Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Inhalation Route

Two Generation Toxicity to Reproduction – Inhalation Route: Read-Across From Grieve (2017)

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L. The No Observed Effect Level (NOEL) for reproductive toxicity was determined to be 20 µg/L.

Dermal Route

The registered substance is very poorly  water soluble (IUCLID section 4.8), hence minimal amount of the potential substance is made available for systemic absorption via the dermal route. Even if minimally available from exposure, the physiological properties of the registered substance do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies (IUCLID section 7.3) . Moreover, this is not the most likely route of systemic exposure. Therefore, according to Column 2, Section 8.6 of Regulation No 1907/2006 , this test is not considered necessary.  

Oral Route

MnO is mostly used in industrial settings where good industrial hygiene is employed.  Outside these industrial settings, the registered substance is used by trained professionals.  Exposure via the oral route is therefore implausible. Therefore, this is not a likely route of exposure. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO (IUCLID 5 section 7.2.1) confirms no evidence of toxicity via this route. Therefore in accordance with Annex XI section 1.1, this test is not considered necessary.  

Link to relevant study records

Referenceopen allclose all

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:
Justification for type of information:
Inhalation: The particle size distribution test indicates that inhalation is a possible route of exposure . However, an investigation on the bioavailability of the registered substance in gastric and artificial lung fluid demonstrates the low bioavailability of the substance in both compartments (see Andersen K, section 7.12). To support this, MnO is not acutely toxic by the oral route (IUCLID section 7.2.1) and not acutely harmful by the inhalation route ( IUCLID section 7.2.2). A literature review of available human and animal data on reproductive toxicity to manganese-based compounds showed equivocal evidence of reproductive toxicity with no report or incidence on MnO specifically. Therefore, taking into account both the low bioavailability of MnO and animal welfare, this test is not considered scientifically necessary in accordance with Annex XI, section 1.1. Dermal: The registered substance is very poorly water soluble (IUCLID section 4.8), hence minimal amount of the potential substance is made available for systemic absorption via the dermal route. Even if minimally available from exposure, the physiological properties of the registered substance do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies (IUCLID section 7.3) . Moreover, this is not the most likely route of systemic exposure. Therefore, according to Column 2, Section 8.6 of Regulation No 1907/2006 , this test is not considered necessary. Oral: MnO is mostly used in industrial settings where good industrial hygiene is employed. Outside these industrial settings, the registered substance is used by trained professionals. Exposure via the oral route is therefore implausible. Therefore, this is not a likely route of exposure. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO (IUCLID 5 section 7.2.1) confirms no evidence of toxicity via this route. Therefore in accordance with Annex XI section 1.1, this test is not considered necessary.
Reproductive effects observed:
not specified
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Inhalation: The particle size distribution test indicates that inhalation is a possible route of exposure . However, an investigation on the bioavailability of the registered substance in gastric and artificial lung fluid demonstrates the low bioavailability of the substance in both compartments (see Andersen K, section 7.12). To support this, MnO is not acutely toxic by the oral route (IUCLID section 7.2.1) and not acutely harmful by the inhalation route (IUCLID section 7.2.2). A literature review of available human and animal data on reproductive toxicity to manganese-based compounds showed equivocal evidence of reproductive toxicity with no report or incidence on MnO specifically. Therefore, taking into account both the low bioavailability of MnO and animal welfare, this test is not considered scientifically necessary in accordance with Annex XI, section 1.1.
Dermal: The registered substance is very poorly water soluble (IUCLID section 4.8), hence minimal amount of the potential substance is made available for systemic absorption via the dermal route. Even if minimally available from exposure, the physiological properties of the registered substance do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies (IUCLID section 7.3) . Moreover, this is not the most likely route of systemic exposure. Therefore, according to Column 2, Section 8.6 of Regulation No 1907/2006 , this test is not considered necessary.
Oral: MnO is mostly used in industrial settings where good industrial hygiene is employed. Outside these industrial settings, the registered substance is used by trained professionals. Exposure via the oral route is therefore implausible. Therefore, this is not a likely route of exposure. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO (IUCLID 5 section 7.2.1) confirms no evidence of toxicity via this route. Therefore in accordance with Annex XI section 1.1, this test is not considered necessary.
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 July 2012 to 04 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.
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. Bedding material was sterilised white wood shavings.
- Diet: ad libitum
- Water: Water taken from the public supply was available 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 periods of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 26 °C
- Humidity: 30 - 69 %
- Air changes: at least 10 air changes per hour
- Photoperiod: 12 hours light / 12 hours dark

IN-LIFE DATES:
From: 02 July 2012
To: 04 March 2013
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 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 powder cake 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 an appropriately sized modular nose only stainless steel flow past exposure chamber (in-house design). 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 substrate collection plates (34 mm stainless steel) and back up filter (34 mm Westech) were weighed before and after sampling to determine the total amount of test and/or vehicle aerosol collected in each particle size range.
After weighing, the substrate collection plates and back up filters of were retained in amber glass jars and stored in a refrigerator set to maintain 4 °C.
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 the testing facility.
Duration of treatment / exposure:
F0 animals were dosed for 10 weeks prior to mating; for F0 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F0 females were dosed throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.
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; for F1 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F1 females were dosed throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.
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 gestation 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. 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.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked 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 pre-trial, 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 of 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
- Time schedule: Monitoring of water consumption was limited to a visual inspection of the water bottles on a regular basis throughout the study.

OTHER:
- Bioanalytical Sample Collection
Blood (1 mL) was collected from the tail vein of all animals, after careful cleaning of the sample site to avoid any possible contamination, into tubes containing lithium heparin. Samples were collected at the following time-points:
F0 generation: samples were collected from all animals pre-dose, prior to mating and prior to weaning/necropsy
F1 generation: samples were collected from all animals after selection (timing and volume dependent on weight of animal), prior to mating and at necropsy (or shortly prior to, as appropriate).
The samples were stored at -80 °C prior to analysis of manganese in whole blood performed with a validated ICP-MS method.

- 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.

- Sexual Maturation in F1 Animals
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 was decapsulated and homogenised. 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 number 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 gland, pituitary gland, prostate gland, thyroid glands, kidneys, liver, lungs, ovaries, spleen, testes and 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.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the following tissues were collected from all animals and preserved in 10 % neutral buffered formalin: brain, epididymides, adrenal glands, pituitary gland, prostate gland, seminal vesicle gland, thyroid glands, kidneys, larynx, liver, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, spleen, testes (preserved in modified Davidson’s fixative), anterior and posterior trachea, uterus and vagina.

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) and 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.

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, i.e. pairwise comparison 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 stabilise 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
Description (incidence and severity):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
MORTALITY (PARENTAL ANIMALS)
- F0 animals
Animal 138 (Group 1F) was killed prematurely on Day 97 of the study. The animal was sacrificed at the time of parturition as the animal had difficulty giving birth and there was a pup protruding from the vagina (the animal gave birth to one live pup). The uterus also contained live foetuses and one late death. Animal 330 (Group 3F) was killed prematurely on Day 94 of the study. The animal had a prolonged parturition and had given birth to 3 live pups. One dead foetus was found in the right uterine horn at necropsy. There were no abnormalities detected at histological evaluation.
Animals 228 (Group 2M) and 236 (Group 2F) were killed prematurely on Day 85 and Day 83, respectively due to clinical signs. The male animal had shavings stained red, a cold body, reduced activity, rolling gait, staggering and weight loss. Necropsy findings for this animal included yellow froth filled duodenum, ileum and jejunum, pale foci on kidney, pale foci left lung lobe, enlargement of adrenal gland, small thymus, urinary bladder adhesions. Histological findings included a mild ulcer in the larynx. The female had partially closed eyes, dilated pupils, tremors, unkempt coat, walking on tip toes, irregular respiration, staggering and subdued. Necropsy findings included pale extremities and fluid accumulation in both horns of the uterus (the animal was sacrificed prior to having a clear indication of mating). There were no abnormalities detected at histological evaluation.
There was no treatment related pattern to these deaths and these were not positively attributed to treatment.
- F1 animals
Animal 521 (Group 1M), animal 717 (Group 3M), animal 748 (Group 3F), Animal 752 (Group 3F) and animal 816 (Group 4M) were killed prematurely. However, none of these premature deaths were considered to be related to treatment but were considered to be due to accidental injury.

CLINICAL SIGNS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there were 2/28 males noted as having wheezing respiration. Animal 422 had this sign recorded on only one day (Day 14 of the study) and Animal 424 had wheezing recorded on 5 occasions (Days 91 and 112-115 of the study). Animal 333 (Group 3F) had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over ca. Days 83-90 of the study. Due to the signs dosing for the animal was stopped for a few days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals, these signs were considered to be incidental.
Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
- F1 animals
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).

BODY WEIGHT (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was a decrease in body weight gain in males over Days 0-21 of the study. From Day 21 of the study, the body weight gains were generally comparable to the controls but the group mean weights remained lower than the controls throughout the study. At target 20 μg/L, there was a group mean body weight gain in females prior to mating were similar to the controls, however body weight gains over Days 0-20 of gestation were slightly lower than the controls. Gains over lactation were similar to the controls.
- F1 animals
At target 20 μg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study, however gains over the following week were greater than the controls and then remained comparable with the controls throughout the remainder of the treatment period. Slight intergroup differences in group mean body weight gains in the F1 females prior to mating were too small to be attributed to treatment. At 20 μg/L, there was a slight reduction in body weight gains throughout gestation compared to the controls.
There were no effects of treatment noted in the lactation females.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls. At target 20 μg/L, there was a transient reduction in food consumption in the females on commencement of treatment compared with the controls; however, consumption for the remainder of the pre-mating period was similar to the controls. Slight intergroup differences in the group mean food consumption in the males at target 5 and 10 μg/L were not attributed to treatment. Slight intergroup differences in group mean food consumption throughout gestation and lactation were not attributed to treatment.
- F1 animals
At target 20 μg/L, there was a slight reduction in group mean food consumption in the males over Days 40-68 of the study; these reductions achieved statistical significance. Slight intergroup differences in group mean food consumption at target 5 and 10 μg/L were not attributed to treatment. Group mean food consumption in the females prior to mating and throughout gestation and lactation were comparable to the controls.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
The stages of the oestrus cycles and their mean duration were similar in all groups for both generations.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects on the sperm motility, count or morphology at any of the dose levels applied, in either generation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on mating performance, fertility or duration of gestation in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, reduced brain weights in males achieved statistical significance (P<0.05) compared with controls. However, the lower body weight was also statistically significant (P<0.05); following covariance analysis, brain weight did not achieve significance and therefore was not positively attributed to treatment. In all treated females, there was a statistically significant increase in lung weights, compared with the controls; these increases were still present following covariance analysis (P<0.01 at target 5 μg/L and P<0.001 at target 10 and 20 μg/L). Other slight differences in organ weights such as an increased thyroid weight in males at target 5 μg/L and an increase in kidney weights of females at target 10 μg/L were not attributed to treatment.
- F1 animals
At target 5 and 10 μg/L, kidney weights in males were statistically higher than the control, however there was no dose relationship to this increase and following covariance analysis, these findings were no longer evident. At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights in females (P<0.05 at target 10 μg/L and P<0.001 at target 20 μg/L) following covariance analysis. Other slight differences in organ weights such as an increased adrenal weight in females at target 20 μg/L were not attributed to treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment related findings observed in the reproductive tract in the F0 or F1 generations.
Histological findings were confined to the respiratory tract. 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.

- F0 animals
In the larynx there was a broadly dosage-related minimal to moderate squamous metaplasia with minimal to moderate submucosal inflammation. Minimal to marked ulceration of the laryngeal epithelium was associated with the squamous metaplasia in several animals from all treated groups. Occasional incidences of mineralisation, intraluminal necrotic debris or intra-epithelial pustules were seen in some of the treated animals.
In the lungs the principal test material related change was seen in centroacinar regions where there was minimal or mild inflammation and focal or diffuse minimal or mild bronchoalveolar hyperplasia. This latter finding was considered reactive. Minimal or mild goblet cell hyperplasia in the bronchial or bronchiolar epithelium was present in animals exposed to target 20 μg/L together with occasional incidences of degeneration and/or squamous metaplasia of the bronchiolar epithelium. Minimal inflammatory findings (inflammatory cell foci and perivascular inflammatory cell infiltration) were also present with a greater incidence in animals exposed to the test material than in controls.
In the nasal cavity, minimal or mild goblet cell hyperplasia and minimal to moderate eosinophilic globules in the olfactory epithelium were observed in all the male treated groups and in females exposed to target 10 or 20 μg/L. At all dose levels, there was a greater incidence of minimal or mild submucosal inflammatory cell infiltration compared to controls.
In males, inflammation of the nasolacrimal duct and squamous metaplasia of the ductal epithelium was seen in most animals exposed to target 10 or 20 μg/L. In addition to these changes, incidences of minimal or mild focal degeneration of the olfactory, respiratory or transitional epithelia, minimal or mild atrophy of the olfactory epithelium, ulceration and focal squamous metaplasia were observed, mainly in animals exposed to target 10 or 20 μg/L, but occasionally in animals at target 5 μg/L. Deposits of crystalline material, presumed to be test material, was seen in the nasolacrimal ducts of a few animals in the treated groups.
Minimal goblet cell hyperplasia was observed in the pharynx of most males exposed to target 20 μg/L and there were occasional incidences of minimal or mild focal epithelial degeneration, focal inflammation and focal squamous metaplasia in males exposed to target 10 or 20 μg/L.
In the trachea, minimal or mild focal squamous metaplasia and inflammation at the carina and minimal or mild focal epithelial degeneration at sites other than the carina were observed at all dose levels.
Other microscopic findings observed were considered incidental, or of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
- F1 animals
Crystals were occasionally observed in the nasal cavity and in the pharynx from animals exposed to target 10 or 20 μg/L. They consisted of small amounts of needle-shaped crystals either deposited on the olfactory epithelium in the nasal cavity, or free in the lumen of the pharynx. These crystals were considered to result from deposition of test material in some parts of the respiratory tract.
Squamous metaplasia in the nasal cavity was observed mainly in the nasolacrimal duct and to a lesser extent in the transitional and respiratory epithelia.
In the trachea, findings such as epithelial degeneration, squamous metaplasia and submucosal inflammation were observed predominantly in the carina.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.

OTHER FINDINGS (PARENTAL ANIMALS)
- Sexual Maturation
The age and body weight at preputial separation or vaginal opening of the F1 generation animals in all treated groups was similar to the controls.

- Blood analysis
In all treated groups of the F0 generation, the levels of test material in the blood increased significantly on commencement of dosing 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 material 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.
Dose descriptor:
NOAEL
Remarks:
(toxicity)
Effect level:
20 other: µg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
(reproductive performance)
Effect level:
20 other: µg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed.
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
LITTER SIZE AND PUP MORTALITY
- F0 generation, F1 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 20 μg/L, there was an increase in the number of animals losing more than 2 pups at birth (total pups born/no. of implantation sites). However, the mean birth index (%) was well within the background range and these increases were considered to be incidental.
- F1 generation, F2 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 10 and 20 μg/L, pup survival (no. losing >3 pups) over Days 0-4 of lactation was slightly lower than the controls. However, the number of animals losing the entire litter was comparable with controls and the remaining animals generally lost 4 pups. In addition, there was no clear dose related response to these reductions and these were considered not to be an effect of treatment.

LITTER AND PUP WEIGHTS
- F0 Generation
In all treated groups, group mean litter and pup weights were comparable to the controls.
- F1 Generation
At target 20 μg/L, group mean litter weights were slightly lower than the controls which reflected the smaller litter size at this level. However, although the litter weights were slightly lower than the controls, the mean pup weights in both males and females were comparable to the controls.

ABNORMALITIES AMONG PUPS
The type and distribution of observations amongst pups did not indicate any association with treatment.

ORGAN WEIGHTS
- F0 generation, F1 production
At target 20 μg/L, there was a reduction in thymus weight of the females, compared with the controls (P<0.01). Following covariance analysis, this reduction did not achieve statistical significance. There were no effects on organ weights at target 5 and 10 μg/L.
- F1 generation, F2 production
Slight intergroup differences in organ weights did not achieve statistical significance and were not attributed to treatment.

GROSS PATHOLOGY
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to treatment with the test material.

HISTOPATHOLOGY
There were no treatment related findings observed in the tissues examined of the F1 or F2 weanlings.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 other: µg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
20 other: µg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed.
Reproductive effects observed:
not specified

Table 1: F0 Blood Analysis Results

F0 Males

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

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))

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

Control animals were exposed via the food and water.

At target 20 μg/L, manganese levels prior to mating were 350 % higher than controls in males and 550 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 383 and 371 % higher for males and females, respectively.

At target 10 μg/L, manganese levels prior to mating were 283 % higher than controls in males and 367 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 350 and 243 % higher for males and females, respectively.

At target 5 μg/L, manganese levels prior to mating were 117 % higher than controls in males and 167 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 217 and 129 % higher for males and females, respectively.

Table 2: F1 Blood Analysis Results

F1 Males

Time-point

Blood Mn conc. (ppb w/v (ng/mL))

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))

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

At target 20 μg/L, manganese levels prior to mating were 217 % higher than controls in males and 283 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 250 and 200 % higher for males and females, respectively.

At target 10 μg/L, manganese levels prior to mating were 117 % higher than controls in males and 167 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 133 and 129 % higher for males and females, respectively.

At target 5 μg/L, manganese levels prior to mating were 50 % higher than controls in males and 66 % higher than controls in females at the pre-mating time-point. At terminal necropsy, these values were 50 and 43 % higher for males and females, respectively.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

Table 3: F0 Group Mean Body Weight Values (g)

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

-7

212

212

207

204

123

128

130

124

0

253

255

246

245

152

156

156

151

7

285

287

275

267*

176

179

178

172

14

314

316

304

288***

195

201

198

191

21

337

343

330

305***

212

218

220

213

28

347

354

339

315***

216

223

229

221

35

366

371

360

338**

232

241

246

237

42

375

382

373

350*

240

252

256

247

49

388

402

384

363*

249

262*

266**

256

56

401

415

397

374*

257

268

271

260

63

419

430

410

389**

262

272

274

265

70

428

441

422

395**

265

276

278

267

77

434

445

433

402**

-

-

-

-

84

442

457

441

414*

-

-

-

-

91

448

465

447

417*

-

-

-

-

98

455

473

453

427*

-

-

-

-

105

463

483

459

436*

-

-

-

-

112

468

485

462

435*

-

-

-

-

119

458

488

478

451

-

-

-

-

Change 0 - 70

-

-

-

-

113

120

122

116

Change 0 - 112

215

231

215

190*

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

***Significantly different from Group 1: p<0.001

Day 0 = first day of treatment

 

Table 4:F0 Females Group Mean Body Weight Values (g) During Gestation and Lactation

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

269

271

281

266

7

294

297

306

290

14

326

327

334

318

20

379

377

388

366

Weight Gain² (% of control)

110 (-)

106 (96)

107 (97)

100 (91)

Day of Lactation³

1

323

273

282

271

7

321

323

326

314

14

344

345

349

336

21

329

330

337

331

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

 

Table 5: F0 Group Mean Body Weight Values (g)

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

0

59

62

60

63

56

58

58

60

5

110

121*

98*

84***

76

79

77

76

12

123

135

126

123

106

110

110

106

19

166

174

167

164

135

137

139

135

26

206

221

215

206

157

161

163

157

33

246

263

254

241

178

180

183

177

40

277

295

285

268

193

197

197

193

47

302

323*

311

291

206

209

211

207

54

319

341*

327

308

216

217

219

214

61

340

364*

344

324

226

225

226

224

68

346

369

351

332

231

231

232

228

75

353

376

358

341

-

-

-

-

82

365

390*

374

256

-

-

-

-

89

374

399

385

365

-

-

-

-

96

376

404*

390

369

-

-

-

-

103

384

415*

399

380

-

-

-

-

110

382

418**

402

384

-

-

-

-

Change 0 - 68

-

-

-

-

175

172

174

168

Change 0 - 110

322

356**

343

322

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

***Significantly different from Group 1: p<0.001

Day 0 = first day of treatment

 

Table 6:F0 Females Group Mean Body Weight Values (g) During Gestation and Lactation

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

232

230

229

229

7

259

257

258

253

14

289

287

289

281

20

339

337

339

329

Weight Gain² (% of control)

107 (-)

107 (100)

110 (103)

100 (93)

Day of Lactation³

1

243

243

237

240

7

290

287

282

280

14

321

317

307

307

21

315

311

304

304

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

Conclusions:
Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L. The No Observed Effect Level (NOEL) for reproductive toxicity was determined to be 20 µg/L.
Executive summary:

The reproductive toxicity of the test material 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.

Male and female Sprague-Dawley rats were exposed to the test material via the inhalation route at target concentrations of 0, 5, 10 and 20 µg/L. F0 animals were randomised into 4 test groups, each containing 28 males and 28 females. These animals were dosed with the test material 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 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 and this accounts for the lower litter weights. 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. 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.

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 dose groups of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

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 material 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 (NOEL) for adult effects was not established due to effects on the respiratory tract. However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of the test material.

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L. The NOEL for reproductive toxicity was determined to be 20 µg/L.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Remarks:
Toxicity
Effect level:
20 other: μg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See "Remark".
Dose descriptor:
NOEL
Remarks:
Reproductive performance
Effect level:
20 other: μg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 other: μg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
20 other: μg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed.
Reproductive effects observed:
not specified
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:
subchronic
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

Two Generation Toxicity to Reproduction – Inhalation Route: Read-Across From Grieve (2017)

The reproductive toxicity of the test material 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.

Male and female Sprague-Dawley rats were exposed to the test material via the inhalation route at target concentrations of 0, 5, 10 and 20 µg/L. F0 animals were randomised into 4 test groups, each containing 28 males and 28 females. These animals were dosed with the test material 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 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 and this accounts for the lower litter weights. 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. 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.

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 dose groups of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

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 material 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 (NOEL) for adult effects was not established due to effects on the respiratory tract. However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of the test material.

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L. The NOEL for reproductive toxicity was determined to be 20 µg/L.

Effects on developmental toxicity

Description of key information

Inhalation Route

Key Study - Inhalation Route: Read-Across From MnCl2 (Dettwiler, 2016) Rat

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.

Dermal Route

The registered substance is very poorly  water soluble (IUCLID section 4.8), hence minimal amount of the potential substance is made available for systemic absorption via the dermal route. Even if minimally available from exposure, the physiological properties of the registered substance do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies (IUCLID section 7.3) . Moreover, this is not the most likely route of systemic exposure. Therefore, according to Column 2, Section 8.6 of Regulation No 1907/2006 , this test is not considered necessary. 

Supporting Study – Subcutaneous Route: Read-Across From MnCl2 (Sanchez et al., 1993)

The author concluded that from 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.

Oral Route

Oral Route: Read-Across From MnCO3 (Stannard, 2020) Pilot Study in Rabbits

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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
Developmental toxicity in rats
Data waiving:
study waived due to provisions of other regulation
Justification for data waiving:
other:
Justification for type of information:
Inhalation: The particle size distribution test indicates that inhalation is a possible route of exposure . However, an investigation on the bioavailability of the registered substance in gastric and artificial lung fluid demonstrates the low bioavailability of the substance in both compartments (see Andersen K, section 7.12). To support this, MnO is not acutely toxic by the oral route (IUCLID section 7.2.1) and not acutely harmful by the inhalation route ( IUCLID section 7.2.2). A literature review of available human and animal data on reproductive toxicity to manganese-based compounds showed equivocal evidence of reproductive toxicity with no report or incidence on MnO specifically. Therefore, taking into account both the low bioavailability of MnO and animal welfare, this test is not considered scientifically necessary in accordance with Annex XI, section 1.1.

Dermal: The registered substance is very poorly water soluble (IUCLID section 4.8), hence minimal amount of the potential substance is made available for systemic absorption via the dermal route. Even if minimally available from exposure, the physiological properties of the registered substance do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies (IUCLID section 7.3) . Moreover, this is not the most likely route of systemic exposure. Therefore, according to Column 2, Section 8.6 of Regulation No 1907/2006 , this test is not considered necessary.

Oral: MnO is mostly used in industrial settings where good industrial hygiene is employed. Outside these industrial settings, the registered substance is used by trained professionals. Exposure via the oral route is therefore implausible. Therefore, this is not a likely route of exposure. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO (IUCLID 5 section 7.2.1) confirms no evidence of toxicity via this route. Therefore in accordance with Annex XI section 1.1, this test is not considered necessary.
Species:
rabbit
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Remarks:
Developmental toxicity in rabbits
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Inhalation: The particle size distribution test indicates that inhalation is a possible route of exposure . However, an investigation on the bioavailability of the registered substance in gastric and artificial lung fluid demonstrates the low bioavailability of the substance in both compartments (see Andersen K, section 7.12). To support this, MnO is not acutely toxic by the oral route (IUCLID section 7.2.1) and not acutely harmful by the inhalation route ( IUCLID section 7.2.2). A literature review of available human and animal data on reproductive toxicity to manganese-based compounds showed equivocal evidence of reproductive toxicity with no report or incidence on MnO specifically. Therefore, taking into account both the low bioavailability of MnO and animal welfare, this test is not considered scientifically necessary in accordance with Annex XI, section 1.1. Dermal: The registered substance is very poorly water soluble (IUCLID section 4.8), hence minimal amount of the potential substance is made available for systemic absorption via the dermal route. Even if minimally available from exposure, the physiological properties of the registered substance do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies (IUCLID section 7.3) . Moreover, this is not the most likely route of systemic exposure. Therefore, according to Column 2, Section 8.6 of Regulation No 1907/2006 , this test is not considered necessary. . Oral: MnO is mostly used in industrial settings where good industrial hygiene is employed. Outside these industrial settings, the registered substance is used by trained professionals. Exposure via the oral route is therefore implausible. Therefore, this is not a likely route of exposure. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO (IUCLID 5 section 7.2.1) confirms no evidence of toxicity via this route. Therefore in accordance with Annex XI section 1.1, this test is not considered necessary.
Species:
rat
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:
no
Remarks:
No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.
Limit test:
no
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:
no
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):
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.

Group Receiving Arachis Oil Only (Group 4): 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.

Groups Receiving Test Material in 1 % Methylcellulose (Groups 2 and 3): Treatment of female rabbits at 600 or 1 000 mg/kg/day resulted in no premature deaths.

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.

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; these premature deaths were related to the test material administration.
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

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

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

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

Dark area(s)

 

0

0

1

0

 

Liver

Lobular pattern accentuated

 

0

0

0

1*

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*

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 (%)

Early

Late

Total

Male

Female

Total

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.

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.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
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:
no effects observed
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.
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
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 January 2014 to 30 June 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
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:
no
GLP compliance:
yes
Limit test:
no
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:
15 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Post-natal effects and increased thyroid size.
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Summary of Performance of Mated Females

Group

1

2

3

4

Dose (μg/L air)

0

5

15

25

Number of mated females

22

22

22

22

Not pregnant

4

3

0

2

Resorptions only

0

1

0

0

No. females with live foetuses at termination*

18

18

22

20

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

 

Inhalation Technical Data

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

- Nominal Aerosol Concentration

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

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

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

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

 

-Gravimetric Aerosol Concentrations

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

 

Table 2: Gravimetric Aerosol Concentrations

Group

Group Gravimetric Aerosol Concentration [μg/L]

Corrected Gravimetric Aerosol Concentration [μg/L]

1

7.8 ± 1.8 (n = 24, CV = 23.5 %)

4.7 ± 1.1

2

25.2 ± 3.0 (n = 24, CV = 12.0 %)

15.1 ± 1.8

3

43.4 ± 2.9 (n = 24, CV = 6.7 %)

26.0 ± 1.7

 

- Particle Size Determination

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

Table 3: Gravimetric determination of particle size distribution

Group

Mean MMAD [μm] (mean GSD)

Range of MMAD [μm]

Range of GSD

Number of Determinations

Mass Percentage of Particles <3.0 μm

2

1.63 (2.40)

1.53 - 1.73

2.24 - 2.59

3

75.7

3

2.04 (2.36)

1.86 - 2.14

2.20 - 2.52

3

67.3

4

1.58 (2.26)

1.46 - 1.67

2.23 - 2.26

3

78.4

Conclusions:
Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) as well as the NOEL (No Observed Effect Level) for the toxicity in pregnant females were considered to be 5 µg/L air. In non-pregnant females, the NOEL for systemic toxicity was established at 15 µg/L air, whereas the NOAEL was established at 25 µg/L air.
Although foetal thyroids were increased in size at 25 µg/L air, a dose which caused adverse maternal toxicity, the causal correlation for these observations was unclear. Also foetal findings at 25 µg/L for the postnatal live young could not be conclusively established as non-treatment related. Therefore the NOEL as well as NOAEL for prenatal developmental toxicity was considered to be 15 µg/L air.
Executive summary:

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

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

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

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

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

- Main study animals

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

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

Treatment caused a dose dependent reduction in food consumption in groups 3 and 4. This reduction was statistically significant during the most of the study and was accompanied by reduced body weights, reduced body weight gain during the study and reduced corrected body weight gain at termination at both dose levels and therefore the effect was considered to be adverse. No test material-related effects on food consumption were noted in group 2.

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

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

- Foetal Data

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

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

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

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

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

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

- Recovery Females

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

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

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

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

Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) as well as the NOEL (No Observed Effect Level) for the toxicity in pregnant females were considered to be 5 µg/L air. In non-pregnant females, the NOEL for systemic toxicity was established at 15 µg/L air, whereas the NOAEL was established at 25 µg/L air.

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

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
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
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity
Dose descriptor:
NOEL
Effect level:
15 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity
Dose descriptor:
NOAEL
Effect level:
25 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity
Dose descriptor:
NOAEL
Effect level:
15 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Post-natal effects and increased thyroid size.
Abnormalities:
not specified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Good level of detail within the reporting of the study, the study follows good basic scientific principles.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
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:
no
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 analytical verification of doses or concentrations:
Not reported
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
Details on study design:
Not reported
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.
Indices:
Not reported
Historical control data:
Not reported
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: Acute subcutaneous LD50
Effect level:
320 other: mg/kg
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day
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:
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
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± 6.0f

44.8± 4.4f

Gravid uterine weight (g)a

20.7± 1.8

19.6± 5.3

18.3± 7.8

9.9± 6.0f

3.4± 1.4f

Corrected body weight (g)a

40.5± 1.8

41.1± 3.9

41.0± 2.7

37.3± 3.6d

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± 0.3e

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± 0.3d

Kidney weight (g)

0.48± 0.05

0.48± 0.04

0.5± 0.05

0.45± 0.04

0.53± 0.07d

Relative kidney weight (%)c

1.18± 0.12

1.17± 0.08

1.21± 0.07

1.21± 0.05

1.28± 0.13d

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, fSignificantly 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± 4.0b

0.3± 0.6b

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± 0.8a

4.1± 2.8b

11.6± 4.1b

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± 0.11b

0.82± 0.10b

Values indicate mean ± SD

a, bSignificantly 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

6b

-

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

5c

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

25c

36c

7c

Litters affected

0

0

11b

18c

6c

Parietal bone, reduced ossification

Foetuses affected

0

0

0

6a

6c

Litters affected

0

0

0

5

6c

Occipital bone, reduced ossification

Foetuses affected

0

0

0

6a

6c

Litters affected

0

0

0

5

6c

a, b, c Significantly different from controls (P < 0.05; P < 0.001, respectively)

Conclusions:
The author concluded that from 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.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
other: Acute subcutaneous LD50
Effect level:
320 other: mg/kg
Based on:
test mat.
Basis for effect level:
other: Acute subcutaneous LD50
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Developmental toxicity
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 µg/m³
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral Route: MnCO3 Stannard (2020) Pilot Study in Rabbits

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.

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.

 

Key Study - Inhalation Route: MnCl2 Dettwiler (2016) Rat

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.

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.

 

Supporting Study – Subcutaneous Route: Read-Across From MnCl2 (Sanchez et al., 1993)

The author concluded that from 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.

Justification for classification or non-classification

Classification via the inhalation route is not justified because the bioavailability of MnO in artificial lung fluid demonstrates the low bioavailability of the substance (see IUCLID section 7.12). To support this, MnO not acutely harmful by the inhalation route ( IUCLID section 7.2.2). A literature review of available human and animal data on reproductive toxicity to manganese-based compounds showed equivocal evidence of reproductive toxicity with no report or incidence on MnO specifically.

Classification via the dermal route is not justified because MnO is very poorly water soluble (IUCLID section 4.8), hence minimal amount of the potential substance is made available for systemic absorption via the dermal route. Even if minimally available from exposure, the physiological properties of MnO do not indicate a significant rate of absorption through the skin. Furthermore, there were no systemic effects or any other evidence of absorption seen in the skin and eye irritation studies IUCLID section 7.3) .

Classification via the oral route is not justified because MnO is mostly used in industrial settings where good industrial hygiene is employed. Outside these industrial settings, the registered substance is used by trained professionals. Exposure via the oral route is therefore implausible. Therefore, this is not a likely route of exposure. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO (IUCLID section 7.2.1) confirms no evidence of toxicity via this route. Leaching studies in simulated gastric juice (see IUCLID section 7.12) show a low level of leaching and combined with the low oral absorption of Mn due to the homeostatic mechanism, add further support for this justification.

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

Additional information