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Toxicological information

Toxicity to reproduction: other studies

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Administrative data

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.
Cross-reference
Reason / purpose:
other: read-across target
Reference
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): manganese dichloride
- Physical state: solid
- Storage condition of test material: Stored in cool place (room temperature (15 - 25 °C); Kept in tightly closed container. Moisture sensitive.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Results and discussion

Effect levels

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

Observed effects

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.

Any other information on results incl. tables

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. (-)

Applicant's summary and conclusion

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.