 or 10 mice at 9 and 15 months.

Survival rates of exposed male and female mice in the 2-year study were similar to those of the control groups. The mean body weights of exposed male mice were similar to that of the control group. Compared to controls, female mice had exposure-related lower mean body weights 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 group. Feed consumption by all exposure groups was similar to that by the control groups. No clinical findings were attributed to the administration of manganese (II) sulfate monohydrate. 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-month interim evaluation and in 5000 and 15000 males and all exposed females at the 15-month interim evaluation.

Incidences of thyroid follicular dilatation and hyperplasia were significantly greater in 15000 ppm male and female mice than in controls. Follicular cell adenomas occurred in one 15000 ppm male at the 15-month interim evaluation and in three 15000 ppm males at the end of the study but not in the lower exposure groups or the control group. 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 manganese (II) sulfate monohydrate. The incidences of focal hyperplasia of the forestomach epithelium were significantly greater in the 15000 ppm male and exposed female groups. The hyperplasia was associated with ulcers and inflammation in some mice, particularly males.

Under the conditions of the study, there was equivocal evidence of carcinogenic activity (according to the NTP Levels of Evidence of Carcinogenic Activity) of manganese (II) sulfate monohydrate in male and female mice, based on the marginally increased incidences of thyroid gland follicular cell adenoma and the significantly increased incidences of follicular cell hyperplasia.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 000 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

Two studies conducted on the read-across substance MnSO4, one on rats and one on rabbits, in compliance with GLP.

Organ:

thyroid gland

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the material does not require classification with respect to carcinogenicity.

Additional information

WoE Read-Across Source MnSO4: NTP (1993) - Rat

Groups of 70 male and 70 female F344/N rats were fed diets containing 0, 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate for 2 years, to determine the carcinogenic potential of the substance. Based on average daily feed consumption, these doses resulted in the daily ingestion of 60, 200, or 615 mg/kg body weight (males) or 70, 230, or 715 mg/kg (females). Interim sacrifices were carried out on groups of 8 to 10 rats at 9 and 15 months. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Survival of 15000 ppm male rats in the 2-year study was significantly lower than that of the control group. The deaths of males in the control and exposure groups were attributed to a variety of spontaneous neoplastic and nonneoplastic lesions; however, the greater number of deaths in the 15000 ppm group resulted from increased incidences of advanced renal disease related to ingestion of manganese (II) sulfate monohydrate. The decreased survival of the 15000 ppm males did not occur until approximately week 93 of the study; before week 93, survival was similar in all groups. Survival of exposed females was similar to that of the controls. The mean body weight of 15000 ppm male rats was within 5% of the control group until week 89; by week 104, the mean body weight of 15000 ppm males was 10% lower than that of the control group. The mean body weights of 1500 and 5000 ppm male rats and all exposed female groups were similar to those of the controls throughout the study. Feed consumption by all exposure groups was similar to that by the control groups. No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion.

The ingestion of diets containing 15000 ppm manganese (II) sulfate monohydrate was associated with a marginal increase in the average severity of nephropathy in male rats (0 ppm, 2.9; 1500 ppm, 3.0; 5000 ppm, 3.0; 15000 ppm, 3.2). The increased severity of nephropathy in the 15000 ppm male rats was accompanied by significantly increased incidences of mineralization of the blood vessels and glandular stomach, parathyroid gland hyperplasia, and fibrous osteodystrophy of the femur. These lesions are manifestations of renal failure, uremia, and secondary hyperparathyroidism. The increased incidence of advanced renal disease caused reduced survival of the high-dose male rats. No increase in the incidence of neoplasms in male or female rats was attributed to the ingestion of diets containing manganese (II) sulfate monohydrate. Under the conditions of the study, there was no evidence of carcinogenic activity (according to the NTP Levels of Evidence of Carcinogenic Activity) of manganese (II) sulfate monohydrate in male or female rats.

 

WoE Read-Across Source MnSO4: NTP (1993) - Mouse

Groups of 70 male and 70 female B6C3F1 mice received diets containing 0, 1500, 5000, or 15000 ppm manganese (II) sulfate monohydrate for 2 years, to determine the carcinogenic potential of the substance. These levels resulted in an average daily ingestion of 160, 540, or 1800 mg/kg body weight (males) or 200, 700, or 2250 mg/kg (females). Interim sacrifices were carried out on groups of 9 or 10 mice at 9 and 15 months. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Survival rates of exposed male and female mice in the 2-year study were similar to those of the control groups. The mean body weights of exposed male mice were similar to that of the control group. Compared to controls, female mice had exposure-related lower mean body weights 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 group. Feed consumption by all exposure groups was similar to that by the control groups. No clinical findings were attributed to the administration of manganese (II) sulfate monohydrate. 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-month interim evaluation and in 5000 and 15000 males and all exposed females at the 15-month interim evaluation.

Incidences of thyroid follicular dilatation and hyperplasia were significantly greater in 15000 ppm male and female mice than in controls. Follicular cell adenomas occurred in one 15000 ppm male at the 15-month interim evaluation and in three 15000 ppm males at the end of the study but not in the lower exposure groups or the control group. 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 manganese (II) sulfate monohydrate. The incidences of focal hyperplasia of the forestomach epithelium were significantly greater in the 15000 ppm male and exposed female groups. The hyperplasia was associated with ulcers and inflammation in some mice, particularly males.

Under the conditions of the study, there was equivocal evidence of carcinogenic activity (according to the NTP Levels of Evidence of Carcinogenic Activity) of manganese (II) sulfate monohydrate in male and female mice, based on the marginally increased incidences of thyroid gland follicular cell adenoma and the significantly increased incidences of follicular cell hyperplasia.

Carcinogenicity: via oral route (target organ): glandular: thyroids