Manganese sulphate

Endpoint summary

Description of key information

Please refer to the RAAF report attached to section 13 of this dataset which discusses the cateogory approach for sharing of data between the three soluble manganese salts Mn chloride, Mn nitrate and Mn sulphate.

There is a body of publicly available literature data on repeated application of MnCl2 however, these data are not conducted according to standardised test guidelines or the principles of GLP and are of varying quality and reliability.  Whilst the literature for repeated dose toxicity of MnCl2 shows a variety of effects on repeated application of MnCl2, these studies are not consistent in their dosing methods, duration, or with the species of test animals that are used.  As a consequence of this, the results are variable and not aligned with observations from more reliable studies conducted to standardised international guidelines and GLP.  Therefore the effects observed in the reproductive and developmental toxicity studies on MnCl2 via the inhalation route are given more weight than the lower reliability literature references and are considered to be the better indicator of the long term toxicological effects of this substance via the inhalation route rather than the older literature data.

In the reproductive toxicity study by Grieve (2017) subsequently published by McGough & Jardine (2016) clinical signs of reaction to treatment to inhalation exposure of MnCl2 were confined to a few animals with wheezing respiration in the F0 generation exposed to 10 and 20 μg/L.  At 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls.  At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs.  In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls which was not evident in the F1 females.  The NOEL for adult effects was not established due to effects on the respiratory tract.  However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of MnCl2 and therefore, when the local irritant effects are disregarded, the parental NOAEL was considered to be 20 μg/L (Grieve, 2017).

In the developmental toxicity study (Dettwiler, 2015) conducted in accordance with OECD Guideline 414, treatment with MnCl2 caused breathing noises in eight females in exposed to 15 μg/L air and eighteen females exposed to 25 μg/L air.  Treatment caused a dose dependent reduction in food consumption in groups exposed to 15 μg/L air and 25 μg/L air. This reduction was statistically significant during the most of the study and was accompanied by reduced body weights, reduced body weight gain during the study and reduced corrected body weight gain at termination at both dose levels and therefore effect on food consumption was considered to be adverse.  Histopathology examination performed on the lungs from six selected pregnant females per group revealed lesions with a dose dependent frequency and severity in groups exposed to 15 μg/L and 25 μg/L.  The macroscopically identified foci in two females in groups exposed to 25 μg/L were correlated to alveolar haemorrhage or phagocytic alveolar macrophage foci. Based on these results, the NOAEL as well as the NOEL for the toxicity in pregnant females were considered to be 5 μg/L air.  In non-pregnant females, the NOEL for systemic toxicity was established at 15 μg/L air, whereas the NOAEL was established at 25 μg/L air.  These observations are consistent with a number of literature references indicating a loss of body weight during administration of MnCl2 and a developmental neurotoxicity study which also noted this loss and a dose-dependent reduction in food consumption (Dettwiler, 2016).

The repeated dose studies on MnSO4 were from the US National Toxicology Program technical report on the toxicology and carcinogenesis of manganese(II) sulphate monohydrate in F344/N rats and B6C3F1 mice (feed studies) (NTP, 1993).  The studies were not conducted to then-standardised guidelines but to peer reviewed methods as specified by the NTP and as such are considered wholly reliable. The high level of MnSO4 consumed by rats on a daily basis for 13 weeks in this study, without mortality, supports the lack of acute toxicity.  In the two-year study survival of male rats exposed to 15 000 ppm was significantly lower than that of the controls attributed to increased incidences of advanced renal disease relating to the ingestion of MnSO4. The final mean bodyweight of male rats exposed to 15 000 ppm was 10 % lower than that of the controls. The mean body weights of all the other exposed groups were similar to the controls (NTP, 1993).  At both the 9- and 15-month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of male and female rats exposed to 5 000 and 15 000 ppm, with an accompanying depression of hepatic iron.  The ingestion of diets containing 15 000 ppm MnSO4 was associated with a marginal increase in the average severity of nephropathy in male rats. In these rats, lesions associated with renal failure, uraemia, and secondary hyperparathyroidism were observed.  No increased incidence of neoplasms in male or female rats was attributed to ingestion of MnSO4.  There was no evidence of carcinogenic activity of manganese sulphate monohydrate in male or female F344/N rats (NTP, 1993).

In mice, no clinical findings were attributed to manganese sulphate ingestion at 13 weeks (NTP, 1993).  Concentrations of manganese were significantly elevated in the livers of mice exposed to 5 000 and 15 000 ppm at the 9 and 15 month evaluations. It was uncertain if the slightly increased incidence of follicular cell adenoma is related to the ingestion of MnSO4. 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 (NTP, 1993).

There are no data available on manganese dinitrate itself.

As stated above, given that the toxicity of the soluble manganese salts is generally accepted to be attributed to the Mn2+ ion it is proposed to use read across to maximise the use of the available data.

The lack of GLP, guideline studies performed on MnCl2 to specifically investigate the repeated dose effects warrants the application of the information generated in the reproductive and developmental  toxicity studies on this substance.  These studies are conducted under GLP conditions to modern standardised guidelines and are considered to provide adequate information with regards to the long term effects of the substance via the inhalation route.  Similarly it is considered appropriate to use these same studies as the source of data to address the repeated dose toxicity of MnSO4 via the  inhalation route as no data are available on the toxicity of the sulphate via this route  of exposure.

The repeated dose toxicity studies conducted on MnSO4 in support of the NTP programme are well conducted and reliable studies and are considered to be the most appropriate source of data on the long term effects of exposure to the Mn2+ ion via the oral route.  As such it is considered appropriate to use these studies to support this endpoint in both the MnSO4 dossier and as read across in the MnCl2 dossier.  These studies on MnSO4 via the oral route do not result in the classification of the substance and in conjunction with studies via the inhalation route conducted on MnCL2 are considered to represent the “worst case scenario” in relation to the effects of long term exposure to soluble manganese salts.

No repeated dose toxicity studies are available on Mn(NO3)2 manganese dinitrate  and read-across is proposed for these endpoints. As described above, given that the toxicity of the soluble manganese salts is generally accepted to be attributed to the Mn2+ ion in conjunction with the lack of any modern studies performed on Mn(NO3)2 , it is considered appropriate to utilise read-across to the key studies performed on MnCl2 (inhalation exposure) and MnSO4 ( Oral exposure). Furthermore, it is considered that these guideline studies can be considered to give an accurate representation of the toxicity of the Mn2+ ion in Mn(NO3)2 and should therefore be considered adequate to address these endpoints. These studies via the oral route (MnSO4) do not result in the classification of the substance and as such the studies via the inhalation route as conducted on MnCL2 are considered to represent the “worst case scenario” in relation to the effects of long term exposure to soluble manganese salts.

Read-across between the three salts is considered to be justified based on the high water solubility and ultimate identical physiological fate all three salts share once the anions disassociate into the respective endogenous physiological pools.  In the case of both MnCl2 and MnSO4, evidence suggests that repeated dosing triggers a strong homeostatic response which reduces the toxicity by presumably increasing excretion/decreasing absorption (Vrcic and Kello, 1988).  This can be taken as further evidence that read-across between these three soluble manganese salts  is justified.

Toxicokinetic and toxicity data confirm  that the manganese cations are the driving factor for any human health effects observed.  The respective anions, chloride (Cl-), sulphate (SO42-) and nitrate (NO3 ), are readily absorbed and excreted from the human body, and their contribution on toxicological effects is not considered significant.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to GLP and according to peer reviewed methods.

Qualifier:

according to guideline

Guideline:

other: NTP peer reviewed methods

Principles of method if other than guideline:

Groups of 10 male and 10 female rats were fed diets containing 0, 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.

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Route of administration:

oral: feed

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 3130, 6250, 12500, 25000 or 50000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 females and 10 males 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:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

-CLINICAL SIGNS AND MORTALITY: 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 GAIN: Mean bodyweight 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): Feed consumption was similar to that of the controls.

-HAEMATOLOGY: The percent haematocrit, haemoglobin concentrations and mean erythrocyte volumes of 50000 ppm male and female mice were significantly lower than those of the controls.

-ORGAN WEIGHTS: The absolute and relative liver weights of the 50000 ppm males were significantly lower than the controls.

Dose descriptor:

NOAEL

Effect level:

25 000 ppm

Sex:

male

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Sex:

female

Critical effects observed:

not specified

No clinical findings were attributed to manganese sulphate ingestion..

Conclusions:

No clinical findings were attributed to manganese sulphate ingestion .

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to GLP and according to peer reviewed methods.

Qualifier:

according to guideline

Guideline:

other: NTP peer reviewed methods

Principles of method if other than guideline:

Groups of 10 male and 10 female rats were fed diets containing 0, 1600, 3130, 6250, 12500 or 25000 ppm manganese sulphate. Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses. A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.

GLP compliance:

yes

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals 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

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 1600, 3130, 6250, 12500 or 25000 which was equivalent to doses from 110 to 1700 mg/kg in males and 115 to 2000 mg/kg in females
Basis:
nominal in diet

No. of animals per sex per dose:

10 male and 10 female per dose group

Control animals:

yes

Observations and examinations performed and frequency:

Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses.

Sacrifice and pathology:

A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

-BODY WEIGHT AND WEIGHT GAIN: The mean bodyweight gain in males receiving 3130 ppm was marginally lower than that of the controls and was significantly lower in the three highest female dose groups than the controls.
-FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption by exposed rats was similar to that of the controls. Females ingested an average of 20% more manganese sulphate than males in the corresponding exposure groups.
-HAEMATOLOGY: Neutrophil counts were significantly higher in all exposed male groups. Lymphocyte counts were significantly lower in the three highest dose groups.
In females: leukocyte counts were significantly lower in the three highest dose groups.
A significant increase in the percent haematocrit and erythrocyte counts occurred in males exposed to the three highest dose levels.
-ORGAN WEIGHTS: Absolute and relative liver weights of all exposed males and of the female 25000 ppm group were significantly lower than the controls.

Dose descriptor:

NOAEL

Effect level:

1 700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

food consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

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)	Survival^a	Mean body weight and weight changes^brelative			Final Weight Feed to controls (%)	Consumption^c
		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.

Reference 3

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to GLP and according to peer reviewed methods.

Qualifier:

according to guideline

Guideline:

other: NTP peer reviewed methods

Principles of method if other than guideline:

Male and Female mice received diets containing varying concentrations of MnSO4 for 103 weeks. Groups of 9 or 10 animals were evaluated after 9 and 15 months of chemical exposure.

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mice were approximately 41 days old at the beginning of the study.

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

daily

Remarks:

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

No. of animals per sex per dose:

70 male and 70 female per dose group.

Control animals:

yes

Observations and examinations performed and frequency:

All animals were observed twice daily. Clinical findings were recorded weekly for the first 13 weeks and monthly thereafter.

Sacrifice and pathology:

All animals were necropsied. At necropsy all organs were examined for gross lesions and all major tissues were fixed and preserved for histopathological examinations.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

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

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

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

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

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

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

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

OTHER FINDINGS: The study suggests equivocal evidence of carcinogenicity in male and female mice. Based on marginally increased incidences of thyroid gland follicular cell adenoma and significantly increased incidences of follicular cell hyperplasia.

Conclusions:

No clinical findings were attributed to MnSO4 treatment.

Reference 4

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: To GLP, studies performed according to peer-reviewed methods.

Qualifier:

according to guideline

Guideline:

other: NTP peer-reviewed methods

Principles of method if other than guideline:

Male and female rats were fed diets containing varying concentrations of MnSO4 for 103 weeks.

GLP compliance:

yes

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

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 1500, 5000, 15000 ppm, equivalent to 0, 60, 200 or 615 mg/kg in male and 0, 70, 230 or 715 mg/kg in females
Basis:
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

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

other: food intake and concentration in diet

Sex:

male

Dose descriptor:

NOAEL

Effect level:

230 mg/kg bw/day (nominal)

Based on:

other: food consumption and concentration in feed

Sex:

female

Critical effects observed:

not specified

CLINICAL SIGNS AND MORTALITY: Survival of male rats exposed to 15000 ppm was significantly lower than that of the controls. This was attributed to increased incidences of advanced renal disease relating to the ingestion of MnSO4.

BODY WEIGHT AND WEIGHT GAIN: The final mean bodyweight of male rats exposed to 15000 ppm was 10% lower than that of the controls. The mean body weights of all the other exposed groups were similar to the controls.

HAEMATOLOGY AND CLINICAL CHEMISTRY: No differences in haematology and clinical parameters attributable to the ingestion of MnSO4 were observed.

TISSUE MANGANESE CONCENTRATIONS: At both the 9 and 15 month interim evaluations, tissue concentrations of manganese were significantly elevated in the livers of male and female rats exposed to 5000 and 15000 ppm, with an accompanying depression of hepatic iron.

GROSS PATHOLOGY: The ingestion of diets containing 15000 ppm MnSO4 was associated with a marginal increase in the average severity of nephropathy in male rats. In these rats, lesions associated with renal failure, uremia, and secondary hyperparathyroidism were observed.

No increased incidence of neoplasms in male or female rats was attributed to ingestion of MnSO4.

Conclusions:

MnSO4 is relatively non-toxic at the doses examined, and the parameters assessed in the study, over a 2 year period of exposure, via the oral route.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study focussing mainly on tissue concentrations with some other parameters investigated. However, a lack of histopathology significantly reduces the usefulness for this endpoint.

Principles of method if other than guideline:

Monkeys were exposed to an atmosphere containing manganese sulphate for a maximum period of 65 days, with exposure taking place for 5 days per week, 6 hrs per day. Tissue concentrations and body weights were assessed together with selected clinical and haematolgical parameters. Some gross pathology was undertaken, and organ weights assessed. Control animals were used.

GLP compliance:

not specified

Species:

monkey

Strain:

other: Rhesus

Sex:

male

Details on test animals or test system and environmental conditions:

monkeys were 17 - 22 months old at time of arrival and were medically examined prior to start of the study. Animals were between 20 and 24 months of age at the start of the inhalation exposure. Animals were fed a certified primate chow diet twice a day. Mean manganese concentrations determined in the feed were 133 ± 14 ppm. Therefore manganese intake from the base diet was approximately 6.2 mg/ kg/ day. Mn levels in dietary supplements and drinking water were also determined. During non exposure periods monkeys were individually housed in domiciliary stainless steel cages . On each exposure day animals were transferred to stainless steel cages that were designed to fit within the 8m3 inhalation chambers. Animals were moved back to their domiciliary cages after each 6 hour exposure.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: 1.73, 1.89, 2.12 and 1.72 µm for the target concentrations of 0.18, 0.92 and 4.62 (group 1) and 4.62 (group 2) mg MnSO4 / m3 respectively

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

65 days (group 1), 15 or 33 days (group 2)

Frequency of treatment:

5 days per week, 6 hours per day

Remarks:

Doses / Concentrations:
0.18, 0.92 and 4.62 mg MnSO4 / m3
Basis:

No. of animals per sex per dose:

Group 1:
control : 6
0.18 mg MnSO4 / m3: 6
0.92 mg MnSO4 / m3: 4
4.62 mg MnSO4 / m3: 4

Post-exposure recovery groups:
4.62 mg MnSO4 / m3 (held for 45 or 90 days post-exposure): 8

Group 2:
4.62 mg MnSO4 / m3: 4

All animals were male.

Control animals:

other: Yes: filtered air

Sacrifice and pathology:

NECROPSY: food was withheld overnight prior to necropsy. Monkeys were anesthetised with ketamine and blood was collected from a peripheral vein. A smalll volume was used to determined in the PCV and additional samples were collected for complete blood cell counts, clinical chemistry, evaluation of basal levels of lutenizing hormone (LH) and red blood cell glutathione (GSH) concentrations.

SACRIFICE: monkeys were euthanised with pentobarbital and exsanguinated.

The lungs and other thoraic organs were removed, weighed and inspected for gross lesions and manganese concentration was analysed. Brain was removed and dissected for the purpose of determining manganese concentrations. Mn concentration was also determined in the following tissues: olefactory epithelium, heart, femur, skullcap, liver, pancreas, kidney, skeletal muscle, testes, gall bladder contents and urine.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL OBSERVATIONS: Clinical signs observed were of minimal veterinary concern and were not related to MnSO4 exposure.
BODY WEIGHT AND WEIGHT GAIN: Subchronic inhalation exposure to MnSO4 did not effect bodyweight gain or terminal bodyweight.
HAEMATOLOGY: Mean corpuscular hemoglobin concentration (MCHC%) was decreased in monkeys exposed to MnSO4 at 4.62 mg MnSO4 /m3 for 15 days and monkeys evaluated 45 days after a 13-week exposure to MnSO4 at 4.62 mg MnSO4 /m3.
CLINICAL CHEMISTRY: A statistically significant decrease in the difference between pre- and post-exposure total bilirubin concentrations was observed in monkeys exposed to MnSO4 at 4.62 mg MnSO4/m3 for 65 exposure days. However, end-of-exposure total bilirubin concentrations in the air-and MnSO4-exposed monkeys were virtually identical. A twofold higher pre-exposure total bilirubin concentration was present in the monkeys assigned to the high-dose MnSO4 group. Alkaline phosphatase activity was approx 1.6 fold higher in monkeys exposed to MnSO4 at 4.62 mg MnSO4/m3 for 33 exposure days and monkeys evaluated 90 days after a 13-week exposure to MnSO4 at 4.62 mg MnSO4/m3. The pre-exposure and post-exposure total bilirubin, alkaline phosphatase and MCHCs were in the normal reference range reported for male rhesus monkeys (Wolford et al 1986).
Additional selected post-exposure clinical chemistry parameters were recorded. These were LH, iron, transferrin, total iron binding capacity, total GSH, reduced GSH and ratio GSSG:GSH.
ORGAN WEIGHTS: No statistically significant difference from control in absolute organ weights was observed with any organ in animals exposed to MnSO4 for 65 days and then assessed immediately thereafter. Because the animals continue to grow, evaluation of post-exposure organ weights was confounded by the animals increase in body weight. There was a statistically significant decrease (approx 17%) in relative heart weight in monkeys evaluated 90 days after the end of a 13 week exposure to MnSO4 at 4.62 mg MnSO4 / m3. No other statistically significant differences in relative organ weight
were observed in MnSO4 exposed animals versus controls.
GROSS PATHOLOGY
OTHER FINDINGS: issue manganese concentrations were recorded. Subchronic exposure at the lowest concentration resulted in increased Mn concentration in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, white matter, cerebellum and heart.
Monkeys at the mid-dose exposed for 65 exposure days developed increased Mn concentrations in all the above tissues, as well as in the olfactory tract, caudate, pituitary gland, kidney, pancreas, lung, bile, blood, and urine. Monkeys exposed to the highest dose for 65 exposure days additionally had increased Mn concentrations in the frontal cortex, trigeminal nerve, liver, skeletal muscle and parietal bone.
The group of monkeys exposed to the top dose but assessed 45 or 90 days later showed tissue Mn concentrations remained elevated (vs controls) in the olfactory cortex, globus pallidus, putamen, pituitary gland and blood 45 days after the end of the 13-week exposure period. All tissue Mn concentrations had returned to levels observed in the air-exposed control animals by 90 days after the end of the exposure.
Elimination of Mn from the monkey brain varied from region to region with the shortest halftime occurring in the olfactory bulb (4.9 days) and longest in the cerebellum (32.3 days)

Critical effects observed:

not specified

Clinical Observations:

Subchronic inhalation exposure to MnSO4 did not affect body weight gain (data not shown) or terminal body weight. Clinical signs observed in the monkeys were of minimal veterinary concern (e.g., alopecia or pulling of hair on the arms and legs, intermittent abnormal stool) and were not related to MnSO4 inhalation.

No statistically significant difference from control in absolute organ weights was observed with any organ in animals exposed to MnSO4 for 65 days and then assessed immediately thereafter. Because the animals continued to grow, evaluation of post-exposure organ weights was confounded by the animal’s increase in body weight. There was a statistically significant decrease (approximately 17%) in the relative heart weight (relative to body weight) in monkeys evaluated 90 days after the end of a 13-week exposure to MnSO4 at 1.5 mg Mn/m3.

No other statistically significant differences in relative organ weight (relative to either body weight or brain weight) were observed in the MnSO4-exposed animals versus controls.

Haematology and Clinical Chemistry:

A statistically significant decrease in the difference between pre- and post-exposure total bilirubin concentrations was observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 65 exposure days when compared to air-exposed controls.

However, end-of-exposure total bilirubin concentrations in the air- and MnSO4-exposed monkeys were 0.15 ± 0.02 and 0.15 ±

0.03 mg/dl, respectively. A twofold higher pre-exposure total bilirubin concentration was present in the monkeys assigned to the high-dose MnSO4 exposure group.

Alkaline phosphatase activity was approximately 1.6-fold higher in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 33 exposure days and monkeys evaluated 90 days after a 13-week exposure to MnSO4 at 1.5 mg Mn/m3, when compared to controls (524 ± 53

IU/l). Mean corpuscular hemoglobin concentration (MCHC %) was decreased in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 15 days (post-exposure value ¼ 33.5 ± 0.3%) and monkeys evaluated 45 days after a 13-week exposure to MnSO4 at 1.5mg Mn/m3 (post-exposure value ¼ 33.6 ± 0.3%) versus controls (post-exposure value ¼ 35.1 ± 0.1%).

Observed differences in clinical chemistry or haematology parameters are unlikely to be toxicologically significant or related to MnSO4 exposure.

Tissue manganese concentrations:

Subchronic exposure to MnSO4 at the lowest exposure concentration (≥ 0.06 mg Mn/m3) resulted in increased manganese concentrations in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, white matter, cerebellum, and heart. Monkeys exposed to MnSO4 at the mid-dose (≥0.3 mg Mn/m3) for 65 exposure days developed increased manganese concentrations in all the above tissues, as well as in the olfactory tract, caudate, pituitary gland, kidney, pancreas, lung, bile, blood,

and urine. Monkeys exposed to MnSO4 at the highest exposure concentration (1.5 mg Mn/m3) for 65 exposure days additionally

had increased manganese concentrations in the frontal cortex, trigeminal nerve, liver, skeletal muscle, and parietal bone.

Conclusions:

MnSO4 inhalation affected the haematology and resulted in increased Mn concentrations in the brain of the monkey. Due to the lack of histopathology however, the study is of limited value in fulfilling the sub-chronic inhalation endpoint.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

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

Justification for classification or non-classification

There is no strong evidence to suggest that MnSO4 should be classified for oral toxicity by repeat dose. It is not acutely toxic and there are no unequivocal human studies or animal studies that would support such a classification. MnSO4 is currently classified as STOT RE2 and, based on current data, this should be for the inhalation route only. No dermal classification is justified by the expected lack of dermal absorption of MnSO4, therefore no systemic exposure and no effect possible for that route.