Manganese dichloride

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

Endpoint:

repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

other: read-across target

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:

other: food intake and concentration in food.

Sex:

male

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

other:

Sex:

female

Dose descriptor:

NOAEL

Effect level:

ca. 553 other: mg Mn/kg bw/day

Based on:

element

Sex:

male

Dose descriptor:

NOAEL

Effect level:

ca. 650 other: mg Mn/kg bw/day

Based on:

element

Sex:

female

Critical effects observed:

not specified

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

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

 

Concentration (ppm)	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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

553 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study was to GLP and to an internationally recognised guideline, but used another water soluble form of inorganic manganese. The NOAEL is expressed as manganese only.
.

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

No histopathology was conducted, however the most sensitive endpoint (neurolochemical changes) which formed the basis for proposed STOT RE2 classification were observed.

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

other: read-across target

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)

Dose descriptor:

NOAEC

Effect level:

ca. 0.92 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Neurochemical changes in the brain.

Dose descriptor:

NOAEC

Effect level:

ca. 0.3 other: mg Mn/m3 air

Based on:

element

Sex:

male

Basis for effect level:

other: Neurochemical changes in the brain.

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

Dose descriptor:

NOAEC

0.3 mg/m³

Study duration:

subchronic

Species:

monkey

Quality of whole database:

The study was not to GLP or a specific guideline but the methodology was well described.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to sound scientific principles with a sufficient level of detail to assess the quality of the relevant results. The focus of the study was on biological changes in lung; not a traditional repeated dose study as required observations not made.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rabbits were exposed to MnCl2 via aerosol for 6 hours a day, 5 days a week, for a period of 4 to 6 weeks. Within 3 days after the end of the exposure period the rabbits were sacrificed and the lungs excised. The right lung was lavaged and the alveolar macrophages collected. The macrophage concentration was measured in a Burker chamber and the cell viability tested by staining with eosin-y. Smears of lung macrophage were air dried, fixed in methanol and stained. Size distribution was determined by measuring the diameters of 100 -200 cells from each rabbit in a Lanameter. The upper left lobe was studied using light microscopy. Three tissue pieces from the middle part of the left lower lobe were sampled for electron microscopy and the remainder of the lobe was used for lipid analysis. The functionality, phagocytic activity and bacteriocidal capacity of the macrophages was investigated. Lipid analysis was also performed.

GLP compliance:

not specified

Species:

rabbit

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.1 -2.2 ± 0.3 kg
- Housing: During the exposure period animals were kept in 0.6 m exposure chambers (4 rabbits per chamber)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: 1 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: MnCl2 aerosols were produced using an ultrasonic nebulizer
- Method of particle size determination: Mass median aerodynamic diameter of both aerosols was estimated with an impactor

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Manganese concentration was measured daily for 3 hours by air suction through a membrane filter (Gelman GN-4, 0.8 µm) and the amount of metal deposited on the filter was measured by atomic absorption spectroscopy (Varian AA6).

Duration of treatment / exposure:

6 hours/day, 5 days/week for 4 to 6 weeks

Frequency of treatment:

daily (5 days a week)

Remarks:

Doses / Concentrations:
1.1 mg/m3
Basis:
other: manganese (as MnCl2)

Remarks:

Doses / Concentrations:
3.9 mg/m3
Basis:
other: manganese (as MnCl2)

No. of animals per sex per dose:

8 males per group

Control animals:

yes

Observations and examinations performed and frequency:

No in-life examinations were reported.

Sacrifice and pathology:

SACRIFICE
Within 3 days after the end of the exposure period the rabbits were sacrificed by an overdose of sodium pentobarbital and the lungs excised.
The right lung was lavaged and the alveolar macrophages collected. The macrophage concentration was measured in a Bürker chamber and the cell viability tested by staining with eosin-y. Smears of lung macrophages were air dried, fixed in methanol and stained with Giemsa solution. Size distribution was determined by measuring the diameters of 100 -200 cells from each rabbit in a Lanameter. The upper left lobe was studied using light microscopy. Three tissue pieces from the middle part of the left lower lobe, about 1 mm³ each, were sampled for electron microscopy and the remainder of the lobe was used for lipid analysis.

LIGHT MICROSCOPY
The left upper lobe was fixed in 10% formalin and routine paraffin sections were stained with hematoxylin and eosin.

ELECTRON MICROSCOPY
Morphometric measurements were performed on 21 randomly selected fields from each rabbit in the control group and in the group exposed to the high Mn(II) concentrations, at a primary magnification of 1000. The area occupied by type II cell profiles divided by the area occupied by alveolar tissue profiles, was determined for each rabbit. the size of the type II cells was estimated on toluidine blue-stained Epon sections by means of a Lanameter.

FUNCTIONAL TESTS
The oxidative metabolic activity of the macrophages was estimated by measuring their ability to reduce nitroblue tetrazolium to formazan at rest and in the presence of Escherichia coli. The phagocytic activity of the macrophages was measured. A suspension of cells in Eagle's medium was incubated with yeast cells (Saccharomyces cerevisiae) labelled with fluorescein isothiocyanate and opsonized with pooled rabbit serum. After 30 and 60 minutes the phagocytosis was interrupted and the preparation stained with crystal violet. Ingested particles were recognised by their fluorescence and the attached ones being stained with the dye.
Bacterial capacity was tested by incubating the macrophages with Staphylococcus aureus "Oxford" in a suspension in Eagle's medium containing 0.1% gelatin and diluted pooled rabbit serum. After 90 minutes, colony forming units in this and in the original suspension were determined.

LIPID ANALYSIS
The left lower lobe was homogenised at 4°C and extracted with chloroform:methanol 2:1 (v/v). After filtration, 0.58% sodium chloride in water was added. The lower phase was dried and the lipids were separated by reverse-phase chromatography. The quantities of phospholipids were estimated by phosphorus determinations.

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

LUNG MORHPHOLOGY
The gross appearance of the lungs was normal both in MnCl2- exposed rabbits and in controls. The weight of the left lower lung weight was similar in all three groups.
Light microscopy showed focal infiltration of eosinophils (indicative of inflammation) in 4 controls, 3 in the low-dose group and 4 in the high-dose experimental animals. A few alveoli with increased accumulation of macrophages were found in 2 high-dose animals, 1 low-dose animal and 1 control. The majority of both control and experimental animals showed scattered areas of atelectasis. Slight inflammatory changes were non-significant and therefore were concluded to be unrelated to the experimental protocol.
Electron microscopy showed apparently normal alveolar septa, with the exception of 1 control and 1 exposed rabbit which showed focal oedema of alveolar type I cells. Values for volume density of type II cells were similar in all groups.

MACROPHAGE DATA
The cell diameter was significantly larger in the high dose group animals compared to the controls. The cell viability was above 90% in all animals.
By electron microscopy, macrophages from both exposed rabbits and controls had an undulating surface with some protrusions and their cytoplasm was rich in lysosomes. Some macrophages from exposed and control rabbits contained one or a few laminated inclusions. The oxidative metabolic activity of the macrophages was similar in the three groups both at rest and after stimulation with E. coli, as were the number of yeast particles ingested or attached to the macrophage surface. The bacteriacidal capacity was similar in exposed animals and controls.

PHOSPHOLIPID DATA
Phospholipids did not differ between controls and experimental animals.

Dose descriptor:

NOAEC

Effect level:

3.9 mg/m³ air (analytical)

Based on:

other: concentration of manganese in test material

Sex:

male

Basis for effect level:

other: There was an increase in the size of alveolar macrophages, although this may be expected in lungs being dosed with foreign material. Inflammation did not appear to be apparent.

Critical effects observed:

not specified

Conclusions:

No abnormalities were found in Mn(II) exposed animals, except for an increase in the size of alveolar macrophages in the high-dose group.

Executive summary:

Rabbits were exposed to MnCl2 via aerosol for 6 hours a day, 5 days a week, for a period of 4 to 6 weeks. Within 3 days after the end of the exposure period the rabbits were sacrificed and the lungs excised. The right lung was lavaged and the alveolar macrophages collected. The macrophage concentration was measured in a Burker chamber and the cell viability tested by staining with eosin-y. Smears of lung macrophage were air dried, fixed in methanol and stained. Size distribution was determined by measuring the diameters of 100 -200 cells from each rabbit in a Lanameter. The upper left lobe was studied using light microscopy. Three tissue pieces from the middle part of the left lower lobe were sampled for electron microscopy and the remainder of the lobe was used for lipid analysis. The functionality, phagocytic activity and bacteriocidal capacity of the macrophages was investigated. Lipid analysis was also performed.

Under the conditions of the study, no abnormalities were found in Mn(II) exposed animals, except for an increase in the size of alveolar macrophages in the high-dose group.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

3.9 mg/m³

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The study was not to GLP or a specific guideline but the methodology was well described.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Read-across is to the substance MnSO4, chloride ions are not intrinsically more toxic than sulphate ions or vice versa, and in the milleu in the stomach the anion takes on little importantance, paticularly give the excess of chloride. Therefore in accordance with Annex XI section 1.1, because existing data can be used, a new study specifically with MgCl2 is not scientifically necessary.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Repeat dose study studying the most sensitive endpoint for manganese which is neurological effects.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
study examining local effects after inhalation exposure.

Repeated dose toxicity: inhalation - systemic effects (target organ) neurologic: central nervous system

Justification for classification or non-classification

There is no strong evidence to suggest that MnCl2 should be classified for oral toxicity by repeat dose. Although it is acutely toxic by the oral route, there is evidence (Vrcic (1998) section 7.2.1), repeated dosing triggers a strong homeostatic response which reduces the toxicity by presumably increasing excretion/decreasing absorption. Also repeat exposure would be to lower doses allowing even more homeostatic control. Thus it is considered acceptable for repeat dose classification to be read-across from MnSO4. MnSO4 is currently classified as STOT RE2, based on current data (such as the NTP report for oral exposure for two years to MnSO4) hence the same classification is proposed for MnCl2.

No dermal classification is justified based on no significant rate of dermal absorption (see IUCLID section 7.1.2, Jaeger M (2010))