Registration Dossier

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Administrative data

Description of key information

No data on repeated dose toxicity is available for the substance. Since the substance is used in cosmetic products, tests on animals are banned. Therefore, a weight of evidence approach is proposed based on information on the anions and cations present in this molecule.

Based on the available oral subchronic data on pyrophosphates, the no observed effect level (NOEL) and no observed adverse effect level (NOAEL) were considered to be 250 and 500 mg/kg /day respectively.

Based on the available oral chronic data on ammonium, the NOAEL was estimated to be 256 and 284 mg/kg/day in males and females, respectively.

In relation to the manganese cation, the bioavailability of different forms of Mn is a matter for consideration. There have been unsubstantiated claims that the higher valence states of Mn (Mn+3, Mn+4) and the higher oxides in ores (Mn2O3 and Mn3O4) are more toxic (Oberdoerster and Cherian, 1988). At present, however, insufficient information exist by which to determine the relative toxicities of different forms of Mn, and thus, no distinction is made among various compounds of Mn (IRIS, US EPA, 1996). Furthermore, manganese may undergo metabolism from Mn2+ to Mn3+ in the liver (Gibbons et al., 1976). Based on the available data on bibliography, absorption of manganese from soluble salts after oral exposure is low (3-13%) and oral absorption of insoluble compounds is slow, probably due to excretion before complete dissolution. In animal experiments, oral and inhalation bioavailability of insoluble Mn-based substances is less efficient compared to soluble compounds. The oral route is unlikely for human exposure and the dermal exposure is relevant although subsequent systemic exposure is deemed unlikely due to the low absorption of inorganic substances, especially insoluble compounds, which is the case of the substance under registration.

Based on the available oral chronic data on manganese in rats and mice, the lowest NOAEL was estimated to be 200 mg/kg/day.

Regarding the inhalation route, based on the data available in the bibliography from human occupational studies, three chronic studies have been identified and considered as reliable to derive a NOAEL value for manganese exposure by the inhalation route:

Gibbs et al ., 1999: NOAEL = 66 µg/m3

Deschamps et al., 2001: NOAEL = 57 µg/m3

Young et al., 2005: NOAEL = 58 µg/m3

Roels et al., 1992: NOAEL = 20 µg/m3

Based on the epidemiological evidence for MnO2 (Roels et al., 1992) and the animal evidence for MnSO4, these two substances could be classified as STOT RE 2 for neurotoxicity. The evidence for classification of all other manganese based inorganic compountds is equivocal and subject to confounders. Based on the lack of evidences regarding the inhalation route for the substance under registration, a classification for repeated dose toxicity is not proposed for this insoluble inorganic manganese compound.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
A 13 week sub-chronic toxicity study of potassium pyrophosphate was carried out in male and female F344 rats at the dose levels of 10, 5, 2.5, 1.25, 0.6 and 0% in the diet. Sixty animals of both sexes were divided into the 6 dosage groups. The purpose of the study was to determine the maximumtolerable dose dose of test substance for use in rats in a long-term carcinogenicity study. Observations were limited to mortality, clinical abnormalities, body weights, food intake, haematology, clinical chemistry, gross pathology (incl. organ weight) and histopathology.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd Japan.
- Age at study initiation: At least 6 weeks old.
- Fasting period before study: No. Fasting is recorded to have been carried out after the last dosing until the animals were sacrificed.
- Housing: 5 animals per plastic cage with soft chip on the floor which was changed every 2 weeks.
- Diet (e.g. ad libitum): CRF-1 (powder), Charles River Ltd. The test substance was mixed in the diet and provided ad libitum.
- Water (e.g. ad libitum): Mains water, ad libitum.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 25°C
- Humidity (%): 50 - 60%
- Air changes (per hr): 18 times per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of darkness.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Up to 91 days.
Frequency of treatment:
Ad libitum during the exposure period.
Dose / conc.:
0 other: % (nominal in diet)
Dose / conc.:
0.6 other: % (nominal in diet)
Dose / conc.:
1.25 other: % (nominal in diet)
Dose / conc.:
2.5 other: % (nominal in diet)
Dose / conc.:
5 other: % (nominal in diet)
Dose / conc.:
10 other: % (nominal in diet)
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily.
- Cage side observations checked: Mortality, general abnormalities.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Daily.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 92.
- Anaesthetic used for blood collection: Yes , ether.
- Animals fasted: Yes
- How many animals: All animals.
- Parameters checked: GOT, GPT, Alp, TTT, T-bil, T-cho, TG, beta-lipopro, T-pro, A/G, BUN, creatinine, uric acid, ZTT, Ca, erythrocyte, Ht, MCV, leukocyte and Hb.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two males and one female from the 10% groups as a result of renal failure.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males and one female from the 10% groups as a result of renal failure.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gains of both male and female rats treated with 10% and of male rats treated with 5% were significantly decreased as compared with controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was effected in the 10% and 5% groups. No further details are provided.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards higher uric acid levels in both sexes at higher doses. There were significant changes of the hepato-related parameter levels in the dosed groups.
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were significant deviations in organ weights.
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
KIDNEYS: In the 10% groups both sexes exhibited a mild to severe tubulorrhexis accompanied by calcification, interstitial fibrosis and mononuclear cell infiltration in the area of the cortico-medullary border.
The 5% group exhibited a slight to mild tubulorrhexis accompanied by calcification in all animals.
The 2.5% group atrophy and deciduation of the uriniferous tubule epithelial cells was observed in 7/10 males and 8/10 females.
No other effects were reported at the lower doses.

TONGUE: Ulceration and/or granuloma formation was reported in the mucosal tissue at the root of the tongue for 8/8 males and 7/9 females in the 10% group; and for 6/10 males and 4/10 females the 5%.

SALIVARY GLAND: Hypertrophy and basophilisation of acinar cells of the sublingual gland and submandibular gland. Also noted was a compression/atrophy of the striated duct, intercalated duct and excretory duct. These effects were observed in 7/8 males and 4/9 females in the 10% group and in 2/10 males in the 5% group.

OTHER: Hyperplasia of RES cells and lymphoblasts was reported. A lesion in the submandibular lymph node is recorded in 2/10 males and 1/10 female in the 10% group. No other effects are reported for the other organs
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
LOEL
Effect level:
0.6 other: %
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
organ weights and organ / body weight ratios
Key result
Dose descriptor:
LOEL
Effect level:
308 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: Extrapolated from level of compound in diet assuming 0.35 kg rat eats 18 g food/day.
Key result
Critical effects observed:
not specified

Reduction in the body weight gain and the trend of higher uric acid levels may be due to the decreased food consumption and the effects in the kidneys. The effects in the tongue would be due to the high alkaline property of the test substance in the solution.

Table 1 - Serum chemistry and hematology results (male)

Dose

10%

5%

2.5%

1.25%

0.6%

control

Effective No.

7

10

10

10

10

8

GOT (KU)

92.0±9.6a

70.0±7.3

66.2±8.1

67.7±4.8

68.5±8.1

67.5±6.1

GPT (KU)

39.5±9.0a

27.9±1.4b

26.2±1.9

25.8±2.8

24.3±2.8

25.8±2.0

Alp (KAU)

21.1±2.0a

13.3±1.2

11.3±1.2a

11.8±0.9a

11.8±1.3b

13.3±1.0

TTT(SHU)

0.99±0.54a

0.40±0.16

0.27±0.11

0.45±0.16

0.42±0.29

0.34±0.17

T-bil.(mg/dL)

0.55±0.08b

0.47±0.08

0.51±0.07

0.52±0.08

0.48±0.06

0.46±0.07

T-cho.(mg/dL)

96.3±5.5a

51.0±5.4

49.9±2.7

54.2±2.7

50.1±3.8

51.7±3.8

TG(mg/dL)

69.1±16.8

77.6±17.6

77.5±14.3

116.0±18.6a

94.7±30.7

85.3±17.5

β lipo-pro.(mg/dL)

131.4±26.8

117.5±20.8

106.0±27.1

195.1±147.1

132.4±36.8

127.2±19.8

T-pro.(g/dL)

6.25±0.22b

6.54±0.13

6.57±0.18

6.65±0.16b

6.5±0.12

6.48±0.10

A/G

1.70±0.19

1.75±0.05

1.81±0.07

1.85±0.07

1.89±0.09

1.93±0.08

BUN(mg/dL)

34.6±2.1a

22.7±1.8a

17.9±2.1

17.6±1.4

17.0±1.3

17.5±1.1

Creatine(mg/dL)

0.85±0.21

0.65±0.25

0.84±0.23

0.94±0.15

0.72±0.45

0.91±0.35

Uric acid(mg/dL)

2.00±0.037a

1.19±0.29

1.01±0.20

1.29±0.56

1.47±0.63

0.98±0.29

ZTT(KU)

2.79±1.02a

1.62±0.32a

1.24±0.19b

1.41±0.36b

1.07±0.23

1.08±0.12

Ca(mg/dL)

9.2±0.4a

10.5±0.3a

10.7±0.2

10.8±0.4

11.1±0.6

10.9±0.3

Erythrocytes(x104/mm2­­)

706.6±43.7a

893.1±17.3a

926.1±24.0

953.4±33.0

942.8±15.1

949.0±22.4

Ht.(%)

375.0±16.4a

443.6±9.1a

454.6±11.8

468.4±15.6

465.1±9.5

465.5±10.8

MCV(µ2)

53.3±1.1a

49.7±0.7b

49.1±0.3

49.2±0.4

49.0±0.5

49.0±0.0

Leukocyte(x102/mm2)

71.9±14.5

59.9±10.5

85.7±38.5

146.6±41.4a

98.9±14.2a

59.3±15.5

Hb.(g/dL)

121.1±20.7a

154.0±3.7a

153.8±15.1

160.3±6.1

159.9±3.2

161.3±3.6

ap<0.01;bp<0.05

 Table 1 continued - Serum chemistry and hematology results (female)

Dose

10%

5%

2.5%

1.25%

0.6%

control

Effective No.

8

9

10

10

9

10

GOT (KU)

90.3±3.6a

69.0±3.9a

70.7±5.8a

68.8±8.7

69.0±5.2b

63.3±4.1

GPT (KU)

35.2 ±9.0a

26.5±4.2b

24.1±2.8

23.4±1.4

23.0±2.6

23.1±2.0

Alp (KAU)

15.0±1.8a

8.6±1.1

8.6±0.8

10.0±1.7

9.0±1.2

8.5±1.4

TTT(SHU)

0.43±0.17

0.39±0.14

0.45±0.22

0.48±0.19

0.31±0.09b

0.44±0.16

T-bil.(mg/dL)

0.47±0.16

0.53±0.12

0.62±0.06

0.55±0.11

0.59±0.07

0.57±0.11

T-cho.(mg/dL)

112.0±10.0a

83.9±9.2

85.0±5.5

86.4±8.5

80.8±5.1b

87.7±6.2

TG(mg/dL)

65.0±26.2

53.2±9.7

59.0±10.3

57.0±12.2

54.3±4.6

58.3±9.4

β lipo-pro.(mg/dL)

79.0±30.7b

52.9±13.7a

93.3±16.4

92.6±21.8

90.4±8.5

102.8±16.9

T-pro.(g/dL)

6.01±0.20a

6.19±0.18b

6.41±0.24

6.45±0.09

6.32±0.19

6.37±0.16

A/G

2.00±0.18

1.90±0.26

1.88±0.09

1.84±0.33

2.02±0.10

1.93±0.12

BUN(mg/dL)

35.6±5.3a

25.8±2.3a

19.4±1.4a

18.3±1.4a

16.8±1.1

16.1±1.2

Creatine(mg/dL)

0.66±0.14

0.70±0.25

0.76±0.34

0.91±0.22

0.86±0.31

0.64±0.33

Uric acid(mg/dL)

1.75±0.32

1.71±0.35

1.66±0.22

1.73±0.29

1.74±0.37

1.78±0.26

ZTT(KU)

0.80±0.38a

1.67±0.71

2.18±0.39

2.37±1.58

1.56±0.47

1.98±0.82

Ca(mg/dL)

9.5±0.5a

10.8±1.1

10.4±0.9

9.8±0.4a

10.0±0.2a

10.4±0.4

Erythrocytes(x104/mm2­­)

685.8±34.9a

862.2±25.6

861.1±23.0

856.6±23.4

846.6±21.4

876.9±32.6

Ht.(%)

372.1±15.5a

445.4±14.1

444.8±13.5

441.1±14.3

448.3±10.1

452.7±16.4

MCV(µ2)

54.1±0.8a

51.6±0.5

51.8±0.6

51.6±0.7

51.9±0.3

51.7±0.7

Leukocyte(x102/mm2)

66.8±8.3a

127.9±64.8

207.5±51.1a

239.0±33.6a

160.3±55.3

116.5±23.3

Hb.(g/dL)

116.9±24.5a

159.3±5.3

160.1±5.6

157.8±5.4

158.8±4.7

159.5±4.0

ap<0.01;bp<0.05

Table 2 - Absolute and relative organ weights (male)

Relative organ weights are shown in parenthesises (%)

Dose

10%

5%

2.5%

1.25%

0.6%

Control

Effective No.

8

10

10

10

10

10

Brain

1.86±0.04a(1.1)a

1.98±0.06

(0.7)b

2.01±0.06 (0.6)

1.98±0.05 (0.6)b

2.02±0.04 (0.6)b

2.02±0.08 (0.6)

Pituitary

0.007±0.003 (0.004)

0.011±0.004 (0.003)

0.008+0.003 (0.002)

0.008±0.003 (0.002)

0.007±0.002 (0.002)

0.011±0.006 (0.003)

Saliv. Glds.

0.79±0.21b(0.48)a

0.82±0.28b(0.28)b

0.68±0.12 (0.20)

0.60±0.06 (0.18)

0.62±0.05 (0.19)

0.59±0.06 (0.19)

Thymus

0.12±0.05a(0.07)

0.20±0.04 (0.07)

0.23±0.03 (0.07)

0.22±0.03 (0.07)

0.26±0.04 (0.08)

 

0.23±0.05 (0.07)

Lung (R)

0.48±0.03a(0.29)a

0.71±0.07 (0.24)

0.77±0.04 (0.24)

0.77±0.07 (0.23)

0.77±0.10 (0.23)

0.75±0.11 (0.24)

Lung (L)

0.26±0.03a(0.15)a

0.36±0.04 (0.12)

0.39±0.03 (0.11)

0.38±0.03 (0.12)

0.41±0.06 (0.12)

0.37±0.03 (0.12)

Heart

0.58±0.05a

(0.36)a

0.87±0.14 (0.30)

1.01±0.06 (0.31)

0.96±0.06 (0.29)

1.01±0.08 (0.30)

0.95±0.12 (0.30)

Spleen

0.40±0.05a(0.25)a

0.61±0.05 (0.21)b

0.64±0.04 (0.19)

0.61±0.04 (0.19)

0.65±0.06 (0.19)

0.62±0.05 (0.20)

Liver

4.13±0.37a(2.5)a

7.00±0.58 (2.4)a

7.62±0.60 (2.3)

7.77±0.47b

(2.4)b

7.67±0.45 (2.3)

7.22±0.54 (2.3)

Adrenal (R)

 

0.019±0.002b

(0.012)a

0.21±0.006 (0.007)

0.022±0.001 (0.006)

0.020±0.005 (0.006)

0.021±0.005 (0.006)

0.024±0.004 (0.008)

Adrenal (L)

 

0.021±0.005 (0.012)a

0.021±0.003 (0.007)

0.023±0.001 (0.007)

0.023±0.001 (0.007)

0.023±0.003 (0.007)

0.023±0.008 (0.007)

Kidney (R)

0.78±0.05a

(0.48)a

1.09±0.05a

(0.38)a

1.03±0.07 (0.31)

0.99±0.09 (0.30)

1.02±0.05 (0.31)

0.99±0.06 (0.31)

Kidney (L)

0.84±0.08a

(0.52)a

1.10±0.06a

(0.37)a

1.04±0.08 (0.32)

1.00±0.07 (0.30)

1.02±0.06 (0.31)

0.99±0.05 (0.31)

Testis (R)

1.17±0.07a

(0.71)a

1.41±0.08 (0.49)b

1.48±0.06

(0.45)

1.43±0.10 (0.44)

1.48±0.09 (0.44)

1.42±0.07 (0.45)

Testis (L)

1.22±0.06a

(0.75)a

1.44±0.08 (0.50)a

1.50±0.05 (0.46)

1.47±0.06

(0.45)

1.52±0.05

(0.46)b

1.49±0.07

(0.47)

ap<0.01;bp<0.05

Table 2 continued - Absolute and relative organ weights (female)

Relative organ weights are shown in parenthesises (%)

Dose

10%

5%

2.5%

1.25%

0.6%

Control

Effective No.

9

10

10

10

10

10

Brain

1.75±0.07a

(1.5)a

1.84±0.05 (1.0)

1.86±0.06 (1.0)

1.84±0.05 (1.0)

1.89±0.05b(1.1)

1.86±0.05 (1.0)

Pituitary

0.006±0.002a

(0.005)

0.010±0.003 (0.006)

0.012±0.003 (0.007)

0.011±0.003 (0.006)

0.010±0.003 (0.006)

0.010±0.003 (0.005)

Saliv. Glds.

0.61±0.18a

(0.54)a

0.44±0.04b

(0.25)a

0.40±0.05 (0.22)

0.43±0.08 (0.23)

0.41±0.05 (0.23)

0.40±0.03 (0.22)

Thymus

0.13±0.04a

(0.12)

0.17±0.03 (0.09)

0.17±0.06 (0.09)

0.20±0.05 (0.11)

0.20±0.04 (0.11)

0.19±0.03 (0.10)

Lung (R)

0.40±0.04a

(0.35)a

0.57±0.07 (0.32)b

0.54±0.04 (0.30)

0.56±0.06 (0.30)

0.54±0.04 (0.30)

0.54±0.06 (0.29)

Lung (L)

0.21±0.03a(0.19)a

0.29±0.03 (0.16)

0.27±0.03 (0.15)

0.28±0.03 (0.15)

0.27±0.02 (0.15)

0.29±0.03 (0.16)

Heart

0.48±0.04a

(0.43)a

0.63±0.03 (0.35)a

0.65±0.07 (0.35)b

0.60±0.05 (0.32)

0.60±0.04 (0.33)

0.61±0.04 (0.33)

Spleen

0.34±0.06a

(0.30)a

0.48±0.06b

(0.27)a

0.43±0.03

(0.24)

0.43±0.04 (0.23)

0.42±0.05 (0.23)

0.43±0.03 (0.23)

Liver

3.16±0.24a

(2.8)a

4.07±0.34b

(2.3)a

3.97±0.16

(2.2)b

3.94±0.17

(2.1)

3.77±0.25 (2.1)

3.77±0.28

(2.0)

Adrenal (R)

 

0.018±0.002b

(0.016)a

0.022±0.005

(0.012)

0.024±0.004 (0.01)

0.023±0.005 (0.012)

0.024±0.004 (0.013)

0.23±0.005 (0.012)

Adrenal (L)

 

0.020±0.003a

(0.018)b

0.25±0.003 (0.014)

0.025±0.006 (0.014)

0.024±0.005 (0.013)

0.027±0.004 (0.015)

0.026±0.004 (0.014)

Kidney (R)

0.64±0.05a

(0.57)a

0.98±0.06a

(0.55)a

0.72±0.06a

(0.40)a

0.63±0.05b

(0.34)b

0.59±0.03

(0.33)a

0.57±0.04 (0.31)

Kidney (L)

0.67±0.05a

(0.59)a

0.97±0.04a

(0.56)a

0.71±0.05a

(0.39)a

0.62±0.05

(0.33)

0.60±0.03 (0.33)

0.60±0.04

(0.32)

Testis (R)

0.020±0.005b

(0.018)

0.033±0.010

(0.019)

0.041±0.019 (0.023)

0.042±0.020 (0.023)

0.040±0.009 (0.022)

0.042±0.022 (0.023)

Testis (L)

0.012±0.003 (0.011)

0.040±0.020

(0.022)

0.046±0.021 (0.025)

0.039±0.022 (0.021)

0.045±0.016

(0.025)b

0.033±0.009 (0.18)

ap<0.01;bp<0.05

Table 3 - Summary of histopathological findings in F344 rats (both sexes) after 13 weeks treatment.

Findings

Concentration of potassium pyrophosphate in diet

10%

5%

2.5%

1.25%

0.6%

0%

Kidney

Tubular necrosis, with calcification and interstitial fibrosis

+++

++

±

-

-

-

Tongue

Ulceration and/or granuloma formation

+

+ ~ ±

-

-

-

-

Salivary glands (serous)

hypertrophy

+

± ~ -

-

-

-

-

+++ severe, ++ moderate, + mild, ± slight

Conclusions:
LOEL = 0.6%
Executive summary:
A 13 week sub-chronic toxicity study of potassium pyrophosphate was carried out in male and female F344 rats at the dose levels of 10, 5, 2.5, 1.25, 0.6 and 0% in the diet. Sixty animals of both sexes were divided into the 6 dosage groups. The purpose of the study was to determine the maximum tolerable dose of test substance for use in rats in a long-term carcinogenicity study. Observations were limited to mortality, clinical abnormalities, body weights, food intake, haematology, clinical chemistry, gross pathology (incl. organ weight) and histopathology. By the end of the test, two male and one female from the 10% dosage group had died of renal failure. Bodyweight gains of both male and female rats treated with 10% and of male rats treated with 5% were significantly decreased as compared with controls. On histological examination, necrosis and calcification in renal tubules, ulceration and/or granuloma formation in tongue mucosa and hypertrophy of salivary glands were observed at high incidence. Based on these findings, a concentration of 5% in diets was considered to be the maximum tolerable dose for use in a long-term carcinogenicity study.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
Continuous administration of Sodium Pyrophosphate over 16 weeks at dietary levels of 0, 1, 2.5 and 5%.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Birmingham Wistar
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): diets were prepared in 3 kg batches
- Mixing appropriate amounts with (Type of food): whole wheat flour, full cream dried milk, salt mix and casein
- Storage temperature of food: cool room
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
100 - 110 days
Frequency of treatment:
Continuous
Dose / conc.:
5 other: % (nominal in diet)
Remarks:
of Disodium hydrogen phosphate
Dose / conc.:
0 other: % (nominal in diet)
Remarks:
of Sodium pyrophosphate
Dose / conc.:
1 other: % (nominal in diet)
Remarks:
of Sodium pyrophosphate
Dose / conc.:
2.5 other: % (nominal in diet)
Remarks:
of Sodium pyrophosphate
Dose / conc.:
5 other: % (nominal in diet)
Remarks:
of Sodium pyrophosphate
No. of animals per sex per dose:
20 (10 individual caging, 5 and 5 group caging)
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

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

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: No data

URINALYSIS: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
LOEL
Effect level:
< 514 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Extrapolated from level of compound in diet assuming 0.35 kg rat eats 18 g food/day.
Key result
Critical effects observed:
not specified

Body weight and feed consumption:

The body weight gain and feed consumption of the 5 % pyrophosphate group was decreased, the effect is not so obvious because the control group had decreased values.

Table 1: Body weight and feed consumption in g

  males  females   
  bw gain  feed intake  bw gain  feed intake 
control  210 + 20  1309 + 28   137 + 7   1184 + 28
1% Pyrophosphate  215 + 16  1359 + 27   129 + 6   1110 + 28
2.5% Pyrophosphate  229 + 15  1305 + 24   129 + 6   1116 + 16
5% Pyrophosphate  187 + 15  1213 + 48   114 + 7*   1055 + 26
5% Orthophosphate  234 + 14  1363 + 18   140 + 8   1168 + 23

* significantly diferent from control group p<0.05

Balance studies:

Sodium pyrophosphate and orthophosphate are absorbed in a similar manner. Rat feces are capable of hydrolysing pyrophosphate to orthophosphate.

Function tests:

BSP Test: no changes due to treatment noted

The phenol red test was highly variable, no significant differences.

Concentration test: males dosed with 2.5 and 5% pyrophosphate (PP), 5% orthophosphate (OP) and females dosed with 5% PP or OP had impaired renal function.

Table 2: Values of the kidney concentration test:

 Group males   females 
Control   1.0622 + 0.0026  1.0592 + 0.0024 
1% Pyrophosphate  1.0547 + 0.0047  1.0626 + 0.0026 
2.5% Pyrophosphate  1.0429 + 0.0061*  1.0561 + 0.0015 
5% Pyrophosphate  1.0432 + 0.0051*  1.0445 + 0.0014** 
5% Orthophosphate  1.0488 + 0.0018**  1.0513 + 0.0025* 

* significantly different from control group p<0.05

** significantly different from control group p<0.01

Hematology:

Despite some differences no significant trends towards abnormalites with increasing pyrophosphate doses.

Organ weights:

The main increases in the relative weights of heart, liver, spleen, stomach intestines kidneys and testes were seen in the 5% PP groups, reaching often statistical significance.

Table 3a: Relative organ weights males ( in mg/100g live weight + standard error)

  Control  1% PP  2.5% PP  5% PP  5% OP 
Heart  244 + 4  255 + 6  251 + 77  295 + 19*  256 + 7 
Liver  3170 + 133  3097 + 75  3029 + 87  3452 + 234  3112 + 57 
Spleen  271 + 14  233 + 7  261 + 16  286 + 15  256 + 13 
Stomach  378 + 12  365 + 15  388 + 12  608 + 39**  407 + 14 
Intestine  2229 + 112  2108 + 111  1964 + 83  2716 + 239  1940 + 99 
Adrenals  13 + 1  11 + 0.6  12 + 0.9  13 + 0.7  12 + 0.4 
Kidneys  634 + 21  598 + 17  648 + 24  769 + 31*  741 + 15** 
Testes  962 + 42  900 + 29  905 + 42  1233 + 79*  1005 + 37 

Table 3b: Relative organ weights females ( in mg/100g live weight+standard error)

  Control  1% PP  2.5% PP  5% PP  5% OP 
Heart  278+4  273 +5  295 +10  311 +9*  285 +9 
Liver  3146 +33  3045 +80  3048 +129  3292 +92  3021+65 
Spleen  297+14  304 +21 305 +20  334 +20  285 +30 
Stomach  451 +16 459+15  506 +22  721 +63**  486 +23 
Intestine  2521 +73  2928 +149  2626 +85  3203 +149**  2696 +103
Adrenals  27+1.5  26 +1.7  26 +1.0  26 +1.7  25 +2.0 
Kidneys  601 +12  629 +21  743 +24**  893 +42**  838 +35** 

* significantly different from control group p<0.05

** significantly different from control group p<0.01

Gross and histopathology:

Macroscopic findings were limited to kidneys, heart and stomach, see Table 4. Only the kidneys showed macroscopical changes.

Table 4: Macroscopical findings (of 10 animals per group)

  Control  1% PP  2.5% PP  5% PP  5% OP  Control  1% PP  2.5% PP  5% PP  5% OP 
Pale, pitted kidneys 
Calcification of kidneys 
Hypertrophy of cardiac/pyloric border of stomach  
Hemorrhages at this site 

The histopathology revelad that the changes in the lower doses were mainly in the cortex, whereas at higher doses the medullary zone was more affected.

Table 5: Microscopical findings in kidneys (% of each group with abnormalities)

  Controls  1% PP  2.5% PP  5% PP  5% OP 
Renal damage  25  95  100  100  100 
Cortical atrophy  15  60  60  15  15 
Cortical hyaline degeneration   10  55  55  20  10 
Cortical calcification   10  10 
Medullary calcification  30  70  75 
Medullary necrosis  10  60  65 
Tubular casts  30  55  55 
Hemorrhages and exsudate  45  50  60  60 
Chronic inflammatory changes  70  70 

Conclusion

The administation of pyrophosphate to rats caused effects at various levels. At 5 % PP feed intake and bw gain was affected. Kidney concentration function was impaired at 2.5% PP and above. Organ weights had changes at 2. 5 and 5 % PP. Levels of 1 % PP and higher and 5% of OP caused resulted in demonstrable changes in the kidneys.

Conclusions:
LOEL < 514 mg/kg bw/day (lowest dose tested)
Executive summary:

Continuous administration of Sodium Pyrophosphate over 16 weeks at dietary levels of 0, 1, 2.5 and 5%

.

The administation of pyrophosphate to rats caused effects at varoius levels. At 5 % pyrophosphate feed intake and bw gain was affected. Kidney concentration function was impaired at 2.5% pyrophosphate and above. Organ weights had changes at 2.5 and 5 % pyrophosphate. Levels of 1 % PP and higher and 5% of orthophosphate caused resulted in demonstrable changes in the kidneys.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 6 weeks old.
- Housing: The animals were housed in groups of 3 or 4 in plastic cages, on sterilised soft wood chips.
- Diet (e.g. ad libitum): Diet (CRF-1, Oriental Yeast, Japan), ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%): 55±5%
- Air changes (per hr): 18 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Ammonium sulphate was mixed into powdered diet at concentrations of 0%, 0.1%, 0.6%, 1.5% and 3.0%, the diet was then pelleted. Diets were provided ad libitum, and were replaced regularly within 2 week intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical methods were not given in the paper. Recapture rates for ammonium sulfate from the admixed diet at each concentration level were confirmed to be 95.4-98.7%. Stability of ammonium sulfate in the solid pellet diet was evaluated and no decomposition was confirmed after storage under refrigeration or at room temperature for 2 weeks.
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
Daily - ad libitum in diet
Dose / conc.:
0 other: % (nominal in diet)
Dose / conc.:
0.1 other: % (nominal in diet)
Dose / conc.:
0.6 other: % (nominal in diet)
Dose / conc.:
3 other: % (nominal in diet)
No. of animals per sex per dose:
10 rats/sex/group
Control animals:
yes, plain diet
Details on study design:
The study was a combined chronic toxicity and carcinogenicity study. The chronic toxicity study used 10 rats/sex/dose, exposed for 52 weeks. A 13-week preliminary study was conducted in which rats were administered diets containing 0%, 0.38%, 0.75%, 1.5% and 3.0%. Reduced body weight gain and diarrhoea were ntoed in the 3.0% males, no effects were seen in the females.
Observations and examinations performed and frequency:
Clinical signs and mortality were observed daily, and body weight and food consumption were recorded every 2 weeks until week 10, and every 5 weeks thereafter.
Sacrifice and pathology:
After 52 weeks exposure, all surviving rats were euthanised. Rats were fasted overnight prior to terminal necropsy.
Blood samples were collected from the abdominal aorta at the time of sacrifice. Haematological examinations were performed using an automatic haematology analyser, and the following parameters determined: red blood cell count, heamoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, platelet count, white blood cell count, differential leukocyte counts and reticulocyte counts. The following serum chemistry parameters were evaluated: total protein, albumin, albumin/globulin ratio, total bilirubin, total cholesterol, triglyceride, blood urea nitrogen, creatinine, calcium, inorganic phosphorous, sodium, potassium, chloride, aspartate transaminase, alanine transaminase, alkaline phosphatase and gamma-glutamyl transpeptidase.
The rats were subject for a complete necropsy at sacrifice. Animals that were found dead during the study were also subjected to a complete necropsy as soon as they were found. The brain, lungs, heart spleen, liver, adrenals, kidneys and testes were weighed. In addition to these organs, the nasal cavity, trachea, aorta, pituitary, thyroids, parathyroids, salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, urinary bladder, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, mammary glands, skin, mesenteric and submandibular lymph nodes, thymus, sternum, femur including bone marrow, sciatic nerve, trigeminal nerve, spinal cord (cervical, thoracic and lumber cords), eye, Harderian gland and thigh muscle were excised and specimens from all these organs or tissues were fixed in 10% buffered formalin for paraffin-embedding sectioning and staining with haematoxylin and eosin for histopathological examination. All organs and tissues in the control and 3.0% group animals were histopathologically examined. Additionally, macroscopically abnormal sites in the 0.1% and 0.6% group animals were also histopathologically examined.
Statistics:
Bartlett test followed by ANOVA or Kruskal-Wallis as appropriate, Dunnett's multiple test was used for subsequent comparison when significance was achieved. Survival rates were compared with Fisher's exact probability test or the Mann-Whitney U-test.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed, including diarrhoea which was noted in the preliminary study.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects on body weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no effects on food consumption, except for a tendency towards increased food intake in the 3.0% males (Table 1).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No group differences were detected in haematology parameters. Some slight changes were found in white blood cell parameter, but there was no dose-relationship and they were therefore considered to be incidental.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were also no effects on serum chemistry parameters.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative kidney weights were increased or showed a tendency for increase at 3.0% in both sexes. Absolute spleen weights were decreased and relative liver weights were increased in the 3.0% male group. No dose-related changes were found in the other organs.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no obvious macroscopic findings at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Several non-neoplastic lesions such as bile duct proliferation in the liver and focal myocarditis in the heart were noted in the control and 3.0% group, and there were no significant differences in their incidences between the groups (in either sex).
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Malignant pheochromocytoma of the adrenal in the 3.0% males, two adenomas in the anterior pituitary in 3.0% females, and uterine endometrial stromal poly in 1 female control, were noted.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
0.6 other: %
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: The dietary level of 0.6% is equivalent to 256 and 284 mg/kg/day in males and females, respectively.
Key result
Critical effects observed:
not specified

Table 1. Final body weight, food consumption and test substance intake.

Sex

Dose level (%)

Final body weight (g)

Food consumption (g/rat/day)

Intake of ammonium sulphate (mg/kg bw./day)

Male

0

410.9±12.3

13.9

-

0.1

428.6±17.6

13.6

42

0.6

416.7±23.7

13.4

256

3.0

400.5±15.1

15.7

1527

Female

0

207.4±13.5

8.4

-

0.1

220.3±8.7

8.6

48

0.6

219.2±13.6

8.4

284

3.0

212.7±24.4

8.6

1490

Each value represents the mean throughout the administration period

Conclusions:
Based on effects on organ weights, the NOAEL was estimated to be 0.6%, which is equivalent to 256 and 284 mg/kg/day in males and females, respectively.
Executive summary:

Chronic toxicity studies of ammonium sulphate, used as a food additive in fermentation, were performed in male and female Fisher 344 rats at dietary concentrations of 0%, 0.1%, 0.6% and 3.0% in a 52-week toxicity study. Treatment caused significant increase in kidney and/or liver weights in males and females of the 3.0% diet group, but no effects were found on survival rate, body weights, and hematological, serum biochemical or histopathological parameters at any dose levels in the chronic toxicity study. It was concluded that the no observed adverse effect level of ammonium sulphate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg b.w./day in males and females, respectively, and the compound is non-carcinogenic under the conditions of the study.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
(limited histopathology)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Nippon
- Age at study initiation: 6 weeks old
- Diet (e.g. ad libitum): Animals were maintained on CRF-1 powder diet.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ºC
- Humidity (%): 55 ± 5%
- Air changes (per hr): 18 air exchanges/day
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
0 other: % (nominal in diet)
Dose / conc.:
0.38 other: % (nominal in diet)
Dose / conc.:
0.75 other: % (nominal in diet)
Dose / conc.:
1.5 other: % (nominal in diet)
Dose / conc.:
3 other: % (nominal in diet)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, plain diet
Details on study design:
The dose levels were set on the basis of results from a previous 2-week study with a dose level of 5% (no further details reported).
Observations and examinations performed and frequency:
CLINICAL CHEMISTRY, HEMATOLOGY: The following clinical parameters were determined: total protein (TP), albumin/globulin (A/G), albumin, total cholesterin, BUN, Na, Cl, K , Ca, P, GOT, GPT, alkaline phosphatase. Hematology included red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hk), mean corpuscular volume (MCV), mean erythrocyte hemoglobin (MCH), mean erythrocyte hemoglobin concentration (MCHC), platelet count, and leukocyte count.
Sacrifice and pathology:
At necropsy, the following organs were weighed and absolute and relative organ weights determined:

males: brain, lung, heart, spleen, liver, adrenal, kidney, testis
females: brain, lung, heart, spleen, liver, adrenal, kidney

ORGANS EXAMINED HISTOPATHOLOGICALLY: males: brain, lung, heart, spleen, liver, adrenal, kidney, testis. females: brain, lung, heart, spleen, liver, adrenal, kidney. Organs were fixed in formalin and hematoxylin-eosin slides were prepared and examined histologically.
Statistics:
Bartlett,and Kruskal - wallis tests, and parametric Dunnett and Scheffe tests.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male animals in the 3% group exhibited diarrhea during the administration period.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At study end final body weights were in males 298, 273, 287, 282 and 284 g for the 0, 0.38, 0.75, 1.5 and 3% groups, respectively. In females, final body weights were 151, 157, 152, 161 and 158 g for the 0, 0.38, 0.75, 1.5 and 3% groups, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
14.2, 14.0, 14.3, 14.1, 13.8 in the males of the 0, 0.38, 0.75, 1.5 and 3% groups, respectively. In females, the values were 9.2, 9.1, 9.3, 9.3 and 8.4 for the 0, 0.38, 0.75, 1.5 and 3% groups, respectively.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No biologically significant changes were observed in any of the investigated parameters. Though there were statistical differences in some of the parameters, there was no consistent dose-effect relationship and/or all values were within the normal ranges of values normally found in the rat strain used in this study. In particular, there were no signs indicative of a metabolic acidosis.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in absolute or relative organ weights were observed for brain, lung, heart, spleen, liver, adrenals, kidney and testis weights. Increases (<15%) in the relative and absolute kidney weights in high dose male and females, and in liver weight in high dose females (+11%), were not accompanied by any functional (clinical parameters) or histopathological changes, and were therefore not considered as adverse effects by the authors.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in absolute or relative organ weights were observed for brain, lung, heart, spleen, liver, adrenals, kidney and testis weights. Increases (<15%) in the relative and absolute kidney weights in high dose male and females, and in liver weight in high dose females (+11%), were not accompanied by any functional (clinical parameters) or histopathological changes, and were therefore not considered as adverse effects by the authors.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No significant pathological effects were found. In the 3% male group myofibrosis cordis, basophilic kidney tubulus as well as splenic melanosis were observed; in the female group basophilic kidney tubulus as well as splenic melanosis were observed. However the rate of occurrence was similar to controls.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % (equivalent to 886 mg/kg bw/day)
Sex:
male
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
3 other: % (equivalent to 1975 mg/kg bw/day)
Sex:
female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
not specified
Conclusions:
Ammonium sulphate was shown to be of low toxicity. Based on these results, the NOEL was judged to be 1.5% in males (886 mg/kg bw/day) and 3% in females (1975 mg/kg bw/day).
Executive summary:

A 13-week subchronic oral toxicity study with ammonium sulphate was performed in both sexes of F344 rats by feeding them diet containing concentrations of 0%, 0.38%, 0.75%, 1.5%, and 3.0% of the substance. Rats were randomly divided into 5 groups each consisting of 10 males and 10 females. Male animals in the 3% group exhibited diarrhoea during the administration period. No changes indicating obvious toxicity were observed in the body weights, organ weights, hematological, serum biochemical, or histopathological examinations. Based on these results, the NOEL (no-observed-effect level) of ammonium sulphate in F344 rats was judged to be 1.5% in males (equivalent to 886 mg/kg bw/d) and 3% in females (1975 mg/kg bw/d). The MTD for 2-year carcinogenicity studies in F344 rats was concluded to be 3.0% or more in the diet.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Principles of method if other than guideline:
28-day repeat dose via oral route.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
200 000 ppm
Remarks:
nominal in diet
Dose / conc.:
20 000 ppm
Remarks:
nominal in diet
Dose / conc.:
2 000 ppm
Remarks:
nominal in diet
Dose / conc.:
200 ppm
Remarks:
nominal in diet
Dose / conc.:
0 ppm
Remarks:
nominal in diet
Dose / conc.:
70 mg/kg bw (total dose)
Remarks:
70-110 mg/kg bw over weeks 1-4 (Group 3). Nominal in diet
Dose / conc.:
1 750 mg/kg bw (total dose)
Remarks:
Week 1. Nominal in diet
Dose / conc.:
7 500 mg/kg bw (total dose)
Remarks:
7500 to 8900 mg/kg bw during Weeks 2,3,4 (Group I). Nominal in diet.
Dose / conc.:
630 mg/kg bw (total dose)
Remarks:
630-1220 mg/kg bw over weeks 1-4 (Group 2). Nominal in diet.
Dose / conc.:
4 mg/kg bw (total dose)
Remarks:
4-13 mg/kg bw over weeks 1-4 (Group 4). Nominal in diet.
No. of animals per sex per dose:
4 males per dose group
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
-Bodyweight was recorded twice weekly.

-Food consumption and signs of clinical abnormalities.
Sacrifice and pathology:
All rats were sacrificed after 28 days. Sections of tissues (lung, heart, spleen, liver, kidney, brain, adrenal gland, intestine, muscle and spinal cord) were fixed and examined microscopically for histopathological changes.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rats in highest dose group were emaciated, lethargic and hypersensitive to handling. After 28 days of exposure these conditions were more pronounced.
Mortality:
no mortality observed
Description (incidence):
No mortalities in any dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Rats in highest dose group steadily lost weight throughout the experiment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Rats in the highest dose group consumed less than one-half as much feed as rats in other groups.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
8 900 mg/kg bw/day (nominal)
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
Key result
Critical effects observed:
not specified

Mn3O4 fed to rats for 28 days showed low oral toxicity, even at doses as high as 8900 mg Mn3O4/kg bw/day.

Conclusions:
Mn3O4 fed to rats for 28 days showed low oral toxicity, even at doses as high as 8900 mg Mn3O4/kg bw/day.
Executive summary:
Mn3O4 fed to rats for 28 days showed low oral toxicity, even at doses as high as 8900 mg Mn3O4/kg bw/day.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
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: 50 days
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
nominal in diet
Dose / conc.:
1 600 ppm
Remarks:
equivalent to 110 mg/kg in males and 115 mg/kg in females. Nominal in diet
Dose / conc.:
3 130 ppm
Remarks:
nominal in diet
Dose / conc.:
6 250 ppm
Remarks:
nominal in diet
Dose / conc.:
12 500 ppm
Remarks:
nominal in diet
Dose / conc.:
25 000 ppm
Remarks:
equivalent to doses 1700 mg/kg in males and 2000 mg/kg in females. 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
Description (incidence and severity):
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
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.
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:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative liver weights of all exposed males and of the female 25000 ppm group were significantly lower than the controls.
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 700 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
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. No clinical or histopathologic findings in rats were chemical related.
Executive summary:
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. No clinical or histopathologic findings in rats were chemical related.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
nominal in diet
Dose / conc.:
3 130 ppm
Remarks:
nominal in diet
Dose / conc.:
6 250 ppm
Remarks:
nominal in diet
Dose / conc.:
12 500 ppm
Remarks:
nominal in diet
Dose / conc.:
25 000 ppm
Remarks:
nominal in diet
Dose / conc.:
50 000 ppm
Remarks:
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. Mice were weighed at the beginning of the studies and twice 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:
mortality observed, non-treatment-related
Description (incidence):
One control male mouse and one female mouse receiving 3130 ppm died of unknown causes during this study. No deaths were thought to be chemical related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gains of all exposed males were significantly lower than that of the control group and the final mean bodyweight of the 50000 ppm group was 13% lower than that of the controls. The mean bodyweight gain of the females receiving 50000 ppm was significantly lower than that of the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption was similar to that of the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The percent haematocrit, haemoglobin concentrations and mean erythrocyte volumes of 50000 ppm male and female mice were significantly lower than those of the controls.
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:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative liver weights of the 50000 ppm males were significantly lower than the controls.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Epithelial hyperplasia and hyperkeratosis of the forestomach occurred in three 50.000 ppm males.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
25 000 ppm
Sex:
male
Basis for effect level:
body weight and weight gain
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
50 000 ppm
Sex:
female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
not specified
Conclusions:
No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion. Epithelial hyperplasia and hyperkeratosis of the forestomach occurred in three 50,000 ppm males.
Executive summary:
Groups of 10 male and 10 female mice were fed diets containing 0, 3130, 6250, 12500, 25000 or 50000 ppm manganese sulphate. Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Mice were weighed at the beginning of the studies and twice 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. No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion. Epithelial hyperplasia and hyperkeratosis of the forestomach occurred in three 50,000 ppm males.
Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
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) sulfate monohydrate. 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.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals or test system and environmental conditions:
Rats were approximately 41 days old at the start of the study.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Dose / conc.:
1 500 ppm
Remarks:
equivalent to 60 mg/kg in male and 70 mg/kg in females. Nominal in diet.
Dose / conc.:
5 000 ppm
Remarks:
equivalent to 200 mg/kg in male and 230 mg/kg in females. Nominal in diet.
Dose / conc.:
15 000 ppm
Remarks:
equivalent to 615 mg/kg in male and 715 mg/kg in females. Nominal in diet.
No. of animals per sex per dose:
70 male and 70 female rats per 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:
effects observed, treatment-related
Description (incidence and severity):
No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion.
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival of 15,000 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 15,000 ppm group resulted from increased incidences of advanced renal disease related to ingestion of manganese (II) sulfate monohydrate. The decreased survival of the 15,000 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.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of 15,000 ppm male rats was within 5% of the control group until week 89, by week 104, the mean body weight of 15,000 ppm males was 10% lower than that of the control group. The mean body weights of 1,500 and 5,000 ppm male rats and all exposed female groups were similar to those of the controls throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption by all exposure groups was similar to that by the control groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No differences in hematology parameters attributable to the ingestion of manganese (II) sulfate monohydrate occurred between exposed and control groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No differences in clinical chemistry parameters attributable to the ingestion of manganese (II) sulfate monohydrate occurred between exposed and control groups. At both the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of 5,000 and 15,000 ppm male and female rats, with an accompanying depression of hepatic iron.
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:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The ingestion of diets containing 15,000 ppm manganese (II) sulfate monohydrate was associated with a marginal increase in the average severity of nephropathy in male rats (0 ppm, 2.9; 1,500 ppm, 3.0; 5,000 ppm, 3.0; 15,000 ppm, 3.2). The increased severity of nephropathy in the 15,000 ppm male rats was accompanied by significantly increased incidences of mineralization of the blood vessels (4/52, 10/51, 6/51,17/52) and glandular stomach (8/52,13/51, 9/51, 23/52), parathyroid gland hyperplasia (14/51, 14/46, 12/49, 23/50), and fibrous osteodystrophy of the femur (12/52,14/51,12/51, 24/52). 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.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No increase in the incidence of neoplasms in male or female rats was attributed to the ingestion of diets containing manganese (II) sulfate monohydrate.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
230 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
mortality
Key result
Critical effects observed:
not specified
Conclusions:
No increase in the incidence of neoplasms in male or female rats was attributed to the ingestion of diets containing manganese (II) sulfate monohydrate.
Executive summary:
Groups of 70 male and 70 female rats were fed diets containing 0, 1,500, 5,000, or 15,000 ppm manganese (II) sulfate monohydrate. 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.

Survival of 15,000 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 15,000 ppm group resulted from increased incidences of advanced renal disease related to ingestion of manganese (II) sulfate monohydrate. The decreased survival of the 15,000 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 15,000 ppm male rats was within 5% of the control group until week 89, by week 104, the mean body weight of 15,000 ppm males was 10% lower than that of the control group. The mean body weights of 1,500 and 5,000 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.


No differences in hematology and clinical chemistry parameters attributable to the ingestion of manganese (II) sulfate monohydrate occurred between exposed and control groups. At both the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of 5,000 and 15,000 ppm male and female rats, with an accompanying depression of hepatic iron.


The ingestion of diets containing 15,000 ppm manganese (II) sulfate monohydrate was associated with a marginal increase in the average severity of nephropathy in male rats (0 ppm, 2.9; 1,500 ppm, 3.0; 5,000 ppm, 3.0; 15,000 ppm, 3.2). The increased severity of nephropathy in the 15,000 ppm male rats was accompanied by significantly increased incidences of mineralization of the blood vessels (4/52, 10/51, 6/51,17/52) and glandular stomach (8/52,13/51, 9/51, 23/52), parathyroid gland hyperplasia (14/51, 14/46, 12/49, 23/50), and fibrous osteodystrophy of the femur (12/52,14/51,12/51, 24/52). 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.

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
Principles of method if other than guideline:
Groups of 70 male and 70 female mice received diets containing 0, 1,500, 5,000, or 15,000 ppm manganese (II) sulfate monohydrate. These levels resulted in an average daily ingestion of 160, 540, or 1,800 mg/kg body weight (males) or 200, 700, or 2,250 mg/kg (females). Nine or 10 mice from each group were evaluated at the 9-month and 15-month interim evaluations.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Mice were approximately 41 days old at the beginning of the study.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
nominal in diet
Dose / conc.:
1 500 ppm
Remarks:
nominal in diet
Dose / conc.:
5 000 ppm
Remarks:
nominal in diet
Dose / conc.:
15 000 ppm
Remarks:
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
Description (incidence and severity):
No clinical findings were attributed to the administration of manganese (II) sulfate monohydrate.
Mortality:
no mortality observed
Description (incidence):
Survival rates of exposed male and female mice in the 2-year study were similar to those of the control groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 1,500, 5,000, and 15,000 ppm groups were 6%, 9%, and 13% lower than that of the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption by all exposure groups was similar to that by the control groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No chemical-related differences between exposed and control groups occurred in hematology parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No chemical-related differences between exposed and control groups occurred in clinical chemistry parameters. At the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of the 5,000 and 15,000 ppm groups. Hepatic iron levels were significantly lower in exposed females at the 9-month interim evaluation and in 5,000 and 15,000 males and all exposed females at the 15-month interim evaluation.
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:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Incidences of thyroid follicular dilatation and hyperplasia were significantly greater in 15,000 ppm male and female mice than in controls. Follicular cell adenomas occurred in one 15,000 ppm male at the 15-month interim evaluation and in three 15,000 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 1,500, and five 15,000 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 15,000 ppm male and exposed female groups. The hyperplasia was associated with ulcers and inflammation in some mice, particularly males.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
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.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
not specified

No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion. Epithelial hyperplasia and hyperkeratosis of the forestomach occurred in three 15,000 ppm males.

Conclusions:
No clinical findings were attributed to manganese (II) sulfate monohydrate ingestion. Epithelial hyperplasia and hyperkeratosis of the forestomach occurred in three 15,000 ppm males.
Executive summary:
Groups of 70 male and 70 female mice received diets containing 0, 1,500, 5,000, or 15,000 ppm manganese (II) sulfate monohydrate. These levels resulted in an average daily ingestion of 160, 540, or 1,800 mg/kg body weight (males) or 200, 700, or 2,250 mg/kg (females). Nine or 10 mice from each group were evaluated at the 9-month and 15-month interim evaluations.


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 1,500, 5,000, and 15,000 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.


No chemical-related differences between exposed and control groups occurred in hematology or clinical chemistry parameters. At the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of the 5,000 and 15,000 ppm groups. Hepatic iron levels were significantly lower in exposed females at the 9-month interim evaluation and in 5,000 and 15,000 males and all exposed females at the 15-month interim evaluation.


Incidences of thyroid follicular dilatation and hyperplasia were significantly greater in 15,000 ppm male and female mice than in controls. Follicular cell adenomas occurred in one 15,000 ppm male at the 15-month interim evaluation and in three 15,000 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 1,500, and five 15,000 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 15,000 ppm male and exposed female groups. The hyperplasia was associated with ulcers and inflammation in some mice, particularly males.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
no
Remarks:
Scientific publication
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Co., Ltd. (Gyeonggi-do, Korea)
- Age at study initiation: 6 weeks old
- Diet (e.g. ad libitum): commercial rodent chow (2.0 Mrad ɣ-ray sterilized EP pellet, Cargill Agri Purina Korea Ltd, Korea), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±3 ºC
- Humidity (%): 55±5%
- Air changes (per hr): 10~18 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial lighting from 08:00 to 20:00

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 day
Frequency of treatment:
5 days/week
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the results of the acute toxicity study and a 14-day dose range study. No toxicity or
mortality was observed in any of the five male and female rats treated for 14 days.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed thoroughly for the onset of any immediate signs of toxicity, and throughout the observation period to record any delayed acute effects and mortality. All animals were observed once a day for 90 days after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were monitored for body weight changes once a week after administration. The mean body weight of animals that survived up to the end of the administration period (90 days) was calculated.

FOOD CONSUMPTION: Yes
Food consumption by each group was measured at the start of treatment and during the 90-day administration period.

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were fasted overnight.
- Parameters: Hematology parameters in EDTA 3K-treated blood (Sewon Medical, Korea) were measured using a hematological autoanalyzer (Advia 120E, Bayer, UK), including total white blood cell (WBC) count, differential leukocyte percentage, total red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte ratio (Retic), and platelet count (PLT). Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured in sodium citrate-treated blood using an automated hematocoagulation analyzer (Coagrex-100S, Japan).

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes. Animals were fasted overnight.
- Parameters: total protein (TP), albumin (ALB), A/G ratio, total bilirubin (TBIL), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRN), blood urea nitrogen (BUN), total cholesterol (CHOL), triglycerides (TG), glucose (GLU), calcium (Ca), phosphorus (IP) and creatine kinase (CK). Serum electrolytes such as chloride (Cl), sodium (Na), and potassium (K) were measured using an ion autoanalyzer (Bayer 644 Na/K/Cl analyzer, USA).

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of administration, urinalysis of five animals of each sex per group was conducted to determine glucose, bilirubin, ketone body, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocyte levels, using the Multistix 10SG (Bayer, U.S.A.) and urine analyzer (Clinitek 500, U.S.A.).

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At scheduled termination, all surviving animals were anesthetized by isoflurane inhalation for blood sample collection, and then sacrificed by exsanguination of the abdominal aorta. Complete gross postmortem examinations were performed on all terminated animals. All organs were fixed and stored individually for histopathological examination.

ORGAN WEIGHTS: Yes
The absolute and relative (organ-to-body weight ratios) weights of the following organs were measured in all survivors following sacrifice: liver, kidneys, adrenals, testis, ovaries, and brain.

HISTOPATHOLOGY: Yes
The following tissues were obtained from all animals: brain, pituitary, eye, thyroids, heart, lung, liver, kidney, spleen, adrenals, stomach, testis, urinary bladder, femur, and ovaries. Eyes and testes were preserved in Davidson’s fixative and Bouin’s fixative, respectively. Other tissues were fixed with 10% neutral buffered formalin solution. The liver, kidney, adrenal, heart, and spleen from the high dosage and control groups were routinely processed, embedded in paraffin, and sectioned to 3-5 µm. The sections were stained with hematoxylin and eosin for microscopic examination.
Statistics:
The differences in parameters (body weights, organ weights, and the results of the blood biochemistry and hematology) between groups were assessed by a standard two-way analysis of variance (ANOVA). If these showed statistical significance, Duncan’s or Dunnett’s multiple range test were used to compare groups (SPSS version 12.0). P-values < 0.05 were considered statistically significant.
Clinical signs:
no effects observed
Description (incidence and severity):
Female rats treated with 500 and 1,000 mg/kg of test substance showed hair loss. No significant clinical signs were observed in any other group.
Mortality:
no mortality observed
Description (incidence):
There was no treatment-related mortality in the animals treated with the test substance during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The terminal body weight of male rats treated with 1,000 mg/kg of test substance was lower than that of the control group, while there were no differences in body weight between the 250, 500 mg/kg, and control group. There was no treatment-related change in body weight in female rats.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in food consumption was observed in the 1,000 mg/kg group of males at week 8 and 9. Food consumption in females increased significantly in the 1,000 mg/kg group at week 4. There were no significant changes in food consumption in the other groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no significant changes in hematology in the 250 mg/kg group. WBC and neutrophil counts were significantly increased in males and females of the 1,000 mg/kg group, whereas the lymphocyte count was significantly lower in males and females of the 1,000 mg/kg group. RBC, HB, HCT, PT, and APTT were significantly reduced in the males of the 1,000 mg/kg group, and PT was significantly reduced in males of the 500 mg/kg group
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no significant changes in the serum biochemistry of males and females in the 250 mg/kg group. Total protein was significantly reduced in both sexes in the 500 and 1,000 mg/kg group. Albumin was significantly reduced in males of the 500 and 1,000 mg/kg group, and in females of the 1,000 mg/kg group. The A/G ratio was significantly increased in females of the 500 and 1,000 mg/kg group. T-BIL was significantly increased in females of the 1,000 mg/kg group, and ALP was significantly reduced in females of the 1,000 mg/kg group. AST was significantly increased in males of the 1,000 mg/kg group, but ALT was significantly reduced in females of the 1,000 mg/kg group. TG were significantly increased in both sexes in the 1,000 mg/kg group, while Ca, IP, Na, K, and Cl were significantly reduced in both sexes in this group. IP was also reduced in males of the 500 mg/kg group
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no specific symptoms in urinalysis at the doses of 250, 500, and 1,000 mg/kg.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No exposure-related histopathological changes were observed in any of the organs examined in SD rats, with the exception of kidney lesions. Cortical tubular basophilia of the renal tubule was more evident in males of the 1,000 mg/kg group and mineralization of the kidney was evident in females of the 1,000 mg/kg group. Other background lesions were observed in the 0, 250, 500, and 1,000 mg/kg groups in both sexes.
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
haematology
histopathology: non-neoplastic
Key result
Critical effects observed:
not specified

Tubular basophilia of the renal tubule was evident in males and females, with mineralization of the kidney in females. Tubular basophilia is a relatively frequent finding in young control rats, and can be induced by nephrotoxic agents; the lesion is regenerative in nature.

Conclusions:
Based on these results, the no observed effect level (NOEL) and no observed adverse effect level (NOAEL) were considered to be 250 and 500 mg/kg /day respectively under the conditions of this study.
Executive summary:

A repeated dose 90-day oral toxicity study was conducted in accordance with OECD guidelines for the Testing of Chemical No. 408 ‘Repeated Dose 90-day Oral Toxicity Study in Rodents’. Groups of 10 randomly assigned rats (6 weeks old) of each sex were injected by oral gavage with the test substance suspended in distilled water at doses of 250, 500, and 1,000 mg/kg/BW/day five times a week for 90 days. In the repeated dose toxicity study, there were no significant changes in body weight in the 1,000 mg/kg treatment group, or food consumption, urinalysis, and hematology in any group. With regards serum biochemistry, the levels of total protein, albumin, A/G ratio, triglyceride, calcium and inorganic phosphate were altered at doses of 500 and 1,000 mg/kg. However, no changes were observed at the dose of 250 mg/kg. With regards histopathological findings, cortical tubular basophilia of the kidney increased at the dose of 1,000 mg/kg, but not at doses of 250 and 500 mg/kg. No significant changes were observed in other organs at doses of 250, 500, and 1,000 mg/kg. Based on these results, the no observed effect level (NOEL) and no observed adverse effect level (NOAEL) were considered to be 250 and 500 mg/kg /day respectively under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
See discussion.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

References:

- Deschamps FJ, Guillamot M, Raux S. 2001. Neurological effects in workers exposed to manganese. J Occup Environ Med 43(2): 127-132.

- Gibbons, R.A., S.N. Dixon, K. Hallis, et al. 1976. Manganese metabolism in cows and goats. Biochim. Biophys. Acta 444:1-10.

- Gibbs JP, Crump KS, Houck DP, et al. 1999. Focused medical surveillance: A search for subclinical movement disorders in a cohort of U.S. workers exposed to low levels of manganese dust. Neurotoxicology 20:299-313.

- Oberdoerster and Cherian. 1988. Biological Monitoring of Toxic Metals. Manganese. Rochester Series on Environmental Toxicity, pp 283 -301.

- Roels, H.A., P. Ghyselen, J.P. Buchet, et al. 1992. Assessment of the permissible exposure level to manganese in workers exposed to manganese dioxide dust. Br. J. Ind. Med. 49(1):25-34.

- Young T, Myers JE, Thompson ML. 2005. The nervous system effects of occupational exposure to manganese--measured as respirable dust--in a South African manganese smelter. Neurotoxicology 26(6):993-1000.

Justification for classification or non-classification

The substance is not classified.