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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Dosing was conducted between 24 September 1984 - 14 September 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted under the conditions of GLP and according to an appropriate methodology. The reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. This study is selected as the key study as it is considered to be most relevant for assessment of repeated dose toxicity via the oral route (test animas and duration of study considered to be ost appropraite. In addition, the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP) Rationale for read-across The study was performed to assess the repeated dose toxicity and the carcinogenicity of manganese (as Mn2+). The substance tested (manganese(II)sulphate monohydrate) was chosen as the specific compound for testing due to stability, solubility, availability, and use of the sulphate as a food supplement for humans and animals. It is therefore considered that these data are reliable and justified for use in read-across to other Mn2+ containing compounds in which the manganese is considered to be the toxicologically relevant moiety. As manganese sulphate is considered to be classified as STOT-RE 2, H373; with regards to neurotoxic effects observed following inhalation exposure it is considered to be appropriate to read this classification across to soluble manganese phosphates and as such this is considered to be the leading health effect. This conclusion is supported by the relatively low oral toxicity observed in a 2-year rat and mouse study for manganese sulphate. As such it is not considered to be scientifically justified to further investigate the repeated dose toxicity of manganese bis (dihydrogen phosphate) in accordance with Regulation (EC) No. 1907/2006 (REACH).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993

Materials and methods

Principles of method if other than guideline:
Groups of 70 male and 70 female rats were fed diets containing 0, 1,500, 5,000, or 15,000 ppm manganese (II) sulphate monohydrate for 103 weeks. 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). Eight to 10 rats from each group were evaluated at 9 and 15 months.

Rationale for choice of substance: Manganese (II) sulphate monohydrate was chosen as the specific compound for testing due to stability, solubility, availability, and use of the sulphate as a food supplement for humans and animals.
GLP compliance:
yes
Remarks:
performed in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58).
Limit test:
no

Test material

Constituent 1
Reference substance name:
manganese (II) sulphate monohydrate
IUPAC Name:
manganese (II) sulphate monohydrate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): manganese (II) sulphate monohydrate
- Analytical purity: Chelometric titration indicated a purity of 97.7% ± 0.4%. The overall data indicated that the manganese was in the divalent state and supported a purity of greater than 97%.
- Stability under test conditions: Periodic monitoring of the chemical by the study laboratory using chelometric titration and elemental analyses (Galbraith Laboratories, Inc., Knoxville, TN) indicated no degradation of the bulk chemical during the studies.

Test animals

Species:
rat
Strain:
other: F344N
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Stone Ridge, NY)
- Age at study initiation: approximately 41 days old when recieved
- Acclimation period: quarantined for 12 days

Before initiation of the studies, five male and five female rats and mice were randomly selected for parasite evaluation and gross observation for evidence of disease.
Rats and mice were housed five per cage. Feed and water were available ad libitum. Feed consumption was measured for 7 days, once a month. Cages were rotated every 2 weeks; racks were rotated every 2 weeks. Further details of animal maintenance are given in Table 1. Information on feed composition is provided in Appendix K

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared by mixing manganese (II) sulphate monohydrate with feed. 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 once for the 14-day studies and weekly for the 13-week and 2-year studies. Dose formulations were discarded 21 days after the date of preparation.




Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of manganese (II) sulphate monohydrate were conducted at the study laboratory and at the analytical chemistry laboratory using spectrophotometric methods. Dose formulations were analysed once during the 14-day studies, three times during the 13-week studies, and every 2 months during the 2-year studies. All dose formulations for rats and mice were within the specified 10% of the target concentrations throughout the studies.
Duration of treatment / exposure:
2 years / 103 weeks
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1500, 5000, or 15000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Groups of 70 male and 70 female rats.
Control animals:
yes, plain diet
Details on study design:
Controls: The level of manganese in the diet received by controls was approximately 92 ppm.
Administration of test diet: The appropriate feed was supplied twice weekly and was available ad libitum. Clinical findings were recorded weekly. Feed consumption was recorded weekly by cage.
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).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded weekly for the first 13 weeks and monthly thereafter.

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

Blood was collected at the 9- and 15-month interim evaluations for hematology and clinical chemistry determinations. Samples of blood plasma (rats), kidneys, livers, and pancreas were collected at the 9- and 15-month evaluations for tissue metal concentration analyses.

HAEMATOLOGY: Yes
- Parameters checked: Erythrocytes, hemoglobin, hematocrit, platelets, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, reticulocytes, nucleated erythrocytes, and leukocyte count and differential.

CLINICAL CHEMISTRY: Yes

- Parameters checked: alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, blood urea nitrogen, and creatinine.

Tissue metal concentration analyses: manganese, iron, copper, and zinc concentrations.

Sacrifice and pathology:
sacrificed via carbon dioxide asphyxiation.
Necropsy performed on all animals.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All animals were necropsied. 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 hematoxylin and eosin for microscopic examination. Complete histopathologic examinations were performed on 0 and 15,000 ppm animals at the 9- and 15-month interim evaluations and gross lesions were examined for the 1,500 and 5,000 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. Tissues examined are as below:

Complete histopathologic examinations were performed on all 0 and 15,000 ppm animals at the 9- and 15-month interim evaluations and gross lesions examined for the 1,500 and 5,000 ppm groups. Complete histopathologic examinations were performed on all animals at the end of the studies and on all animals that died or were killed moribund during the studies. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, bone, bone marrow, brain, cecum, colon and rectum, esophagus, gallbladder (mice), 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.

Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered into the Toxicology Data Management System. The microscopic slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. The slides, individual animal data records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual animal records and tables were compared for accuracy, the slide and tissue counts were verified, and the histotechnique was evaluated. For the 2-year studies, a quality assessment pathologist reviewed the pancreas of male and female rats, the kidney of male rats, the forestomach and thyroid gland of male and female mice, and the pituitary gland of female mice.
Other examinations:
The brain, kidneys, and liver from animals selected for the 9- and 15-month evaluations were weighed at necropsy. Tissues examined in metal concentration analyses for copper, iron, manganese, and zinc were blood plasma (rats), brain, kidney, liver, and pancreas.
Statistics:
See below

Results and discussion

Results of examinations

Details on results:
Survival
Estimates of survival probabilities are presented in table 4 and in the Kaplan-meier survival curves (figure 1) -both are attached below. Survival of 15,000 ppm male rats was significantly lower than of controls. All other groups were similar to controls. The decreased survival in the top dose males was attributed to increased severity of nephropathy and renal failure. Decreased survival did not occur until approximately week 93 of the study.

Body Weights, Feed Consumption, and Clinical Findings
The mean body weights of 1,500 and 5,000 ppm male rats exposed to manganese (II) sulphate monohydrate were similar to those of controls throughout the 2-year study (Table 5 and Figure 2 - attached). The mean body weights of 15,000 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 (Table 6). Feed consumption by exposed groups was similar to that by control groups (Tables J1 and J2). Rats exposed to 1,500, 5,000, or 15,000 ppm manganese (II) sulphate 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.

Haematology, Clinical Chemistry, and Tissue Metal Concentration Analyses
Values for haematology and clinical chemistry parameters were generally similar among exposed and control groups at the 9- and 15-month interim evaluations (Tables G3 and G4). Slight differences in some parameters between exposed and control groups were not considered related to the ingestion of manganese (II) sulphate monohydrate. At both the 9- and 15-month interim evaluations, the manganese levels in the liver of 5,000 and 15,000 ppm males and females were significantly greater than those in controls. The hepatic iron concentrations for these exposure groups were lower than for controls (Tables H1 and H2). The concentrations of manganese in the brain, kidney, and pancreas of exposed and control rats were variable; 15,000 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 15,000 ppm males at 9 months and in 15,000 ppm females at 9 and 15 months were significantly greater than those of the controls.

Pathology
No chemical-related lesions were observed at the 9- or 15-month interim evaluations.
Summaries of the incidences of neoplastic and nonneoplastic nonneoplastic lesions, individual animal tumour diagnoses, statistical analyses of primary neoplasms that occurred with an incidence of at least 5% in at least one animal group, and historical incidences for the neoplasms mentioned in this section are presented in Appendixes A for male rats and B for female rats.

Pancreas: 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; 1,500 ppm, 2/50; 5,000 ppm, 2/51; 15,000 ppm, 3/51; adenoma: 0/52, 3/50, 4/51, 3/51; Tables A5 and A1). In addition, a carcinoma of the pancreatic islets was found in one 15,000 ppm male. However, neither the trend test nor pairwise comparisons were significant (Table A3),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%; Table A4a).

Kidney: At the 9- and 15-month interim evaluations, the absolute kidney weights of exposed rats were similar to those of the controls (Tables F3 and F4). 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 (Tables 7 and A5). 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 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 15,000 ppm male rats. These lesions included mineralization of blood vessels (4/52, 10/51, 6/51, 17/52), mineralisation 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) (Table A5).

Adrenal Gland: In females, medullary hyperplasia occurred with a significant negative trend and a significantly decreased incidence in the 15,000 ppm group (control 12/50, 1,500 ppm 11/50, 5,000 ppm 6/51, and 15,000 ppm 1/48; Table B4). 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; Table A3); the incidence of medullary hyperplasia in exposed males was similar to that of the controls (Table A5).

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calculated on the basis of food intake and concentration in diet
Sex:
male
Dose descriptor:
NOAEL
Effect level:
230 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calculated on the basis of food intake and concentration in diet
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Tables attached below as too large for freetext field.

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of manganese (II) sulphate monohydrate in male or female F344/N rats receiving 1,500, 5,000, or 15,000 ppm. The ingestion of diets containing manganese (II) sulphate monohydrate was associated with an increased severity of nephropathy in male rats.