Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

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.

The potential reproductive toxicity of MnCl2 was assessed in three studies, two one-generation studies and a single two-generation study.  All of these studies are considered reliable with Klimisch scores of 1 or 2.  The key study (McGough & Jardine, 2016 published and Grieve, 2017 report unpublished) was conducted according to OECD Guideline 416 with administration via the inhalation route (nose only).  In this two-generation study the dose levels were selected based on results from a preliminary reproduction study in rats.  In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.  There were no treatment-related effects on litter weights, sperm motility, sperm morphology or the ovary follicle scoring and no findings were observed in the reproductive tract of any of the generations tested.  There were no effects of treatment on the sexual maturity of the F1 animals.  The NOEL for reproductive performance was considered to be 20 μg/L, the highest dose tested (Grieve, 2017).  

Two supporting studies are also available on MnCl2.  Both are one-generation studies conducted via the oral route.  In the first supporting study no treatment-related effects on reproduction or development were noted (Ali et al., 1983).  In the second supporting study, mice were administered the MnCl2 at very high doses up to 8 000 mg MnCL2/L in the drinking water for 12 weeks.  Fertility of males was significantly reduced in the highest dose group.  Whilst the study is considered reliable with regards to documentation, the doses that were administered were extremely high, with the high dose equivalent to a 70 kg adult human consuming almost 50 g of the test material.  The study concluded that it was unclear whether effects noted on fertility were caused by males that were so severely intoxicated that successful pairing was no longer possible (Elbetieha et al., 2001).

There is a significant body of publicly available data on the effects of MnCl2 on developmental toxicity, some of which demonstrate teratogenicity or embryotoxicity.  However, much of the data from the literature references are via non-standard routes of administration or have low reliability due to methodological deficiencies or lack of reported information.  

There are also studies conducted according to OECD Test Guidelines and the principles of GLP, of which the OECD 414 study conducted by Dettwiler (2016) is the key study.  MnCl2 was administered by the inhalation route (nose only).  An increase in the incidence of large foetal thyroids at the highest dose was observed in the presence of maternal toxicity.  The increase in size correlated with diffuse follicular hypertrophy/hyperplasia and an increase in mitotic figures in follicular epithelial cells when the thyroids were examined microscopically.  The possible relationship between the changes to the foetal thyroids and maternal toxicity remained unclear.  The NOEL and NOAEL for prenatal developmental toxicity was therefore considered to be 15 μg/L air.  The initial doses for this study were based on the results of a Developmental Neurotoxicity study (Dettwiler, 2015) due to clinical signs observed at the highest dose level.  This study was conducted according to OECD Test Guideline 426 and in compliance with the principles of GLP and included an examination of reproduction and developmental effects in pups.  However, no test material-related differences between the control and the dose groups were observed for any of the reproductive or developmental parameters assessed (Dettwiler, 2015). Certain development effects seen in the publicly available literature were not replicated in these modern GLP studies conducted to the appropriate OECD guidelines. Based on the scores assigned in line with the Klimisch scale, the results of the standardised, GLP studies are considered the more reliable.

When assessing reproductive and developmental toxicity care is needed not to attribute effects on reproduction to the test material that may be the result of secondary non-specific consequences of other toxic effects.  On reviewing all available data it was concluded that there was no reliable evidence linking MnCl2 with specific direct reproductive or developmental toxicity via any relevant routes of exposure.

There is a smaller body of literature data available on the reproductive/developmental toxicity of effects  MnSO4 and no studies performed to standardised guidelines. One literature reference (Batineh et al, 1998) on the reprotoxic potential of MnSO4 was awarded a Klimisch reliability rating of 2.  Adult male rats ingested MnSO4 along with drinking water at a concentration of 1 000 ppm for 12 weeks.  Male rat sexual behaviour was suppressed, particularly with ejaculation latency.  Male rat aggression was also abolished after the ingestion of MnSO4.  The total number of resorptions was increased in female rats impregnated by males ingesting MnSO4.  Body, absolute or relative testes, and seminal vesicles weights were dropped in adult male rats ingesting MnSO4.  These results suggest that the long-term ingestion of MnSO4 would have adverse effects on sexual behavior, territorial aggression, fertility and the reproductive system of the adult male rat (Batineh et al, 1998) and were not considered sufficient to lead to classification.  Despite the available literature showing some reproductive and developmental effects, other long-term repeated dose studies on MnSO4 have shown no such effects.  A two-year carcinogenicity and repeated dose toxicity report (NTP, 1993) showed no effect on the testes of rats exposed orally for up to 2 years.

Järvinen and Ahlström (1975) examined the effect of dietary manganese level in female rats and their foetuses up to 1 004 mg Mn/kg.  There was no impact on foetuses at any of the doses of MnSO4 studied and no impact on reproductive parameters at any dose level (Järvinen & Ahlström, 1975).  

As with MnCl2 despite some literature references demonstrating reproductive or developmental toxicity, there is no reliable unequivocal animal evidence to link MnSO4 with reproductive toxicity via relevant routes of exposure.  

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 modern studies performed on MnSO4 under GLP and to standardised OECD guidelines where all appropriate parameters are investigated and the deficiencies in those studies conducted on MnSO4, it is considered appropriate to utilise read-across to the key studies performed on MnCl2. It is considered that the reliability of these guideline studies can be considered to give a more accurate representation of the toxicity of the Mn2+ ion than the studies performed on MnSO4 itself.

No reproductive or developmental toxicity studies are available on Mn(NO3)2 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  under GLP and to standardised OECD guidelines where all appropriate parameters are investigated, it is considered appropriate to utilise read-across to the key studies performed on MnCl2. 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

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.

The potential reproductive toxicity of MnCl2 was assessed in three studies, two one-generation studies and a single two-generation study.  All of these studies are considered reliable with Klimisch scores of 1 or 2.  The key study (McGough & Jardine, 2016 published and Grieve, 2017 report unpublished) was conducted according to OECD Guideline 416 with administration via the inhalation route (nose only).  In this two-generation study the dose levels were selected based on results from a preliminary reproduction study in rats.  In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.  There were no treatment-related effects on litter weights, sperm motility, sperm morphology or the ovary follicle scoring and no findings were observed in the reproductive tract of any of the generations tested.  There were no effects of treatment on the sexual maturity of the F1 animals.  The NOEL for reproductive performance was considered to be 20 μg/L, the highest dose tested (Grieve, 2017).  

Two supporting studies are also available on MnCl2.  Both are one-generation studies conducted via the oral route.  In the first supporting study no treatment-related effects on reproduction or development were noted (Ali et al., 1983).  In the second supporting study, mice were administered the MnCl2 at very high doses up to 8 000 mg MnCL2/L in the drinking water for 12 weeks.  Fertility of males was significantly reduced in the highest dose group.  Whilst the study is considered reliable with regards to documentation, the doses that were administered were extremely high, with the high dose equivalent to a 70 kg adult human consuming almost 50 g of the test material.  The study concluded that it was unclear whether effects noted on fertility were caused by males that were so severely intoxicated that successful pairing was no longer possible (Elbetieha et al., 2001).

There is a significant body of publicly available data on the effects of MnCl2 on developmental toxicity, some of which demonstrate teratogenicity or embryotoxicity.  However, much of the data from the literature references are via non-standard routes of administration or have low reliability due to methodological deficiencies or lack of reported information.  

There are also studies conducted according to OECD Test Guidelines and the principles of GLP, of which the OECD 414 study conducted by Dettwiler (2016) is the key study.  MnCl2 was administered by the inhalation route (nose only).  An increase in the incidence of large foetal thyroids at the highest dose was observed in the presence of maternal toxicity.  The increase in size correlated with diffuse follicular hypertrophy/hyperplasia and an increase in mitotic figures in follicular epithelial cells when the thyroids were examined microscopically.  The possible relationship between the changes to the foetal thyroids and maternal toxicity remained unclear.  The NOEL and NOAEL for prenatal developmental toxicity was therefore considered to be 15 μg/L air.  The initial doses for this study were based on the results of a Developmental Neurotoxicity study (Dettwiler, 2015) due to clinical signs observed at the highest dose level.  This study was conducted according to OECD Test Guideline 426 and in compliance with the principles of GLP and included an examination of reproduction and developmental effects in pups.  However, no test material-related differences between the control and the dose groups were observed for any of the reproductive or developmental parameters assessed (Dettwiler, 2015). Certain development effects seen in the publicly available literature were not replicated in these modern GLP studies conducted to the appropriate OECD guidelines. Based on the scores assigned in line with the Klimisch scale, the results of the standardised, GLP studies are considered the more reliable.

When assessing reproductive and developmental toxicity care is needed not to attribute effects on reproduction to the test material that may be the result of secondary non-specific consequences of other toxic effects.  On reviewing all available data it was concluded that there was no reliable evidence linking MnCl2 with specific direct reproductive or developmental toxicity via any relevant routes of exposure.

There is a smaller body of literature data available on the reproductive/developmental toxicity of effects  MnSO4 and no studies performed to standardised guidelines. One literature reference (Batineh et al, 1998) on the reprotoxic potential of MnSO4 was awarded a Klimisch reliability rating of 2.  Adult male rats ingested MnSO4 along with drinking water at a concentration of 1 000 ppm for 12 weeks.  Male rat sexual behaviour was suppressed, particularly with ejaculation latency.  Male rat aggression was also abolished after the ingestion of MnSO4.  The total number of resorptions was increased in female rats impregnated by males ingesting MnSO4.  Body, absolute or relative testes, and seminal vesicles weights were dropped in adult male rats ingesting MnSO4.  These results suggest that the long-term ingestion of MnSO4 would have adverse effects on sexual behavior, territorial aggression, fertility and the reproductive system of the adult male rat (Batineh et al, 1998) and were not considered sufficient to lead to classification.  Despite the available literature showing some reproductive and developmental effects, other long-term repeated dose studies on MnSO4 have shown no such effects.  A two-year carcinogenicity and repeated dose toxicity report (NTP, 1993) showed no effect on the testes of rats exposed orally for up to 2 years.

Järvinen and Ahlström (1975) examined the effect of dietary manganese level in female rats and their foetuses up to 1 004 mg Mn/kg.  There was no impact on foetuses at any of the doses of MnSO4 studied and no impact on reproductive parameters at any dose level (Järvinen & Ahlström, 1975).  

As with MnCl2 despite some literature references demonstrating reproductive or developmental toxicity, there is no reliable unequivocal animal evidence to link MnSO4 with reproductive toxicity via relevant routes of exposure.  

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 modern studies performed on MnSO4 under GLP and to standardised OECD guidelines where all appropriate parameters are investigated and the deficiencies in those studies conducted on MnSO4, it is considered appropriate to utilise read-across to the key studies performed on MnCl2. It is considered that the reliability of these guideline studies can be considered to give a more accurate representation of the toxicity of the Mn2+ ion than the studies performed on MnSO4 itself.

No reproductive or developmental toxicity studies are available on Mn(NO3)2 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  under GLP and to standardised OECD guidelines where all appropriate parameters are investigated, it is considered appropriate to utilise read-across to the key studies performed on MnCl2. 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 January 2014 to 30 June 2014
Reliability:
2 (reliable with restrictions)
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.
Justification for type of information:
Read-across study. See report attached to Section 13 for justification.
Reason / purpose:
other: read-across target
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines (MAFF, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: RccHan™: WIST(SPF)
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 203 to 262 g (Day 0 post coitum)
- Housing: Group A females (mated) were housed in groups of three to five animals in cages with wire mesh tops up to the day of mating and afterwards individually in cages with wire mesh tops. Group B females (not mated) were housed individually in cages with wire mesh tops. Cages were equipped with sterilised standard softwood bedding with paper enrichment.
- Diet: Pelleted standard rodent maintenance diet (ad libitum)
- Water: Community tap water in water bottles (ad libitum)
- Acclimation period: Animals were acclimated under test conditions after a health examination. Dams were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 % (relative)
- Air changes: 10 - 15 air changes per hour
- Photoperiod: There was a 12-hour fluorescent light / 12-hour darkness cycle with music during the light period.

IN-LIFE DATES:
From: 28 Jan 2014
To: 28 April 2014
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 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 aerosol was discharged constantly through the exposure system. The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. Before commencement of the exposure of the group(s), technical trials were conducted (without animals) using the inhalation system foreseen for the study.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes.
- System of generating particulates/aerosols: A dust aerosol was generated from the test material using a rotating brush aerosol generator connected to a micronising jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutraliser. Furthermore, the aerosol concentrations of the test material of the low dose group were achieved by serial dilution with compressed, filtered, dry air of the higher aerosol concentration of the mid dose group using an air vacuum device.
- Temperature, humidity, pressure in air chamber: Aerosol concentration, particle size distribution, relative humidity and temperature were measured on test aerosol samples taken at a representative exposure port. The relative humidity and temperature in the chamber were measured continuously during each exposure using a calibrated device. Additionally, values were recorded hourly during each exposure.
- Oxygen concentration: The oxygen concentration was measured on test aerosol samples taken at a representative exposure port. The oxygen concentration in the chamber was measured during each exposure using a calibrated device. Additionally, values were recorded hourly by hand during each exposure. The oxygen concentration was maintained above 19 % during the exposure period.
- Air flow rate: The flow of air at each tube was 1 L/min, which is sufficient to minimise re-breathing of the test aerosol as it is more than twice the respiratory minute volume of a rat. All airflow rates (including those for concentration and particle size measurements) were determined using calibrated gas meters and pressure gauges or flow meters. The exposure airflow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and/or pressure gauges. The actual airflow rate was monitored hourly during each exposure. Additional measurements were performed if considered necessary.
- Method of particle size determination: The particle size distribution was determined gravimetrically three times for the low, mid and high dose groups. The cumulative particle size distribution of the test aerosol was determined using a Mercer 7 stage cascade impactor Model 02-130 (In-Tox. Products Inc., Albuquerque, New Mexico, USA). The test aerosol was impacted at each stage onto stainless steel slips and the particle size distribution of the test material in the generated aerosol was measured by gravimetrically analysing the test material deposited on each stage of the cascade impactor. 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 were 1 to 3 μm for the MMAD and 1.5 to 3 for the GSD.
- Treatment of exhaust air: The aerosol was exhausted using a tubing/filter system.

TEST ATMOSPHERE
- Brief description of analytical method used:

>Determination of Nominal Aerosol Concentration
The test material usage was measured during each exposure in the mid and high dose groups by weighing the generator cylinders containing the test material before and after each exposure to determine the quantity of test material used. The weight used was then divided by the total air-flow volume to give the nominal concentration. The nominal concentration of the low dose group was calculated from the value of the mid dose group under consideration of the dilution factor. These data were used for the purpose of monitoring the performance of the generation system.

>Gravimetric Determination of Aerosol Concentration
Gravimetric determination of the aerosol concentration was performed twice to four times per exposure for the low, mid and high dose groups. Additional samples were collected for monitoring purposes.

VEHICLE
- Composition of vehicle: Compressed, filtered, dry air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test aerosol samples were collected onto Millipore® durapore filters, type HVLP using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port. The filters were weighed before and at least 10 minutes after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test material present on the filter and the sample volume. A correction factor of 1.67 (determined from the technical trials) was applied to correct for the adsorption of water during sampling due to the hygroscopic properties of the test material. This factor was determined during technical trials by AAS analysis on the Mn content and was confirmed by additional AAS analysis of filters taken during exposure. For AAS analysis filter samples were sent to the person responsible for dose formulation analysis.

FORMULATION ANALYSIS
- Analytical Standard
Manganese 1000 μg/mL AAS/ICP

- Study Samples and Storage
Filter samples were dispatched to the analytical laboratories internally (at room temperature) and directly analysed.

- Purified water
Prepared in-house with an ELGA water purification system (Ultra Bio No. UBH 279651)

ANALYTICAL PROCEDURE
- Preparation of Calibration Solutions
A stock solution of analytical standard in 1 M chloride acid (HCl) with a concentration of 2.56 μg/mL was prepared (solution A) by dissolving 256 μL of the analytical standard in 100 mL of 1 M chloride acid. Standard solutions were prepared by successive dilution of solution A with 1 M chloride acid. The resulting concentrations ranged from 0.040 to 1.280 μg/mL. These standard solutions as well as solution A were used to calibrate the atomic absorption spectrometer.

- Work up of Samples
An appropriate volume of 1 M chloride acid was added to each filter sample and dissolution was achieved by sonication for at least 5 minutes.

-Atomic Absorption Spectrometry with Flame Assembly
Instrument: Perkin-Elmer Model PE 2100 (software 4100) atomic absorption spectrometer
Flame: Acetylene flame/air
Slit Width: 0.2
Wavelength: Calcium: 279.5 nm

- Evaluation of Results
Samples were quantified by atomic absorption spectrometry (AAS) of manganese with reference to the respective calibration curve (with zero intercept). The calibration curve (non-linear) and the concentration (in μg/mL) were calculated using the Perkin Elmer software.
The concentration of precipitated test material in the filter samples was calculated using the following equation:
Filters: A(filter) = (Cs ∙ V ∙ D ∙ F) / 1000
where
A(filter) = Actual amount of test material on filter [μg/filter]
Cs = Measured concentration of manganese in sample [μg/mL]
V = Volume solvent for dissolution [mL]
D = Dilution factor
F = Correction factor of 2.2906
Details on mating procedure:
- Impregnation procedure: Cohoused. After acclimatisation, females were housed with sexually mature males in special automatic mating cages i.e. with synchronised timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females.
- M/F ratio per cage: 1:1
- Length of cohabitation: Not reported
- Proof of pregnancy: The females were removed and housed individually if the daily vaginal smear was sperm positive or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- Other: Male rats of the same source and strain were used only for mating. These male rats are in the possession of laboratory and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored. Females in recovery groups were not mated.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
Animals were treated with the test material once daily at approximately 24 hour intervals.
Duration of test:
Females were treated for 15 consecutive days. Mated females were treated from days 6 to 20 post coitum) and recovery animals from day 1 to 15 of a concurrent treatment period.
The recovery period was 8 weeks.
No. of animals per sex per dose:
Females A: 88 mated females, 22 per group
Females B: 24 not mated females, 6 per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous developmental neurotoxicity study in Han Wistar rats conducted at the testing facility using aerosol concentrations of 5, 15 and 25 µg/L air. At a dose level of 25 µg/L laboured breathing and reduced body weight were observed in dams after treatment during gestation. No test material-related effects were recorded in breeding at any aerosol concentration for the test material.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for viability/mortality twice daily. Daily cage-side clinical observations were made once daily during acclimatisation and after treatment up to the day of necropsy.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: For Group A, body weights were recorded daily from day 0 until day 21 post coitum. For Group B, body weights were recorded on treatment days 1, 8 and 15 and recovery days 1, 8, 15, 22, 29, 36, 43, 50 and 57.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: For Group A, food consumption was recorded at 3-day intervals on days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.
For Group B, food consumption was recorded on treatment days 1 - 8 and 8 – 15 and recovery days 1 - 8, 8 - 15, 15 - 22, 22 - 29, 29 - 36, 36 - 43, 43 – 50 and 50 – 57.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice
At the scheduled necropsy on day 21 post coitum, main study females were sacrificed by CO₂ asphyxiation and the foetuses were removed by Caesarean section. Recovery females were sacrificed by intraperitoneal injection of pentobarbitone after 4 (3 females per group) or 8 weeks (3 females per group) of recovery period.

-Necropsy
Group A: Any female sacrificed during the study was subjected to macroscopic examination with emphasis on the uterus and its contents. Post mortem examination, including gross macroscopic examination of all internal organs was performed. The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
When considered appropriate, macroscopic changes in the dams were photographed and samples of tissue fixed in neutral phosphate buffered 4 % formaldehyde solution for possible microscopic examination.
One foetus from 6 different litters of each dose group was removed, weighed and stored at -20 ± 5 °C for possible determination of test material levels. In agreement with the Sponsor, these foetuses were discarded after delivery of the draft report.

- Tissue Preservation
At scheduled sacrifice, the lungs from certain females were preserved; the lungs from 6 pregnant females per dose group, all non-pregnant females per dose group and all 6 recovery females were preserved in neutral phosphate buffered 4 % formaldehyde solution.

-Histotechnique
The lungs from pregnant Group A females in the control and high-dose group as well as all occurring gross lesions were processed, embedded and cut at an approximate thickness of 4 micrometres and stained with haematoxylin and eosin.
Treatment-related changes were observed in the lungs of pregnant females at the high-dose, therefore the lungs of pregnant females in groups 2 and 3 and all recovery females were processed.

- Histopathology
Slides of all organs and tissues collected at terminal sacrifice of the control and high-dose group were examined.
Test material-related morphologic changes were detected in organs of high-dose animals and therefore the lungs from the remaining groups were examined to establish a no-effect level, if possible.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Group A: Any female sacrificed during the study was subjected to macroscopic examination with emphasis on the uterus and its contents. Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible haemorrhagic areas of implantation sites.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Foetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). At least one half of the foetuses from each litter was fixed in Bouin's fixative (one foetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic vials. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. After the staining of the foetuses for skeletal examination, specimens were evaluated. Foetuses were examined in mixed group order. Each litter was examined in sequential order. Foetuses with abnormalities were photographed when considered appropriate.

- Histotechnique and Histopathology
An increased number of large thyroids was found in group 4 during visceral examination of foetuses and therefore this organ was examined histopathologically to establish whether the increase in size is related to any microscopic change.
To this purpose, normal thyroids from ten foetuses in the control group and thyroids with increased size from ten foetuses in the high-dose group were selected as follows:
- in the control group one foetus per litter were randomly selected to represent ten litters;
- in the high-dose group, all five foetuses (from four litters) with large thyroid were selected and additionally five foetuses with slightly large thyroid were selected from five different litters.
Foetal thyroids were trimmed transversely leaving them attached to the trachea. They were embedded on this cut surface and serial section were cut at 4 μm. They were then stained with haematoxylin and eosin.
Statistics:
The following statistical methods were used to analyse food consumption, body weights, reproduction and skeletal examination data:
- Means and standard deviations of various data were calculated and included in the report.
- 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 if the variables could be dichotomised without loss of information.

The skeletal examination data were first assessed using Bartlett’s test for homogeneity of variance. As these data were found to be non-homogenous, non-parametric assessment by Kruskall-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was used.
Historical control data:
Historical control data were included in the report.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
GROUP A
All females survived the scheduled study period.
Treatment with the test material caused breathing noises and dyspnea in females in groups 3 and 4. In group 4, breathing noises were observed on day 8 post coitum in one female. The number of females affected increased and until day 17 post coitum it was recorded in 18 females in this group.
In group 3, one female had dyspnea on day 8 post coitum followed by breathing noises observed in this female for several days. Breathing noises were also recorded for 7 more females in this group; in 4 animals for one day and in 3 animals for four days.
A red secretion from the nose and eyes was noted in several females in all groups including control. This finding was considered to be related to the treatment route.

GROUP B
All females survived until the scheduled necropsy.
Treatment with the test material caused breathing noises in females in groups 3 and 4.
In group 4, breathing noises were observed on day 3 of the treatment in one female. The number of females affected increased and on day 12 of the treatment it was recorded in all 6 females. The breathing noises were observed until the end of the treatment period and on day 1 of the recovery period. No breathing noises were observed in any female during the remaining recovery period days 2 to 57.
In group 3, breathing noises were observed for the first time on day 5 of treatment. Four females were affected in this group, with breathing noises also observed on day 1 of the recovery period, but not thereafter.
A red secretion from the nose and eyes was noted in several females in all groups including the control. This finding was observed during the treatment and on day 1 of the recovery period but not thereafter. It was considered to be related to the treatment route.

BODY WEIGHTS
GROUP A
Mean body weight gain from day 6 to 21 post coitum was 38.2, 37.2, 32.4 and 29.4 % whereas mean corrected body weight gain was 1.4, 1.9, -2.4 and -5.4 % in groups 1, 2, 3 and 4, respectively.
Treatment with the test material caused a dose dependent body weight loss followed by a reduced body weight gain and a reduction in body weights in groups 3 and 4. A body weight loss of 2 and 5 % was noted in groups 3 and 4, respectively, on day 8 post coitum followed by a reduced body weight gain during the remaining study period. The reduction in body weights as well as the reduction in body weight gain was statistically significant from day 7 to 21 post coitum in both groups. Also corrected body weight gain (body weight gain corrected for the gravid uterus weight at termination) was dose dependently reduced in both groups. After subtraction of the gravid uterus weights, a body weight loss was established with a statistical significance in both groups if expressed as absolute values and in group 4 if expressed as a percentage of the body weight at the start of the treatment.
In group 2, body weight was lower if compared to the control values with a statistical significance during most of the study period, as well as before the start of treatment. Body weight gain in this group was however similar to the control values and corrected body weight gain was slightly higher than the control value. For these reasons lower body weights in group 2 were considered not to be related to the treatment with the test material but due to biological variability.

GROUP B
Treatment with the test material caused a reversible reduction in body weights and body weight gain in group 4. Body weight loss of 8.1 % was noted on day 8 and a reduced body weight gain on day 15 of the treatment period. The reduction in body weight gain was statistically significant during the entire treatment period. After the completion of the treatment, body weight gain recovered and was higher than in the control group with a statistical significance during the entire recovery period. As a consequence, body weights were reduced during the treatment period with a statistical significance on day 8 of this period. Although the body weight gain recovered and increased during the treatment, body weights had not recovered to the pre-dose values by the end of the treatment (day 15). Body weights recovered after the completion of the treatment and were higher than the control values during the recovery period with a statistical significance on day 29.
In groups 2 and 3, no statistically significant differences in body weight gain or body weights were noted if compared to the control group during the treatment. During recovery, a slight but statistically significant increase in body weight gain but with no significant changes in body weights was observed on individual days in both groups. These differences were considered to be incidental.
Mean body weight gain in groups 1, 2, 3 and 4 was, respectively: 3.0, -0.6, 0.2 and -2.6 % during the treatment and 17.9, 24.0, 24.3 and 29.4 % during the recovery period.

FOOD CONSUMPTION
GROUP A
Mean food consumption from day 6 to 21 post coitum was 20.4, 18.9, 17.8 and 16.1 g/animal/ day in groups 1, 2, 3 and 4, respectively.
Treatment with the test material caused a dose dependent reduction in food consumption in groups 3 and 4. The reduction was statistically significant from day 6 to 18 post coitum in both groups. Afterwards it remained lower when compared to the control value; however the differences were not statistically significant.
In group 2, lower food consumption was recorded during the entire study with a statistical significance on days 0 - 3, 6 - 12 and 15 - 18. As the differences in food consumption were already recorded before the start of treatment and remained similar during the study, they were considered not to be related to the treatment with the test material but due to biological variability.

REPRODUCTION DATA
GROUP A
Four females in the control group, three females in group 2 and two females in group 4 were not pregnant. One female in group 2 had implantations only. All remaining females were pregnant and had foetuses at termination on day 21 post coitum.
The relevant reproduction data (post-implantation loss and number of foetuses per dam) were not affected by treatment with the test material. Mean incidence of post-implantation loss per dam was 0.5, 0.9, 0.6 and 0.7, whereas mean number of foetuses per dam at termination was 13.4, 12.1 12.3 and 13.3 in order of ascending dose levels.

GROUP B
Treatment with the test material caused a reversible reduction in food consumption in group 4. The reduction was statistically significant from day 1 to 8 of the treatment period. Afterwards food consumption recovered and was similar to the control value from day 8 to 15 of the treatment. During the recovery period, food consumption was higher than the control values during the first four weeks with a statistical significance from day 8 to 29 of this period and similar to the control values during the remaining four weeks of this period.
In groups 2 and 3, food consumption was not affected by the treatment with the test material. Mean food consumption was 15.4, 14.6, 14.6 and 13.4 g/animal/day during the treatment and 16.7, 17.6, 17.5 and 19.0 g/animal/day during the recovery period in groups 1, 2, 3 and 4, respectively.


MACROSCOPIC PATHOLOGY
GROUP A
In group 4, foci on the lungs were found in two females (nos. 67 and 68). This finding was considered to be test material-related. No further findings were noted during the necropsy in any group.

GROUP B
No findings were observed during macroscopic examination at any dose level.

HISTOPATHOLOGY
GROUP A
Histopathology examination was performed on the lungs from six selected pregnant females per group and from 2 females with macroscopically identified findings in the lungs. Treatment with the test material caused lesions with a dose dependent frequency and severity in groups 3 and 4. Phagocytic alveolar macrophage foci were noted in all six females from group 3 at minimal or slight severity and in all six females from group 4 at slight or moderate severity. Further, granulolymphocytic alveolar inflammation was recorded at minimal degree in four females from group 3 and at minimal to moderate degree in all six females from group 4. The granulolymphocytic alveolar inflammation at minimal degree was recorded also for one female in the control group.
The macroscopically identified foci in two females in group 4 were correlated to alveolar haemorrhage or phagocytic alveolar macrophage foci.
No test material related lesions were found in the lungs of females in group 2.

GROUP B
No test material-related findings were noted during the histopathological examination of female lungs after four or eight weeks of the recovery period. All findings were considered to be within the spontaneous background occurrence of the finding in untreated rats.
Dose descriptor:
NOEL
Effect level:
5 mg/m³ air
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: In pregnant females
Dose descriptor:
NOAEL
Effect level:
5 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: In pregnant females
Dose descriptor:
NOEL
Effect level:
15 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: In non-pregnant females
Dose descriptor:
NOAEL
Effect level:
25 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects
Remarks on result:
other: In non-pregnant females
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
EXTERNAL ABNORMALITIES AND VARIATIONS
No test material-related findings were observed during external examination of the foetuses in any group.
The only finding recorded during external examination of foetuses at termination was haematoma found in five foetuses from litter no. 47 and one foetus from litter no. 48 in group 3. Due to the isolated occurrence and lack of dose dependency, this finding was considered to be incidental.

SEX RATIOS
No effects on the sex ratio of the foetuses were noted in any group.
The proportion of male foetuses was 49.2, 54.6, 47.4 and 49.4 % in order of ascending dose levels.

BODY WEIGHTS
Mean foetal body weights calculated on a litter basis were: 4.8, 4.9, 5.0 and 4.5 g whereas calculated on an individual basis, they were 4.7, 4.8, 4.8 and 4.4 g, both cited in order of ascending dose levels
Treatment with the test material caused a reduction in foetal body weights in group 4. This reduction was statistically significant if calculated on an individual basis and not statistically significant if calculated on a litter basis.
Foetal body weight in groups 2 and 3 were not affected by the treatment with the test material.

VISCERAL ABNORMALITIES AND VARIATIONS
During visceral examination of the foetuses, findings were noted in: 54 % OF examined foetuses (in 100 % of litters) in group 1; 60 % of examined foetuses (in 100 % of litters) in group 2; 49 % of examined foetuses (in 91 % of litters) in group 3; and 58 % of examined foetuses (in 95 % of litters) in group 4.
Treatment with the test material caused an increase in the incidence of large or slightly large foetal thyroid in group 4. This finding was recorded in 12 % of foetuses (in 65 % of litters); 4 % of foetuses (from 20 % of litters) had large thyroid and 8 % of foetuses (from 50 % of litters) had slightly large thyroid. In the control group slightly large thyroid was found in 2 % (in 11 % litters).
The incidence of the large thyroid in group 4 was approximately twice as high as in the historical control group with the highest incidence where 5 % of foetuses (in 29 % of litters) were found with this finding; 2 % of the foetuses (from 10 % of litters) had large thyroid and 3 % of the foetuses (from 24 % of litters) had slightly large thyroid.
The frequency of the remaining findings was within the normal biological background.

MICROSCOPIC EXAMINATION OF THYROIDS
During histopathological examination of the foetal thyroids diffuse follicular hypertrophy and/or hyperplasia at minimal to moderate degree was noted in all five male foetuses from group 4 and at slight or moderate degree in four females from group 4. A minimal degree of this finding was recorded in one group 1 female foetus. Mitotic figures in follicular epithelial cells were increased in both male and female group 4 foetuses.

SKELETAL EXAMINATION
The evaluation of foetuses for skeletal development showed treatment-related changes in group 4 including incomplete or lack of ossification of cervical arch, metatarsals, caudal vertebrae and hind paw phalanges. In addition, the percentage of foetuses with one or more wavy ribs was higher in this group if compared to the control group.
In groups 2 and 3, no findings were recorded which were considered to be test material related.
Dose descriptor:
NOAEL
Effect level:
15 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
other: Post-natal effects and increased thyroid size.
Dose descriptor:
NOEL
Effect level:
15 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Summary of Performance of Mated Females

Group

1

2

3

4

Dose (μg/L air)

0

5

15

25

Number of mated females

22

22

22

22

Not pregnant

4

3

0

2

Resorptions only

0

1

0

0

No. females with live foetuses at termination*

18

18

22

20

*Only dams with at least one live foetus at Caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

 

Inhalation Technical Data

The achieved group aerosol concentrations were 4.7, 15.1 and 26.0 µg/L.

- Nominal Aerosol Concentration

Group nominal aerosol concentrations are given below (mean ± SD, n = number of exposures, CV = coefficient of variation):

Group 2: 11.2 ± 1.6 µg/L (n = 45, CV = 14.4 %)

Group 3: 34.6 ± 5.1 µg/L (n = 45, CV = 14.7 %)

Group 4: 58.4 ± 11.4 µg/L (n = 45, CV = 19.6 %)

 

-Gravimetric Aerosol Concentrations

The gravimetric aerosol concentrations were stable in groups 3 and 4 during the whole treatment period, based on the small coefficients of variance. There were variations for the aerosol concentrations for group 2 during the treatment period. These fluctuations were considered to be mainly related to differences in the adsorption of water due to the hygroscopic properties of the test material as well as to the loss of moisture from the filter due to the use of dried air for aerosol generation on different days and, therefore, not reflecting real differences in the actual aerosol concentrations. The extent of the water adsorption can be seen from the difference to the corrected gravimetric values. The results are presented in the following table (mean ± SD, n = number of exposures, CV = coefficient of variation):

 

Table 2: Gravimetric Aerosol Concentrations

Group

Group Gravimetric Aerosol Concentration [μg/L]

Corrected Gravimetric Aerosol Concentration [μg/L]

1

7.8 ± 1.8 (n = 24, CV = 23.5 %)

4.7 ± 1.1

2

25.2 ± 3.0 (n = 24, CV = 12.0 %)

15.1 ± 1.8

3

43.4 ± 2.9 (n = 24, CV = 6.7 %)

26.0 ± 1.7

 

- Particle Size Determination

The values for gravimetrically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) were as stated in the following table. The MMADs were at the lower limit of the target range of 1 to 3 μm, therefore deposition of the particles can be assumed to have occurred mainly in the lower but also in the upper respiratory tract. In addition, the Geometric Standard Deviations (GSD) were within the target range of 1.5 to 3. In conclusion, the particle size distribution obtained was considered to be appropriate for this type of study.

Table 3: Gravimetric determination of particle size distribution

Group

Mean MMAD [μm] (mean GSD)

Range of MMAD [μm]

Range of GSD

Number of Determinations

Mass Percentage of Particles <3.0 μm

2

1.63 (2.40)

1.53 - 1.73

2.24 - 2.59

3

75.7

3

2.04 (2.36)

1.86 - 2.14

2.20 - 2.52

3

67.3

4

1.58 (2.26)

1.46 - 1.67

2.23 - 2.26

3

78.4

Conclusions:
Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) as well as the NOEL (No Observed Effect Level) 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.
Although foetal thyroids were increased in size at 25 µg/L air, a dose which caused adverse maternal toxicity, the causal correlation for these observations was unclear. Also foetal findings at 25 µg/L for the postnatal live young could not be conclusively established as non-treatment related. Therefore the NOEL as well as NOAEL for prenatal developmental toxicity was considered to be 15 µg/L air.
Executive summary:

The potential of the test material (manganese dichloride) to cause prenatal developmental toxicity via the inhalation route was investigated in accordance with the standardised guidelines OECD 414, EU Method B.31, US EPA OPPTS 870.3700, and Japanese Guideline 12 Nohsan No. 8147 (2-1-18) under GLP conditions.

The purpose of this study was to detect effects on the pregnant Han Wistar rat, development of the embryo and foetus consequent to exposure of the pregnant female via inhalation route (by nose-only, flow-past exposure). A recovery group of non-mated females in all dose groups and the control group were observed for reversibility, persistence or delayed occurrence of systemic toxic effects in the lung.

Four groups of 22 mated females (main study animals) and 6 non mated females (recovery animals) were treated with the test material once daily, for 6 hours per day. Mated females were treated from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) and recovery animals from day 1 to 15 of a concurrent treatment period at target dose levels of 0, 5, 15 and 25 µg/L air (Groups 1, 2, 3 and 4, respectively).

All mated females were sacrificed on day 21 post coitum and the foetuses were removed by Caesarean section. For the recovery animals, three females per group were sacrificed after four weeks and three females per group were sacrificed after eight weeks of the recovery period.

The achieved group aerosol concentrations were 4.7, 15.1 and 26.0 µg/L. The mean mass median aerodynamic diameter (MMAD) was between 1.46 and 2.14 μm for all groups. Therefore, the aerosol was considered to be respirable to rats.

- Main study animals

All females survived until the scheduled necropsy. Treatment with the test material caused breathing noises in eight females in group 3 and eighteen females in group 4. Dyspnea was observed in one female in group 3. No further test material- related findings were noted in any group.

Treatment with the test material caused a dose dependent reduction in body weights, body weight loss followed by a reduced body weight gain and a reduction in corrected body weight gain in groups 3 and 4. These effects were considered to be adverse. No test material-related effects on bodyweights or body weight gain were noted in group 2.

Treatment caused a dose dependent reduction in food consumption in groups 3 and 4. 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 the effect was considered to be adverse. No test material-related effects on food consumption were noted in group 2.

The relevant reproduction data (post-implantation loss and number of foetuses per dam) were not affected by the treatment with the test material.

Treatment with the test material caused foci on the lungs in two females in group 4. Histopathology examination performed on the lungs from six selected pregnant females per group revealed lesions in this organ with a dose dependent frequency and severity in groups 3 and 4: phagocytic alveolar macrophage foci and granulolymphocytic alveolar inflammation. The macroscopically identified foci in two females in group 4 were correlated to alveolar haemorrhage or phagocytic alveolar macrophage foci. No macroscopic or microscopic findings were recorded in group 2.

- Foetal Data

No test material-related findings were noted during the external examination of foetuses and no effects on the sex ratio of the foetuses were noted in any group.

Treatment with the test material caused a reduction in foetal body weights in group 4. This effect was considered not to be adverse. No effects on foetal body weights were noted in groups 2 and 3.

Treatment caused an increase in the incidence of large or slightly large foetal thyroid in group 4. This effect was observed in the presence of maternal toxicity. However, the relationship between the maternal effects and the increased thyroid size remained unclear. The frequency of the remaining findings was within the normal biological background.

Histopathological examination of foetal thyroids revealed that the increased size of the organ in group 4 was correlated with a diffuse follicular hypertrophy/hyperplasia and an increase in mitotic figures in follicular epithelial cells.

Treatment with the test material caused an increased frequency of incomplete ossified or lack of ossification of several bones and an increase in the number of foetuses with wavy ribs. These effects were considered to unlikely have any adverse impact on the post-natal growth and development.

In groups 2 and 3, no findings were recorded which were considered to be test material-related.

- Recovery Females

All females survived until the scheduled necropsy. Treatment with the test material caused breathing noises in females in groups 3 and 4. This finding was observed until day 1 of the recovery period but not thereafter. No further test material- related findings were noted in any group.

Treatment with the test material caused a reversible reduction in body weights and body weight gain in group 4. These effects were considered not to be adverse. Body weights and body weight gain in groups 2 and 3 were considered not to be affected by treatment.

Treatment with the test material at the high-dose level caused a reduction in food consumption with recovery being observed during the treatment. This effect was considered not to be adverse. In groups 2 and 3, food consumption was not affected by the treatment with the test material.

No findings were noted during macroscopic examination of females after four or eight weeks of the recovery period. No test material-related findings were noted during the histopathological examination of female lungs after four or after eight weeks of the recovery period.

Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) as well as the NOEL (No Observed Effect Level) 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.

Although foetal thyroids were increased in size at 25 µg/L air, a dose which caused adverse maternal toxicity, the causal correlation for these observations was unclear. Also foetal findings at 25 µg/L for the postnatal live young could not be conclusively established as non-treatment related. Therefore the NOEL as well as NOAEL for prenatal developmental toxicity was considered to be 15 µg/L air.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across study. See report attached to Section 13 for justification.
Reason / purpose:
read-across source
Dose descriptor:
NOEL
Effect level:
5 mg/m³ air
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: In pregnant females
Dose descriptor:
NOAEL
Effect level:
5 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: In pregnant females
Dose descriptor:
NOEL
Effect level:
15 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: In non-pregnant females
Dose descriptor:
NOAEL
Effect level:
25 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects
Remarks on result:
other: In non-pregnant females
Dose descriptor:
NOAEL
Effect level:
15 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
other: Post-natal effects and increased thyroid size.
Dose descriptor:
NOEL
Effect level:
15 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
15 mg/m³
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

Read across to structurally similar substance, MnCl2

NOEL (No Observed Effect Level) for dams and pups was established at 17.6 µg/L air.

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June 2013 to 04 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted 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. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose:
other: read-across target
Qualifier:
according to
Guideline:
other: OECD Guideline 426 (Developmental Neurotoxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)
Deviations:
no
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: 208 - 256 g (Day 0 post coitum)
- Housing:
Dams: During acclimatisation the animals were group housed. After mating, the animals were housed individually in Makrolon type-3 cages.
Pups: Housed by litter in Makrolon type-3 cages until weaning; after weaning a maximum of 4 animals (divided by sex) were housed in Makrolon type-3 cages. Around day 35 post partum the animals were moved in Makrolon type-4 cages.
All animals had standard softwood bedding, nesting material and wood for chewing.
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days (minimum)
Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Dams were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
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 minimise re-breathing of the test aerosol as it was more than twice the respiratory minute volume of a rat.

- Preliminary Investigations: 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 in a GLP certified laboratory, but partly carried out before the study initiation date. Therefore this part is excluded from the statement of compliance.

TEST ATMOSPHERE
dust aerosol was generated from the test material using a rotating brush aerosol generator connected to a micronising jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. Furthermore, the aerosol concentrations of the test material of the low and mid dose group were achieved by serial dilution with compressed, filtered, dry air of the higher aerosol concentration using an air vacuum device.

EXPOSURE SYSTEM MONITORING
Aerosol concentration, particle size distribution, relative humidity, temperature and oxygen concentration were measured on test aerosol samples taken at a representative exposure port.
All airflow rates (including those for concentration and particle size measurements) were determined using calibrated gas meters and pressure gauges or flow meters.

DETERMINATION OF NOMINAL AEROSOL CONCENTRATION
The test material usage was measured during each exposure in groups 3 and 4 by weighing the generator cylinders containing the test item before and after exposure to determine the quantity of test material used. The weight used 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.

GRAVIMETRIC DETERMINATION OF AEROSOL CONCENTRATION
Gravimetric determination of the aerosol concentration was performed two to three times per exposure for groups 2 to 4. Additional samples were collected for monitoring purposes.
Test aerosol samples were collected onto Millipore® durapore filters, type HVLP using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port. The filters were weighed before and at least 10 minutes after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test material present on the filter and the sample volume. A correction factor of 1.3 (as calculated in the technical trials) was applied as necessary in order to correct for the adsorption of water during sampling due to the hygroscopic properties of the test material. This factor was determined during technical trials by AAS analysis on the Mn content and was confirmed by additional AAS analysis of filters taken during exposure. For AAS analysis filter samples were sent to the responsible for dose formulation analysis, G. Heinemann.
Due to a calculation error in the technical trial data, the correction factor was wrongly calculated. Recalculation of the factor after completion of the exposure phase using Mn content determined by ASS and corresponding filter weights gave resulted in a correction factor of 1.9. Consequently, achieved aerosol concentrations were below target.

DETERMINATION OF PARTICLE SIZE DISTRIBUTION
The particle size distribution was determined gravimetrically three times for groups 2 to 4.
The cumulative particle size distribution of the test aerosol was determined using a Mercer 7 stage cascade impactor Model 02-130, In-Tox. Products Inc., Albuquerque, New Mexico, USA). The test aerosol was impacted at each stage onto stainless steel slips and the particle size distribution of the test material in the generated aerosol was measured by gravimetrically analysing the test material deposited on each stage of the cascade impactor. 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 (Microsoft Corporation, USA). The target ranges were 1 to 3 μm for the MMAD and 1.5 to 3 for the GSD.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
> Analytical Standard
- Identity: Manganese 1000 µg/mL AAS/ICP
- Batch no: 17.0760909
- Expiration date: September 2014

> Analytical procedure:
- Preparation of Calibration Solutions
A stock solution of manganese analytical standard in 1 M chloride acid (with a concentration of 2.56 μg/mL) was prepared (solution A) by dissolving 256 μL of the analytical standard in 100 mL of 1 M chloride acid. Standard solutions were prepared by successive dilution of solution A with 1 M chloride acid. The resulting concentrations ranged from 0.040 μg/mL to 1.280 μg/mL. These standard solutions as well as solution A were used to calibrate the atomic absorption spectrometer.

- Work up of Samples
An appropriate volume of 1 M chloride acid was added to each filter sample and dissolution was achieved by sonication for at least 5 minutes.

- Atomic Absorption Spectrometry with Flame Assembly
Instrument: Perkin-Elmer Model PE 2100 (software 4100) atomic absorption spectrometer
Flame: Acetylene flame/air
Slit Width: 0.2 nm
Wavelength: Calcium: 279.5 nm

- Evaluation of Results
Samples were quantified by atomic absorption spectrometry (AAS) of manganese with reference to the respective calibration curve (with zero intercept). The calibration curve (non-linear) and the concentration (in μg/mL) were calculated using the Perkin Elmer software.
The concentration of precipitated manganese (II) chloride (MnCl2) in the filter samples was calculated using the following equation:

A(filter) = (Cs.V.D.F) / 10000

where
A(filter) = Actual amount of manganese (II) chloride on filter [μg/filter]
Cs = Measured concentration of manganese in sample [μg/mL]
V = Volume solvent for dissolution [mL]
D = Dilution factor
F = Correction factor of 2.2906
Duration of treatment / exposure:
Dams were exposed to test aerosols from day 6 to day 19 post coitum and from day 1 to day 20 post partum. Pups were not treated directly but exposed indirectly via maternal blood and milk during pre- and post-natal neurological development.

Duration of treatment period (per exposure):
From day 6 - 19 post coitum: 6 hours per day
From day 1 - 2 post partum: 1 hour per day
From day 3 - 4 post partum: 2 hours per day
From day 5 - 20: 6 hours per day
No treatment during parturition (from day 20 post coitum onwards until parturition)
Frequency of treatment:
Daily during treatment period.
Duration of test:
Approximately 95 days
Dose / conc.:
5 other: µg/L air (target aerosol concentration)
Remarks:
3.5 µg/L air (achieved aerosol concentration)
Dose / conc.:
15 other: µg/L air (target aerosol concentration)
Remarks:
12.3 µg/L air (achieved aerosol concentration)
Dose / conc.:
25 other: µg/L air (target aerosol concentration)
Remarks:
17.6 µg/L air (achieved aerosol concentration)
No. of animals per sex per dose:
27 dams per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study Number D32146, using aerosol concentrations of 0.050, 0.50 and 5.0 μg/L air. No test material-related effects were recorded in dams and pups at any aerosol concentration for manganese dichloride.

MATING PROCEDURE
After acclimatisation, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchroniSed timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- The daily vaginal smear was sperm positive, or
- A copulation plug was observed.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used only for mating. These male ratswere not considered part of the test system. The fertility of these males had been proven and was continuously monitored.

GESTATION AND LACTATION
All dams were allowed to give birth and rear their pups up to day 21 post partum (weaning). Day 0/1 was designated as the day on which a dam had delivered all her pups. Nesting and nursing behaviur of the dams was observed daily. If birth did not occur, the female was sacrificed on day 25 post coitum. Dam no. 19 which lost its litter was killed and necropsied together with the other dams after weaning of their pups.
The offspring was examined as soon as possible after completion of delivery and throughout the lactation period, as described in observations of offspring. Immediately after birth the pups were weighed individually, but without tattooing, on day 0 post partum to prevent cannibalism. On day 4 post partum, the surplus pups were culled by random selection to yield as near as possible 4 males and 4 females per litter. Pups were selected on total randomisation basis without body weight or other bias. Litters of 7 or 8 pups with at least 3 pups of each sex were used for further examinations. Litters not used were killed and discarded after day 4 post partum.

EXAMINATION OF THE DAMS
The following observations were recorded:
- Viability / Mortality: Twice daily
- Clinical Signs: Daily cage-side clinical observations, once daily, during acclimatization until treatment start (day 5 post coitum). Afterwards twice daily up to day of necropsy. During treatment cage side clinical signs were taken before and after treatment, otherwise at the beginning and the end of the working day.
- Food Consumption: Recorded for the following intervals: days 0 - 6, 6 - 11, 11 - 16 and 16 - 21 post coitum, and days 1 - 4, 4 - 7 and 7 - 14 post partum
- Body Weights: Recorded daily from day 0 post coitum until day 21 post partum and on the day of necropsy
- Detailed Clinical Observations: A detailed clinical observation was performed outside of the home cage once prior to the test material administration on day 1 and on day 7, 14 and 19 post coitum and on day 4, 11 and 18 post partum. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

EXAMINATION OF THE PUPS
The following observations were recorded in all pups:
- Litter Size, Sex, Malformations:
> Sex of pups and number of newborns with gross abnormalities
> Number of live and dead or missing pups (daily until weaning)
- Clinical Signs: Abnormal findings and mortalities in the pups (daily)
- Food Consumption: Food consumption was recorded weekly after weaning.
- Body Weights: Individual pup weights and mean body weights by sex on days 0 (if possible), 1, 4, 10, 13, 17, 21, 22 post partum and weekly thereafter
- Developmental Landmarks: The date on which ears opened, lower incisors erupted, hair grew and eyes opened was recorded.

SAMPLING OF BREAST MILK
On day 7 post partum, breast milk was sampled from 6 dams per group about 15 - 30 minutes after application (dams were kept separately from the pups until the end of sampling of the milk).
The milk production was stimulated by an intraperitoneal injection of oxytocin (4 IU/kg body weight) about 5 minutes prior to milk sampling. The milk specimens were obtained by an inhouse built vacuum driven milking pump. A specimen volume of 100 μL was collected, where possible.
The milk samples were frozen at -20 ± 5 °C at Harlan Laboratories and send on dry ice to the responsible for bioanalytics and measurement of Mn levels was performed.

OBSERVATIONS OF SELECTED PUPS
> Sexual Maturation: The age and body weight, on which vaginal opening or preputial separation occurred were recorded in all pups.

> Detailed Clinical Observations: Twenty males and 20 females per group, typically the first male and first female pup from each litter were observed on days 5 and 11 post partum. For each pup following observations were tested: animal defensive during lifting, normal posture, convulsions present, milk in the stomach, abdomen distended, skin cold or discoloured, abnormal excretion, normal respiration, pain response present and righting reflex present. Negative geotaxis was done on day 11 post partum only.

> Functional Observational Battery (FOB)
Twenty males and 20 females per group, typically the first male and first female pup from each litter were observed on day 21, 35, 45 and 60 post partum. Each pup was examined outside the home cage by a technician who was ‘blind’ with respect to the treatment group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Any abnormal findings were recorded and, where appropriate, graded in severity.

> Locomotor Activity
At least 20 males and 20 females per group, typically the first male and first female pup from each litter were observed on days 13, 17, 21 and 60 post partum (±2 days around day 60 post partum). Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). 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.

> Y-Maze - Learning and Memory
Water maze tests (Y maze) were performed on days 25 and 61 post partum with at least 20 males and 20 females per group. On day 25 post partum (±1 day), typically the first male and first female pup from each litter were observed and on day 61 post partum (±1 day) typically the third male and third female pup from each litter were observed.
A "Y" shaped water maze with one escape ladder was used. The time taken by the rats to find the escape ladder was recorded for each trial. All animals were given 6 trials on day 1 (learning phase). The ability to find a ladder in a water labyrinth constituted a positive reaction. On the same day a straight "maze" (channel) was used to evaluate swimming speed.
The rats were retested 7 days later on day 32 and 68 post partum in a memory trial. Memory was demonstrated by a decrease in the mean time taken to complete the memory trial compared with the first learning trial and by a similar mean time when compared to the memory trial with the sixth learning trial.

> Startle Habituation
The motor and sensory function of at least 20 males and 20 females per group, typically the second and fourth male or female pup from each litter were measured on days 25 and 61 post partum.
Automated recording apparatus was used for the test. The mean response amplitude and time to maximum amplitude on each block of 10 trials (5 blocks of 10 trials per session on each day of testing) were calculated. Animal allocation to chambers and the result filename were documented in the raw data.

TERMINAL PROCEDURES
> DAMS
Dams were weighed and sacrificed by intraperitoneal injection with pentobarbitone after weaning of the offspring on day 21 post partum. Any dam sacrificed or found dead during the study was subjected to macroscopic examination with emphasis on the uterus and its contents. Gross lesions and/or target organs were preserved in neutral phosphate buffered 4% formaldehyde solution.
- Blood Sampling
A blood sample was taken from each dam, from which the litter was used, by heart puncture (approx. 2 mL) into tubes containing lithium heparin form measurement of Mn content.

> LITTERS
Surplus pups were sacrificed, examined macroscopically and discarded. After the behaviour tests had been performed, pups not selected for testing were sacrificed by CO2 asphyxiation or intraperitoneal injection of pentobarbitone, necropsied and any macroscopic abnormalities were recorded. Gross lesions and/or target organs were preserved in neutral phosphate buffered 4% formaldehyde solution.

- Pathology
Ten pups/sex/dose level each on days 11 and 22 post partum and 20 pups/sex/dose level on day 63 post partum were selected. All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period and all moribund animals were anesthetised by intraperitoneal injection of pentobarbitone.
On day 11 post partum and on day 63 post partum the animals (not foreseen for perfusion) were killed by intraperitoneal injection of pentobarbitone and their tissues fixed by immersion in neutral phosphate buffered 4% formaldehyde solution.
On day 22 post partum and on day 63 post partum the animals were anaesthetised and perfused in situ with 1 mL of 50 IU heparin followed by rinsing of 0.9% NaCl and neutral phosphate buffered 4% formaldehyde solution for 5 - 10 minutes for 22-day animals and 10 - 15 minutes for 63-day animals.

- Brain Weights
The brain weights (including the olfactory bulb) on day 11 and 22 and 63 post partum were recorded the next day after the perfusion or immersion infixative; in non-perfused animals on day 63 post partum fresh brain weight before fixation was taken.

- Morphometry
From 10 pups fixed on days 11, 22 and 63 post partum, macroscopic morphometry was performed on the brains before trimming (photographs from the dorsal aspect, with a millimeter scale) and the lengths of the cerebrum and cerebellum were determined. In addition, microscopic morphometry was performed as linear measurements on hematoxylin and eosin stained transverse sections of the brain.
The fixed brains from the animals day 63 post partum were also photographed from the dorsal aspect for macroscopic morphometry. These brains were trimmed, processed and examined by microscopic morphometry when false positive results of microscopic morphometry on perfusionfixed brains were suspected.
The evaluation of the brains was performed for the control and high dose animals only and was not extended to groups 2 and 3, since examinations revealed no test item-related changes.
Animals were excluded if they died before the scheduled necropsy, if the brain was damaged, or if the brain exhibited obvious unusual architecture.

- Tissue Preservation
On days 11, 22 and 63 post partum, in addition to the brain from all 10 animals/sex/dose level, all gross lesions from these individuals were taken and preserved in neutral phosphate buffered 4% formaldehyde solution.
The remaining body of the animals on days 11 and 22 post partum was stored in neutral phosphate buffered 4% formaldehyde solution.
From the perfused animals on day 63 post partum, additional tissues were taken, and/or parts of their body preserved in neutral phosphate buffered 4% formaldehyde solution, as described below.
From animals for neuropathology on day 63 post partum the following additional tissues were collected and processed from 6 animals/sex in the control group and the high dose level group, respectively: eyes with with retina and optic nerves, gasserian ganglia, cervical spinal cord including C4-C7, thoracic spinal cord, lumbar spinal cord including L4-L5, gastrocnemius muscle, cervical dorsal root ganglia*, cervical dorsal roots*, cervical ventral roots*, lumbar dorsal root ganglia**, lumbar dorsal roots**, lumbar ventral roots**, proximal sciatic nerve (below sciatic notch), proximal tibial nerve (below knee), distal tibial nerve (lower leg).
* at least 2 from C4-C7
** at least 2 from L4-L5
The tissue in the above list was dissected and further processed for these 6 animals/sex in the control and high dose level group, respectively.
From the remaining 4 animals/sex of these groups and from all 10 animals/sex/dose level of the intermediate groups, the following body parts were taken and preserved in neutral phosphate buffered 4% formaldehyde solution:
• The eyes with the optic nerves (post fixed in Davidson)
• The skull basis containing the Gasserian ganglia
• Portions of the cervical, thoracic and lumbar vertebral column containing the spinal
cord, dorsal root ganglia and spinal nerve roots at levels indicated in the above list
• Hindlimbs devoid of skin, containing the peripheral nerves and skeletal muscles

- Tissue Processing
On days 11, 22 and 63 post partum, the brains of all 10 animals/sex/dose level were trimmed as transverse sections (eight levels on day 63 post partum, on days 11 and 22 post partum). All brains from control and high dose groups were trimmed and simultaneously embedded in paraffin. Since it was essential to label the left/right side of the brain, the sections were notched on one side. The paraffin blocks of the intermediate dose groups were only sectioned and
examined further if the initial examination of the control and high dose animals revealed test material-related changes.
The tissues selected for paraffin embedding were post-fixed in neutral phosphate buffered 4% formaldehyde solution, embedded in paraffin, sectioned and stained with hematoxylin and eosin.
The tissues selected for plastic embedding were post-fixed in 5% glutaraldehyde in 0.1M sodium cacodylate buffer, trimmed for plastic embedding, impregnated for 3 hours with osmic acid, embedded in epoxy resin (such as epon) sectioned to semithin sections and stained with toluidine blue.

- Microscopic Evaluation
All sections were qualitatively examined by light microscopy. Informed evaluation was followed by coded evaluation in order to confirm the findings. The frequency and severity of microscopic changes were assessed, paying particular attention to a dose-effect relationship. A photodocumentation of the test material-related effects was provided.
Quantitative morphometric microscopic examination was evaluated as bilateral values both separately (left/right) and as mean values of both sides, the midline values were evaluated as single values.
Statistics:
The following statistical methods were used to analyse food consumption, body weight, macroscopical findings, reproduction and breeding data:
• Means and standard deviations of various data were calculated
• 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 if the variables could be dichotomised without loss of information.
Dose descriptor:
NOEL
Remarks:
(dams and pups)
Effect level:
17.6 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed
OBSERVATIONS DAMS
> MORTALITY / VIABILITY
At the high dose level, one dam (no. 87) was killed in extremis on day 15 post coitum. This dam showed breathing noises from day 11 post coitum onwards and laboured breathing (highest grading) after exposure from day 12 post coitum onwards until section. Additionally, it was in a weakened condition, with body weight loss, ruffled fur and a hunched posture. All other dams survived until the scheduled necropsy.

> CLINICAL SIGNS
At the high dose level, up to 70% of the dams showed breathing noises during the gestation period (starting on day 8 post coitum) with steadily decreasing numbers of affected dams towards the end of the period. One dam (no. 97) had also slightly ruffled fur during three days. All bar three dams showed no clinical signs during the lactation period. The mentioned three dams had slight breathing noises towards the end of the lactation period. No test material-related clinical signs were observed at the low and mid dose level during the study.

> GESTATION AND LACTATION PERIODS
- Food Consumption
Mean food consumption was dose-dependently decreased following treatment with the test material but recovered from day 11 post coitum (mid dose) and from day 16 post coitum (high dose) onwards. There were no effects on food consumption during gestation and lactation period for females treated at the low dose level. During lactation period, there were no effects on mean food consumption at any dose group.

- Body Weights:
Mean body weight gain was dose-dependently decreased following treatment with the test material in animals of the mid and high dose levels.
At the high dose level, there was a body weight loss up to 4% within the first 2 days following treatment. Afterwards mean body weight gain remained significantly reduced compared to the control group (from day 12 to 17 post coitum). As a consequence, mean body weight was significantly reduced during the gestation period (from day 8 to 17 post coitum).
At the mid dose level, mean body weight gain was reduced following treatment with the test material until day 17 post coitum; body weight gain was significantly reduced compared to the control group (from day 8 - 10 post coitum and on day 12 post coitum). Mean body weights were not significantly different form the control group.
No effects on mean body weight gain and mean body weights were observed at the low dose level during the gestation period. During lactation period, there were no effects on mean body weight gain and mean body weights at any dose level.

> REPRODUCTION AND BREEDING DATA
- Gestation and Parturition:
One female was not pregnant in the control group, in the mid and high dose group, respectively, and two females at the low dose level.
The mean duration of gestation was similar in all dose groups and not affected by treatment with the test material. The mean duration of gestation was 21.6, 21.5, 21.8 and 21.5 in order of ascending dose levels.

- Litter Size at First Litter Check:
Mean number of pups per litter was similar in all groups at first litter check (11.9, 13.4, 12.4 and 12.5 in order of ascending dose level) and not affected by treatment. Sex ratios at first litter check and on day 21 post partum were unaffected by treatment with the test material.
At the low dose level, female no. 35 did not give birth and showed only embryonic resorptions during necropsy. Additionally at this dose level, female no. 34 was observed to start to give birth on day 21 post coitum but on the next day for first litter check, no pups were observed.

- Postnatal Loss Days 0 - 4 Post Partum:
Postnatal loss was not affected by treatment with the test material. The number of pups lost at first litter check until day 4 post partum was 6, 9, 3 and 3 in order of ascending dose level. In the control group one female (no. 19) lost its whole litter (single pup on day 2 post partum).

- Breeding Loss after Day 5 - 21 Post Partum:
Breeding loss was not affected by treatment with the test material. At the mid and high dose level, only one pup was missing on day 8 and 13 post partum, respectively.

> BREAST MILK LEVEL DETERMINATION
Breast mild was collected on day 7 post partum. The amount of test material in the sample was 31, 53, 164 and 271 μg/L, in order of ascending dose level.

> BLOOD SAMPLING
A blood sample was taken from each dam by heart puncture on the day of necropsy. Due to logistical reasons necropsy was performed between day 21 and 27 post partum (either directly after cessation of treatment or up to 6 days later). The manganese concentration in whole blood was below the lowest calibration solution in the control group and in the low dose group (except two animals), around 1 μg/L in the mid dose group and between 1-2 μg/L in the high dose group.

> MACROSCOPIC FINDINGS
There were no test material-related macroscopic findings in any group.

OBSERVATIONS - PUPS
> EXTERNAL EXAMINATIONS AT FIRST LITTER CHECK
No test material-related effects were noted in any of the treatment groups. At the mid dose level, sinlge pups from separate litters, were found to have no milk in the stomach at first litter check; both were found to be dead at this time point.
At the high dose level, single pups, from separate litters, were found dead and autolytic at first litter check.

> MORTALITY / VIABILITY
No test material-related mortality was observed at any dose level. At the high dose level, one female pup was missing on day 8 post partum. Another one had to be killed in extremis on day 26 post partum since it showed malpositioned upper incisors, an abnormal cloudy right eye and ruffle fur. These isolated occurrences were considered to be incidental.

> CLINICAL SIGNS
No test material-related effects were noted in any treatment group.

> FOOD CONSUMPTION
- Post-Weaning (Day 21 - 69 Post Partum):
At the high dose level, mean food consumption was significantly decreased in male pups (4% compared to the control group). In female pups the same tendency was observed, although no statistically significant differences were measured. At the low and mid dose level, mean food consumption was similar to the control group.

> BODY WEIGHTS
- Pre-Weaning (Day 0 - 21 Post Partum):
Mean pup weight and its development were unaffected during the lactation period by treatment with the test material of their dams.
Calculated for combined data of male and female pups mean body weight gain from day 1 to 21 post partum was 634%, 645%, 638% and 651% in order of ascending dose levels.

- Post-Weaning (Day 22 - 69 Post Partum):
After weaning mean pup weight gain was reduced at all dose levels compared to the control group until day 69 post partum (+595%, +567%, +570% and +558% in male pups and +354%, +349% +331% and +335% in female pups, in order of ascending dose levels, respectively). The differences were not statistically different in males at the mid dose level or in females at the low dose level. Furthermore, no clear dose dependency was observed between the low and mid dose group. The lower mean body weight gain resulted in reduced mean body weights from day 57 post partum onwards at the high dose level (-5% and -3% in male and female pups, respectively, on day 69 post partum).
The reduction in mean body weight gain in pups was considered to be a result of the treatment of the dams at all dose levels. However, since the reduction in mean body weight gain did not have an effect on absolute mean body weights at the low and mid dose group, this reduction in mean body weight gain was considered to be not adverse in these groups.

> DEVELOPMENT INDICES
There were no test material-related differences when the developmental indices (pinna unfolding, incisor eruption, onset of coat development and opening of eyes) occurred amongst the control and the dose groups.

> SEXUAL MATURATION
Mean age and mean body weight at balanopreputial separation in males and vaginal opening in females were similar in all groups.

> BEHAVIOURAL TESTS
- Detailed Clinical Observations:
On days 5 and 11 post partum, no complete FOB was possible due to the immature condition of the pups at this age. Therefore for each pup only a detailed clinical observation was performed. During the detailed clinical observation on days 5 and 11 post partum, the righting reflex was not present at one pup in the mid dose on day 5 post partum. This isolated finding was considered to be incidental.

- Functional Observational Battery (FOB):
In male and female pups observations, conducted as part of the functional observational battery on days 22, 35, 45 and 60 post partum, did not indicate any test material-related effects in any group.
During FOB on days 21, 35, 45 and 60 post partum, between 0 - 2 rearings were counted in the open field within 1 minute in all groups for both sexes. On day 21 and 35 post partum more animals were affected as on day 45 and 60 post partum. This observation correlates with the fact that younger animals in general are less active as the adult ones. Since no dose dependency was observed, this finding was considered not to be test material-related. Although incidentally an increase in the number of rearings were counted in single animals.
Mean values of grip strength (fore- and hind paws), body temperature and landing foot splay also gave no indication of test material-related effects. Isolated differences, which were found to be statistically significant, showed no dose dependency. At the high dose level, significant differences were observed in two measurements (male pups on day 60 post partum in landing foot splay and in female pups on day 21 post partum in grip strength of the fore paws), which were also considered to be incidental, since the difference occurred only on single days but did not show a development. Additionally, mean temperature was higher on day 60 post partum in female pups at all dose levels (39.1 °C, respectively, compared to 38.8 °C in the control group). Since no dose dependency was observed and the levels are in the range of the normal values (up to 39.1 °C), this finding was considered to be incidental.

- Locomotor Activity:
The aim of this investigation was to observe the ontogeny of locomotor behaviour and of its habituation. Therefore, the measurements were made in the same animals on several time points during the pre-weaning (day 13, 17 and 21 post partum) and post-weaning period (day 60 post partum).
In general, locomotor activity developed most rapidly between day 13 and 17 post partum, since the beam count was approximately twice as high on day 17 post partum compared to day 13 post partum. A similar development occurred between day 21 and 60 post partum in all groups.
Locomotor activity and its development from day 13 until 60 post partum were not affected by treatment with the test material at any dose level.

- Learning and Memory - Y-Maze Performance:
Learning was demonstrated on days 25 and 61 post partum in both sexes in all groups by a marked decrease in the mean time taken to complete the second trial compared to the first trial and all subsequent trials up to a plateau, which was reached after the third trial.
Memory was demonstrated on day 32 and 68 post partum in both sexes in all groups by a marked decrease in the mean time taken to complete the memory trial compared with the first learning trial and by a similar mean time when comparing the memory trial with the sixth learning trial.
Several animals did not find the ladder during the given time of 30 seconds. The number of animals, which did not find the ladder at all in different trials, was distributed equally over the groups.

- Startle Habituation:
The mean amplitude of startle response on each block of 10 trials (5 blocks with 10 trials per session on each day of testing) was calculated. On days 25 and 61 post partum the same animals were used.
The mean amplitude of startle response and habituation on days 25 and 61 post partum were not affected by treatment with the test material.
On day 25 post partum in male and female pups, habituation to startle showed only a weak learning curve, indicated by a similar mean response amplitude in all calculated block of trials. This behaviour is typical of animals of this age.
On day 61 post partum, a clear habituation to startle was observed in all animals. Male and female pups exhibited a decrease in the mean response amplitude over the 5 blocks of trials.

> NEUROPATHOLOGY (PUPS)
- Brain Weights:
There were no effects on brain weight in any group on ays 11, 22 and 63 post partum (regardless of the fixation method).

- Macroscopic Findings:
No macroscopical findings were observed in any pup at any dose group.

- Macroscopic Brain Morphometry:
No findings were noted on the length of the cerebellum and forebrain of all examined groups on day 11, 22 and 63 post partum.
On day 63 post partum, the length of the cerebellum from the dorsal aspect was slightly shorter compared to the control group in male pups of the low dose level and female pups of the high dose level. The forebrain was significantly longer in female pups of the mid dose group in female pups.
Since the differences showed no dose-dependency and consistency, they were considered to be incidental.

- Microscopic Findings:
* Day 11 Sacrifice
Macroscopic Pathology: There were no macroscopic findings.
Microscopic Pathology: There were no morphological findings in the tissues examined that could be attributed to treatment with the test material. Many of the sections available for morphometric analysis were deemed non-assessable because of processing or sectioning artefacts, particularly for the measurement of cerebellar height. A slightly lower thickness of the corpus callosum was considered more likely to have resulted from minor differences in sectioning than an effect of the test material.

* Day 22 Sacrifice
Macroscopic Pathology: There were no macroscopic findings.
Microscopic Pathology: There were no morphological findings in the tissues examined that could be attributed to treatment with the test material. Many of the sections available for morphometric analysis were deemed non-assessable because of processing or sectioning artefacts, particularly for the measurement of cerebellar height. No notable differences were present between controls and treated animals.

* Day 63 Sacrifice
Macroscopic Pathology: There were no macroscopic findings.
Microscopic Pathology: There were no morphological findings in the tissues examined that could be attributed to treatment with the test material. Many of the sections available for morphometric analysis were deemed non-assessable because of processing or sectioning artefacts, particularly for the measurement of the hippocampal gyrus. No notable differences were present between controls and treated animals.
All of the histopathological findings were considered to have arisen spontaneously or post mortem.

Table 1: Determination of Mn Content by ASS

Date of sampling

Group

Filter No.

Mn content (µg MnCl2)

Recovery (%)

Correction factor

Gravimetric

ASS

03.07.14

2

1

2293

1012

44

2.3

2

2014

973.2

48

2.1

3

1957

935.9

48

2.1

3

1

1705

660.4

39

2.6

2

2165

1050

48

2.1

3

2390

1122

47

2.1

4

1

2922

1691

58

1.7

2

4097

2457

60

1.7

3

3759

2309

61

1.6

Mean

53

1.9

15.07.13

2

1

1794

1047

58

1.7

2

1718

1022

59

1.7

3

1

1845

1024

56

1.8

2

1714

885.3

52

1.9

3

1987

1167

59

1.7

4

1

2009

1129

56

1.8

2

2716

1633

60

1.7

3

3876

2166

56

1.8

Mean

57

1.9

Table 2: Particle Size Distribution

Group

Mean MMAD

(µm)

Range of MMAD

(µm)

Range of

GSD

No of

determinations

Mass % of particles

<3.0 µm

2

1.34 (2.26)

1.28 - 1.43

2.11 - 2.41

3

83.8 %

3

1.99 (2.34)

1.75 - 2.25

2.23 - 2.45

3

68.6%

4

1.59 (2.22)

1.48 - 1.78

1.96 - 2.39

3

78.6%

Exposure conditions:

Temperature, relative humidity and oxygen concentration during exposure were considered to be satisfactory for this type of study. As the test material was a powder the relative humidity values are quite low. Nevertheless these values are acceptable for this study type. The exposure conditions were as follows:

Temperature: 21.3 - 22.9 °C

Relative humidity: 1.4 - 5.6 %

Oxygen concentration: 20.3 - 20.6 °C

Table 3: Body Weight (g) of Dams During Gestation Period

Day

Group 1

Group 2

Group 3

Group 4

0 µg/L air

5 µg/L air

15 µg/L air

25 µg/L air

0

233

228-

231-

231-

1

240

235-

238-

236-

2

245

241-

243-

243-

3

248

244-

245-

245-

4

249

245-

247-

247-

5

250

247-

249-

248-

6

251

248-

250-

249-

7

252

248-

250-

246-

8

253

250-

249-

239**

9

254

252-

250-

238**

10

258

257-

253-

242**

11

261

261-

257-

246**

12

266

265-

261-

249**

13

270

269-

266-

255**

14

274

274-

270-

259**

15

280

280-

276-

265**

16

288

289-

285-

276**

17

298

301-

296-

287*

18

310

315-

309-

300-

19

322

328-

323-

311-

20

334

341-

335-

324-

21

340

348-

344-

333-

* / ** / - : Dunnett-test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)

Table 4: Body Weight Gain (%) of Dams during Gestation Period

Day

Group 1

Group 2

Group 3

Group 4

0 µg/L air

5 µg/L air

15 µg/L air

25 µg/L air

0

-7

-8-

-7-

-7-

1

-4

-5-

-5-

-5-

2

-2

-3-

-3-

-2-

3

-1

-1-

-2-

-1-

4

-1

-1-

-1-

-1-

5

0

0-

0-

0-

6

0

0

0

0

7

0

0-

0-

-1**

8

1

1-

0*

-4**

9

1

2-

0*

-4**

10

3

4-

1**

-3**

11

4

5-

3-

-1**

12

6

7-

5*

0**

13

8

9-

6-

2**

14

9

11-

8-

4**

15

12

13-

11-

7**

16

15

17-

14-

11**

17

19

21-

19-

16**

18

23

27*

24-

21-

19

29

32-

29-

25-

20

33

38*

34-

30-

21

36

40*

38-

34-

* / ** / - : Dunnett-test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)

Conclusions:
Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) for dams was established at 17.6 μg/L air whereas the NOAEL as well as NOEL (No Observed Effect Level) for pups was established at the dose level of 17.6 μg/L.
Executive summary:

The purpose of this study was to demonstrate potential functional and morphological effects on the developing nervous system of the rat offspring that may arise from exposure in utero and during early life. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 426 and EPA OPPTS 870.6300.

During the study the test material was administered by nose only inhalation at actual aerosol concentrations of 3.5, 12.3 and 17.6 μg/L air. Target aerosol concentrations of 5, 15 and 25 μg/L air were selected based on the results of a two generation study and the dose range-finding study. Target aerosol concentrations were monitored by gravimentric analysis during exposure and a factor, to convert gravimetric data to manganese content as determined by ASS (atom absorption spectroscopy), was determined during technical trials. Due to a calculation error in the data obtained during the technical trials, the factor was too low and consequently the achieved aerosol concentrations were below target. Measurement of manganese dichloride in the milk showed clearly that the pups were exposed to the test material via milk during lactation.

One dam treated with the test material at the dose level of 17.6 μg/L air had breathing noises and severely labored breathing. A weakened condition with body weight loss, ruffled fur and a hunched posture were noted for this female on several days after the treatment start and therefore it was killed in extremis on day 15 post coitum. The effects leading to pre-termination were observed only in one animal and therefore they were considered unrelated to the treatment. At the dose level of 17.6 μg/L air, up to 70 % of the dams showed breathing noises during the gestation period (starting on day 8 post coitum the earliest) with steadily decreasing number of affected dams towards the end of the period. During the lactation period no more breathing problems occurred, except for three females towards the end of this period. Dams at dosages of 12.3 and 17.6 μg/L air had reduced mean food consumption immediately after treatment started. At the mid dose level this effect lasted for one week; within this week mean body weight gain was slightly but statistically significantly reduced. Both parameters recovered afterwards and no effects on absolute body weight were observed. At the high dose level, reduction of food consumption lasted for two weeks and was accompanied by a body weight loss of 3 % during the first week of the treatment. At start of the lactation period, food consumption, body weight and body weight gain recovered at this dose level. The transient effects at 12.3 μg/L and 17.6 μg/L air were considered not to be adverse. No test material-related effects were noted at 3.5 μg/L air.

Treatment of the dams and their effects, did not affect the viability and survival of the offspring. Furthermore, pups food consumption, body weights, body weight gain, developmental indices, sexual maturation or morphometric measurements of the brain did not indicate any test material related effect. The daily exposure of the test material, in utero and in early life produced no behavioural abnormalities or neuropathological findings. All of the histopathological findings encountered were considered to have arisen spontaneously or post mortem.

Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) for dams was established at 17.6 μg/L air whereas the NOAEL as well as NOEL (No Observed Effect Level) for pups was established at the dose level of 17.6 μg/L air.

Endpoint:
toxicity to reproduction: other studies
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose:
read-across source
Dose descriptor:
NOEL
Remarks:
(dams and pups)
Effect level:
17.6 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed
Additional information

Read across to structurally similar substance, MnCl2

The purpose of this study was to demonstrate potential functional and morphological effects on the developing nervous system of the rat offspring that may arise from exposure in utero and during early life. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 426 and EPA OPPTS 870.6300.

During the study the test material was administered by nose only inhalation at actual aerosol concentrations of 3.5, 12.3 and 17.6 μg/L air. Target aerosol concentrations of 5, 15 and 25 μg/L air were selected based on the results of a two generation study and the dose range-finding study. Target aerosol concentrations were monitored by gravimentric analysis during exposure and a factor, to convert gravimetric data to manganese content as determined by ASS (atom absorption spectroscopy), was determined during technical trials. Due to a calculation error in the data obtained during the technical trials, the factor was too low and consequently the achieved aerosol concentrations were below target. Measurement of manganese dichloride in the milk showed clearly that the pups were exposed to the test material via milk during lactation.

One dam treated with the test material at the dose level of 17.6 μg/L air had breathing noises and severely labored breathing. A weakened condition with body weight loss, ruffled fur and a hunched posture were noted for this female on several days after the treatment start and therefore it was killed in extremis on day 15 post coitum. The effects leading to pre-termination were observed only in one animal and therefore they were considered unrelated to the treatment. At the dose level of 17.6 μg/L air, up to 70 % of the dams showed breathing noises during the gestation period (starting on day 8 post coitum the earliest) with steadily decreasing number of affected dams towards the end of the period. During the lactation period no more breathing problems occurred, except for three females towards the end of this period. Dams at dosages of 12.3 and 17.6 μg/L air had reduced mean food consumption immediately after treatment started. At the mid dose level this effect lasted for one week; within this week mean body weight gain was slightly but statistically significantly reduced. Both parameters recovered afterwards and no effects on absolute body weight were observed. At the high dose level, reduction of food consumption lasted for two weeks and was accompanied by a body weight loss of 3 % during the first week of the treatment. At start of the lactation period, food consumption, body weight and body weight gain recovered at this dose level. The transient effects at 12.3 μg/L and 17.6 μg/L air were considered not to be adverse. No test material-related effects were noted at 3.5 μg/L air.

Treatment of the dams and their effects, did not affect the viability and survival of the offspring. Furthermore, pups food consumption, body weights, body weight gain, developmental indices, sexual maturation or morphometric measurements of the brain did not indicate any test material related effect. The daily exposure of the test material, in utero and in early life produced no behavioural abnormalities or neuropathological findings. All of the histopathological findings encountered were considered to have arisen spontaneously or post mortem.

Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) for dams was established at 17.6 μg/L air whereas the NOAEL as well as NOEL (No Observed Effect Level) for pups was established at the dose level of 17.6 μg/L air.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Regulation 1272/2008, the substance does not require classification with respect to reproductive or developmental toxicity as the criteria are not met.