Manganese

Endpoint summary

Description of key information

REPEATED DOSE - INHALATION: NOAEL: 0.5 µg/L Mn Metal Powder, rat (male/female), Broich (2016).

Key value for chemical safety assessment

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:

key study

Study period:

29 May 2012 to 29 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-4 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: RccHanTM: WIST(SPF)
- Age at study initiation: 10 weeks
- Weight at acclimatization: Males: 208.1 to 241.2 g (± 8.2 %), Females: 142.7 to 170.6 g (± 10.0 %)
- Housing: In groups of maximally four (allocation A and B) or groups of maximally three (allocation C to E) in cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment.
- Diet: Pelleted standard rodent maintenance diet was available ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations.
- Water: Community tap-water was available ad libitum in water bottles except during the periods when the animals were restrained in the exposure tubes.
- Acclimation period: 14 or 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 % (relative)
- Air changes: 10 to 15 air changes per hour
- Photoperiod: The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours of music during the light period

IN-LIFE DATES
From: 12 June 2012
To: 25 September 2012 (allocation A males), 26 September 2012 (allocation A females), 23 October 2012 (allocation B males), 24 October 2012 (allocation B females), 19 September 2012 (allocation C males), 20 September 2012 (allocation C females), 16 October 2012 (allocation D males), 17 October 2012 (allocation D females), 25 September 2012 (allocation E males) and 26 September 2012 (allocation E females)

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: - Determination of Particle Size Distribution
The particle size distribution was determined gravimetrically six, ten and nine times for groups 2 to 4, respectively. Due to an invalid measurement one particle size determination for group 2 was excluded from the results.
The cumulative particle size distribution of the test aerosol was determined using a Mercer 7 stage cascade Impactor .The test aerosol was impacted at each stage onto aluminium slips and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test material deposited on each stage of the cascade impactor. Impactor samples in group 2 were collected over five days of exposure due to the low aerosol concentration. The airflow rate through the impactor was 1 L/min.
The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the gravimetric results from the impactor, using Microsoft Excel® software. The target ranges are 1 to 3 µm for the MMAD and 1.5 to 3 for the GSD.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Preparation of Test Material:
Prior to use for aerosol generation, the test material was ground twice for 60 minutes (allowing time to cool between runs) using medium balls at 500 rpm in a Retch Planetary Ball Mill, in order to facilitate aerosol generation and an acceptably small particle size. The purity of the material was identified before and after grinding to confirm that changes in physical state did not affect purity of the material. The resulting fine particles were used in the present study.


- Inhalation Exposure System:
Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The animals were confined separately in restraint tubes. The aerosol was discharged constantly through the exposure system and exhausted using a tubing/filter system.
The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1 L/min, which was sufficient to minimize re-breathing of the test atmosphere as it was about twice the respiratory minute volume of a rat.
Before commencement of the exposure of the groups, technical trials were conducted (without animals) using the inhalation system foreseen for the study. The technical trials were conducted using established procedures based on GLP.

- Test Aerosol Generation:
A dust aerosol was generated from the test material using a rotating brush generator. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. Furthermore, the aerosol concentrations of the test item of the low and mid dose group were achieved by serial dilution with compressed, filtered, dry air of the higher aerosol concentration using a air vacuum device.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DETERMINATION OF NOMINAL AEROSOL CONCENTRATION
The test item usage was measured during each exposure in group 4 by weighing the generator cylinders containing the test item before and after exposure to determine the quantity of test item used. The weight was then divided by the total air-flow volume to give the nominal concentration. These data were used for the purpose of monitoring the performance of the generation system.
The nominal aerosol concentrations were 4.919 ± 0.632 µg/L for Group 2, 37.10 ± 4.739 µg/L for Group 3 and 123.9 ± 15.77 µg/L for Group 4.

GRAVIMETRIC DETERMINATION OF AEROSOL CONCENTRATION
The gravimetric aerosol concentration was determined three to four times during each exposure for groups 2, 3 and 4.
Test aerosol samples were collected onto Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. Sampling flow was 1.0 to 1.1 L/min. The duration of sampling was sufficient to ensure reliable results. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
The gravimetrically determined aerosol concentrations of 0.56, 5.05 and 21.3 µg/L Mn Metal Powder were achieved in groups 2 to 4, respectively, and met or were slightly above the respective targets. They were considered to be stable during the whole treatment period.
The achieved aerosol concentrations were 0.559 ± 0.094 µg/L (111.8 ± 18.9 %) for Group 2, 5.047 ± 0.649 µg/L (100.9 ± 13.0 %) for Group 3 and 21.34 ± 2.158 µg/L (106.7 ± 10.8 %) for Group 4.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days/week

Dose / conc.:

0.5 other: µg/L nominal conc.

Dose / conc.:

5 other: µg/L nominal conc.

Dose / conc.:

20 other: µg/L nominal conc.

No. of animals per sex per dose:

Group 1: 25 males and 25 females, Groups 2 to 4: 31 males and 31 females.

The groups were subdivided into the following allocations:
A: Main study animals (10 per group)
B: Recovery animals (10 per group)
C: Bioanalysis animals (3 per groups 2 to 4, none in the control group)
D: Bioanalysis recovery animals (3 per groups 2 to 4, none in the control group)
E: Neurotoxicity animals (5 per group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for Aerosol Concentration Selection: The target concentrations were selected by the Sponsor and the testing laboratory based on data from a 2-week dose range finding study performed at the testing laboratory and are based on guidance values for CLP classification.

Observations and examinations performed and frequency:

VIABILITY / MORTALITY
Recorded twice daily during treatment, once daily during acclimatization and recovery.

CLINICAL SIGNS
Recorded once daily during treatment (after exposure) and once weekly during acclimatization and recovery. Only data from allocation A and B animals were reported. In addition, a careful examination of each animal (allocation A to C) was performed once during acclimatization and once weekly during treatment period in a standard arena.

FUNCTIONAL OBSERVATION BATTERY (FOB)
Animals (allocation A) were observed for behavior, reflexes, activity, responsiveness, urine or feces, posture and general abnormalities once at the end of the treatment period. A description of all test parameters was listed in the report. Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with a DeMe Tec activity measurement system AMS0151. Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

FOOD CONSUMPTION
Recorded weekly (per cage) for the animals of allocation A and B during acclimatization, treatment and recovery.

BODY WEIGHTS
Recorded shortly before the first exposure during acclimatization (each individual animal), twice weekly during the first 4 weeks of treatment and weekly thereafter. Weekly recording throughout recovery and recording at the time of death. Only data from allocation A and B animals were reported.

OPHTHALMOSCOPY
Examinations were performed for all main study animals (allocation A and B), using a direct ophthalmoscope during acclimatization and at week 13 of treatment. As no test item-related findings were observed after 13 weeks of treatment no ophthalmoscopy was performed during recovery.

CLINICAL LABORATORY INVESTIGATIONS
Investigations took place after 13 weeks for the allocation A animals and after 4 weeks of recovery for the allocation B animals.
Blood samples were drawn from the retro-orbital plexus from all allocation A or B animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
Urine was collected during the 18 hour fasting period into a specimen vial, using a metabolism cage.

- Hematology:
The following hematology parameters were determined:

> Complete Blood Cell Count:
Erythrocyte count, hemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index (low, medium, high fluorescence), leukocyte count, total differential leukocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), large unstained cells and platelet count.

> Coagulation:
Prothrombin time (= Thromboplastin time) and Activated partial Thromboplastin time

> Clinical Biochemistry:
The following clinical biochemistry parameters were determined:
Glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transferase, creatine kinase, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin, globulin and albumin/globulin ratio.

> Urinalysis:
The following urine parameters were determined:

- Physical Examination:
Urine volume (18 hour), specific gravity (relative density), color and appearance

> Chemical Examination:
pH value, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes and leukocytes


BIOANALYSIS
- Blood and Organ Sampling
Blood samples (approximately 0.8 mL) were collected from the retro-orbital plexus from all allocation C and D animals under light isoflurane anesthesia.
Blood samples were collected in tubes containing lithium heparin and were stored at room temperature until shipment to the person performing blood cell preparation immediately after completion of each sampling event. The samples were shipped at room temperature within four hours after sampling.
The blood compartments (erythrocytes and platelets) were separated by using Ficoll/Percoll gradient centrifugation and cell count was determined. From last day of acclimatization onwards the blood samples were pooled for each sex and group. Then whole blood, erythrocytes and platelets were sent to the person responsible for bioanalytics and were stored at
-20 ± 5 °C. After receipt of all samples measurement of Mn levels were performed in whole blood, erythrocytes and platelets via AAS at the end of the study.
After the last blood sampling, the animals used for bioanalysis of Mn levels were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. The lungs and the brain were taken, weighed and the different parts of the brain (frontal cortex, striatum, globus pallidus) were separated. The parts of the brain were shipped on dry ice to the person responsible for bioanalytics and stored at -80 ± 10 °C until analysis. The lungs were stored at -80 ± 10 °C until 3 months after finalization of the report for possible further analysis.
Lungs and brain were taken from the reserve animals for implementation of the bioanalytical method.

- Determination of Mn - Levels in Blood, Lung and Brain:
Bioanalysis of Mn levels in blood (whole blood, erythrocytes and platelets) and different parts of the brain (frontal cortex, striatum, globus pallidus) were performed by the person responsible for bioanalytics via atomic absorption spectrometry AAS. Measurements were performed after receipt of all samples at the end of the study. The lungs will be analyzed for Mn content if requested by the sponsor.

Sacrifice and pathology:

PATHOLOGY
- Necropsy:
All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals surviving to the end of the observation period and the animal killed in extremis were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and, unless otherwise indicated, fixed in neutral phosphate buffered 4 % formaldehyde solution. Additional tissues (such as animal identification) will be retained in accordance with standard operating procedures but will not be processed or examined further.

- Organ Weights:
The weights of the organs from allocation A and B animals listed in Table 1 were recorded on the scheduled dates of necropsy listed and their organ to terminal body weight ratios as well as organ to brain weight ratios determined.

- Histotechnique:
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

- Histopathology:
Slides of all organs and tissues which were collected at terminal sacrifices from all animals of the control and high-dose groups (allocation A and B) were examined by the study pathologist. The same applied to all occurring gross lesions (allocation A and B) and to all animals, which died spontaneously or had to be terminated in extremis. A peer review of findings was performed.
As test item-related morphologic changes were detected in the lungs of the high-dose animals, the lungs from the mid- and low-dose group were examined to establish a no-effect level.

- Neuropathology:
Animals were subjected to deep anaesthesia by intraperitoneal injection of Esconarkon (approximately 200 mg/kg body weight) and sacrificed by perfusion fixation in situ with 1 mL of 50 IU heparin followed by 0.9 % saline buffer as the rinsing solution.
For fixation a 4 % formaldehyde solution was used. After perfusion fixation, the brain was removed and post-fixed. All organs and tissues that were not needed for further processing were removed and discarded. The animals fixed by perfusion fixation were kept in situ in a plastic container containing 4 % formaldehyde for approximately 24 hours until post-fixation was performed. The peripheral nerve tissues foreseen for plastic embedding were post-fixed with 4 % paraformaldehyde, 5 % glutaraldehyde in 0.1 M sodium phosphate buffered saline (at pH 7.4) for approximately 2 hours at 5 ± 3 °C. Rinsing with 0.1 M sodium phosphate buffered saline was done 3 times for approximately 10 minutes each.
Furthermore, the control and high dose groups were post-fixed with 2 % osmium tetraoxide and 1.8 % potassiumhexa-cyanoferrate II trihydrate (C6FeK4N6.3H2O) in 0.1 M sodium phosphate buffer for approximately 3 hours at room temperature (20 ± 5 °C). Thereafter the tissues were rinsed three times with 0.1 M sodium phosphate buffered saline for approximately 10 minutes each. Tissues were fixed in 0.1 M sodium phosphate buffered saline and stored at 5 ± 3 °C.
The animals fixed by perfusion were necropsied and the visible organs or organ sections were assessed for gross abnormalities as accurately as possible following perfusion fixation.

- Weight Determinations:
The following organ weight was determined from all animals killed by perfusion fixation after perfusion fixation prior to further processing:
1. Brain: For determination of the relative organ weights the terminal body weights were used.

- Organ / Tissue Preservation List:
In addition, the following organs/tissues were preserved in neutrally buffered 4 % formaldehyde solution:
Brain (remaining material after trimming)
Spinal cord (sections from cervical and lumbar cord)
All gross lesions
The remaining animal body was stored in neutrally buffered 4 % formaldehyde solution.
Samples of the following tissues and organs were collected from all perfusion fixed animals at necropsy and, unless otherwise indicated, postfixed in neutral phosphate buffered 4 % formaldehyde solution:
Brain (forebrain, midbrain, cerebellum and pons, upper brain stem, medulla oblongata, hippocampus)
Spinal cord - cervical segment (C4 - C7), lumbar segment (L4 - L5)
Spinal nerve root - dorsal (cervival & lumbar level), ventral (cervical & lumbar) level
Sciatic nerve (proximal, below sciatic notch)
Tibial nerve at knee (proximal & distal) and gastrocnemius branches
Dorsal root ganglion (spinal ganglion, cervical & lumbar level)
Eyes w/optic nerves and retina (fixed in Davidson's solution)
Gasserian ganglion
Gastrocnemius muscle

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, food consumption, body weight, ophthalmoscopic examinations, macroscopic findings, organ weights and ratios, as well as clinical laboratory data:
- The Dunnett-test: (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test: (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test: was applied to the ophthalmoscopic and macroscopic findings.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

see below

Urinalysis findings:

no effects observed

Description (incidence and severity):

see below

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

not examined

Details on results:

OBSERVATIONS
- Viability / Mortality:
One female of the control group (no. 119, allocation A) was killed in extremis on day 52 due to visible weight loss and a leiomyosarcoma in the eye region. One male of group 2 (no. 38, allocation B) was found dead in the cage on day 28 of recovery.
All other animals survived the scheduled treatment or recovery periods.

- Clinical Signs:
Slight reddish nasal excretion was observed in the recovery male of group 2 (no. 38) on day 28 of the recovery period before spontaneous death of this animal was observed. The cause of death could not be determined. In the female of the control group (no. 119), that was killed in extremis, visible weight loss, slight hair loss, ptosis, slight localized swelling and slight reddening of the right eye were recorded. A leiomyosarcoma was identified throughout histopathological examination as a cause of the bad condition of this female.
Clinical signs of mainly slight to moderate severity were occasionally observed in a few animals of both sexes of all treatment groups throughout the treatment period and consisted of injured eyes with following marked white discoloration in one case, enlarged eyes, scabs and/or wounds of the nose and anterior dorsum and hair loss at eyes, neck, foreleg, thigh, anterior dorsum and shoulders. Except for the injured eye, that was observed from week 1 of the treatment period in one animal, the remaining clinical signs were mainly recorded from week 6 to 12 and lasted on some occasions until the end of the treatment period.
No further clinical signs were observed during the treatment and recovery period.

- Detailed Clinical Observations:
Detailed clinical observations resulted in no findings.

- Functional Observational Battery:
No test item-related findings were noted during performance of the FOB.
Decreased rearings, decreased activity, unkempt fur, increased feaces-balls, decreased pupil size and absence of click response were occasionally observed in single animals of all groups and did not follow a dose-relationship. Therefore these findings were considered not to be related to treatment with Mn Metal Powder.

>Grip Strength, Landing Foot Splay and Body Temperature
Mean values of grip strength (fore and hind leg), landing foot splay and body temperature measurements showed similar values within all treatment groups and demonstrated no effect of the treatment with the test item.

>Locomotor Activity
Measurement of the locomotor activity by low beams count after 6, 12, 18, 24 and 30 min in all animals during week 12 of the treatment period gave no indication of a test item related effect.
Statistical significance was occasionally achieved but did not show a dose-relationship and was therefore considered to be of normal biological variation.

- Food Consumption;
Food consumption in females of groups 2 to 4 was slightly higher than the control over the whole treatment period and statistical significance was occasionally achieved in groups 3 and 4. During the recovery period food consumption was still slightly higher in females of group 4 and statistical significance was seen in week 2 and 3.
There was no effect on food consumption in the male animals of all treatment groups.

- Body Weights:
An increase in body weight and body weight gain was observed in the female animals of all treatment groups when compared to the control group mainly from Day 19 onwards. Statistical significance for body weight was occasionally achieved in the animals of group 2. For body weight gain statistical significance was achieved for the majority of days in groups 3 and 4 and occasionally in group 2. This effect was not observed during the recovery period.
No effect on body weight and body weight gain was observed in the male animals of all treatment groups.

- Ophthalmoscopic Examinations:
There were no ophthalmoscopic findings that were considered to be related to the treatment with the test item.

CLINICAL LABORATORY INVESTIGATIONS
- Hematology:
Statistically significant increased neutrophils (absolute) were recorded for the males and females of group 4. Furthermore, a statistically significant increase in white blood cells was seen for the males of group 4. Neutrophils (absolute) and white blood cell counts returned to normal during the recovery period.
Some inter-group variations occasionally achieved statistical significance but did not show a dose-relationship, were within the historical control range or were considered to be of normal biological variation. Therefore they were considered not to be related to treatment with the test item and are not discussed further.

- Clinical Biochemistry:
There were no changes in clinical biochemistry that were considered to be related to treatment with Mn Metal Powder.
Some inter-group variations occasionally achieved statistical significance but did not show a dose-relationship, were within the historical control range or were considered to be of normal biological variation and were therefore considered not to be related to treatment with the test item.

- Urinalysis:
There were no changes in urinalysis that were considered to be related to treatment with Mn Metal Powder.
Some inter-group variations occasionally achieved statistical significance but did not show a dose-relationship or were considered to be of normal biological variation and were therefore considered not to be related to treatment with the test item.

BIOANALYSIS
- Manganese Level Determination:
The concentration of Mn measured in whole blood samples collected from animals during the acclimatization period was below the detection level in all groups. However, during treatment, a general dose and gender dependent increase in Mn concentration was observed.

Overall, the Mn concentrations detected in whole blood were variable and in females of all groups, did not increase over the treatment period in the same pattern as they did in males.

After a recovery period of 4 weeks, a dose-related increase in levels of Mn compared to the beginning of treatment was still noted in whole blood samples in males. In group 2, the level measured was comparable to the pre-dose sample while in groups 3 and 4, the Mn concentrations were still higher than at the beginning of treatment but notably lower than in week 13 (group 4) and weeks 6 and 13 (group 3), respectively. In females, Mn levels were not measurable in groups 2 and 3. In group 4, the Mn concentration was clearly lower than the concentrations found in group 4 females during treatment. Therefore, there was clear indication of recovery.

In erythrocytes, with the exception of one sample, measurable Mn concentrations in the range between 0.0005 and 0.0015 µg/Billion Cells were detected in all samples from males and in half of the samples from females collected in all groups during the acclimatization period.

In group 2, measurable Mn concentrations were detected in eight of ten samples from males and three of ten samples from females collected during treatment; the values were not significantly different from those measured during the acclimatization period.

In group 3, measurable Mn concentrations were detected in nine of ten samples from males and in seven of ten samples from females collected during treatment. The concentrations were similar to those observed during the acclimatization period and ranged between 0.003 and 0.0016 µg/Billion Cells.

In group 4, measurable Mn concentrations were detected in all samples collected from males during treatment and all samples collected from females except the sample from day 2 during treatment. The concentrations ranged between 0.0008 and 0.134 µg/Billion Cells in males and 0.0008 and 0.0023 µg/Billion Cells in females. The highest concentrations were obtained in week 6 in males and in week 13 in females.

After 4 weeks of the recovery period, Mn concentrations measured in erythrocytes were similar to those during the acclimatization period reaching values of up to 0.0015 µg/Billion Cells – thus showing recovery.

In all samples of the platelets fraction collected during the acclimatization period, Mn concentrations were below the detection level.

There were no measurable concentrations of Mn in the platelet fraction of any of the samples collected from males and females of group 2.

There were no measurable concentrations of Mn in any of the samples collected from males of group 3. In group 3 females, measurable concentrations of Mn were detected in the sample collected on day 2 (0.0042 µg/Thousand Cells) and in one sample collected during week 13 of treatment (0.0210 µg/Thousand Cells).

Mn concentrations slightly above the detection level were observed in samples collected in group 4. In this group, the values of 0.0020 and 0.0025 µg/Thousand Cells were obtained respectively for males and females during week 6. Maximal values were reached during week 13 of treatment for males and females respectively - 0.0050 and 0.0070 µg/Thousand Cells.

After 4 weeks of the recovery, Mn concentrations measured in all male and female groups were below the detection level.

Measurable Mn concentrations were detected in all brain tissues examined: cortex, striatum and pallidus.

In the cortex of males or females a slight dose-related increase of Mn concentration was noted after 13 weeks of treatment. However, the individual results were quite variable in males and did not show large differences between groups 2 and 3. After the 4-week recovery period, the values obtained for groups 3 and 4 were generally slightly lower than after treatment indicating the likelihood for recovery.

In the striatum and pallidus, a dose dependent increase in Mn concentration was generally noted after 13 weeks of treatment. However, in the pallidus, the individual results were highly variable within the groups and there was no clear dose-relation in females. After the 4-week recovery period, the Mn concentrations found in the striatum were generally slightly lower than after treatment indicating recovery. In the pallidus, the Mn concentrations after the 4-week recovery period also showed a tendency for recovery. However, the results were similar for all groups and thus did not show a dose relation.

PATHOLOGY
- Organ Weights:
Increased lung weights (absolute and relative) were recorded in the males and females of group 4 and additionally in females of groups 2 and 3. In groups 3 and 4, the increase was statistically significant for absolute organ weights as well as for organ weights relative to body and to brain weights. In group 2, the increase of absolute lung weights reached statistical significance whereas increase in lung weight relative to body or to brain weight was not statistically significant. Statistically significant increased thymus weights, absolute as well as relative to body or to brain weights, were seen in males of group 4, only. Increased liver and kidney weights were observed in females of groups 3 and 4. The increase was statistically significant with regard to the absolute weights of these organs as well as liver and kidney weights relative to brain weights whereas the increase in organ weights relative to body weights was not statistically significant.

Except for the lung weight, which was still marginally increased after recovery in males of group 4, thymus, liver and kidney weights returned to normal during recovery.

Some further inter-group variations occasionally achieved statistical significance but showed no dose-relationship. Therefore they are considered not to be test item related.

- Macroscopic Findings:
There were no macroscopic findings during necropsy that were considered to be related to treatment with the test item.
All findings were considered to be incidental findings commonly noted in rats of this strain and age and were therefore considered not to be related to treatment with the test item.

- Microscopic Findings:
Minimal to slight alveolar histiocytosis (mainly focal, multifocal to diffuse, and/or diffuse) was observed in males and females of all groups including control group. With increasing dose level, there was a trend from focal histiocytosis to mainly diffuse histiocytosis. Furthermore, slight to moderate microgranulomas at the bronchiolar-alveolar junction were diagnosed in 2 and 7 males of groups 3 and 4, respectively, and in 8 females of group 4. Slight to moderate alveolitis was diagnosed in 3 males and 5 females of group 4.

Control group lesions were within the range of historical controls. The Armitage trend test revealed a positive statistically significant trend for histiocytes, multifocal (females only) and diffuse, microgranuloma, and alveolitis. In the Fisher test, the multifocal histiocytosis was statistically significantly increased in all test item groups, and diffuse histiocytosis increased in group 4; microgranuloma and alveolitis (females only) increased statistically significantly in group 4.

After the recovery period, the dose-dependently increased incidence from focal histiocytosis to multifocal to diffuse or diffuse histiocytosis was still noted in groups 2 to 4. However, multifocal to diffuse histiocytosis was also noted in 2 control males. Generally, the total number of animals per groups and sex showing histiocytosis was slightly decreased compared to the end of treatment while the severity grades were comparable. Minimal alveolitis was still noted after the recovery period in one female of group 4.

All other microscopic findings noted in this study were considered to be incidental findings commonly observed in rats of this strain and age.

NEUROPATHOLOGY
No morphological signs of neurotoxicity were noted histopathologically.

DETERMINATION OF PARTICLE SIZE DISTRIBUTION
The mass median aerodynamic diameters (MMADs) ranged between 2.3 and 4.69 µm. However, more than 45.6 % of the particles were calculated to be below 3 µm, and therefore deposition of a sufficient amount of particles was considered to have occurred mainly in the upper but also in the lower respiratory tract. Therefore, MMAD values above the target of 3 µm were considered to be acceptable. In addition, the Geometric Standard Deviations (GSD) were within the target range of 1.5 to 3 or slightly above (one measurement), which was also considered to be acceptable. In conclusion, the particle size distribution obtained was considered to be appropriate for this type of study.

Particle size distribution data are as follows:
GROUP 2
- Mean MMAD (µm) and (Mean GSD): 3.34 (2.68)
- Range of MMAD (µm): 2.79 to 3.52
- Range of GSD: 2.40 to 3.17
- Mean Percentage of particles <3 µm: 45.6

GROUP 3
- Mean MMAD (µm) and (Mean GSD): 3.29 (2.48)
- Range of MMAD (µm): 2.30 to 4.69
- Range of GSD: 2.06 to 2.71
- Mean Percentage of particles <3 µm: 46.0

GROUP 4
- Mean MMAD (µm) and (Mean GSD): 2.77 (2.41)
- Range of MMAD (µm): 2.36 to 3.40
- Range of GSD: 2.04 to 2.77
- Mean Percentage of particles <3 µm: 53.6

Dose descriptor:

NOAEL

Effect level:

0.5 other: µg/L

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

BIOANALYSIS

Table 4: A summary of the values obtained for Maximum Mn concentrations determined per sampling occasion (µg/g)

Sampling Occasion
	Predose (1 Time Point)	Day 1 (2 Time Points)	Day 2 (1 Time Point)	Week 6 (3 Time Points)	Week 13 (3 Time Points)	Recovery (1 Time Point)
Males
Group 2	0.0046	n.a.	n.a.	0.0053	n.a.	0.0046
Group 3	0.0048	0.0048	n.a.	0.0154	0.0141	0.0074
Group 4	n.a.	0.0059	0.0430	0.0143	0.0270	0.0163
Females
Group 2	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.
Group 3	n.a.	n.a.	n.a.	0.0085	0.0065	n.a.
Group 4	n.a.	0.0174	n.a.	0.0096	0.0104	0.0055

n.a.: not applicable / not measurable

 

Table 5: A summary of the mean values obtained for Mn concentrations determined in the Cortex, Striatum and Pallidus (µg/g)

Tissue analyzed
	Cortex		Striatum		Pallidus
Sampling Occasion	13 Weeks + 1 Day	Recovery	13 Weeks + 1 Day	Recovery	13 Weeks + 1 Day	Recovery
Males
Group 2	0.574	0.553	0.979	0.684	0.752	0.672
Group 3	0.608	0.547	1.557	0.903	1.657	0.993
Group 4	0.992	0.618	2.077	0.997	2.150	0.940
Females
Group 2	0.579	0.555	1.374	0.986	1.307	0.910
Group 3	0.745	0.586	1.543	1.039	1.803	1.337
Group 4	0.895	0.584	1.913	1.051	1.733	1.028

Conclusions:

Under the conditions of the study, the No-Observed-Adverse-Effect-Level (NOAEL) was determined to be 0.5 µg/L Mn Metal Powder.

Executive summary:

A study was conducted to investigate the repeated dose toxicity of the test material via inhalation. The study was conducted in accordance with the standardised guidelines OECD 413 and US EPA OPP 82-4 under GLP conditions.

Manganese (Mn) Metal Powder was administered 6 hours daily for 5 days/week to rats by nose-only, flow-past inhalation exposure. The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 4-week treatment free recovery period following the treatment period. The study comprised four groups containing 25 male and 25 female rats in group 1 and 31 male and 31 female rats in groups 2 to 4. The rats of the control group (group 1) were exposed to air only. Groups 2 to 4 were exposed to target concentrations of 0.5, 5 and 20 µg/L Mn Metal Powder, respectively.

Throughout the study all animals were observed for viability and clinical signs. In addition body weights and food consumption were recorded. Detailed clinical signs and functional observation battery were performed. Ophthalmoscopic examinations were performed and blood and urine samples were taken for clinical laboratory investigations. Blood, brain and lungs were taken for measurements of Mn levels in different blood compartments as well as brain areas. Following completion of the treatment and recovery period, a detailed necropsy was performed on all animals and selected organs were weighed. Various tissues and organs were placed in fixative, processed and examined microscopically. Neuropathology was performed in selected animals.

Exposure to gravimetrically determined aerosol concentrations of 0.56, 5.05 and 21.3 µg/L Mn Metal Powder were achieved in groups 2 to 4, respectively, and met or were slightly above the respective targets. The generated aerosols were considered to be respirable to rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

One female of the control group was killed in extremis on day 52 due to visible weight loss and a leiomyosarcoma in the eye region. One male of group 2 was found dead in the cage on day 28 of recovery. All other animals survived the scheduled treatment or recovery periods.

Clinical signs of mainly slight to moderate severity consisted of injured eyes with following marked white discoloration in one case, enlarged eyes, scabs and/or wounds of the nose and anterior dorsum and hair loss at eyes, neck, foreleg, thigh, anterior dorsum and shoulders and were observed in a few animals of both sexes of groups 2 to 4 mainly from week 6 to 12 and lasted on some occasions until the end of the treatment period. Slight reddish nasal excretion was observed in one recovery male before its spontaneous death and the cause remains unknown. Detailed clinical observations resulted in no findings.

No test item-related findings were noted during performance of the FOB. Grip strength, landing foot splay and body temperature measurements demonstrated no test item-related effects. Measurement of the locomotor activity gave no indication of a test item related effect.

Food consumption in females of groups 2 to 4 was slightly increased over the whole treatment period which was still observed during recovery in females of group 4. There was no effect on food consumption in the male animals of all treatment groups.

An increase in body weight and body weight gain was observed in the female animals of all treatment groups from Day 19 onwards. This effect was not observed during the recovery period. No effect on body weight and body weight gain was observed in the male animals of all treatment groups.

There were no test item-related ophthalmoscopic findings.

Increased neutrophils were recorded for the males and females of group 4. Furthermore, an increase in white blood cells was seen for the males of group 4. Neutrophils and white blood cell counts returned to normal during the recovery period. There were no changes in clinical biochemistry and urinalysis that were considered to be related to treatment with Mn Metal Powder.

No measurable level of Mn was detected in the whole blood or platelets fraction collected from animals during acclimatization. In the erythrocytes fraction, the level of Mn was slightly above the lowest concentration of the calibration solution even before the treatment.

During the treatment, a general time, dose and gender dependent increase in Mn concentration was measured in whole blood samples in all groups, the increase was more pronounced in males. Furthermore, an increase of the Mn concentration during the treatment was noted in erythrocytes in males and females of groups 3 and 4 and in platelets in males and females of group 4.

The concentration of Mn in brain tissue was above the lowest calibration solution in all tissues examined: cortex, striatum and pallidus. There were no noticeable/significant differences in the concentration measured in individual dose groups in the samples collected after 13 weeks of treatment as compared to those collected after four weeks of recovery in the cortex of males or females. In the striatum and pallidus a dose dependent increase of Mn concentration was noted in groups 2, 3 and 4 after 13 weeks of treatment in males. Values measured after the four weeks of recovery were up to 2 times lower than after the treatment. This finding was similar in all groups.

Increased lung weights were recorded in the males of group 4 and additionally in females of groups 2, 3 and 4. Increased thymus weights were seen in males of group 4, only. Increased liver and kidney weights were observed in females of groups 3 and 4. Except for the lung weight, which showed a marginal increase in weight after recovery in males of group 4, all organ weights returned to normal during recovery.

No test item-related macroscopic findings were recorded during necropsy. No morphological signs of neurotoxicity were noted histopathologically.

A dose-dependent increase in incidence of minimal to slight focal to multifocal to diffuse or diffuse alveolar histiocytosis was observed in males and females of all groups including control group and was still noted after the end of recovery. Slight to moderate microgranulomas at the bronchiolar-alveolar junction were diagnosed in males of groups 3 and 4 and in females of group 4. Slight to moderate alveolitis was diagnosed in both sexes of group 4 and in one female of group 4 after recovery. The reactive focal and multifocal/diffuse alveolar histiocytosis was not regarded as adverse in nature. Microgranuloma, alveolitis and inflammatory lesions are indicative for an adverse particle overload reaction.

In accordance to the findings above the lung was considered to be the target organ for the exposure to Mn Metal Powder mainly because it was the port of entry. There was indication of reversibility of the findings, however, the 4 week duration of the recovery period was considered insufficient to demonstrate full reversal of all findings.

Based on the histopathological findings in this study a No-Observed-Adverse-Effect-Level (NOAEL) was established at 0.5 µg/L Mn Metal Powder.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

One key study available, performed to standardised guidelines under GLP conditions. Therefore, the quality of the database is considered to be good.

Additional information

REPEATED DOSE - INHALATION

There is one key study available to address this endpoint; Broich (2016). This study was conducted to investigate the repeated dose toxicity of the test material via inhalation. The study was conducted in accordance with the standardised guidelines OECD 413 and EPA OPP 82-4 under GLP conditions. Therefore, the study was assigned a reliability score of 1 in line with the principles for assessing data quality as defined by Klimisch et al. (1997).

Manganese (Mn) Metal Powder was administered 6 hours daily for 5 days/week to rats by nose-only, flow-past inhalation exposure. The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 4 - week treatment free recovery period following the treatment period. The study comprised four groups containing 25 male and 25 female rats in group 1 and 31 male and 31 female rats in groups 2 to 4. The rats of the control group (group 1) were exposed to air only. Groups 2 to 4 were exposed to target concentrations of 0.5, 5 and 20 µg/L Mn Metal Powder, respectively.

Throughout the study all animals were observed for viability and clinical signs. In addition body weights and food consumption were recorded. Detailed clinical signs and functional observation battery were performed. Ophthalmoscopic examinations were performed and blood and urine samples were taken for clinical laboratory investigations. Blood, brain and lungs were taken for measurements of Mn levels in different blood compartments as well as brain areas. Following completion of the treatment and recovery period, a detailed necropsy was performed on all animals and selected organs were weighed. Various tissues and organs were placed in fixative, processed and examined microscopically. Neuropathology was performed in selected animals.

Exposure to gravimetrically determined aerosol concentrations of 0.56, 5.05 and 21.3 µg/L Mn Metal Powder were achieved in groups 2 to 4, respectively, and met or were slightly above the respective targets. The generated aerosols were considered to be respirable to rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

One female of the control group was killed in extremis on day 52 due to visible weight loss and a leiomyosarcoma in the eye region. One male of group 2 was found dead in the cage on day 28 of recovery. All other animals survived the scheduled treatment or recovery periods.

Clinical signs of mainly slight to moderate severity consisted of injured eyes with following marked white discoloration in one case, enlarged eyes, scabs and/or wounds of the nose and anterior dorsum and hair loss at eyes, neck, foreleg, thigh, anterior dorsum and shoulders and were observed in a few animals of both sexes of groups 2 to 4 mainly from week 6 to 12 and lasted on some occasions until the end of the treatment period. Slight reddish nasal excretion was observed in one recovery male before its spontaneous death and the cause remains unknown. Detailed clinical observations resulted in no findings.

No test item-related findings were noted during performance of the FOB. Grip strength, landing foot splay and body temperature measurements demonstrated no test item-related effects. Measurement of the locomotor activity gave no indication of a test item related effect.

Food consumption in females of groups 2 to 4 was slightly increased over the whole treatment period which was still observed during recovery in females of group 4. There was no effect on food consumption in the male animals of all treatment groups.

An increase in body weight and body weight gain was observed in the female animals of all treatment groups from Day 19 onwards. This effect was not observed during the recovery period. No effect on body weight and body weight gain was observed in the male animals of all treatment groups.

There were no test item-related ophthalmoscopic findings.

Increased neutrophils were recorded for the males and females of group 4. Furthermore, an increase in white blood cells was seen for the males of group 4. Neutrophils and white blood cell counts returned to normal during the recovery period. There were no changes in clinical biochemistry and urinalysis that were considered to be related to treatment with Mn Metal Powder.

No measurable level of Mn was detected in the whole blood or platelets fraction collected from animals during acclimatization. In the erythrocytes fraction, the level of Mn was slightly above the lowest concentration of the calibration solution even before the treatment.

During the treatment, a general time, dose and gender dependent increase in Mn concentration was measured in whole blood samples in all groups, the increase was more pronounced in males. Furthermore, an increase of the Mn concentration during the treatment was noted in erythrocytes in males and females of groups 3 and 4 and in platelets in males and females of group 4.

The concentration of Mn in brain tissue was above the lowest calibration solution in all tissues examined: cortex, striatum and pallidus. There were no noticeable/significant differences in the concentration measured in individual dose groups in the samples collected after 13 weeks of treatment as compared to those collected after four weeks of recovery in the cortex of males or females. In the striatum and pallidus a dose dependent increase of Mn concentration was noted in groups 2, 3 and 4 after 13 weeks of treatment in males. Values measured after the four weeks of recovery were up to 2 times lower than after the treatment. This finding was similar in all groups.

Increased lung weights were recorded in the males of group 4 and additionally in females of groups 2, 3 and 4. Increased thymus weights were seen in males of group 4, only. Increased liver and kidney weights were observed in females of groups 3 and 4. Except for the lung weight, which showed a marginal increase in weight after recovery in males of group 4, all organ weights returned to normal during recovery.

No test item-related macroscopic findings were recorded during necropsy. No morphological signs of neurotoxicity were noted histopathologically.

A dose-dependent increase in incidence of minimal to slight focal to multifocal to diffuse or diffuse alveolar histiocytosis was observed in males and females of all groups including control group and was still noted after the end of recovery. Slight to moderate microgranulomas at the bronchiolar-alveolar junction were diagnosed in males of groups 3 and 4 and in females of group 4. Slight to moderate alveolitis was diagnosed in both sexes of group 4 and in one female of group 4 after recovery. The reactive focal and multifocal/diffuse alveolar histiocytosis was not regarded as adverse in nature. Microgranuloma, alveolitis and inflammatory lesions are indicative for an adverse particle overload reaction.

In accordance to the findings above the lung was considered to be the target organ for the exposure to Mn Metal Powder mainly because it was the port of entry. There was indication of reversibility of the findings, however, the 4 week duration of the recovery period was considered insufficient to demonstrate full reversal of all findings.

Based on the histopathological findings in this study a No-Observed-Adverse-Effect-Level (NOAEL) was established at 0.5 µg/L Mn Metal Powder.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Only one key study available.

Justification for classification or non-classification

Classification by the oral route is not justified based on the lack of toxicity in the acute oral toxicity study (IUCLID section 7.2.1)

Mn metal is mostly used in industrial settings where good industrial hygiene is employed. Therefore, this is not a likely route of exposure. However, in the unlikely event of accidental consumption, the acute oral toxicity test confirms no evidence of toxicity via this route.

Classification by the inhalation route is not justified because leaching in artificial lung fluid is very low , hence systemic exposure will also be very low. The very poor solubility in water (0.70 mg/L, see IUCLID section 4.8) is likely to mean that a significant percentage of any particles inhaled will undergo mucociliary escalation back into the gastro-intestinal tract where there is a low level of oral toxicity.

An investigation on the bioavailability of the registered substance in artificial lung fluid demonstrates the low bioavailability of the substance in the lungs (0.020 %, see Anderson KA, IUCLID section 7.12). To support this, Mn metal is not acutely toxic by the inhalation route ( IUCLID section 7.2.2).

Classification by the dermal route is not justified due to the very poor water solubility of Mn metal and hence a minimal amount of the potential substance is made available for systemic absorption via the dermal route.

Even if minimally available from exposure, the physiological properties of the registered substance do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies .