Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

Manganese carbonate

EC number: 209-942-9 | CAS number: 598-62-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Grieve (2017)

Under the conditions of the study the No Observed Effect Level (NOEL) of the read-across substance, manganese dichloride, for reproductive toxicity, was determined to be 20 µg/L via the inhalation route.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: two-generation reproductive toxicity
Remarks:: based on test type (migrated information)
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: 2 July 2012 to 4 March 2013
Reliability:: 2 (reliable with restrictions)
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 a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.
Justification for type of information:: The data on the read-across substance is considered representative.
Reason / purpose for cross-reference:: other: Target record
Qualifier:: according to guideline
Guideline:: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Age at study initiation: (F0) 6 - 8 weeks
- Weight at study initiation: (F0) Males: 155 - 298 g; Females: 130 - 194 g
- Housing: Animals were initially housed 2 per cage by sex in polycarbonate cages measuring approximately 61 x 43.5 x 24 cm with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a stainless steel grid insert measuring approximately 48 x 37.5 x 25 cm. After mating, the males were rehoused with their original cage-mates in solid bottomed cages. Mated females were transferred to individual solid bottomed cages (approximately 58.6 x 42.5 x 21 cm). White paper tissues were supplied as nesting material from Day 20 of gestation. Females with litters were retained in this cage type until termination after weaning. F1 animals retained after weaning were housed 2 per cage in cages measuring approximately 61 x 43.5 x 24 cm, as described above. The F1 animals then followed the same caging regime as described for the F0 animals.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: F0 animals were acclimatised for 13 days before the commencement of dosing. For at least 7 days prior to commencement of dosing the animals were conditioned to the restraint procedures used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing period of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 26°C
- Humidity (%): 33 - 69%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Route of administration:: inhalation: aerosol
Type of inhalation exposure (if applicable):: nose only
Vehicle:: air
Details on exposure:: GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test aerosols were generated using a Wright Dust Feed generator device. Exposure of the animals to the test material, or vehicle, was achieved utilising a modular nose only stainless steel flow past inhalation chamber.

- Dose formulation Preparation and analysis
Test material formulation was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh size of 100 μm prior to use, except on one occasion where a sieve mesh of 180 μm was used.

- Preliminary Aerosol Characterisation Investigations
Characterisation of the aerosol generating/exposure system was undertaken prior to commencement of the animal exposures to demonstrate satisfactory performance. Preliminary aerosol characterisation investigations demonstrated that aerosol concentrations were stable spatially within the exposure system and over time and that the particle size distribution investigations showed that test formulation particles for Groups 2 to 4 were respirable for the rat.

- Aerosol Generation
Test item aerosols were generated using a Wright Dust Feed generator device (Wright Dust Feed Mark II, BGI Industries, USA). Prior to the commencement of aerosol generation, a reservoir canister was packed with the test material powder formulation. The powdercake was slowly advanced into the scraper blade at an appropriate speed and scraped powder carried in a pressurised air stream.
The Wright Dust Feed generator device was operated at an appropriate target scraper speed, and air flow rate identified during the preliminary aerosol characterisation investigations. The generated test aerosols were then delivered to the flow past exposure chamber via a connecting tube manifold and mixed with dilution air to achieve the target aerosol concentration. A vacuum pump system was used to continuously exhaust test aerosols from the exposure chamber. Each aerosol generation system was operated to sustain a dynamic airflow sufficient to ensure an evenly distributed exposure aerosol.

- Inhalation Exposure (see Figure 1)
Exposure to the test aerosols was performed using appropriately sized modular nose only stainless steel flow past exposure chamber. Separate inhalation exposure systems were used for the delivery of test aerosol to each treatment group. Each inhalation exposure system was located in an extract booth (to prevent cross-group contamination). This exposure technique allowed a continuous supply of test aerosol to be delivered to each animal; the biased flow created using the flow-past chamber design ensured that there was no re-breathing of the test atmosphere.
For all inhalation exposures, the rats were restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ noses protruded through the anterior end of the restraint tubes which were connected to the exposure chamber by way of a push fit through rubber ‘o’ rings in the chamber wall. This exposure technique was used to minimise concurrent exposure by the oral and dermal routes. The exposure system was operated at an appropriate target total airflow. All flow rates (delivered and extracted) were monitored visually using calibrated flow meters. Exposure chamber flow rates, temperature and relative humidity were monitored and recorded at appropriate intervals during each daily exposure period.

TEST ATMOSPHERE
The aerosol concentration of test material formulation (Groups 2 to 4) or air (Group 1) in the animals’ breathing zone was measured gravimetrically for all groups at regular intervals throughout each daily exposure period.
The test aerosols were sampled using glass-fibre filters (47 mm Whatman GF/B) contained in a stainless steel filter holder in-line with a sampling system comprising a vacuum pump, flow meter and gas meter. Filter samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate to ensure that there was no overloading of the filter which would cause a reduction in sampling flow rate. The filters were weighed before and after sampling and the aerosol concentration calculated using the weight of formulation collected and the volume of air sampled.
In addition to the aerosol chamber concentration assessment, blank filter samples were taken to assess background levels of test material and retained for analysis.
All retained filters from Groups 1 to 4 were placed in amber glass jars and stored in a refrigerator set to maintain 4°C prior to analysis for the determination of the aerosol concentration of test material.
A real time aerosol monitor (Casella Microdust, Casella Measurements, UK) was used to assist in monitoring/ assessing the target concentrations at the start of generation each day and provided a continuous overview of any fluctuations in aerosol concentration.

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.
Details on mating procedure:: A few days prior to the initiation of mating, the males were separated into individual grid bottomed cages. Pairings were on a 1 male to 1 female basis. Animals were paired in numerical order within the groups. Each female was transferred to the cage of its appropriate co-group male near the end of the work day, where it remained until mating had occurred or 14 days had elapsed. Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrous observed in each lavage recorded. The presence of sperm in such a lavage and/or a copulatory plug in situ was designated as Day 0 of gestation. If the number of males in a group was reduced by mortality, mating was on a 1 male to 2 female basis.
The time taken for each female to show a positive mating sign was evaluated.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at Charles River, Edinburgh under Study No. 428133 (Method No. 2813). Full details of the analytical methodology are contained within that report.
Duration of treatment / exposure:: F0 animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation. For F0 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation. For F1 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
Frequency of treatment:: Daily (ca 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of lactation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca 1 hour per day
From Day 3-4 of lactation: ca 2 hours per day
From Days 5-20 of lactation until prior to termination (ca Day 21 of lactation): ca 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.
Details on study schedule:: - Selection and Weaning of F1 Animals
From each group, at least 24 males and 24 females were selected for post-weaning assessments. The selected pup(s) were the median’th weight pup(s) of that sex in the litter on Day 21 of lactation. These pups were removed from their mother on Day 21 of lactation, individually identified and housed in a new cage. Pups that were not selected for post-weaning assessments remained with their mother until sacrifice.
Remarks:: Doses / Concentrations:
0, 5, 10, 20 µg/L
Basis:
nominal conc.
Remarks:: Doses / Concentrations:
0, 6, 15, 25 µg/L
Basis:
analytical conc.
(F0 generation)
Remarks:: Doses / Concentrations:
0, 4, 10, 17 µg/L
Basis:
analytical conc.
(F1 generation)
No. of animals per sex per dose:: - F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: The dose levels were selected for use based on results from a preliminary reproduction study in rats (Charles River Study 495849). In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values (TLVs) proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.
Positive control:: na
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: Yes
- All animals were checked for early each morning and as late as possible each day for viability. Furthermore, all animals were examined for reaction to treatment daily during the course of dosing on the study. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Weights of F0 animals were recorded one week prior to the first day dosing, then weekly thereafter until the start of the mating period. Males continued to be weighed weekly until termination; but for females, weighing resumed on Day 0 of gestation (the day of detection of
a positive mating sign), and then on Days 7, 14 and 20 of gestation and Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
Post-weaning F1 animals were weighed weekly, starting on a suitable day within one week of weaning of the majority of the litters and continued until termination for males and until mating commenced for females. Mated F1 females were weighed on Days 0, 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation. Females that did not show a positive mating sign were weighed weekly until parturition or termination. Females who had a positive mating sign but failed to litter reverted to the weekly weighing regimen following their theoretical Day 24 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was quantitatively measured for both sexes weekly, starting one week before treatment commenced (F0 animals) or from a suitable day within one week of weaning of the majority of animals (F1 animals) until placement of males in individual cages prior to mating. Weekly measurements continued after the 14 day mating period. For females, following a clear indication of mating, food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: Yes
- Monitoring of water consumption was limited to a visual inspection of the water bottles on a regular basis throughout the study.

OTHERS:
- Observation of Females with Litters during Lactation
The females were allowed to litter normally. If any animal suffered from a difficult or prolonged parturition, this was recorded. The day of birth of the litter (day on which the first pups are born) was designated Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.

- Seuxal Maturation
Commencing at 28 days of age, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the
body weight on that day. Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.
Oestrous cyclicity (parental animals):: Over a 2 week period prior to the initiation of mating, vaginal lavages were taken early each morning and the stages of oestrous observed were recorded.
Sperm parameters (parental animals):: The tip of the cauda epididymis was placed in Medium 199 containing 0.2% BSA and HEPES. The sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was examined using a Hamilton Thorne sperm motility analyser; sufficient replicates to provide 200 motile sperm were assessed (except where it was obvious that motility was compromised for that animal).
The remaining portion of the cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From a sample of the sperm suspension described above, a sperm smear was prepared and stained with eosin. From the Control and High dose animals, two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce.
One testis wase decapsulated and homogenized. The homogenate may have been sonicated to remove tissue debris etc, as required. The number of homogenisation resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.
Litter observations:: The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined from Day 1 onwards for the presence of milk in the stomach and for any externally visible abnormalities daily. The pups were weighed en masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day 21 all pups were weighed individually.
Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Prior to Day 14 of lactation, any externally abnormal decedent pup was preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.
Postmortem examinations (parental animals):: SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

UNSCHEDULED DEATHS
These animals, including those killed or found dead, had a terminal body weight recorded and were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. The tissues list for animals at scheduled necropsy along with representative samples of abnormal tissues, together with any other tissues considered appropriate, were fixed in neutral 10% formalin. The reproductive tracts of all females were examined for signs of implantation (if they had been paired for mating prior to necropsy), the number of any implantation sites being recorded.

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of external surfaces and orifices; cranial; thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations consisted of an external and internal examination and recording of observations for all animals.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.

OVARIAN AND UTERINE EXAMINATIONS
The reproductive tract was dissected from the abdominal cavity. The uterus was opened and the contents examined. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded.

HISTOPATHOLOGY
Histological examination was conducted on all adults in the Control and High dose groups of the F0 and F1 generation and a selection of the premature decedents. After a review of the data, histological examination of the respiratory tract tissues of the Control and High dose animals, it was considered appropriate to conduct histopathology on the respiratory tract of all adult animals of the F0 and F1 generation.
The following tissues were processed for microscopic evaluation: adrenal glands, larynx, left testis, left epididymis, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, prostate, pituitary gland, seminal vesicles and coagulating glands, trachea (anterior and posterior), uterus (with oviducts and cervix), vagina.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
Postmortem examinations (offspring):: SACRIFICE / GROSS NECROPSY
Pups that were not selected for post-weaning assessments were killed at the same time as their mother.
Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

- Offspring found dead or killed (prematurely) before Day 14 of lactation
Where practicable, these animals were sexed, then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal decedents were discarded.

- Offspring (pre-weaning) found dead or killed (prematurely) on or after Day 14 of lactation
These animals were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin. These carcasses were then discarded.

- F1 and F2 Weanlings at scheduled termination
From each litter, 3 male and 3 female pups (where they were available – if a litter only had females or males, then up to 6 of the relevant sex were selected) were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin. From one of the 3 pups of each sex, the weights of the brain, spleen and thymus were recorded, and these organs were preserved. Representative samples of any abnormal tissues from any of the 6 pups were also
preserved. The carcasses were then discarded.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality were necropsied as described in the preceding paragraph. The remaining carcasses were discarded.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.

HISTOPATHOLOGY
Histological examination was conducted on the brain, spleen and thymus of Control and High dose F1 and F2 weanlings (the selected weanlings at necropsy). A single H&E section was cut, stained and evaluated.
Statistics:: Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.
Reproductive indices:: For each group the following were calculated:

Fertility Index (male) = number siring a litter / Number paired

Fertility Index (female) = Number pregnant / Number paired

Gestation Index = Number bearing live pups / Number pregnant
Offspring viability indices:: For each litter and group the following were calculated:

Birth Index = Total number of pups born (alive and dead) / Number of implantation scars

Live Birth Index = Total number of pups live on Day 0 of lactation / Total number born (live and dead)

Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0

Lactation Index = Number of pups live on Day 21 of lactation / Number live on Day 4

Overall Survival Index = Number of pups live on Day 21 of lactation / Total number born (live and dead)
Clinical signs:: effects observed, treatment-related
Body weight and weight changes:: effects observed, treatment-related
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Other effects:: not examined
Reproductive function: oestrous cycle:: no effects observed
Reproductive function: sperm measures:: no effects observed
Reproductive performance:: no effects observed
Dose descriptor:: NOEL
Effect level:: 20 mg/m³ air
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: No treatment related effects were observed
Remarks on result:: other: Generation: F0 and F1 (migrated information)
Clinical signs:: no effects observed
Mortality / viability:: no mortality observed
Body weight and weight changes:: no effects observed
Sexual maturation:: no effects observed
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: no effects observed
Histopathological findings:: no effects observed
Reproductive effects observed:: not specified
Conclusions:: Under the conditions of the study the No Observed Effect Level (NOEL) of manganese chloride, for reproductive toxicity, was determined to be 20 µg/L.
Executive summary:: The reproductive toxicity of manganese chloride was investigated in a two generation study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800. The study was conducted with manganese dichloride, which represents a more bioavailable form of manganese than the registered substance. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds. During the study, F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed with manganese chrloride for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation. From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation. Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy. Clinical signs of reaction to treatment to inhalation exposure of manganese chloride were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L. At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls. There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls, however this reflected a slightly smaller litter size at this level. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment. There were no effects of treatment on the sexual maturity of the F1 animals. At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L). In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females. There was no effect of treatment on the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation. Inhalation of manganese chloride was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L. No test substance-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations. In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test substance through the mother’s milk during lactation resulted in an increased exposure to the test substance in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period. In conclusion, under the conditions of this study, a No Observed Effect Level for adult effects was not established due to effects on the respiratory tract. The No Observed Effect Level (NOEL) for reproductive performance was considered to be the target dose level 20 μg/L.

Reference 2

Endpoint:: extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: other:
Reproductive effects observed:: not specified

Reference 3

Endpoint:: two-generation reproductive toxicity
Type of information:: read-across from supporting substance (structural analogue or surrogate)
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:: The data on the read-across substance is considered representative.
Reason / purpose for cross-reference:: read-across source

Effect on fertility: via oral route

Endpoint conclusion:: no study available

Effect on fertility: via inhalation route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEC
: 20 mg/m³
Study duration:: chronic
Species:: rat
Quality of whole database:: The study was performed under GLP conditions and in accordance with standardised guidelines; however, since the study was conducted with manganese dichloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2 in line with the criteria of Klimisch (1997). The study was conducted with a more bioavailable form of manganese, manganese dichloride, rather than with the registered substance itself. The results are therefore considered to represent a worst case scenario.

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Grieve (2017)

The reproductive toxicity of manganese was investigated in a two generation study which was conducted with manganese dichloride, which represents a more bioavailable form of manganese than the registered substance. During the study, F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation. From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy.

Clinical signs of reaction to treatment to inhalation exposure of the test substance were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L. At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls.

There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls, however this reflected a slightly smaller litter size at this level. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment. There were no effects of treatment on the sexual maturity of the F1 animals.

At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L). In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females.

There was no effect of treatment on the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation.

Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L. No test material related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations.

In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test material through the mother’s milk during lactation resulted in an increased exposure to the test item in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period.

In conclusion, under the conditions of this study, a No Observed Effect Level for adult effects was not established due to effects on the respiratory tract. The No Observed Effect Level (NOEL) for reproductive performance was considered to be the target dose level 20 μg/L.

The study was conducted under GLP conditions and in accordance with standardised guidelines. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2.

Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario in terms of exposure to inorganic manganese and testing with the registered substance itself is not considered to be required and testing is omitted.

Adaptation for Further Reproductive Toxicity Testing with the Registration Substance

In accordance with REACH Annex XI, Section 1.1; use of existing data, reproductive toxicity testing is not considered to be required since data are available on a more soluble (and therefore more bioavailable) Mn substance, manganese chloride (MnCl2). Because the bioavailability of manganese ions from manganese chloride would be substantially greater, the results can be considered a worst-case for manganese carbonate. An OECD 416 study has been completed for manganese chloride in the rat (Grieve, 2017). Under the conditions of this study, there were no adverse effects on the reproductive performance of the animals up to the target dose level - 20 µg/L.

In addition, not a single available literature source from a literature review (Strupp and Furnes, 2009) dating back 50 years (on human and animal data on reproductive toxicity (all aspects) to manganese-based compounds) hints or suggests that MnCO3 specifically could cause reproductive toxicity. Testing is therefore considered unlikely to provide any additional or useful information and is also considered unjustified on animal welfare grounds, particularly considering the high number of animals that would be required.

Classification and Labelling Discussion

A guideline and GLP compliant two generation rat inhalation reproductive toxicity study was conducted with a more efficient provider of soluble manganese ions, namely manganese dichloride. At dose levels showing evidence of tolerable but clear parental toxicity (manifesting as decreased food intake and body weight performance), there was no effect on oestrous cyclicity, mating performance, sexual maturity, fertility or duration of gestation or litter size, the sperm motility or morphology, nor on the formation and maturation of ovarian follicles in either generation. Manganese chloride is therefore not a reprotoxicant, having no effect on sexual function or fertility, or on post-natal development in the rat. Therefore, soluble and insoluble forms of inorganic manganese compounds by extrapolation are considered not reprotoxicants.

In addition a literature review by Strupp and Furnes (2009) showed no evidence of reproductive toxicity for manganese carbonate.

Hence, there is considered to be no requirement to classify manganese carbonate for reproductive toxicity in the absence of data warranting classification.

Effects on developmental toxicity

Description of key information

Good developmental toxicity data (OECD 414 compliant or equivalent) exists in two species (rat and mouse) for a surrogate compound, manganese dichloride by the inhalation (rat) and subcutaneous (mouse) routes - both of which are considered to bypass the liver, and hence are both considered relevant to inhalation exposure of MnCO3. NOAELs were identified in both studies and identified similar developmental effects.

Read-Across Key Study: Dettwiler (2014)

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.

Read-Across Supporting Study: Sanchez (1993)

Under the conditions of the study the no observable adverse effect level (NOAEL) for maternal toxicity in mice was 4 mg MnCl2.4H2O/kg/day. The NOAEL for embryotoxicity was 2 mg/kg/day, there was no evidence of major malformations at any dosage level used.

Pilot Study: Stannard (2016)

Under the conditions of the study it was concluded that dose levels of 150 mg/kg/day and above clearly exceeded the maximum tolerated dose (MTD) in pregnant female NZW rabbits and are unsuitable for further investigation. There was no evidence of any test material-related maternal or foetal effects at 30 or 65 mg/kg/day.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed to sound scientific principles with a sufficient level of detail to assess the quality of the relevant results. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Justification for type of information:

The data on the read-across substance is considered representative.

Reason / purpose for cross-reference:

other: Target record

Qualifier:

no guideline followed

Principles of method if other than guideline:

Swiss mice were administered subcutaneously with manganese chloride tetrahydrate at doses of 0, 2, 4, 8 and 16 mg/kg/day from gestation day (gd) 6 through to 15. Females were sacrificed on gestation day 18, and foetuses were examined for external, visceral, and skeletal abnormalities.

GLP compliance:

not specified

Limit test:

Species:

mouse

Strain:

Swiss

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna Ibercia (Barcelona, Spain)
- Weight at study initiation: 28.32 g
- Diet: Panlab rodent chow, ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 10 %
- Photoperiod (hrs dark / hrs light): 12:12 hour light/dark cycle

Route of administration:

subcutaneous

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: MnCl2 solutions were prepared fresh daily in 0.9 % saline

VEHICLE
- Amount of vehicle : 0.10 mL

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Proof of pregnancy: vaginal plug

Duration of treatment / exposure:

9 days

Frequency of treatment:

Daily

Duration of test:

18 days

No. of animals per sex per dose:

20 animals per group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule: Daily

FOOD CONSUMPTION: Yes
- Time schedule: Monitored daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18
- Organs examined: Liver and kidney and gravid uterus were examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: All live foetuses
- Soft tissue examinations: Yes: One third per litter
- Skeletal examinations: Yes: Two thirds per litter
- Head examinations: No data
- Other: The body weights of all live foetuses were measured

Statistics:

The unit of comparison was the pregnant female of the litter. Kruskal-Wallis analysis of variance procedures were employed to assess the overall effects of MnCl2. Pairwise comparisons were made by the Mann-Whitney U-test. Statistically significant differences between control and test groups were analysed by a two tailed Student's t-test. Significance levels were chosen at P < 0.05.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment related mortality was observed in the high-dose group, 6 of the 19 dams died prior to scheduled necropsy. Statistically significant reductions in body weight and food consumption were noted in the 8 mg/kg group (gd 15-18) and at 16 mg/kg (gd 6-15). A significant decrease in corrected body weight at 8 and 16 mg/kg relative to the controls was noted. Gravid uterine weights were found to be significantly decreased in the 8 and 16 mg/kg/day groups, although the corrected body weight change appeared unaffected by Mn treatment. Relative maternal liver weights were found to be significantly decreased compared to controls at 16 mg/kg.

Dose descriptor:

other: LD50

Effect level:

320 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No significant treatment related effects on the number of total implants, early resorptions, dead foetuses or sex ratio were noted. There was a significant increase in the number of late resorptions in the 4, 8 and 16 mg/kg/day groups. There were seven litters with 100 % resorptions in the highest dosing group. A dose response relationship was noted between decreased foetal body weight and increasing dose concentrations. These were found to be significantly below control values in the 8 and 16 mg/kg/day dose groups.

There were no significant increase in the number of litters with one or more affected foetuses in any treatment group in comparison to controls for total external and visceral abnormalities. The incidence of wavy ribs and delayed or reduced ossifications in the sternebra, parietal and occipital were statistically significant (P < 0.05).

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Body and organ weights at termination for mice treated with manganese chloride tetrahydrate

	Dose (mg/kg/day)
	0	2	4	8	16
No. of dams	19	17	17	18	13
Body weight (g)	61.2±4.1	60.7± 8.0	59.3± 8.8	47.2± 6.0^f	44.8± 4.4^f
Gravid uterine weight (g)^a	20.7± 1.8	19.6± 5.3	18.3± 7.8	9.9± 6.0^f	3.4± 1.4^f
Corrected body weight (g)^a	40.5± 1.8	41.1± 3.9	41.0± 2.7	37.3± 3.6^d	41.4± 3.6
Corrected body weigh change (g)^b	8.5± 2.2	7.0± 2.1	6.8± 2.2	6.7± 2.8	8.5± 2.5
Liver weight (g)	3.1± 0.4	3.2± 0.6	3.0± 0.4	2.7± 0.3^e	2.9± 0.2
Relative liver weight (%)^c	7.7± 1.3	7.8± 1.0	7.3± 0.9	7.2± 1.0	7.0± 0.3^d
Kidney weight (g)	0.48± 0.05	0.48± 0.04	0.5± 0.05	0.45± 0.04	0.53± 0.07^d
Relative kidney weight (%)^c	1.18± 0.12	1.17± 0.08	1.21± 0.07	1.21± 0.05	1.28± 0.13^d
Values indicate mean±SD ^aCorrected body weight = bodyweight at termination – gravid uterine weight ^bCorrected body weight change = corrected body weight – body weight on gestational day 0 ^cCalculated as percentage of corrected body weight ^{d, e, f}Significantly different from controls (P < 0.05; P < 0.01; P < 0.001, respectively)

Table 2: Gestational parameters and foetal weights in mice foetuses following maternal exposure to manganese chloride tetrahydrate

	Dose (mg/kg/day)
	0	2	4	8	16
No. of dams	19	17	17	18	13
No. of total implants/litter	13.6 ± 2.5	13.1± 3.0	12.5± 5.1	11.7± 4.6	14.0± 1.6
No. of live foetuses/litter	12.7± 3.3	10.6± 3.0	10.0± 4.7	4.2± 4.0^b	0.3± 0.6^b
No. of non-viable implants/litter
Early resorptions	0.7± 0.7	1.2± 1.0	0.6± 0.6	1.6± 2.0	1.9± 3.1
Late resorptions	0.1± 0.3	0.7± 1.1	1.4± 0.8^a	4.1± 2.8^b	11.6± 4.1^b
Dead foetuses	0.0± 0.0	0.6± 0.9	0.5± 0.7	1.7± 2.7	0.2± 0.3
Sex ratio (m/f)	1.17± 0.96	1.24± 0.62	1.07± 0.72	1.01± 0.50	1.13± 0.81
Average foetal body weight/litter (g)	1.18± 0.13	1.17± 0.09	1.13± 0.12	0.97± 0.11^b	0.82± 0.10^b
Values indicate mean ± SD ^{a, b}Significantly different from controls (P < 0.01; P < 0.001 respectively)

Table 3: Morphological defects in mice foetuses following maternal exposure to manganese chloride tetrahydrate

	Dose (mg/kg/day)
	0	2	4	8	16
No. foetuses examined viscerally (No. litters)	103 (19)	70 (17)	70 (17)	34 (18)	0 (0)
Enlarged heart
Foetuses affected	0	0	0	4	-
Litters affected	0	0	0	4	-
Renal hypoplasia
Foetuses affected	0	0	2	6^b	-
Litters affected	0	0	2	6	-
No. foetuses examined skeletally (No. litters)	139 (19)	111 (17)	119 (17)	54 (18)	7 (6)
Asymmetrical sternebrae
Foetuses affected	16	19	11	13	0
Litters affected	9	9	6	9	0
Wavy ribs
Foetuses affected	0	2	0	5^c	0
Litters affected	0	2	0	4	0
Dorsal hyperkiphosis
Foetuses affected	0	4	1	1	0
Litters affected	0	1	1	2	0
Sternebrae, delayed ossification
Foetuses affected	0	0	25^c	36^c	7^c
Litters affected	0	0	11^b	18^c	6^c
Parietal bone, reduced ossification
Foetuses affected	0	0	0	6^a	6^c
Litters affected	0	0	0	5	6^c
Occipital bone, reduced ossification
Foetuses affected	0	0	0	6^a	6^c
Litters affected	0	0	0	5	6^c
a, b, c Significantly different from controls (P < 0.05; P < 0.001, respectively)

Conclusions:

Executive summary:

The study was conducted with manganese dichloride, which represents a more bioavailable form of manganese than the registered substance. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

During the study, groups of female mice were administered with 2, 4, 8, or 16 mg/kg of manganese chloride daily from day 6-15 of gestation. Maternal toxicity was observed at the highest dose level with significant mortality (32%). There was also a significant effect on body weight (decrease), organ weight (liver and kidney), as well as reduced food consumption. The only gestational parameter affected by manganese chloride treatment was an increase in late resorptions. In pups, increasing concentrations of manganese chloride caused decreasing body weight. Relatively minor skeletal effects were also observed.

The conclusions of this study are strengthened by the use of multiple dose groups. Since severe maternal toxicity was observed it cannot be excluded that the effect on late resorptions and on pups is due to this effect.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January 2014 to 30 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The data on the read-across substance is considered representative.

Reason / purpose for cross-reference:

other: Target record

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

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:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Wistar

Details on test animals or test system 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:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

5 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

15 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

25 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

15 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

15 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

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:

25 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose

Dose descriptor:

NOEL

Effect level:

25 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose

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.

Developmental toxicity data in the rat on the read-across substance manganese dichloride has no classification for developmental toxicity.

Executive summary:

The potential of the test material 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.

Reference 3

Endpoint:

developmental toxicity

Remarks:

Pilot study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 February 2016 to 18 August 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

The method followed was similar to that which is outlined in the standardised guideline OECD 414, though fewer animals were utilised per dose group.

GLP compliance:

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

Limit test:

Species:

rabbit

Strain:

New Zealand White

Remarks:

The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation (Day 0 of gestation for preliminary embryo-foetal phase animals):
Pilot Phase:
Group 1: 24 weeks.
Group 4: 24 weeks.
Group 2: 30 weeks.
Group 3: 31 weeks.

Preliminary Embro-Foetal Phase:
Groups 5 - 8: 32 to 36 weeks.
Groups 9 - 11: 31 to 33 weeks.

- Weight at study initiation (Day 0 of gestation for preliminary embryo-foetal phase animals):
Pilot Phase:
Group 1: 3.62 to 3.91 kg.
Group 4: 3.60 to 3.98 kg.
Group 2: 3.69 to 3.80 kg.
Group 3: 3.45 to 3.48 kg.

Preliminary Embro-Foetal Phase:
Groups 5 - 8: 2.64 to 3.76 kg.
Groups 9 - 11: 3.34 to 4.46 kg.

- Housing: Animals were individually housed in suspended cages fitted with perforated floor panels. In the preliminary embryo-foetal phase, during mating one male and one female were housed in each cage.

- Diet:
Pilot phase: Restricted (150 g per rabbit per day during acclimatisation up to one week prior to commencement of treatment for Group 1, and 200 g/animal/day for all animals thereafter.
Preliminary Embro-Foetal Phase: Restricted (initially 150 g/animal/day during acclimatisation up to one week prior to the onset of mating and 200 g/animal/day thereafter).
If an individual animal showed a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered.
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly.

- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided.

- Acclimation period:
Pilot phase:
Group 1: 20 days.
Group 4: 26 days.
Group 2: 69 days.
Group 3: 77 days.

Preliminary Embro-Foetal Phase:
Groups 5 - 8: Approximately 14 weeks until mating commenced.
Groups 9 - 11: Approximately 15 weeks until mating commenced (one week from transfer from stock).

ENVIRONMENTAL CONDITIONS
- Temperature: 15 – 21 °C
- Humidity: 45 - 70 %
- Air changes (per hr): Not specified. Filtered fresh air was passed to atmosphere and not recirculated.
- Photoperiod: 14 hours light : 10 hours dark.

Route of administration:

oral: gavage

Vehicle:

other: Pilot Study - Groups 1 and 4: Arachis oil. Pilot Study & Preliminary Embryo-Foetal Phase - Groups 2, 3 and 5 - 11: 1 % methylcellulose.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The method of preparation was the same with either vehicle.
- The required amount of test material was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. The suspension was transferred to the final container, via syringe, whilst magnetically stirring.
- A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test material.
- Formulations were initially hand-stirred with a non-flexible spatula (or similar), and then stirred using a magnetic stirrer for a minimum of 30 minutes before and throughout the dosing procedure to maintain homogeneity. The speed of stirring was such as to maintain a deep vortex (approximately 1 cm) on the surface of the formulation.
- All formulations were prepared weekly.
- Homogeneity of test material formulations in arachis oil and in 1 % methylcellulose in the concentration range 1 to 200 mg/mL following 10 days ambient (nominally +21 °C) or refrigerated (nominally +4 °C) storage was determined as part of another study. Due to the viscosity of formulations at 200 mg/mL following refrigerated storage, formulations were held at ambient temperature between preparation and use.
- Animals were treated at constant doses in mg/kg/day.
- Volume of the dose was 5 mL/kg body weight.
- Individual dose volumes were calculated from the most recetly recorded scheduled body weight.
- Control groups were administered the vehicle at the same volume dose as treated groups.

Analytical verification of doses or concentrations:

Remarks:

No formulation analysis was performed as part of this study.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1 using identified stock New Zealand White bucks. A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals. Natural mating was observed. After mating, each female was injected intravenously with 25 i.u. luteinizing hormone.
- Day 0 of gestation was on the day of mating.

Duration of treatment / exposure:

Pilot Phase: Groups 1 and 4: Up to 14 days. Treatment from day 1 to 5, with an optional extension to day 14. Groups 2 and 3: Treatment from day 1 to day 14.
Preliminary Embryo-Foetal Phase: Days 6 to 28 after mating, inclusive.

Frequency of treatment:

Once daily

Duration of test:

Pilot Phase: Once a clear reaction to treatment was established or maximum practical or maximum desired dose was attained - up to 14 days.
Preliminary Embryo-Foetal Phase: 29 days after mating

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Pilot phase control (Group 4) and prelminary embryo-foetal phase (Group 5)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Pilot phase (Group 1) and preliminary embryo-foetal phase (Group 6)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

Pilot phase (Group 2) and preliminary embryo-foetal phase (Group 7)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Pilot phase (Group 3) and preliminary embryo-foetal phase (Group 8)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Remarks:

Preliminary embryo-foetal phase (Group 9)

Dose / conc.:

65 mg/kg bw/day (actual dose received)

Remarks:

Preliminary embryo-foetal phase (Group 10)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

Preliminary embryo-foetal phase (Group 11)

No. of animals per sex per dose:

Pilot Phase: 3 females per group
Preliminary Embryo-Foetal Phase: 6 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Prior to this study, there was no repeat dose data available for any test species with the test material; as a consequence, a cautious approach was taken for the pilot phase of this study, where the limit dose required for investigation was 1 000 mg/kg/day. An acute oral dose toxicity study has been conducted in the rat and a dose level of 2 000 mg/kg was tolerated.
Initially, a dose level of 300 mg/kg/day was investigated in Group 1 of the pilot phase.
Further dose levels were determined for the pilot phase of the study on the basis of the effects observed at 300 mg/kg/day.
The dose levels for the preliminary embryo-fetal phase were selected based on the effects observed in the pilot phase.

- Rationale for animal assignment
Pilot Phase: Random animal assigment prior to commencement of treatment, avoiding allocation of more than one sibling per group.
Preliminary Embro-Foetal Phase: Where possible only females mating at least twice were allocated in the sequence of mating to group and cage position. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time Schedule:
Mortality: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
Clinical Observations: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.
Detailed observations were recorded daily at the following times in relation to dose administration: Pre-dose observation one to two hours after completion of dosing and as late as possible in the working day.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: A detailed physical examination was performed on each animal to monitor general health, as follows:
Pilot Phase: Weekly during acclimatisation and then daily from day -3.
Preliminary Embryo-Foetal Phase: Weekly during acclimatisation and on days 0, 6, 12, 18, 23 and 29 after mating.
Clinical signs and post-dosing observations were presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT: Yes.
- Time schedule for examinations: The weight of each adult was recorded, as follows:
Pilot Phase: Weekly during acclimatisation and then daily from day -3.
Preliminary Embryo-Foetal Phase: Weekly during acclimatisation and on days 0, 3 and 6 – 29 after mating.

FOOD CONSUMPTION: Yes.
- Time schedule for examinations: The weight of food supplied to each animal, that remaining, and an estimate of any spilled was recorded, as follows:
Pilot Phase: Daily from day -3.
Preliminary Embryo-Foetal Phase: Daily from day 1 after mating.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes.
All animals were sacrificed via an intravenous injection of sodium pentobarbitone. A complete necropsy was performed in all cases.
- Sacrifice in a sequence to allow satisfactory inter-group comparison:
Pilot Phase: Once a clear reaction to treatment was established or maximum practical or maximum desired dose was attained.
Preliminary Embryo-Foetal Phase: Animals surviving until the end of the scheduled study period were killed on Day 29 after mating.
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes, including cervix and ovaries for pregnant females surviving to term.
The following were recorded for all animals (including those prematurely sacrificed, where possible):
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Numder of foetuses (live and dead): Yes

Apparently non-pregnant animals: The absence of uterine implantation sites was confirmed by a second person.

Fetal examinations:

All animals were sacrificed via a subcutaneous injection of sodium pentobarbitone.
All foetuses and placentae were dissected from the uterus and weighed individually. Foetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each placenta and foetus was externally examined and an internal examination of the neck and the contents of the thoracic and abdominal cavities was performed and any abnormalities were recorded, sampled as appropriate and retained in appropriate fixative. The sex of each foetus was recorded. Grossly normal foetuses were discarded.

Statistics:

STATISTICAL ANALYSIS
No statistical analyses were performed on this study.

DATA EVALUATION
This report contains serial observations from Day -3 for the pilot phase and observations pertaining to the period from Day 0 after mating for the preliminary embryo-foetal phase.
For the preliminary embryo-foetal phase, where appropriate, group mean values, each with standard deviation (SD), were calculated from individual data. Summary tabulated data was normally restricted to data derived from females/litters with live young at day 29 after mating (groups 5 and 9 - 11 only).
Standard deviations were not calculated for derived data, such as levels of pre- and post-implantation loss, or for the incidence of resorbing foetuses where the distribution of these findings commonly does not conform to the normal statistical distribution.
For the pilot phase individual weight changes were presented.
For the preliminary embryo-foetal phase group mean weight changes were calculated from the weight changes of individual animals in Groups 5 and 9 - 11 only. Weight changes were calculated and plotted graphically with respect to Day 6 of gestation.
Adjusted body weights on Day 29 after mating were calculated from the body weight at termination minus the gravid uterine weight. Body weight change values for the period Day 6 - 29 were also presented, after being adjusted for the contribution of the gravid uterus.
Group mean food consumptions and standard deviations were derived from unrounded cage values for Groups 5 and 9 - 11 only.
Individual food consumption values are presented for all periods collected. Due to the tolerance limits of the balances used when measuring the diet fed and that remaining, the data capture system may present minor negative food consumption values for animals showing inappetance.

Indices:

REPRODUCTIVE ASSESSMENT
Prenatal losses were separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilisation of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100.

Where the number of implantations exceeded the number of corpora lutea observed, pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = [(Number of implantations – Number of live foetuses) / Number of implantations] x 100.

All group values and SD (as appropriate) were calculated from the individual litter values.

FOETAL, LITTER AND PLACENTAL WEIGHTS
Mean foetal weights were calculated for each litter. Values were presented for male, female and overall foetal weight. Litter weight was calculated as the sum of all foetal weights. Mean placental weight was also calculated for each litter.
Group mean values and SD were calculated using individual litter mean values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

PILOT PHASE
Group Receiving Test Material in Arachis Oil (Group 1): Treatment of female rabbits at 300 mg/kg/day in arachis oil was not tolerated, with all females killed for reasons of animal welfare prior to dose administration on day 4 of the study due to a marked decline in clinical condition. From Day 3 of study clinical signs comprising changes in excreta (loose, liquid and/or few faeces, reduced faecal pellet size, reduced urine output) were observed.

Group Receiving Arachis Oil Only (Group 4): Since it could not be definitively ascertained whether the effects seen in Group 1 animals were attributable to the test material or whether they were vehicle-mediated, a Control group receiving arachis oil was assigned to the study. Prior to dosing on the morning of day 3 of treatment it was clear that the previously observed effects were attributable to the administration of arachis oil, and this group of females was killed for reasons of animal welfare due to a marked decline in clinical condition. On Day 2 - 3 of study clinical signs comprising loose or liquid faeces (3/3 females) was observed.

Groups Receiving Test Material in 1 % Methylcellulose (Groups 2 and 3): As the effects seen at 300 mg/kg/day in arachis oil were of the same magnitude as those seen with vehicle only, it was clear that there was no direct effect of treatment at 300 mg/kg/day. Therefore, following identification of an alternative vehicle (1 % methylcellulose), two further groups were assigned to the study to receive doses of 600 or 1 000 mg/kg/day. Treatment of female rabbits at 600 or 1 000 mg/kg/day was tolerated. There were some clinical signs of possible relationship to treatment. At 1 000 mg/kg/day signs were limited to pale faeces (1/3 females at 1 000 mg/kg/day) and reduced faecal pellet size (1/3 females at 1 000 mg/kg/day). These signs generally only persisted for 24 - 72 hours.

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 300, 600 or 1 000 mg/kg/day (Groups 6 - 8): Treatment at 600 or 1 000 mg/kg/day revealed 3/6 and 5/6 females with clinical signs.
At 300 mg/kg/day, treatment was not tolerated, with three females (numbers 20, 22 and 24) being killed for reasons of animal welfare during days 16 - 18 after mating due to a decline in clinical condition. Signs including underactive behaviour, reduced body temperature, few
faeces, reduced size faecal pellets, and red staining on cage tray paper). The remaining three females (Numbers 19, 21 and 23) survived to scheduled termination on Day 29 after mating, however all three females showed clinical signs of reduced body temperature.

Maternal Responses Groups receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Since suitable dose levels for investigation in a main embryo-foetal development study had not been established, three further groups were subsequently added to the study at dose levels of 30, 65 or 150 mg/kg/day.
Treatment at 150 mg/kg/day was not tolerated, with three of the six females requiring premature termination due to a marked decline in clinical condition during Days 22 - 26 after mating.
Female No. 50 was killed on Day 22 after mating. Signs of reduced body temperature (cold to touch), shallow respiration, thin build, piloerection, colourless/watery nasal discharge, dilated pupils and whole body pallor were observed.
Female No. 52 was killed on Day 26 after mating. Signs of underactive behaviour, slow respiration, few/loose faeces and partially closed eyelids were observed.
Female No. 53 was killed on Day 25 after mating. Signs of deep respiration and few/loose faeces were observed.
Among females surviving to scheduled termination on day 29 after mating, there were no test material-related clinical signs apparent at 30, 65 or 150 mg/kg/day. Signs related to dose administration were limited to one female in the 30 mg/kg/day group (number 37) which showed fast respiration on day 27 after mating.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

PILOT PHASE
Group Receiving Test Material in Arachis Oil (Group 1): Following the commencement of treatment, all females showed progressive weight loss.

Group Receiving Arachis Oil Only (Group 4): As had been seen in the 300 mg/kg/day test material group, all control females showed progressive weight loss and negligible food intake following the start of treatment.

Groups Receiving Test Material in 1 % Methylcellulose (Groups 2 and 3): There was no clear effect of treatment on body weight performance.

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Following the start of treatment on day 6 of gestation, the body weight performance of females given 150 mg/kg/day was similar to control from day 6 to day 23 of gestation. Between day 23 and day 29 of gestation, the period of maximal foetal growth, the surviving pregnant females in this group showed mean weight loss of 20 g compared to mean weight gain of 120 g in the control group.
Female No. 50 was killed on Day 22 after mating. This female had shown progressive weight loss of 190 g from Day 16 - 21 after mating.
Female No. 52 was killed on Day 26 after mating. This female had shown progressive weight loss of 80 g from Day 24 - 26 after mating.
Female No. 53 was killed on Day 25 after mating. This female had shown progressive weight loss of 100 g from Day 22 - 24 after mating.

The mean body weight performance of females given 30 or 65 mg/kg/day was unaffected by test material administration.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 300, 600 or 1 000 mg/kg/day (Groups 6 - 8): At 300 mg/kg/day, treatment was not tolerated, with three females (numbers 20, 22 and 24) being killed for reasons of animal welfare during days 16 - 18 after mating due to a decline in clinical condition with signs including underactive behaviour.

Maternal Responses Groups receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Treatment at 150 mg/kg/day was not tolerated, with three of the six females requiring premature termination. One of these, female number 52, showed signs including underactive behaviour.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean gravid uterine weight on day 29 of gestation was similar in all groups. When overall mean bodyweight gain was adjusted for the contribution of the gravid uterus, net bodyweight loss was recorded in all groups of females, however the magnitude of the weight loss in the 150 mg/kg/day group was substantially greater than that recorded in the control group (470 g compared to 210 g); net weight loss in the 30 or 65 mg/kg/day groups was similar to control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

PILOT PHASE
Group Receiving Test Material in Arachis Oil (Group 1): Macroscopic examination revealed fluid caecal contents (3/3 females), reddened caecum (1/3 females), pale jejunum contents (1/3 females), rectum devoid of contents (1/3 females) and accentuated lobular pattern of the liver (1/3 females).

Group Receiving Arachis Oil Only (Group 4): Macroscopic examination revealed fluid caecal content (3/3 females), reddened caecum (2/3 females), rectum devoid of contents (1/3 females) and dark bladder contents (1/3 females).

Groups Receiving Test Material in 1 % Methylcellulose (Groups 2 and 3): There were no test material-related macroscopic abnormalities detected at scheduled termination.

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 300, 600 or 1 000 mg/kg/day (Groups 6 - 8): Macroscopic examination on Day 29 after mating revealed 3/6 and 5/6 females with dark lungs and bronchi at 600 and 1 000 mg/kg/day, respectively; in addition, the liver of two females at 600 mg/kg/day was pale, with one being tinged green and with an accentuated lobular pattern, and one of these females also showed pale kidneys.
At 300 mg/kg/day, of the six females in this group, three showed macroscopic abnormalities in the liver (irregular surface and/or pale), one had pale kidneys and one showed dark lungs and bronchi.

Maternal Responses Groups receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): There were no test material-related macroscopic abnormalities detected among the adult females or foetuses at scheduled termination on day 29 after mating at dose levels up to and including 150 mg/kg/day.
Treatment at 150 mg/kg/day was not tolerated:
Female No. 50 was killed on Day 22 after mating. Macroscopic examination revealed pale kidneys, pale liver with accentuated lobular pattern and nodular adipose tissue.
Female No. 52 was killed on Day 26 after mating. Macroscopic examination revealed pale kidneys and liver.
Female No. 53 was killed on Day 25 after mating. Macroscopic examination revealed pale kidneys and liver.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

FOOD CONSUMPTION
PILOT PHASE
Group Receiving Test Material in Arachis Oil (Group 1): Following the commencement of treatment, all females showed negligible food intake. For 2/3 females low hay intake was observed.

Group Receiving Arachis Oil Only (Group 4): All control females showed negligible food intake following the start of treatment. On Day 2 - 3 of the study low hay intake was observed for 2/3 females.

Groups Receiving Test Material in 1 % Methylcellulose (Groups 2 and 3): Clinical signs of possible relationship to treatment included low hay intake (1/3 females in each of the 600 or 1 000 mg/kg/day groups) which generally only persisted for 24 - 72 h.
At 1 000 mg/kg/day all females had periods of low food intake (below 50 g/day) lasting for 2 - 5 days, however there was no clear pattern as to when these reductions occurred in relation to the start of dosing (i.e. these occurred for one female on Day 2 - 3 of study, one female on Day 2 - 6 of study and one female on Day 5 - 9 of study). There was no evidence of an effect of treatment on food consumption at 600 mg/kg/day.

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 300, 600 or 1 000 mg/kg/day (Groups 6 - 8): At 300 mg/kg/day treatment three females (numbers 20, 22 and 24) which were killed for reasons of animal welfare had negligible food intake. The remaining three females (numbers 19, 21 and 23) had periods of low food intake.

Maternal Responses Groups receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): At 150 mg/kg/day mean food consumption was essentially similar to control from day 6 to day 17 of gestation. Thereafter, a clear decrease in food intake was evident until scheduled termination on day 29 of gestation.
Female No. 50 was killed on Day 22 after mating due to signs including low food/diet intake with negligible food intake from Day 18 - 21 after mating.
Female No. 52 was killed on Day 26 after mating due to signs including negligible food intake from Day 24 - 25 after mating.
Female No. 53 was killed on Day 25 after mating due to signs including negligible food intake from Day 23 - 24 after mating.
The mean food intake of females given 30 or 65 mg/kg/day was considered unaffected by test material administration.

WATER CONSUMPTION
PILOT PHASE
Group Receiving Test Material in Arachis Oil (Group 1): From day 3, all females showed low water intake.

Group Receiving Arachis Oil Only (Group 4): On day 2 - 3 of the study low water intake (1/3 females) was observed.

Groups Receiving Test Material in 1 % Methylcellulose (Groups 2 and 3): Clinical signs of possible relationship to treatment included low water intake (2/3 females at 1 000 mg/kg/day).

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Three of the six females requiring premature termination were observed to have low water intake.

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

See details on maternal toxic effects.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

See details on maternal toxic effects.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

See details on maternal toxic effects.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

See details on maternal toxic effects.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 300, 600 or 1 000 mg/kg/day (Groups 6 - 8): Treatment at 600 or 1 000 mg/kg/day resulted in apparent non-pregnancy in all females. This may reflect either (a) implantation failure or (b) death of the conceptuses shortly after successful implantation into the uterine wall.

Other effects:

no effects observed

Description (incidence and severity):

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): At 150 mg/kg/day mean placental weights were not clearly affected by maternal test material administration.

Details on maternal toxic effects:

PRELIMINARY EMBRYO-FOETAL PHASE
At 300 mg/kg/day, treatment was not tolerated, with three females (numbers 20, 22 and 24) being killed for reasons of animal welfare during days 16 - 18 after mating. Two of these females showed a total litter resorption and the other female had eight implantation sites but only three live embryos. The remaining three females (numbers 19, 21 and 23) survived to scheduled termination on day 29 but at scheduled necropsy, one female was found to be not pregnant, one female had a total litter resorption (only 2 implantation sites) and the remaining female had five implantation sites but only one live foetus.

Maternal Responses Groups Receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Treatment at 150 mg/kg/day was not tolerated, with three of the six females requiring premature termination due to a marked decline in clinical condition during days 22 - 26 after mating. Uterine examination of female number 50 revealed six implantation sites, all of which were late resorptions; female number 52 was had eight implantation sites, with two dead and six live foetuses and female number 53 eight implantation sites, with two resorptions, five normal live foetuses and one foetus with acephaly and abdominal omphalocele.

Key result

Dose descriptor:

other: Maximum Tolerated Dose (MTD).

Effect level:

>= 65 - < 150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Test material-related maternal or foetal effects.

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

Treatment at 150 mg/kg/day resulted in three of the six females requiring premature termination due to a marked decline in clinical condition. In one uterine examination revealed six implantation sites, all of which were late resorptions; in the second uterine examination revealed eight implantation sites, with two dead and six live foetuses; and in the third eight implantation sites, with two resorptions, five normal live foetuses and one foetus with acephaly and abdominal omphalocele.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): At scheduled termination on day 29 after mating, one female in each of the dose groups were not pregnant. The following assessment is therefore based on 5, 5, 5 and 2 litters in the 0, 30, 65 and 150 mg/kg/day groups, respectively.
At 150 mg/kg/day, mean foetal weights in the two surviving litters were lower than the control; mean foetal weight was considered unaffected by the test material at 30 or 65 mg/kg/day.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and considered unaffected by maternal treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Mean litter size and litter weight were higher than the control in all treated groups, however this was attributable to atypically low litter size in 3/5 Control litters, and no effect of treatment with the test material was considered to be inferred. There was no clear evidence that maternal treatment with the test material at 30 or 65 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of corpora lutea, implantations, resorptions, live young and pre- and post-implantation losses. Although there was no increase in resorptions or in pre- or post-implantation losses among females with live young in the 150 mg/kg/day group, it must be noted that one female in the group had a total litter resorption.

Changes in postnatal survival:

not specified

External malformations:

effects observed, treatment-related

Description (incidence and severity):

PRELIMINARY EMBRYO-FOETAL PHASE
Maternal Responses Groups Receiving 30, 65 or 150 mg/kg/day (Groups 9 - 11): Treatment at 150 mg/kg/day was not tolerated; one female (number 53) had one foetus with acephaly and abdominal omphalocele.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Abnormalities:

effects observed, treatment-related

Localisation:

other: Treatment at 150 mg/kg/day was not tolerated; one female had eight implantation sites, with two resorptions, five normal live foetuses and one foetus with acephaly and abdominal omphalocele.

Developmental effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical Signs: Group Distribution of Observations Embryo-Foetal Phase

Group	5	9	10	11	6	7	8
Compound	Control	Test Material
Dose (mg/kg/day)	0	30	65	150	300	600	1000

Days 0-29 Category	Observation	Group/Sex	Number of Animals Affected
		Group/Sex	5F	9F	10F	11F
		Number in Group	6	6	6	6
Behaviour	Little diet eaten		0	0	0	1*
	Little hay eaten		0	0	0	1*
	Little water drunk		0	0	0	3*
	Underactive		0	0	0	2*

Body Temperature	Reduced		0	0	0	1*

Breathing	Deep		0	0	0	1*
	Shallow		0	0	0	1*
	Slow		0	0	0	1*

Build (Conformation)	Thin		0	0	0	1*

Coat	Hair loss, dorsal body surface		1	0	0	0
	Hair loss, hindlimb (right)		0	0	0	1
	Hair loss, Perigenital		1	1	0	0
	Hair loss, ventral body surface		0	0	0	1
	Piloerection		0	0	0	1*

Discharge	Watery, colourless, nose		0	0	0	1*

Excreta	Faeces loose		0	0	0	2*
	Few faeces		0	0	0	2*

Eye	Pupils dilated, eye (left)		0	0	0	1*
	Pupils dilated, eye (left)		0	0	0	1*

Eyelids	Partially closed		0	0	0	1*

Skin colour	Pallor, whole body		0	0	0	1*

Staining	Abnormal colour, brown, dorsal body surface		0	0	0	1
	Abnormal colour, brown, forelimbs		0	0	0	1
	Abnormal colour, brown, forepaws		0	1	0	0
	Abnormal colour, brown, hindlimbs		0	0	0	1
	Abnormal colour, brown, hindpaws		1	2	0	1
	Abnormal colour, brown, tail		0	0	1	3
	Abnormal colour, yellow, forepaws		0	3	1	0
	Abnormal colour, yellow, hindpaws		1	4	1	1
	Abnormal colour, yellow, tail		0	5	4	4

* Signs seen in premature decedent animals.

Signs Associated with Dosing: Group Distribution of Observations Embryo-Foetal Phase

Sign	Day Number	Group/Sex and Number of Animals Showing Sign
Sign	Day Number	5F	9F	10F	11F
Breathing fast	27	0	1	0	0

Body Weight and Body Weight Change: Group Mean Values (kg) Embryo-Foetal Phase

Group/Sex		Day 0	Day 3	Day 6	Day 7	Day 8	Day 9	Day 10	Day 11	Day 12	Day 13	Day 14	Day 15
5F	Mean	3.31	3.31	3.28	3.27	3.26	3.26	3.27	3.26	3.27	3.30	3.32	3.34
	SD	0.285	0.299	0.291	0.310	0.294	0.286	0.291	0.276	0.256	0.254	0.248	0.248
	N	5	5	5	5	5	5	5	5	5	5	5	5
9F	Mean	3.92	3.94	3.93	3.92	3.92	3.93	3.93	3.93	3.94	3.96	3.98	4.00
	SD	0.325	0.315	0.296	0.262	0.274	0.256	0.238	0.262	0.255	0.246	0.297	0.298
	N	5	5	5	5	5	5	5	5	5	5	5	5
10F	Mean	3.64	3.66	3.62	3.63	3.62	3.62	3.61	3.60	3.61	3.64	3.67	3.70
	SD	0.290	0.265	0.292	0.295	0.299	0.297	0.284	0.288	0.274	0.275	0.274	0.270
	N	5	5	5	5	5	5	5	5	5	5	5	5
11F	Mean	3.75	3.73	3.73	3.72	3.71	3.71	3.73	3.72	3.72	3.74	3.77	3.81
	SD	0.190	0.163	0.178	0.174	0.166	0.190	0.178	0.175	0.169	0.168	0.157	0.171
	N	5	5	5	5	5	5	5	5	5	5	5	5
Group/Sex		Day 16	Day 17	Day 18	Day 19	Day 20	Day 21	Day 22	Day 23	Day 24	Day 25	Day 26	Day 27
5F	Mean	3.34	3.33	3.32	3.34	3.33	3.33	3.32	3.34	3.36	3.38	3.41	3.43
	SD	0.244	0.240	0.261	0.273	0.262	0.262	0.251	0.239	0.232	0.233	0.245	0.244
	N	5	5	5	5	5	5	5	5	5	5	5	5
9F	Mean	3.99	4.00	4.00	3.99	4.00	4.02	4.04	4.03	4.05	4.06	4.08	4.10
	SD	0.293	0.285	0.293	0.271	0.279	0.272	0.245	0.215	0.220	0.237	0.238	0.240
	N	5	5	5	5	5	5	5	5	5	5	5	5
10F	Mean	3.70	3.72	3.72	3.71	3.71	3.72	3.72	3.72	3.72	3.75	3.78	3.82
	SD	0.264	0.265	0.247	0.249	0.230	0.244	0.245	0.248	0.247	0.229	0.217	0.210
	N	5	5	5	5	5	5	5	5	5	5	5	5
11F	Mean	3.82	3.81	3.79	3.77	3.75	3.76	3.78	3.83	3.84	3.83	3.80	3.82
	SD	0.177	0.168	0.172	0.195	0.196	0.182	0.198	0.167	0.173	0.179	0.189	0.278
	N	5	5	5	5	5	5	5	4	4	4	3	2

Body Weight and Body Weight Change: Group Mean Values (kg) Embryo-Foetal Phase (continued)

Group/Sex		Day 28	Day 29	Change 0-6	Change 6-12	Change 12-19	Change 19-23	Change 23-29
5F	Mean	3.45	3.46	- 0.02	- 0.02	0.07	0.01	0.12
	SD	0.248	0.246	0.055	0.037	0.022	0.065	0.021
	N	5	5	5	5	5	5	5
9F	Mean	4.13	4.13	0.01	0.01	0.05	0.04	0.09
	SD	0.253	0.239	0.046	0.074	0.027	0.069	0.078
	N	5	5	5	5	5	5	5
10F	Mean	3.82	3.85	- 0.02	- 0.01	0.10	0.01	0.14
	SD	0.143	0.162	0.044	0.023	0.038	0.046	0.092
	N	5	5	5	5	5	5	5
11F	Mean	3.80	3.75	- 0.02	- 0.01	0.04	0.03	- 0.02
	SD	0.252	0.306	0.052	0.037	0.080	0.044	0.040
	N	2	2	5	5	5	4	2

Gravid Uterine Weight, Adjusted Body Weight and Adjusted Body Weight Change: Group Mean values (kg) Embryo-Foetal Phase

Group/Sex		Body Weight on Day 6	Terminal Body Weight on Day 29	Body Weight Change Days 6-29	Gravid Uterine Weight	Adjusted Body Weight Day 29	Adjusted Body Weight Change Days 6-29
5F	Mean	3.28	3.46	0.18	0.38	3.08	- 0.21
	SD	0.291	0.245	0.095	0.082	0.279	0.043
	N	5	5	5	5	5	5
9F	Mean	3.93	4.13	0.20	0.52	3.61	- 0.32
	SD	0.296	0.235	0.137	0.074	0.203	0.191
	N	5	5	5	5	5	5
10F	Mean	3.62	3.84	0.22	0.48	3.36	- 0.26
	SD	0.292	0.170	0.132	0.118	0.256	0.086
	N	5	5	5	5	5	5
11F	Mean	3.73	3.75	0.02	0.49	3.26	- 0.47
	SD	0.325	0.303	0.022	0.030	0.274	0.051
	N	2	2	2	2	2	2

Food Consumption: Group Mean Values (g/animal/day) Embryo-Foetal Phase

Group/Sex		Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9	Day 10	Day 11	Day 12
5F	Mean	110	92	98	96	119	91	78	79	80	85	95	91
	SD	23.0	16.9	8.3	12.7	49.1	12.1	22.2	16.4	24.3	19.3	19.0	14.9
	N	5	5	5	5	5	5	5	5	5	5	5	5
9F	Mean	133	131	126	126	134	137	122	122	119	111	109	105
	SD	19.5	25.3	35.0	25.9	21.6	40.6	16.8	10.8	11.9	15.5	18.3	14.4
	N	5	5	5	5	5	5	5	5	5	5	5	5
10F	Mean	114	93	94	105	96	97	100	93	84	80	81	90
	SD	29.8	28.3	20.5	14.9	19.4	21.4	19.2	20.0	12.4	17.2	7.3	10.5
	N	5	5	5	5	5	5	5	5	5	5	5	5
11F	Mean	136	108	111	101	100	106	86	93	97	92	88	88
	SD	19.0	11.4	10.3	8.6	20.9	6.6	12.6	19.0	14.0	15.8	22.1	27.8
	N	5	5	5	5	5	5	5	5	5	5	5	5
Group/Sex		Day 13	Day 14	Day 15	Day 16	Day 17	Day 18	Day 19	Day 20	Day 21	Day 22	Day 23	Day 24
5F	Mean	92	94	87	79	66	74	82	83	85	86	89	93
	SD	10.5	17.8	17.3	29.2	22.9	21.0	30.9	28.0	21.5	9.1	7.8	20.9
	N	5	5	5	5	5	5	5	5	5	5	5	5
9F	Mean	97	100	87	95	84	88	87	99	104	101	94	89
	SD	23.3	20.3	24.0	28.7	21.2	31.7	33.0	22.3	16.4	21.3	19.4	9.1
	N	5	5	5	5	5	5	5	5	5	5	5	5
10F	Mean	84	80	74	88	88	82	83	83	85	85	75	109
	SD	10.7	12.8	11.3	16.9	14.4	21.6	18.0	16.8	24.1	23.7	21.4	53.8
	N	5	5	5	5	5	5	5	5	5	5	5	5
11F	Mean	80	83	84	75	79	45	39	55	70	79	58	41
	SD	22.1	31.4	36.4	42.5	34.0	48.1	49.2	48.6	48.2	4.6	37.4	34.3
	N	5	5	5	5	5	5	5	5	5	4	4	4

Food Consumption: Group Mean Values (g/animal/day) Embryo-Foetal Phase (continued)

Group/Sex		Day 25	Day 26	Day 27	Day 28
5F	Mean	98	97	90	77
	SD	21.5	15.1	25.3	22.6
	N	5	5	5	5
9F	Mean	89	83	85	94
	SD	10.4	10.8	20.8	25.8
	N	5	5	5	5
10F	Mean	74	78	79	86
	SD	40.1	40.6	23.7	24.8
	N	5	5	5	5
11F	Mean	40	51	52	46
	SD	36.2	32.0	19.3	14.6
	N	3	2	2	2

Macropathology: Group Distribution of Findings Embryo-Foetal Phase

Tissue and Finding		Group/Sex	Number of Animals Affected
		Group/Sex	5F	9F	10F	11F
		Number Examined	6	6	6	6
Skin	Hair loss		1	0	0	1

Lungs and bronchi	Dark		1	0	0	0
Lungs and bronchi	Dark area(s)		0	0	1	0

Liver	Lobular pattern accentuated		0	0	0	1*
Liver	Pale		0	0	0	3*

Kidneys	Pale		0	0	0	3*

General comments	Fur stained		0	1	2	3

Adipose tissue	Nodular		0	0	0	1*
Adipose tissue	Nodule(s)		0	0	0	1

* Findings apparent in premature decedent animals.

Litter Data: Group Mean Values Embryo-Foetal Phase

Group/Sex		Corpora Lutea	Implantations	Resorptions			Live Young			Sex Ratio (%M)	Implantation Loss (%)
Group/Sex		Corpora Lutea	Implantations	Early	Late	Total	Male	Female	Total	Sex Ratio (%M)	Pre-	Post-
5F	Mean	9.0	8.0	2.2	0.2	2.4	2.4	3.2	5.6	37.1	10.9	27.9
	SD	1.00	1.00				1.82	0.45	1.95
	N	5	5	5	5	5	5	5	5	5	5	5
9F	Mean	10.6	8.6	0.8	0.4	1.2	2.6	4.8	7.4	36.1	18.3	12.4
	SD	1.67	1.52				1.14	1.64	0.55
	N	5	5	5	5	5	5	5	5	5	5	5
10F	Mean	10.0	8.4	0.8	0.2	1.0	2.6	4.8	7.4	35.7	17.3	10.0
	SD	2.24	2.70				1.14	2.05	2.51
	N	5	5	5	5	5	5	5	5	5	5	5
11F	Mean	8.5	8.0	0.0	0.0	0.0	2.5	5.5	8.0	31.3	5.6	0.0
	SD	0.71	0.00				0.71	0.71	0.00
	N	2	2	2	2	2	2	2	2	2	2	2

Placental, Litter and Foetal Weights: Group Mean Values (g) Embryo-Foetal Phase

Group/ Sex		Placental Weight	Litter Weight	Litter Size	Male Foetal Weight	Female Foetal Weight	Overall Foetal Weight
5F	Mean	5.5	243.1	5.6	43.9	44.7	44.5
	SD	0.35	64.49	1.95	6.28	3.33	4.60
	N	5	5	5	4	5	5
9F	Mean	5.8	321.5	7.4	43.2	44.0	43.4
	SD	0.79	43.30	0.55	4.99	2.80	3.81
	N	5	5	5	5	5	5
10F	Mean	5.2	309.8	7.4	44.5	41.9	42.8
	SD	0.67	79.87	2.51	4.81	4.45	4.59
	N	5	5	5	5	5	5
11F	Mean	5.7	312.7	8.0	38.2	39.6	39.1
	SD	0.66	8.35	0.00	0.39	1.88	1.04
	N	2	2	2	2	2	2

Conclusions:

Under the conditions of the study and specifically the results obtained in the preliminary embryo-foetal phase, it was concluded that dose levels of 150 mg/kg/day and above clearly exceeded the maximum tolerated dose (MTD) in pregnant female New Zealand White rabbits and are unsuitable for further investigation. There was no evidence of any test material-related maternal or foetal effects at 30 or 65 mg/kg/day.
Although identifying findings of concern in a limited data set, this preliminary study does not provide sufficient data for basis of classification owing to the excessive maternal toxicity apparent at all dose levels where embryo-foetal effects are apparent. In order to clarify this concern it is recommended that a full OECD Guideline 414 study is conducted in the rabbit. This would allow a conclusion on the appropriate C&L to be reached.

Executive summary:

There is no literature available relating to the potential effects of the test material exposure in rabbits. Consequently, the purpose of the pilot phase of this study was to assess the tolerability of the test material when administered orally (by gavage) to the non-pregnant rabbit, to establish suitable doses for investigation in the preliminary embryo-foetal phase. The preliminary embryo-foetal phase assessed the influence of the test material on embryo-foetal survival and development in the New Zealand White rabbit in order to establish suitable doses for a main embryo-foetal toxicity study. Although no claim for compliance with Good Laboratory Practice was made, the work performed generally followed Good Laboratory Practice principles.

The study was split into two phases. Initially for the pilot phase, three non-pregnant females received the test material at a dose level of 300 mg/kg/day in arachis oil vehicle by oral gavage administration at a volume-dose of 5 mL/kg body weight. As a result of effects observed, a similarly constituted control group received the vehicle only in order to ascertain if the effects seen previously were treatment or vehicle-related. It was concluded that effects were vehicle related and therefore a replacement vehicle, 1 % methylcellulose, was used for the remainder of the study. Three females received the test material at a dose level of 600 mg/kg/day for 14 days. Based on the results seen at 600 mg/kg/day, a further three females were assigned to the pilot phase and received the test material at a dose of 1 000 mg/kg/day for 14 days.

For the preliminary embryo-foetal phase, initially three groups of six females received the test material at dose levels of 300, 600 or 1 000 mg/kg/day by oral gavage administration at 5 mL/kg/day from day 6 to 28 after mating. A control group of six females received the vehicle, 1 % methylcellulose, at the same volume-dose and for the same duration. Animals were killed on day 29 after mating for reproductive assessment and foetal examination. As a result of clear test material-related effects, three further treated groups were added to the preliminary embryo-foetal phase. These groups comprised six females and received the test material at dose levels of 30, 65 or 150 mg/kg/day by oral gavage administration at 5 mL/kg/day from day 6 to 28 after mating. Animals were killed on day 29 after mating for reproductive assessment and foetal examination.

Clinical and post-dosing observations, body weight and food consumption were recorded. All adult females were examined macroscopically at necropsy. For the preliminary embryo-foetal phase, the gravid uterus weight was recorded for pregnant females only and all foetuses were examined macroscopically at necropsy.

In the pilot phase of the study, administration of arachis oil formulations (with or without the test material) to female rabbits was not tolerated and all animals were prematurely killed on day 3-4 of the study due to a marked decline in general clinical condition, weight loss, negligible food intake; marked macroscopic disturbance to the lower gastro-intestinal tract was evident at necropsy. Non-pregnant rabbits tolerated the test material administration at dose levels up to and including 1 000 mg/kg/day when formulated in 1 % methylcellulose. Possible treatment-related effects were limited to short periods of reduced food intake at 1 000 mg/kg/day (although there was no consistent pattern for when these reductions occurred) and a low incidence of minor transient changes in clinical condition, manifest as low water intake (2/3 females at 1 000 mg/kg/day, low hay intake (1/3 females in each of the 600 or 1 000 mg/kg/day groups), pale faeces (1/3 females at 1 000 mg/kg/day) and reduced faecal pellet size (1/3 females at 1 000 mg/kg/day).

In the preliminary embryo-foetal phase, the test material administration to mated female rabbits at dose levels of 150 mg/kg/day and above was not tolerated. At 600 or 1 000 mg/kg/day, although there was no evidence of toxicity to the mated females, treatment resulted in apparent non-pregnancy in all six females in each group. Treatment at 150 or 300 mg/kg/day was also not tolerated, with three of the six females in each dose group prematurely terminated for reasons of animal welfare due to deteriorating clinical condition on day 16 - 18 after mating at 300 mg/kg/day and on day 22 - 26 at 150 mg/kg/day, with common clinical signs including underactive behaviour, reduced body temperature and changes in excreta output, negligible food intake and weight loss. Two of the decedent females at 300 mg/kg/day and one decedent female at 150 mg/kg/day showed a total litter resorption; the other decedent female at 300 mg/kg/day had eight implantation sites but only three live embryos, one decedent female at 150 mg/kg/day had eight implantation sites with two dead and six live foetuses and the other decedent female at 150 mg/kg/day had eight implantation sites, with two resorptions, five normal live foetuses and one foetus with acephaly and abdominal omphalocele. Among surviving females in the 150 or 300 mg/kg/day groups, only two females in the 150 mg/kg/day group were pregnant with live foetuses on day 29 of gestation, and one female had one live foetus in the 300 mg/kg/day group.

Treatment at 30 or 65 mg/kg/day was well tolerated. There were no premature deaths, no the test material-related changes in maternal clinical condition apparent, body weight performance and food intake was not affected by test material administration and there were no treatment-related macroscopic abnormalities detected at scheduled termination on day 29 after mating. There was no effect of maternal treatment with the test material at doses up to and including 65 mg/kg/day on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and post-implantation losses. Placental, litter and foetal weights were similar to the control.

Although identifying findings of concern in a limited data set, this preliminary study does not provide sufficient data for basis of classification owing to the excessive maternal toxicity apparent at all dose levels where embryo-foetal effects are apparent. In order to clarify this concern it is recommended that a full OECD Guideline 414 study is conducted in the rabbit. This would allow a conclusion on the appropriate C&L to be reached.

Reference 4

Endpoint:: developmental toxicity
Type of information:: read-across from supporting substance (structural analogue or surrogate)
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:: The data on the read-across substance is considered representative.
Reason / purpose for cross-reference:: read-across source

Reference 5

Endpoint:: developmental toxicity
Type of information:: read-across from supporting substance (structural analogue or surrogate)
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:: The data on the read-across substance is considered representative.
Reason / purpose for cross-reference:: read-across source

Reference 6

Endpoint:: developmental toxicity
Type of information:: experimental study planned
Study period:: To be confirmed
Justification for type of information:: TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Manganese carbonate

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no GLP studies available for this substance for this endpoint.
- Available non-GLP studies: There are no non-GLP studies available for this substance for this endpoint.
- Historical human data: There are no historical human data available for this endpoint.
- (Q)SAR: For this endpoint there is limited availability of (Q)SAR models whose scientific validity has been established, particularly with applicability domains relevant for this type of substance. As such the documentation from such a prediction may not prove adequate or reliable and the results may not be sufficient for classification, labelling and risk assessment purposes.
- In vitro methods: No validated in vitro methods are available for this endpoint.
- Weight of evidence: There are insufficient data to use a weight of evidence approach to fulfil this endpoint. Currently there are no OECD 414 compliant developmental toxicity studies conducted on the substance itself. A Combined Pilot Study and Preliminary Embryo-Foetal Development Study was conducted but the purpose of the pilot phase of this study was to assess the tolerability of Manganese carbonate when administered orally (by gavage) to the non-pregnant rabbit and to establish suitable doses for investigation in the preliminary embryo-foetal phase. The preliminary embryo-foetal phase assessed the influence of Manganese carbonate on embryo-foetal survival and development in the New Zealand White rabbit in order to establish suitable doses for a main embryo-foetal toxicity study should the need arise for work safety reasons and for future regulatory compliance. The study was not designed to provide dose descriptors such as No Observed Adverse Effect Levels and as such is insufficient for assessment for classification, labelling and hazard assessment purposes. Prior to the pilot study there were no repeat dose data available for any test species with this test material. Furthermore, there are no literature data available relating to the potential effects of manganese exposure in rabbits. Consequently, there is insufficient data to conduct a weight of evidence assessment.
- Grouping and read-across: Read-across from MnCl2 studies via the inhalation and subcutaneous routes are used as supporting data, however these studies are conducted in rats and mice respectively. Given the lack of long-term toxicity data on the manganese substances in rabbits this is considered insufficient justification to waive developmental toxicity testing in rabbits.
- Substance-tailored exposure driven testing: Exposure-based waiving of some tests may be permitted in cases where it can be shown that exposure is insignificant or absent for the substance concerned. The substance is not used or transported under strictly controlled conditions throughout the life cycle and the potential for human contact exists. Therefore exposure-based waiving is not applicable.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Column 2 outlines the rules for adaptation of the tests specified in Column 1. In the case of the proposed pre-natal developmental toxicity study required under Annex IX, no adaptation is available for this endpoint.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: In accordance with the principles of reduction, refinement and replacement, the proposed pre-natal developmental toxicity study, will be performed in one species, via the most appropriate route of administration and having regard to the likely route of human exposure. The test proposed will be performed according to OECD Test Guideline 414 as specified in Column 1 of Annex IX.
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Species:: rabbit

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
: 5 mg/m³
Study duration:: subacute
Species:: rat
Quality of whole database:: Good developmental toxicity data in two species exists. No further data is considered necessary.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 200 mg/kg bw/day
Study duration:: subacute
Species:: mouse
Quality of whole database:: The subcutaneous study was performed to sound scientific principles with a sufficient level of detail to assess the quality of the relevant results. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2.
Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds, such as the registered substance.

Additional information

Adaptation of Developmental Toxicity Testing with the Registration Substance

In accordance with REACH Annex XI, Section 1.1; use of existing data, developmental toxicity testing is not considered to be required since data are available on a more soluble (and therefore more bioavailable) Mn substance, manganese chloride (MnCl2). Because the bioavailability of manganese ions from manganese chloride would be substantially greater, the results can be considered a worst-case for manganese carbonate. Also given that exposure to manganese ions from MnCl2 was not teratogenic at up to 8 mg/kg introduced subcutaneously in Sanchez et al, 1993 (Klimisch score 2) it is unlikely that exposure to manganese ions deriving from manganese carbonate administered orally will be teratogenic either.

ECHA had requested a study via the oral route for the carbonate, rather than the inhalation route, because of the possibility to maximise systemic exposure, and identify the absolute hazard with respect to the developmental toxicity. However, systemic exposure to manganese can be considered to have been maximised in the Sanchez 1993 subcutaneous exposure study with the very soluble chloride, and it can be considered to be addressed from this point of view. Systemic exposure of upto 8 mg/kg of hydrated MnCl2 in Sanchez equates to 1.7 mg Mn /kg, which in turn equates to an equivalent of >71 mg/kg of carbonate. Some common minor skeletal effects were observed at 8 mg of the hydrated chloride/kg, but these are known to be reversible before or after birth. Embryotoxicity and resorptions were observed at lower doses, and this is likely to be a secondary result of manganese ions displacing iron, rather than an intrinsic developmental toxicity of manganese. Further support for the absence of any relevant developmental toxicity for manganese and manganese carbonate comes from a literature review (See Strupp and Furness, 2009 attached) covering the literature dating back 50 years (on human and animal data on all aspects of development toxicity to manganese-based compounds). Being an essential nutrient, necessary for the formation of bones, coupled with its poor absorption and the efficient homeostatic control of manganese in the body, it is very unlikely that MnCO3 will cause developmental effects.

Further testing is therefore considered unlikely to provide any additional or useful information and its absolute necessity would therefore be questionable on animal welfare grounds. The registrant is aware that ECHA requested a pre-natal developmental toxicity study via the oral route, however, a study via the inhalation route was already conducted by the International Manganese Institute in their global worker safety program. This study has been used to comply with this endpoint and is available in this dataset. To investigate via the oral route – in order to maximize the investigation on hazard potential and to comply with the route specified in the final decision - oral, the registrant is in the process of conducting a global research study in rabbits as no rabbit studies exist within the manganese data pool. The study will be completed by Q4 2017 and the dossier will then be updated accordingly.

Read-Across Key Study: Dettwiler (2014)

Data from the International Manganese Institutes worker safety program on MnCL2 (more soluble and more bioavilable) via inhalation OECD 414, EU Method B.31, US EPA OPPTS 870.3700, and Japanese Guideline 12 Nohsan No. 8147 (2-1-18) under GLP conditions was available. Treatment caused an increase in the incidence of large or slightly large foetal thyroid and diffuse thyroid follicular hypertrophy and/or hyperplasia at the top dose. This effect has a significant background incidence in control animals, and the maximum severity seen in top dose animals did not exceed the maximimum seen in control. It was also seen in the presence of maternal toxicity. In the 2-generation reproduction study (Jardine, 2015), no thyroid effect was apparent in pups sacrificed at weaning. Overall it is considered to be a transient effect and of no toxicological relevance. Under the conditions of the Dettwiler 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 mg/m3 air based on reduced food consumption and body weight gain, and a reduction in corrected body weight gain in pregnant females. In non-pregnant females, the NOEL for systemic toxicity was established at 15 mg/m3 air, whereas the NOAEL was established at 25 µg/L air, based on the same effects. The NOEL for prenatal developmental toxicity was considered to be 15 µg/L air based on bone ossification effects and wavy ribs which are reversible after birth.

Read-Across Supporting Study: Sanchez (1993)

In a developmental toxicity study broadly compliant with OECD 414, female mice were administered either 2, 4, 8, or 16 mg/kg of manganese chloride subcutaneously daily from day 6-15 of gestation. Maternal toxicity was observed at the highest dose level with significant mortality (32%). There was also a significant effect on body weight (decrease), organ weight (liver and kidney), as well as reduced food consumption. The only gestational parameter affected by manganese chloride treatment was an increase in late resorptions. In pups, increasing concentrations of manganese chloride caused decreasing body weight. Relatively minor skeletal effects were also observed. Since severe maternal toxicity was observed it cannot be excluded that the effect on late resorptions and on pups is due to this effect. The NOAEL for embryotoxicity (late resorptions) was 2 mg/kg bw/day (equivalent to a 200 mg/kg bw/day external dose given dermal absorption of 1%). The NOAEL for maternal effects (reduced body weight gain) was 4 mg/kg bw/day (equivalent to 400 mg/kg bw/day external dose). The NOEL for developmental toxicity (wavy ribs and delayed or reduced ossifications in the sternebra, parietal and occipital bones) was 4 mg/kg bw/day (equivalent to 400 mg/kg bw/day external dose).

Pilot Study: Stannard (2020)

The developmental toxicity of the test material was assessed in a study which was not designed to meet any particular regulatory requirements and although no claim for compliance with Good Laboratory Practice was made, the work performed generally followed Good Laboratory Practice principles. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al (1997).

There is no literature available relating to the potential effects of the test material exposure in rabbits. Consequently, the purpose of the pilot phase of this study was to assess the tolerability of the test material, an industrial chemical, when administered orally (by gavage) to the non-pregnant rabbit, to establish suitable doses for investigation in the preliminary embryo-foetal phase. The preliminary embryo-foetal phase assessed the influence of the test material on embryo-foetal survival and development in the New Zealand White rabbit in order to establish suitable doses for a main embryo-foetal toxicity study.

The study was split into two phases. Initially for the pilot phase, three non-pregnant females received the test material at a dose level of 300 mg/kg/day in arachis oil vehicle by oral gavage administration at a volume-dose of 5 mL/kg body weight. As a result of effects observed, a similarly constituted control group received the vehicle only in order to ascertain if the effects seen previously were treatment or vehicle-related. It was concluded that effects werevehicle related and therefore a replacement vehicle, 1 % methylcellulose, was used for the remainder of the study.Three females received the test material at a dose level of 600 mg/kg/day for 14 days. Based on the results seen at 600 mg/kg/day, a further three females were assigned to the pilot phase and received the test material at a dose of 1000 mg/kg/day for 14 days.

of clear test material-related effects, three further treated groups were added to the preliminary embryo-foetal phase. These groups comprised six females and received the test material at dose levels of 30, 65 or 150 mg/kg/day by oral gavage administration at 5 ml/kg/day from day 6 to 28 after mating. Animals were killed on day 29 after mating for reproductive assessment and foetal examination.

In the pilot phase of the study, administration of arachis oil formulations (with or without the test material)to female rabbits was not tolerated and all animals were prematurely killed on day 3-4 of the study due to a marked decline in general clinical condition, weight loss, negligible food intake; marked macroscopic disturbance to the lower gastro-intestinal tract was evident at necropsy. Non-pregnant rabbits tolerated the test material administration at dose levels up to and including 1000 mg/kg/day when formulated in 1 % methylcellulose. Possible treatment-related effects were limited to short periods of reduced food intake at 1000 mg/kg/day (although there was no consistent pattern for when these reductions occurred) and a low incidence of minor transient changes in clinical condition, manifest as low water intake (2/3 females at 1000 mg/kg/day, low hay intake (1/3 females in each of the 600 or 1000 mg/kg/day groups), pale faeces (1/3 females at 1000 mg/kg/day) and reduced faecal pellet size (1/3 females at 1000 mg/kg/day).

In the preliminary embryo-foetal phase, the test material administration to mated female rabbits at dose levels of 150 mg/kg/day and above was not tolerated. At 600 or 1000 mg/kg/day, although there was no evidence of toxicity to the mated females, treatment resulted in apparent non-pregnancy in all six females in each group. Treatment at 150 or 300 mg/kg/day was also not tolerated, with three of the six females in each dose group prematurely terminated for reasons of animal welfare due to deteriorating clinical condition on day 16-18 after mating at 300 mg/kg/day and on day 22-26 at 150 mg/kg/day, with common clinical signs including underactive behaviour, reduced body temperature and changes in excreta output, negligible food intake and weight loss. Two of the decedent females at 300 mg/kg/day and one decedent female at 150 mg/kg/day showed a total litter resorption; the other decedent female at 300 mg/kg/day had eight implantation sites but only three live embryos, one decedent female at 150 mg/kg/day had eight implantation sites with two dead and six live foetuses and the other decedent female at 150 mg/kg/day had eight implantation sites, with two resorptions, five normal live foetuses and one foetus with acephaly and abdominal omphalocele. Among surviving females in the 150 or 300 mg/kg/day groups, only two females in the 150 mg/kg/day group were pregnant with live foetuses on day 29 of gestation, and one female had one live foetus in the 300 mg/kg/day group.

Classification and Labelling Discussion

There are two developmental toxicity studies available from which classification and labelling is derived. Dettwiler (2014) conducted in Han Wistar rats according to OECD Test Guideline 414 via the inhalation route and Stannard (2020) a combined pilot study and preliminary embryo-foetal development study in New Zealand White rabbits via the oral (gavage) route. As the Stannard study was a pilot study it was not conducted to a specific guideline.

Developmental toxicity data in the rat on the read-across substance manganese dichloride has no classification for developmental toxicity.

In rabbits, the pilot study was conducted because there was no literature available relating to the potential effects of manganese exposure in this species. It identified exposure up to the limit dose of 1 000 mg/kg/day in non-pregnant rabbits was generally well tolerated for 14 days.

The preliminary study identified clear embryo-foetal toxicity at dose levels ≥ 150 mg/kg/day (decreased embryo-foetal survival) but only in the presence of overt, excessive maternal toxicity which manifested as premature mortality, with an associated decline in clinical condition, body weight loss and inappetence (150 mg/kg/day was associated with maternal mortality of 50 %). ECHA CLP guidance (See reference below: Annex I:3.7.2.4.4) defines that ‘maternal mortality greater than 10 % is considered excessive and the data from that dose level shall not normally be considered further for evaluation’.

Hence, it is justified to consider that although identifying findings of concern in a limited data set, the preliminary study does not provide sufficient data for basis of classification owing to the excessive maternal toxicity apparent at all dose levels where embryo-foetal effects are apparent. In order to clarify this concern it is recommended that a full OECD Guideline 414 study is conducted in the rabbit. This would allow a conclusion on the appropriate C&L to be reached.

In order to clarify this concern it is recommended that a full OECD Guideline 414 study is conducted in the rabbit. This would allow a conclusion on the appropriate C&L to be reached.

Reference: Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 5.0 July 2017, ECHA.

Toxicity to reproduction: other studies

Description of key information

Senn (2014)

The purpose of the Senn 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 resulting from inhalation. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 426 and EPA OPPTS 870.6300.

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2013 to 4 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:

The data on the read-across substance is considered representative.

Reason / purpose for cross-reference:

other: Target record

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 426 (Developmental Neurotoxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Deviations:

GLP compliance:

yes

Type of method:

in vivo

Species:

rat

Strain:

other: HanRcc: WIST(SPF)

Sex:

female

Details on test animals or test system 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

Remarks:

Doses / Concentrations:
0, 5, 15, 25 µg/L air
Basis:
other: target aerosol concentration

Remarks:

Doses / Concentrations:
0, 3.5, 12.3, 17.6 µg/L air
Basis:
other: 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:

12.3 mg/m³ air

Based on:

test mat.

Sex:

male/female

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
Date of sampling	Group	Filter No.	Gravimetric	ASS	Recovery (%)	Correction factor
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	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
Day	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
Day	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 the study the NOEL (No Observed Effect Level) for dams and pups was established at 12.3 μg/L air.

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.

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) with steadily decreasing numbers of affected dams towards the end of the period. During the lactation period no breathing problems occurred, except for three females towards the end of this period. One dam was observed to show signs of severely laboured breathing together with a weakened condition, body weight loss, ruffled fur and a hunched posture; this dam was subsequently killed in extremis on day 15 post coitum.

Dams at dosages of 12.3 and 17.6 μg/L air had reduced mean food consumption directly after treatment commencement. At the mid dose level this effect lasted for one week; within this week mean body weight gain was slightly 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 4% during the first week after 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 air were considered to be not adverse, whereas the effects at 17.6 μg/L air, despite recovery, were considered to be adverse due to the severe clinical signs at one animal. No test material-related effects were noted at 3.5 μg/L air.

Treatment of the dams did not affect the viability or survival of the offspring. However, pups from the high dose group showed slightly reduced mean food consumption from day 21 post partum onwards. Mean body weight gain was dose-dependently reduced in all dose groups after weaning, although this was not always statistically significantly different in the low and mid dose group. 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, the reduction in mean body weight gain was considered to be not adverse in contrast to the high dose group, in which a reduction of 5% was measured.

The retardation in body weight development was not reflected at any other parameters such as developmental indices, sexual maturation or morphometric measurements of the brain.

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.

Based on these findings the NOEL (No Observed Effect Level) for dams and pups was established at 12.3 μg/L air.

Additional information

Senn (2014)

During the study manganese dichloride 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. 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) with steadily decreasing numbers of affected dams towards the end of the period. During the lactation period no breathing problems occurred, except for three females towards the end of this period. One dam was observed to show signs of severely laboured breathing together with a weakened condition, body weight loss, ruffled fur and a hunched posture; this dam was subsequently killed in extremis on day 15 post coitum. Dams at dosages of 12.3 and 17.6 μg/L air had reduced mean food consumption directly after treatment commencement. At the mid dose level this effect lasted for one week; within this week mean body weight gain was slightly 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 4% during the first week after 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 air were considered to be not adverse, whereas the effects at 17.6 μg/L air, despite recovery, were considered to be adverse due to the severe clinical signs at one animal. No test material-related effects were noted at 3.5 μg/L air. Treatment of the dams did not affect the viability or survival of the offspring. However, pups from the high dose group showed slightly reduced mean food consumption from day 21 post partum onwards. Mean body weight gain was dose-dependently reduced in all dose groups after weaning, although this was not always statistically significantly different in the low and mid dose group. 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, the reduction in mean body weight gain was considered to be not adverse in contrast to the high dose group, in which a reduction of 5% was measured. The retardation in body weight development was not reflected at any other parameters such as developmental indices, sexual maturation or morphometric measurements of the brain. 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. Based on these findings the NOEL (No Observed Effect Level) for dams and pups was established at 12.3 μg/L air.

Justification for classification or non-classification

A guideline and GLP compliant 2 generation rat inhalation reproductive toxicity study was conducted with a more efficient provider of soluble manganese ions, namely manganese dichloride. At dose levels showing evidence of tolerable but clear parental toxicity (manifesting as decreased food intake and body weight performance), there was no effect on oestrous cyclicity, mating performance, sexual maturity, fertility or duration of gestation or litter size, the sperm motility or morphology, nor on the formation and maturation of ovarian follicles in either generation. Manganese chloride is therefore not a reprotoxicant, having no effect on sexual function or fertility, or on post-natal development in the rat. Therefore, soluble and insoluble forms of inorganic manganese compounds by extrapolation are considered not reprotoxicants.

In addition, a literature review by Strupp and Furnes (2009) showed no evidence of reproductive toxicity for manganese carbonate.

Hence, there is considered to be no requirement to classify manganese carbonate for reproductive toxicity in the absence of data warranting classification.

Developmental toxicity data in the rat on the read-across substance manganese dichloride has no classification for developmental toxicity.

Hence, it is justified to consider that although identifying findings of concern in a limited data set, the preliminary study does not provide sufficient data for basis of classification owing to the excessive maternal toxicity apparent at all dose levels where embryo-foetal effects are apparent. In order to clarify this concern, it is recommended that a full OECD Guideline 414 study is conducted in the rabbit. This would allow a conclusion on the appropriate C&L to be reached.

In order to clarify this concern, it is recommended that a full OECD Guideline 414 study is conducted in the rabbit. This would allow a conclusion on the appropriate C&L to be reached.

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to either reproductive or developemental toxicity.

Reference: Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 5.0 July 2017, ECHA.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

F0 Males
Time-point	Blood Mn conc (ppb w/v (ng/mL))
Time-point	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	7	7	7	6
Prior to mating	6	13	23	27
Prior to Necropsy	6	19	27	29
F0 Females
Time-point	Blood Mn conc (ppb w/v (ng/mL))
Time-point	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	7	7	7	7
Prior to mating	6	16	28	39
Prior to Necropsy	7	16	24	33

F1 Males
Time-point	Blood Mn conc (ppb w/v (ng/mL))
Time-point	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	12	16	16	17
Prior to mating	6	9	13	19
Prior to Necropsy	6	9	14	21
F1 Females
Time-point	Blood Mn conc (ppb w/v (ng/mL))
Time-point	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	13	12	15	15
Prior to mating	6	10	16	23
Prior to Necropsy	7	10	16	21

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

Manganese carbonate

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Referenceopen allclose all

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Toxicity to reproduction: other studies

Description of key information

Link to relevant study records

Reference

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all

Referenceopen all close all