Potassium permanganate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Please refer to the RAAF report attached to section 13 of this dataset which discusses the cateogory approach for sharing of data between the three soluble manganese substance KMnO4, Mn chloride and Mn sulphate.

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  The key study (McGough & Jardine, 2016 published and Grieve, 2017 report unpublished) was conducted according to OECD Guideline 416 with administration via the inhalation route (nose only).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

One supporting study on the registered substance -one generation oral study was also used. This reliability 2 study on Rat (Wistar Han) male/female (10 males and 25 females/group) with Oral: gavage, daily 7 days/week exposure at 0, 20, 80, 320 mg/kg body weight/day (nominal conc.), water as vehicle for a treatment period for the males was 13 weeks (10 weeks of pre-mating and 3 weeks of mating period).Treatment period of females was at least 8 weeks (2 weeks of pre-mating, at least 1 day of mating, 3 weeks of pregnancy and 3 weeks of lactation) report:

-       NOAEL (general toxicity) (male/female): 80 mg/kg bw/day based on decreased body weight in males, inflammation and/or erosion of digestive tract in both sexes at 320 mg/kg bw/day

-       NOAEL (reproduction) (male/female): 80 mg/kg bw/day based on decreased absolute prostate weight, various damage of spermiogenesis, decreased fertility, conception and gestation index at 320 mg/kg bw/day

-       NOAEL (development) (male/female) < 20 mg/kg bw/day based on vacuolation of brain cell nuclei in pups.

From 80 mg/kg bw/day late opening of eyes at all doses.

In addition at 320 mg/kg bw/day: decreased viability index and increase of relative and absolute brain weight of pups.

However, the effects reported in the high dose group in the presence of severe toxicity are considered to be secondary non-specific consequences of parental toxicity. Also the study was considered of poor quality - very limited statistical analysis and no historical data.

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

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

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

There is a smaller body of literature data available on the reproductive/developmental toxicity of effects  MnSO4 and no studies performed to standardised guidelines.  A two-year carcinogenicity (section 7.7) and repeated dose toxicity report (NTP, 1993) showed no effect on the testes of rats exposed orally for up to 2 years.

 

Link to relevant study records

Reference

Reference 1

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material.

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.

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

other: Read-across target

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 (manganese dichloride) 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.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Remarks:

no certificate included - study is published literature from OECD 416, GLP compliance study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD1(SD))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: F0 animals commenced treatment at 6–8 weeks of age; F1 animals commenced treatment shortly after weaning for 11 weeks.
- Housing: Rats were housed in cages. Cages were racked by treatment group with males and females racked separately.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The F0 animals were allowed to acclimate for 13 days before the commencement of dosing.
- For at least 7 days prior to the commencement of dosing, all animals were conditioned to the restraint procedures (to adapt to the inhalation chamber) 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—6 h daily.
- Each batch of diet was routinely analysed by the supplier for various nutritional components and chemical and microbiological contaminants, including manganese, which is a known essential element in the SDS Rat and Mouse diet and the levels of manganese were (84–94 mg/kg) in each batch of diet. The drinking water was periodically analysed for dissolved materials, heavy metals, pesticide residues, pH, nitrates, nitrites and selected bacteria. Results of these analyses did not provide evidence of contamination; however, Mn is a known component of potable water and was present at a concentration of 1.9 mg Mn/L. Therefore additional Mn exposure from diet and water intake is expected.

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose only

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE
- System of generating particulates/aerosols: The test material was a pure pink powder, it was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh the size of 100 or 180 mm prior to use. Test material aerosols were generated using a Wright Dust Feed generator device.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- 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.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy. 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Target Concentration (mg/L Air) - Nose only: 0, 5, 10 and 20 µg/L air
F0 Actual Concentration (mg/L Air) - Nose only: 0, 6, 15 and 25 µg/L air
F1 Actual Concentration (mg/L Air) - Nose only: 0, 4, 10 and 17 µg/L air

The overall aerosol concentrations aerosols were 6, 15 and 25 µg/L for the F0 Generation and 4, 10 and 17 µg/L for the F1 Generation. This was considered to be satisfactorily close to target concentrations of 5, 10 and 20 µg/L for Groups 2, 3 and 4, respectively. For the air-alone control group (Group 1), the test material was nondetectable or non-quantifiable for all samples collected over the course of the study.
The particle size distribution (mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD)) investigations assured that the test aerosols were respirable to the animals and that good pulmonary exposure was achieved. Particle size distribution investigations assured that the test aerosols were respirable to the animals and that good pulmonary exposure was achieved.

Duration of treatment / exposure:

6 hours per day/ 7 days a week

Frequency of treatment:

F0 animals were treated for 10 weeks prior to pairing for mating. Daily exposure was for 6 h, 7 days a week. Meanwhile F1 animals commenced treatment shortly after weaning for 11 weeks (prior to mating). Daily exposure was for 6 h, 7 days a week. For F0 and F1 males, this treatment continued until the day prior to termination (a total of 17 weeks per generation).
For F0 and F1 females, the animals 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: for 1 h per day
From Day 3–4 of lactation: for 2 h per day
From Days 5–20 of lactation until prior to termination (ca Day 21 of lactation): for 6 h per day.
Animals that did not produce litters, continued on a 6 h dosing until the scheduled termination. Animals that had a litter loss continued on a 6 h dosing regimen until scheduled sacrifice.

Details on study schedule:

- From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on the study (F1) and were dosed for approximately 11 weeks (6 h daily) after weaning, and throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Dose / conc.:

5 other: µg/L air

Dose / conc.:

10 other: µg/L air

Dose / conc.:

20 other: µg/L air

No. of animals per sex per dose:

F0: 28 animals per sex per dose
F1: at least 24 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected after a nose-only inhalation exposure dose range finder study. Ten females and 10 males per group were exposure to the test material at 5, 20 and 30 µg/L. The study was scheduled for 9 weeks however; exposure was stopped at 3 weeks for the high dose as adverse clinical signs included crackling/gasping respiration resulted in the premature sacrifice of 3 animals. Necropsy findings included distended intestines, froth filled trachea, discoloured lungs. Exposure continued to the end of the study for the low and mid dose animals with minimal effects. The high dose for the main study was therefore reduced to 20 µg/L.
- Rationale for animal assignment: In the main study, F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: YES

OTHER:
Blood samples were taken from the tail vein of all adult animals for analysis of Mn concentrations prior to dosing, prior to mating and prior to weaning/necropsy. Samples were collected from all F0 generation animals at pre-dose, prior to mating and prior to weaning/necropsy. Meanwhile for the F1 generation, samples were collected from all animals (timing and volume depended on weight of animals, prior to mating and at necropsy. All samples were stored at -80 °C and analysed for Mn in whole blood via ICP-MS.

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. Computer assisted sperm analysis was also conducted to assess the motility of the sperm. Sperm counts using a haemocytometer to obtain a total sperm count was assessed. A sperm smear was prepared and stained with eosin and evaluated for morphological abnormalities. One testis was decapsulated and homogenized and the homogenisation resistant spermatids were counted using a haemocytometer.

Litter observations:

- 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.
- The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined from Day 1 onwards for the presence of milk in the stomach and for any externally visible abnormalities daily. The pups were weighed en masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day 21 all pups were weighed individually. Where practicable, any pups that were found dead or were killed during lactation were sexed and 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
- ca Day 21 of lactation
- Animals that did not produce litters, continued on a 6 h dosing until the scheduled termination. Animals that had a litter loss continued on a 6 h dosing regimen until scheduled sacrifice.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues assigned to histological examination were processed; sections were cut 4, stained with haematoxylin and eosin (H&E) and evaluated by light microscopy from animals in the Controls and High dose groups. Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
- The following organs were collected and preserved in 10 % neutral buffered formalin, unless otherwise indicated: Animal identification, Lymph node, bronchial, Brainb Lymph node, cervical, Epididymis x 2, Nasal cavity, Gland, adrenal x 2,Ovary x 2, Gland, pituitary, Pharynx, Gland, prostate, Spleen, Gland, seminal vesicle, Testes (Davidson's fixative), Gland, thyroid x 2, Trachea (anterior), Kidney x 2, Trachea, (posterior), Larynx, Uterus, Liver, Vagina and Lung.

Postmortem examinations (offspring):

SACRIFICE
- Where practicable, any pups that were found dead or were killed during lactation were sexed and examined. 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.

HISTOPATHOLOGY / ORGAN WEIGTHS
- 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 Haematoxylin and Eosin (H&E) section was cut, stained and evaluated

Statistics:

- Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between Control and groups receiving the test material. Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in house (validated) software. Pairwise comparisons were only performed against the control group. Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to 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, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female animal in Group 3 had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over Days 83–90 of the study. Due to the signs dosing for the animal was stopped for a two days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals in this group or in group 4, these signs were considered to be incidental. Blood concentrations correlated with exposure levels in males and females) confirming incremental systemic test material availability.
At target 20 µg/L (group 4) there were 2/28 males noted as having wheezing respiration.
Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four animals in the F0 generation (one Control, 2 at target 5 µg/L and one at 10 µg/L) were killed prematurely for various reasons –difficulties giving birth, reduced activity, partially closed eyes, unkempt coat, open lesion on tail etc.
There was no treatment-related pattern to these deaths and these were not attributed to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At target dose 20 µg/L, there was a decrease in body weight gain in males over Days 0–21 of the study (29 % lower gains than those of the Controls). 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 dose 20 µg/L, the 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 (9 % of the Controls). Gains over the lactation period were similar to the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At target dose 20 µg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls.
At target dose 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 dose 5 µg/L and target 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.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Pre treatment manganese levels in all F0 animals were similar to controls.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related findings observed in the reproductive tract in the F0 generation. Histological findings related to treatment were confined to the respiratory tract as follows:

- Larynx: In the larynx there was a broadly dose-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.

- F0 lungs: 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.

- Nasal cavity: 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 the test material, was seen in the nasolacrimal ducts of a few animals in the treated groups.

- Pharynx: 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 the test material at 10 or 20 µg/L.

- Trachea: 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 the inhalation of the test material.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The stages of the oestrus cycles and their mean duration were similar in all Test groups compared to the control group for both generations.
There was no treatment-related effect on the follicle type or incidence in either generation. The scoring methodology included the quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the sperm motility, count of morphology at any of the dose levels applied, or in either generation.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on mating performance, fertility or duration of gestation in either F0 or F1 generation.

Litter size and pup mortality
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 historical background range, hence these increases were considered to be incidental.

Litter and pup weights
- In all treated groups, group mean litter and pup weights were comparable to the controls.

Dose descriptor:

NOAEL

Effect level:

20 other: µg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects observed

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

5 animals in the F1 generation were (one Control, 3 at target 10 µg/L and 1 at target 20 µg/L) killed prematurely for various reasons –difficulties giving birth, reduced activity, partially closed eyes, unkempt coat, open lesion on tail etc. There was no treatment-related pattern to these deaths and these
were not attributed to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At target dose 20 µg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study (p < 0.001), 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 lactating females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At target dose 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 (ranges from p < 0.05 to p < 0.001).
Slight intergroup differences in group mean food consumption at target 5 µg/L and target 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.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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 F1 generation pre-treatment Mn blood levels were higher than the control with dose dependent correlation indicating exposure prior to treatment via lactation. Milk Mn levels were not measured directly but Mn blood pre-treatment levels confirm Mn exposure prior to weaning.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Kidneys: At target doses 5 and 10 µg/L, kidney weights in males were statistically higher than the control (p < 0.01 and p < 0.05, respectively), however there was no dose-response relationship to this increase and following covariance analysis with body weight, these findings were no longer evident.
- At target doses 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 with body weight.

Weanling 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 with body weight, this reduction did not achieve statistical significance. There were no effects on any organ weights at target doses of 5 and 10 µg/L.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related findings observed in the reproductive tract in the F1 generation.
Histological findings related to treatment were confined to the respiratory tract as follows:

- Nasal cavity: 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 the 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.

- Trachea: 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The stages of the oestrus cycles and their mean duration were similar in all Test groups compared to the control group for both generations.
There was no treatment-related effect on the follicle type or incidence in either generation. The scoring methodology included the quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on the sperm motility, count of morphology at any of the dose levels applied, or in either generation.

Reproductive performance:

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on mating performance, fertility or duration of gestation in either F0 or F1 generation.

Dose descriptor:

NOAEL

Effect level:

20 other: µg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects observed

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

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 historical background range, hence these increases were considered to be incidental.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In all treated groups, group mean litter and pup weights were comparable to the controls.

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

Sexual maturation:

no effects observed

Description (incidence and severity):

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.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related findings observed in the tissues examined of the F1 or F2 weanlings.

Other effects:

no effects observed

Description (incidence and severity):

The type and distribution of observations amongst pups did not indicate any association with treatment if compared to species historic data.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

20 other: µg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were observed

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Description (incidence and severity):

At target 20 µg/L, group mean litter weights were slightly lower than the controls; this was considered a reflection of the smaller litter size and not an adverse effect. This interpretation was supported by the mean pup weights in both males and females being comparable to the controls.

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Weanling organ weights
- F1 generation, F2 production: Slight intergroup differences in organ weights did not achieve statistical significance and were not attributed to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

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.
- The type and distribution of observations amongst pups did not indicate any association with treatment if compared to species historic data.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

20 other: µg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were observed

Critical effects observed:

no

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of this study, the No Observed Effect Level (NOEL) for reproductive performance was considered to be the target dose level of 20 µg/L.
Based on these findings, the test material could not be considered a reprotoxicant under these conditions of exposure. Therefore, soluble and insoluble forms of inorganic manganese compounds by extrapolation cannot be considered as reprotoxicants.

Executive summary:

The reproductive toxicity of the test material (manganese dichloride) was investigated in accordance with the standardised guidelines OECD 416 and OPPTS 870.3800.

Doses were selected after a nose-only inhalation exposure dose range finder study. Ten females and 10 males per group were exposure to manganese chloride at 5, 20 and 30 µg/L. The study was scheduled for 9 weeks however; exposure was stopped at 3 weeks for the high dose as adverse clinical signs included crackling/gasping respiration resulted in the premature sacrifice of 3 animals. Necropsy findings included distended intestines, froth filled trachea, discoloured lungs. Exposure continued to the end of the study for the low and mid dose animals with minimal effects. The high dose for the main study was therefore reduced to 20 µg/L.

In the main study, F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed for 10 weeks (6 h daily) 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 the study (F1) and were dosed for approximately 11 weeks (6 h daily) after weaning, and 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 sperms.

There were no deaths related to treatment, though respiratory tract effects were observed in F0 animals in the mid and high dose animals. Body weight and food consumption were affected at high dose in both generation. There were no treatment-related effects on the oestrous cycles, mating performance, sexual maturity, fertility or duration of gestation or litter size, the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation.

Under the conditions of this study, the No Observed Effect Level (NOEL) for reproductive performance was considered to be the target dose level of 20 µg/L.

Based on these findings, the test material could not be considered a reprotoxicant under these conditions of exposure. Therefore, soluble and insoluble forms of inorganic manganese compounds by extrapolation cannot be considered as reprotoxicants.

Reference 3

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.2.-25.9.2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationaly valid GLP principles.

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: males 5 - 6 weeks, females - 9 weeks
- Fasting period before study: no
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood. During mating period, one male with two females were housed together in one cage. Females that mated were individually housed in labelled cages. Offspring was kept with mother until wearing.
- Diet: ad libitum, Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used. Diet was sterilised before using.
Manufacturer: Ing.Mrkvička Miroslav - Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy.
Nutrient content of the diet: crude protein – min. 21%, drip – max. 14%, fat – min. 3%, fiber – max. 4.1%, ash – max. 7%, calcium – min. 1%, phosph orus – min. 0.8%, magnesium – min. 0.2%, sodium – max. 0.25%;
- Water: ad libitum, Free access to drinking water. Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry.
Water was sterilised before using.
- Acclimation period: at least 5 days
- Identification: Identification of animals was made by colour marks on fur, each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and mark of group. The offspring were not individually identified.
- Additional Information: The standard pelleted laboratory animal diet was analysed for nutrients (once a year) and contaminants (each batch) on a regular basis. Results were retained in the CETA archives. Certificates of analysis of water (performed twice a year) were retained in the CETA archives. Bedding (sterilised clean shavings of soft wood) was examined for bacteriological contaminants once a year and results were also retained in the CETA archives.
Analysis of diet, water and bedding, did not reveal any findings that could affect study integrity.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): a relative humidity of 30-70%
- Air changes (per hr): Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle.

Study Time Schedule
Test substance delivery: 3. 3. 2006
Date of animal arrival: males - 13. 2. 2008
females – 30.4.2008
Start of administration: males - 11. 3. 2008
females – 6. 5. 2008
End of administration: males – 12. 6. 2008
females – 18. 7. 2008
Clinical examination: 13. 2. – 17. 7. 2008
Necropsy and biometry of organs: males 10 – 13. 6. 2008
females 5. 6. – 19. 7. 2008
Histopathological examination: 7. 8. – 25. 9. 2007
Evaluation of results and final report elaboration: 30. 7. – 10. 10. 2008

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The concentrations of solutions in all three dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (test substance solution in water for injectione) was prepared daily just before administration. The vehicle control group was administered by water for injectione in the same volume. The application form of the test substance was prepared daily before administration; these solutions were mixed for 10 minutes by magnetic stirrer. The procedure was based on the results of analyses of test substance application form homogeneity and stability (Annex 2 of the Study No. 15/06/7, VUOS-CETA Report No. 0680, 2006).

DIET PREPARATION
- Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used. Diet was sterilised before using.
- Manufacturer: Ing.Mrkvička Miroslav - Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy.
- Nutrient content of the diet: crude protein – min. 21%, drip – max. 14%, fat – min. 3%, fiber – max. 4.1%, ash – max. 7%, calcium – min. 1%, phosphorus min. 0.8%, magnesium – min. 0.2%, sodium – max. 0.25%;
- Composition of food: wheat, oats, fish meal powder, dried snail-clover, soya extracted
groats, wheat sprouts, dehydrated yeast, calcium carbonate, vitamin and mineral complex
- Storage temperature of food:laboratory temeperature, bacteriological contaminants are checked per two month


VEHICLE: water
Water for injection
Batch No. 0102101007
Batch No. 0305031207
Batch No. 0204200308

Details on mating procedure:

- M/F ratio per cage: During mating period, one male with two females were housed together in one cage.
- Length of cohabitation: until the presence of spermatozoa
- Proof of pregnancy:sperm in vaginal smear, vaginal smear were prepared daily during mating period, referred to as [day 0 ] of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females that mated were individually housed in labelled cages.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The procedure was based on the results of analyses of test substance application form homogeneity and stability (Annex 2 of the Study No. 15/06/7, VUOS-CETA Report No. 0680, 2006).

Duration of treatment / exposure:

The treatment period for the males was 13 weeks (10 weeks of pre-mating and 3 weeks of mating period). Treatment period of females - mothers was at least 8 weeks (2 weeks of pre-mating, at least 1 day of mating, 3 weeks of pregnancy and 3 weeks of lactation).

Frequency of treatment:

The treated and control groups were administered daily by gavage. The animals were treated 7 days per week at the specified time (8.00 – 10.00 am).

Details on study schedule:

Body weight: males – the first day of administration and than weekly
females – the first day of administration and then weekly,
during pregnancy: 0., 7th., 14th., 21st., day,
during lactation. 1st., 4th., 7th., 14th., 21st. day
pups – litters: 1st., 4th., 7th., 14th., 21st day of life,
pups - individually: 21st day of life
Food consumption: during premating period weekly,
during pregnancy and lactation - on the same days as body weight (daily consumption was counted)
Mortality control: daily
Health condition control: daily - during the acclimatization and the experimental part
Clinical observations: daily - during the administration period
Laboratory examinations:
- vaginal smears: daily in mating period
- pathological examination: males – after ending of the mating period
mated females without delivery (nonpregnant or aborted)
– 25th day after mating
parental females and pups – on the 21st day of lactation
- biometry of organs: during necropsy

Remarks:

Doses / Concentrations:
20 mg/kg body weight/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
80 mg/kg body weight/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
320 mg/kg body weight/day
Basis:
nominal conc.

No. of animals per sex per dose:

3 treated groups (dose 20, 80, 320 mg/kg body weihgt/day) and one control group (vehicle only). Each group consisted of 10 males and 25 females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses for the one-generation study were determined on the basis of scientific literature information and with respect to the results of acute oral toxicity study, repeated dose oral toxicity study and in vivo micronucleus test performed at CETA test facility. This information was summarized and evaluated in the Expert Report No. 07289, VUOS-CETA, 2007.
- Rationale for animal assignment (if not random): random

Positive control:

none

Parental animals: Observations and examinations:

BODY WEIGHT: Yes
- Time schedule for examinations:The body weight of animals was recorded on automatic balances with group average computing module. Pregnant females were weighted in specified days. All animals were weighed immediately before euthanasia too.
Weight increment was computed as an average per group per time interval (in grams). Nonpregnant females and aborted females were not included in calculation of average weight increment of pregnant females.

FOOD CONSUMPTION AND COMPOUND INTAKE :
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
Average values were calculated for each week of the study. Food consumption for animal/day was calculated of average values of each group. Nonpregnant females and aborted females were not included in calculation of average food of pregnant females.

MORTALITY CONTROL: Yes
All rats were examined for vitality impairment or mortality daily during the whole study.

HEALTH CONDITION CONTROL: Yes
The health condition was controlled daily. Pre-experimental observation of all rats was performed to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. In administration period this observation was performed before application.

CLINICAL OBSERVATIONS: Yes
All rats were observed daily during the administration period.
The observation of parental males and females was made in order to record possible clinical effects after application and all changes in behaviour of animals. This was made at specified time after application every day – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

EXAMINATION OF VAGINAL SMEARS: Yes
The pregnancy was determined by presence of spermatozoa in vaginal smear. The vaginal smears were carried out in female’s mornings every day during mating procedures. The smears were stained and the presence of sperm was watched. Day 0 of pregnancy was defined as the day when sperm are found.

Oestrous cyclicity (parental animals):

It was not monitored.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, Detailed histological examination was performed on the testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

Litter observations:

BODY WEIGHT: Yes
The pups or litters were weighted in specified day too. Individual weights of pups were taken at the end of lactation. According to these individual weights of pups from birth to weaning the lactation ability was evaluated.

CLINICAL OBSERVATION: Yes
Each litter was examined as soon as possible after delivery and number and sex of pups, still births, live births and presence of gross anomalies were recorded. Changes of physical condition and behaviour abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Parental males were killed at the end of the mating period – 93rd day of study (after 92 days of application).
- Maternal animals: Parental females were killed on the 21st day of lactation. Mated females without delivery (nonpregnant or aborted) were killed 25th day after mating.

PATHOLOGICAL EXAMINATION: Yes
Parental males were killed at the end of the mating period – 93rd day of study (after 92 days of application). Parental females were killed on the 21st day of lactation. Mated females without delivery (nonpregnant or aborted) were killed 25th day after mating.
Then they were macroscopically examined for any structural abnormalities or pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.

BIOMETRIC OF REPRODUCTIVE ORGANS: Yes
The absolute weights of testes, epididymides, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries, uterus (incl. uterine tube and cervix) and pituitary gland were recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

Postmortem examinations (offspring):

PATHOLOGICAL EXAMINATION
Dead pups or pups killed before the 14th day of lactation were sexed and externally examined; the stomach was examined for the presence of milk. Pups found dead or killed on or after 14th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

HISTOPATHOLOGICAL TECHNIQUE
All gross lesions of pups were collected and fixed in 4% neutral formaldehyde solution for eventual histological examination.
For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used. Other special staining was used in indicated cases.

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight and biometry of organs. Control group with vehicle was compared with three treated groups.

Reproductive indices:

Percentage mating, Fertility index, Conception index, Gestation index, Percentage of live males at first litter check, Percentage live females at first litter check, Percentage of postnatal loss days 0-4 post partum, Percentage of pre-weaning loss day 5 until weaning, Percentage live males at weaning, Percentage live females at weaning, Viability index, Weaning index

Offspring viability indices:

Development of pups (ability of sucking, growth of hair, teeth and dugs, opening of eyes and ears) was observed in the reported days.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

MORTALITY (PARENTAL ANIMALS)
MALES:
There were no unscheduled deaths during all the study.

FEMALES:
There were no unscheduled deaths at the dose levels 20 and 80 mg/kg/day and at the control group during all the study. One nonpregnant female at the highest dose level died in the first week after mating.

FOOD CONSUMPTION
MALES:
Food consumption at the dose levels 20 and 80 mg/kg/day was well-balanced with the control group until 4th week of application from 5th week to the end of study the consumption was slightly decreased. At the highest dose level 320 mg/kg/day the food consumption was decreased from 3rd week of the application period to the end of study. This change was marked since 8th week.

FEMALES:
Pre-mating period
Average food consumption at the dose levels 20 and 80 mg/kg/day was well-balanced with control group during whole pre-mating period. Food consumption of females at dose level 320 mg/kg/day was lower then in control group at the end of the 1st week of application.
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in evaluation of food consumption during pregnancy.
Average food consumption at all treated groups during pregnancy was relative well-balanced with the control group.
Lactation
Only mothers (females with live pups) were included in evaluation of food consumption during lactation period. Average food consumption of all treated groups was lower than at the control group during whole lactation period. More marked decrease was registered at the dose levels 20 and 320 mg/kg/day.


BODY WEIGHT
MALES:
The animal body weight in the dose levels 20 and 80 mg/kg/day was relatively well-balanced with the control group till 6th week of application and than it was slightly decreased. Markedly lower body weight was recorded at the dose level 320 mg/kg/day since the 1st week and at the end of study (since 11th week) the body weight increment was negative – the animals grew slim. Body weight of males at the highest dose level was statistically significantly decreased at the end of study

FEMALES:
Pre-mating period
Average body weight increments of females at dose level 320 mg/kg/day were markedly lower then in control group at the end of the 1st week of application (tabescence of animals).
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in evaluation of body weight increment during pregnancy.
Body weight increment of pregnant females at the dose level 20 mg/kg/day was relative well-balanced compared to the control group. Slight decrease of increment was recorded at dose level 320 mg/kg/day (only at the end of 1st and 2nd week of pregnancy) and at the dose level 80 mg/kg/day (at the end of 2nd week of pregnancy).
Lactation
Only mothers (females with live pups) were included in evaluation of body weight increment during lactation period. Till 14th day of lactation all groups of animals (treated and control) showed increase of body weight (positive increment). The body weight increment of treated groups was relative well-balanced with control group. Only from 1st to 4th day of lactation it was slightly decreased at the dose levels 20 and 320 mg/kg/day. Marked decrease of body weight was recorded at the end of lactation (from 14th to 21st day of study) in all groups of animals (treated and control). The body weight increment of all groups was negative. Marked tabescence was recorded at the dose level 80 mg/kg/day. On the contrary the tabescence of animals at the dose level 320 mg/kg/day was not so marked.


HEALTH CONDITION
MALES:
The health condition of control males and males treated by the test substance at the dose level 20 mg/kg/day was very good during whole study. No signs of diseases or toxic effect owing to application of the test substance were found out only one male from control group showed diarrhoea and one male from the lowest dose level showed red secretion from nostrils.
At the dose level 80 mg/kg/day sporadic dyspnoea, red secretion and salvation were recorded more often than at the control and lowest dose level. These changes were incidental and did not affect all animals in the group.
The health condition of animals at the highest dose level 320 mg/kg/day was not very good since the first week of application period. The signs of toxic effects related to application of the test substance were found out. These changes (dyspnoea, decreased activity, red secretion around nose or eyes, rigidity, piloerection, salivation) were gradually registered at most of animals.

FEMALES:
The health condition of control females and females at the dose levels 20 and 80 mg/kg/day was very good during the whole study. No signs of diseases or toxic effects related to application of the test substance were found out. One female from control group showed piloerection (only at 6th week). At the lowest dose level one female showed sporadic dyspnoea (at 5th week) and two females showed red secretion around nose (1st and 3rd week). Alopecia was also recorded in one female from 1st to 7th week of application. At the middle dose level only one female showed red secretion around nose from 2nd to 4th week. At the highest dose level 320 mg/kg/day dyspnoea was recorded in females (at 1st week – three females, at 2nd week – two females, at fourth week – one female and seventh week – two females). In the absence of a dose-related response this was considered to be incidental.
ORGAN WEIGHTS (PARENTAL ANIMALS)
MALES:
Absolute and relative weights of all observed organs were relatively well-balanced at the dose level 20 and 80 mg/kg/day compared to control group.Animals at the dose level 320 mg/kg/day showed decrease of absolute weight of prostate gland with statistical significance. Slight decreased absolute weight of testes and epididymides (without statistical significance) was recorded also at the highest dose level.

FEMALES:
Nonpregnant females and females with abortion were not used for calculation of means and evaluation of organs weight. Relative and absolute weight of all measured organs was relative well-balanced (without statistical differences) at the all dose levels.
HISTOPATHOLOGY (PARENTAL ANIMALS)
MALES:
Incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-20-80-320 further in the text.
Incidence of histopathological affections of reproductive system in male genital tract was sporadic at the control group and the lower dose level. Marked changes were recorded at the highest dose level. Detailed examination of testes revealed various damage of spermiogenesis in 1-3-0-7 males, atrophy of germinative epithelium in 3-0-3-1 males and atrophy or decreased quantity of Leydig cells in 0-1-0-1 males. Damage of spermiocytes (decreased number or presence of necrotic cells) was observed in epididymides of 0-1-0-3 males, vacuolar dystrophy in 0-2-0-0 and inflammation in 1-0-2-0 males. Signs of inflammation in prostate gland (oedema of interstitium, lymphocyte infiltration or inflammation) were recorded in 2-2-5-3 males.
In digestive system stomach, forestomach and duodenum were affected. Erosions, ulcerations and haemorrhage of stomach mucosa or submucosa were described in 0-0-1-8 males. Inflammation (inclusive inflammatory infiltration of mucosa and/or submucosa) was diagnosed in stomach of 0-0-1-10 males, in forestomach of 0-0-1-6 males and in duodenum of 0-0-0-2 males.

FEMALES:
Incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0-20-80-320 further in the text.
In digestive system stomach and forestomach were affected. Erosions, ulcerations and haemorrhage of stomach mucosa or submucosa were described in 0-0-0-5 females. Inflammation (inclusive inflammatory infiltration of mucosa and/or submucosa) was diagnosed in stomach of 0-0-0-9 females and in forestomach of 0-0-0-3 females.
In reproductive system ovaries and uterus were markedly affected. Follicular cysts of ovaries (or cystic degeneration of follicles) were recorded in 20-16-22-13 females and cellular hyperplasia of strome in 2-0-2-3 females. In uterus cellular hyperplasia of endometrium was found out in 13-15-14-9 females, and hydrometra in 4-0-1-7 females. Degenerative changes (atrophy of endometrium, extinction of endometrial glands, fibrosis of endometrium or atrophic epithelium in vagina) were recorded in 3-0-3-7 females.
Cysts in pituitary gland were recorded only in 0-1-1-0 females.

Dose descriptor:

NOAEL

Remarks:

for Reproduction

Effect level:

ca. 80 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

NUMBER AND SEX RATIO OF PUPS
The total number of pups was decreased with dose level and the lowest number was at the dose level 320 mg/kg/day. Number of pups per litter was relatively well balanced in all treated groups. Slightly lowered number but without statistically significance was recorded at the dose level 320 mg/kg/day.

DEVELOPMENT OF PUPS
Death of pups was minimal and similar in all groups:
- until 4th day after birth died 3 pups at the control group (from 1 mother), 3 pups at the dose level 80 mg/kg/day (from 3 mothers) and 4 pups at the dose level 320 mg/kg/day (from 2 mothers)
- until 7th day after birth no other pups died
- until 14th day after birth only one pups died at the dose level 320 mg/kg/day
- until 21st day after birth no other pups died
No differences in development of pups were observed at the dose level 20 mg/kg/day. Observation of opening of eyes (until 14 day after birth) showed late opening at the dose level 80 mg/kg/day (2 litters from 16) and at the dose level 320 mg/kg/day (3 litters from 10).


BODY WEIGHT (OFFSPRING)
The average body weigh of pups all treated groups was well balanced with the control group and had an increasing trend during whole lactation period.

MACROSCOPIC EXAMINATION
The pathological examination was performed in all pups. All died new-born pups were examined for presence of air in lungs and divided in stillbirth and live birth pups. Presence of macroscopic abnormalities could not be detected in died pups with autolysis. No pathologic findings were recorded at the control group and at the dose levels 20 and 80 mg/kg/day. Sporadic pathologic findings were recorded at the dose level 320 mg/kg/day: missing testes and epididymides (one pup), one testis reduced (one pup) and stomach mucous membrane congested and chyme with blood (two pups).

EXAMINATION OF BRAIN
Absolute weight of brain was increased at all treated groups with statistical significance at the dose level 320 mg/kg/day. Relative weight of brain was increased al all treated groups with depending on dose level and statistical significance at the dose level 320 mg/kg/day.
During histopathological examination the vacuolisation of cell nuclei in cortex and/or hippocampus was recorded at all dose levels but it was more marked in treated groups. Only mild vacuolisation was recorded in 3 control pups. Number of pups with vacuolisation in brain was well-balanced at all treated groups but marked vacuolisation was recorded at the middle and highest dose level.

REPRODUCTION PARAMETERS
All females (at all groups) were mated so the number of females mated was identical at all groups.
Number of pregnant females and accompanying number of mothers bearing live pups was markedly lower at the highest dose level. Duration of pregnancy of treated groups was similar to control group.
Significant decrease of fertility index and conception index was detected only at the highest dose level. Percentage of postnatal loss was slightly increased at the middle dose level. All other calculated parameters were relative well-balanced.

Dose descriptor:

NOAEL

Remarks:

for Development

Generation:

F1

Effect level:

ca. 20 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: development

Reproductive effects observed:

not specified

Conclusions:

Administration of the test substance Potassium permanganate affected clinical status, growth of parental animals, biometrics and microscopic structure of reproductive male organs and development of pups.
In males of the highest dose level these effects manifested in significant damage of spermatogenesis with negative effect on fertility. These changes resulted in decreased ability of animals to achieve a pregnancy. Application of the test substance at the highest and middle dose levels had a negative developmental effect on pups (pathological changes of brain, late opening of eyes).

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION was established as 80 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT was established as 20 mg/kg body weight/day.

Executive summary:

Study Performance

   Wistar rats of SPF quality were used for testing. The test substance was administered as water solution using a stomach tube; oral application of rats was made daily. The volume administered was adjusted to 1 mL per 100 g of body weight in all doses. The 4 groups of animals were included in the study - 3 treated groups (doses 20, 80, 320 mg/kg of body weight /day) and one control group (vehicle only). Each group consisted of 10 males and 25 females.

   The doses for the one-generation study were determined on the basis of scientific literature information and with respect to the results of acute oral toxicity study, repeated dose oral toxicity study and in vivo micronucleus test performed at CETA test facility. This information was summarized and evaluated in the Expert Report No. 07289, VUOS-CETA, 2007.       

   The treatment period for the males was 13 weeks (10 weeks of pre-mating and 3 weeks of mating period). Treatment period of females - mothers was at least 8 weeks (2 weeks of pre-mating, at least 1 day of mating, 3 weeks of pregnancy and 3 weeks of lactation).

   

   During the study the clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, body weight, sex or vitality) were also recorded.

    The study was finished by gross necropsy of animals. The selected organs from parental animals were removed for weighing and histopathological examination.

 

 

Results

  The application of the test substance at the dose level 320 mg/kg/day had negative influence on clinical status of both sexes (more marked in males) and caused death of one nonpregnant female. The negative effect of the treatment on growth of males and on food consumption of both sexes was found out. Toxicity of the test substance at the highest dose level wasconfirmed also during necropsy and histological examination of males and females (numerous bleeding erosions of stomach or duodenum).

   Histopathological examination of reproductive system of parental males showed high incidence of affected males at the dose level 320 mg/kg/day. Detailed examination of testes revealed various damage of spermatogenesis. Significant decreased weight of prostate gland was recorded also at the highest dose level and could reflect changes in testicular function.

  Examination of reproductive system of parental females showed relative high incidence of findings at all dose levels including the control group but without dose dependence. There was no significant pathological affections indicated damage of reproductive female organs at any of dose levels.

 

  Observation of pups– the number of pups, sex ratio, average weight of litter and average body weight of pups were unaffected and well-balanced with the control group.

  Presence of macroscopic abnormalities – oedematous appearance of brain was recorded in pups at the highest dose level. Statistical examination showed significant increased absolute and relative weight of brain at the highest dose level. During histological examination the high incidence of vacuolisation of cell nuclei in cortex and hippocampus was recorded at all dose levels. This change was more intensive at the middle and highest dose level.

  Observation of development (until 14 day after birth) showed late opening of eyes at the middle and highest dose level. The change was marked at the highest dose level and could be related with biometrical and histopathological findings. 

  Sporadic but significant pathologic changes of stomach and genital organs were recorded in pups at the highest dose level – missing or reduced testis and epididymis, congested stomach mucosa and chyme with blood. These changes could beconsidered as changes of toxicological significance.

 Reproduction parameters– number of pregnant females and accompanying fertility index and conception index were lowered at the highest dose level. This result showed decreased ability of the animals to achieve a pregnancy.

  Duration of pregnancy in treated groups was similar to control group. Gestation index was relatively well-balanced at all dose levels. Administration of the test substance did not cause significant postnatal loss and pre-weaning loss (viability index and weaning index). The other observed parameters (percentage of live males and females - at first check of litter and also at weaning) were unaffected and were in normal range.

Reference 4

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

See the read-across report attached in Section 13

Reason / purpose for cross-reference:

read-across source

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

20 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed.

Critical effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

the effects reported in the high dose group in the presence of severe toxicity are considered to be secondary non-specific consequences of parental toxicity. Also the study was considered of poor quality - very limited statistical analysis and no historical data.

Effect on fertility: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

20 µg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Reliability 1 ( published) and reliability 2 (unpublished). To OECD 416 guideline and GLP. data point addressed by recommended guideline study

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproduction: Several regulatory authorities – SCOEL, 2011; German MAK, 2012, ATSDR, 2011 examined a suite of published literature pre – 2011/2012 and concluded that from a reproductive effects perspective, Mn and its inorganic compounds including KMnO4 and its degradation substances are not reproductive toxicants.

In pubished work is an extract from the two-generation study according to OECD 416 guideline was performed with manganese chloride by inhalation. This route of exposure was chosen because the systemic bioavailability of inhaled manganese is expected to be higher than after ingestion. Indeed, a low oral absorption is likely due to the homeostatic control the body exerts on the amount of manganese absorbed following oral exposure to avoid an excess of manganese. Sprague-Dawley rats received manganese chloride as a powder aerosol by inhalation nose-only. The target concentrations were 0, 5, 10, 20 μg/L. The overall aerosol concentration aerosols were 6, 15 and 25 μg/L for the F0 generation and 4, 9 and 17 μg/L for the F1 generation. Particle size measurements indicated that test aerosol was respirable to rats (MMAD about 2 μm). Daily exposure was ca 6 hours, 7 days a weeks. F0 and F1 males were treated for a duration of ca 17 weeks. F0 and F1 females were dosed from ca 10 weeks until day 21 of lactation.

Blood measurement showed that the level of manganese chloride increased significantly in both males and females and were correlated to tested concentrations. The concentrations recorded prior mating and prior necropsy were comparable in all groups, indicating that no bioaccumulation is expected. Pre-treatment level in F1 generation is higher than pre-treatment level in F0 generation indicating an exposure during lactation.

Inhalation of manganese chloride was associated with microscopical findings in the

respiratory tract. All F0 treated groups presented irritation of the larynx (inflammation, metaplasia, ulceration), lung (hyperplasia, inflammation), nasal cavity (hyperplasia, inflammation, degeneration, atrophy, ulceration, metaplasia of the olfactory epithelium), pharynx (hyperplasia, degeneration, inflammation, metaplasia) and trachea (metaplasia, inflammation, degeneration). In F1, irritation of the respiratory tract was reported from 10 μg/L in nasal cavity, pharynx and trachea. Based on these effects, no NOAEC can be derived.

Regarding systemic effects, a statistically significant decrease of body weight gain in F0 males was reported at 20 μg/L, associated with a reduced food consumption. No consistent effect was reported in F0 females and F1 males and females. Some effects on organ weight were recorded at different dose levels: reduced absolute brain weight and relative lung weight in F0 males, increased absolute and relative lung weight in F0 females, increased relative kidney weight and relative lung weight in F1 males and females. These effects are not consistent between generations and sexes and not associated with histopathological findings. In this context, a NOAEC of 20 μg/L (corresponding to an analytical concentration of 25 μg/L for F0 and 17 μg/L for F1) was set for systemic toxicity.

There was no effect on oestrous cycle, mating performance, fertility, duration of gestation, litter size, sexual maturity, sperm motility, sperm count or sperm morphology and ovary follicle scoring in either generation. In F0 generation exposed to 20 μg/L, there was an increase in the number of animals losing more than 2 pups at birth4 (2, 6, 5, 6 at 0, 5, 10, 20 μg/L) without impact on the mean birth index (86%, 80%, 84%, 90%). In F1 generation exposed to 10 and 20 μg/L, the pup survival over days 0-4 of lactation was slightly lower without a clear dose related response (88%, 94%, 78%, 86% at 0, 5, 10, 20 μg/L). In addition, at the highest dose, the group mean litter weights of F1 generation were slightly lower than the controls (420g, 432g, 407g, 391g at 0, 5, 10, 20 μg/L on day 21) but the mean pup weight in both males and females remained comparable to controls. These effects are not considered treatment-related. Thus a NOAEC of 20 μg/L (corresponding to an analytical concentration of 25 μg/L for F0 and 17 μg/L for F1) was set for reproductive and developmental toxicity.

Based on available data no classification for reproductive toxicity is proposed

Effects on developmental toxicity

Description of key information

Please refer to the RAAF report attached to section 13 of this dataset which discusses the cateogory approach for sharing of data between the three soluble manganese substance KMnO4, Mn chloride and Mn sulphate.

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

One supporting study on the registered substance -prenatal developmental toxicity study- This reliability 2 study on Rat (Wistar Han) females 24 - 25 per group. Oral: gavage at doses 0, 20, 100, 500 mg/kg bw/day (nominal conc.) with water as the vehicle.

Exposure: The females were administered daily from the 5th to the 19th day of pregnancy.

-       NOAEL maternal = 20 mg/kg bw/day based on erosion of digestive tract.

-       In addition, at 500 mg/kg bw/day, there was a decrease in body weight.

-       NOAEL developmental < 20 mg/kg bw/day based on a decreased number of ossification sites in the sternum and an incomplete ossification of cervical vertebrae.

-       In addition, at 500 mg/kg bw/day, there was an increase of post-implantation losses and a decreased pup body weight.

However, the effects reported in the high dose group in the presence of severe toxicity are considered to be secondary non-specific consequences of parental toxicity. Also the study was considered of poor quality - very limited statistical analysis and no historical data.

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

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

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

There is a smaller body of literature data available on the reproductive/developmental toxicity of effects  MnSO4 and no studies performed to standardised guidelines.  A two-year carcinogenicity (section 7.7) and repeated dose toxicity report (NTP, 1993) showed no effect on the testes of rats exposed orally for up to 2 years.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.10. 2008-28.1.2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Han

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 9 weeks
- Weight at study initiation:
- Fasting period before study: no
- Housing: Animals were housed in SPF animal room in plastic cages containing sterilised clean shavings of soft wood. Before mating 2 rats of the same sex in one cage, during mating period – one male and two females in one cage were housed. During pregnancy 1 female was housed in one cage.
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using.
Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.
- Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted
groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.
- Water (e.g. ad libitum): Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.
- Acclimation period: 5 days
- Identification: Identification of females was made by colour marks on fur (system 1 – 10), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance, mark of group and date of planned euthanasia.
- Additional information: The standard pelleted laboratory animal diet was analysed for nutrients (once a year) and bacteriological contaminants (each batch) on a regular basis. Results were retained in the CETA archives. Certificates of drinking water analysis (performed twice a year) were retained in the CETA archives. Bedding (sterilised clean shavings of soft wood) was examined for bacteriological contaminants once a year and results were also retained in the CETA archives.
Analysis of diet, water and bedding, did not reveal any findings that could affect study integrity.


ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment
- Temperature (°C): 22+/-3°C
- Humidity (%): 30-70%
- Air changes (per hr): in which optimal conditions were considered to be approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle.



Study time schedule
Main study
Date of animal arrival: 22. 10. 2008
Mating: 29. 10. - 6. 11. 2008
Start of administration: 4. 11. 2008
End of administration: 25. 11. 2008
Clinical observation: 4. – 25. 11. 2008
Necropsies: 19. – 26. 11. 2008
Microscopical examination: 6. – 28. 1. 2009
Evaluation of results and final report elaboration: 10. 1. – 20. 4. 2009

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The application form (test substance solution in water for injection) was prepared daily just before administration.
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
The application form of the test substance was prepared daily before administration; these solutions were mixed for 10 minutes by magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used, manufacturer: Ing. Mir oslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using.
- Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosp horus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.
- Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbo nate, Vitamin and Mineral complex.
- Storage temperature of food: laboratory temperature, bacteriological contaminants are checked once per two month


VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua pro injectione
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): 0101210308

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The determination of test substance was performed by a spectrophotometry.
Test substance stability and homogeneity were determined by measuring an absorbance of its water solution in visible range of spectrum.
The procedure was based on the results of analyses of test substance application form homogeneity and stability (Annex 2 of the Study No. 15/06/7: Potassium permanganate - Repeated Dose (28 days) Toxicity (Oral), VUOS-CETA Report No. 0680, 2006).

Details on mating procedure:

- Impregnation procedure: After acclimatisation females were mated with males (1 male and 2 females).
Vaginal smears were carried out daily in the morning to control fertilization (first time: 24 hours after the first removing to male).
Presence of sperms was examined.
Day 0 of pregnancy was the day on which sperms in vaginal smears were observed.
Pregnant females were randomly distributed to experimental groups – 24 or 25 probably gravid females were at each group (2 females from beginning total number 100 were not used for study).
- If cohoused:
- M/F ratio per cage: 1 male and 2 females
- Mating: 29. 10.- 6. 11. 2008
- Proof of pregnancy: sperm in vaginal smear referred to as day 0
- Vaginal smears: daily in mating period

Duration of treatment / exposure:

4.11.-25.11.2008

Frequency of treatment:

The treated and control females were administered daily by gavage – from the 5th to the 19th day of pregnancy. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).

Duration of test:

22.10.2008-28.1.2009

No. of animals per sex per dose:

1. Control: 25 probably pregnant females No. 101 - 125
2. 20 mg/kg: 24 probably pregnant females No. 126 - 149
3.100 mg/kg: 24 probably pregnant females No. 151 - 174
4. 500 mg/kg: 25 probably pregnant females No. 176 - 200

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were determined on the basis of results of Study No. 15/06/7: Potassium permanganate – Repeated Dose (28 days) Toxicity (Oral); VUOS-CETA Report No. 0680, 2006 and Study No. 15/06/15: Potassium permanganate – One-Generation Reproduction Toxicity Test; VUOS-CETA Report No. 08142, 2008.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
- In the given day the remainder of pellets of each cage was weighed, the new food was weighed out and the food consumption for one female and one day was computed (average value for each cage).

MORTALITY CONTROL: Yes
- Time schedule: daily-during the acclimatization, mating and pregnancy

HEALTH CONDITION CONTROL
- Time schedule: daily-during the acclimatization, mating and pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on the 20th day of pregnancy
- Organs examined: biometry of organs, microscopical examination,

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes half per litter

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight of pregnant females at the end of pregnancy, absolute and relative weight of uterus, number of foetuses, number of male foetuses, number of female foetuses, weight of foetuses,
weight of male foetuses and weight of female foetuses. Control group with vehicle was compared with three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of averages.

Indices:

Preimplantation loss, postimplatation loss were calculated from number of implantations, corpora lutea and resorptions.

Historical control data:

none

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
cachexia, anemia, cough or hoarse breath, secretion from nostrils and eyes, piloerrection, apathy

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Preimplantation loss, postimplatation loss.
Decreased average body weight of foetuses with dependence on the dose level was recorded at all dose levels

Dose descriptor:

LOAEC

Effect level:

20 mg/kg bw/day

Basis for effect level:

other: overall effects litter size and weights; number viable (number alive and number dead); sex ratio; postnatal growth; postnatal survival; grossly visible abnormalities; external, soft tissue and skeletal abnormalities.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Administration of the test substance Potassium permanganate affected growth, clinical status and macroscopic structure of organs in treated maternal animals. These effects were significantly manifested in the results of health condition controls and clinical observations (cachexia, anemia, cough or hoarse breath, secretion from nostrils and eyes, piloerrection, apathy). During maternal animal necropsies the pathologic changes in stomach and uterus and slightly lower relative weight of pregnant uterus were found. Above mentioned changes were recorded markedly at the highest dose level and sporadically at the middle dose. Important effect on the significant decreased maternal weight was detected at the highest dose level.
In individual foetuses examination the effect to the weight of foetuses was found especially at the highest dose level. Increased number of aborted females and related negative changes of reproduction parameters (postimplantation losses) was also recorded in treated groups with higher incidence at highest and middle dose levels. The increased incidence of some of skeletal variations was found out in foetuses of all treated groups and could be an adverse effect of treatment.
Developmental effect of the test substance together with toxicity to the maternal animals was detected at the highest and middle dose levels. At the lowest dose level 20 mg/kg/day developmental effect (the decreased weight of foetuses, the increased incidence of delayed ossification of vertebrae) occurred in the absence of toxicity to the maternal animals.

Executive summary:

Introduction

   The test substance,Potassium permanganatewastested for prenatal developmental toxicity using the EUMethod B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008.

 

Study performance

     Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with males. The test substance was then administered to pregnant females - daily from the 5^thto the 19^thday of pregnancy. Thestudyincluded four groups of females – 3 treated groups and 1 control group (vehicle only).The test substance was administered dissolved in water by stomach tube and the concentrations of solutions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. 

 

    The dose levels for study – 20, 100 and 500 mg/kg/day were chosen on the basis of results of Study No. 15/06/7: Potassium permanganate – Repeated Dose (28 days) Toxicity (Oral); VUOS-CETA Report No. 0680, 2006 and Study No. 15/06/15: Potassium permanganate – One-Generation Reproduction Toxicity Test; VUOS-CETA Report No. 08142, 2008.

       

   The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during developmental toxicity study. On the 20^thday of pregnancy the maternal animals were killed, the uterine contents were examined and the foetuses were evaluated for soft tissue and skeletal changes.

Results

    At the dose level of 20 mg/kg/day the weight increments, mortality, health condition, clinical status after application, macroscopical structure of organsof pregnant females, values of reproduction parameters (number of live and dead foetuses, intra uterine death early), development of foetuses (foetal weight, evaluation of external and visceral affections)were unaffected by treatment of the test substance.    

    At the dose level of 100 mg/kg/day the mortalityof pregnant females and development of foetuses (evaluation of external and visceral affections)were unaffected by treatment of the test substance.    

    At the dose level of 500 mg/kg/day the mortality,values of some reproduction parameters (number of dead foetuses, intra uterine death early) of pregnant femalesand foetal development judged according to the external and visceral observationwere unaffected by treatment of the test substance.    

    

    The following effects could be attributed to the administration of the test substance:

    At the dose level of 20 mg/kg/day food consumption (decrease) anduterus biometry (decrease of relative weight of uterus) was very slightly influenced by administration of the test substance. Effect of the test substance on the weight of foetuses (slightly decrease) and the increased incidence of delayed ossification of vertebrae (skeletal variations) was recorded.

    At the dose level of 100 mg/kg/day body weight increment (slight decease at the end of application), food consumption (decrease), health condition, clinical status after application (sporadic occurrence of clinical symptoms of toxicity), biometry of uterus (decrease of relative weight of uterus), macroscopical structure of organsofmaternal animals were influenced by administration of the test substance. Some of reproduction parameters (intra uterine death earlyand late, decreased number of live foetuses), weight of foetuses (decrease) and increased incidence of skeletal variation (delayed ossification of vertebrae) were also affected.

   At the dose level 500 mg/kg/day body weight increment (significantly decreased), food consumption (markedly decreased), health condition, clinical status after application (occurrence of clinical symptoms of toxicity), biometry of uterus (decrease of relative weight) and macroscopical structure of organs of maternal animals (findings in digestive system) were influenced by administration of the test substance. Important effect on reproduction parameters (high incidence of aborted females, hight incidence of resorptions and accompanying high postimplantation losses), number of live foetuses (slight decrease of number), weight of male and female foetuses (significantly decrease in female foetuses) and increased incidence of skeletal variation (delayed ossification of vertebrae) were also recorded.

Administration of the test substance Potassium permanganateaffectedgrowth,clinical status and macroscopic structure of organs in treated maternal animals. These effects were significantly manifested in the results of health condition controls and clinical observations (cachexia, anemia, cough or hoarse breath, secretion from nostrils and eyes, piloerrection, apathy). During maternal animalnecropsies the pathologic changesin stomach and uterus and slightly lower relative weight of pregnant uterus were found. Above mentioned changes were recorded markedly at the highest dose level and sporadically at the middle dose. Important effect represented by the significantly decreased maternal weight was detected at the highest dose level. 

    In individual foetuses examination the effect to the weight of foetuses was found especially at the highest dose level. Increased number of aborted females and related negative changes of reproduction parameters (postimplantation losses) was also recorded in treated groups with higher incidence at highest and middle dose levels. The increased incidence of some of skeletal variations was found out in foetuses of all treated groups and could be attributed to an adverse effect of treatment.

  Developmental effect of the test substance together with toxicity to the maternal animals was detected at the highest and middle dose levels. At the lowest dose level 20 mg/kg/day developmental effect(the decreased weight of foetuses, the increased incidence of delayed ossification of vertebrae)occurred in the absence of toxicity to the maternal animals.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January 2014 to 30 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Remarks:

Study conducted on read-across material

Justification for type of information:

See report attached to Section 13 for justification.

Reason / purpose for cross-reference:

other: Read-across target

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliability 2, however, there is a large bod of published literature reporting developmental effects via oral route - weight of evidence

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5 µg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

data point addressed by recommended guideline and GLP study on analogue substance

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental: Manganese is an essential trace element and therefore is also of significance during pregnancy for bone growth and development of the inner ear and the reproductive organs in the embryo. Manganese crosses the placental barrier in man and in animals, accumulates in the foetus and crosses the blood-brain barrier four times as readily in newborn babies as in adults. It is secreted in the milk. Moreover, the gastrointestinal absorption of manganese is significantly more effective in infant/children than in adults, it is not effectively eliminated from the body and even less effectively from the brain (MAK, 2012). All the data indicate that foetus and infant/children can be particularly sensitive to the toxicity of an excess of manganese.

Toxicity to reproduction: other studies

Description of key information

One Prenatal study exists on the registered substance.Potassium permanganate was administered via oral exposure at the dose levels 20, 100 and 500 mg/kg/day in treated maternal animals.

 

Results

-          decreased body weight of maternal animals

-          clinical changes (cachexia, anemia, cough or hoarse breath, secretion from nostrils and eyes, piloerrection, apathy)

-          pathologic changes in stomach and uterus

-          lower relative weight of pregnant uterus

-          reproductive parameters – increased number of aborted females, increase postimplantation losses

-          development of organism in uterus – markedly decreased average body weight of foetuses

-          increased incidence of skeletal variations (incomplete ossification of sternum or cervical vertebrae) in foetuses

One prenaa developmental inhalation study exist on the analogue substance MnCl2.

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

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

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

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

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

One developmental neurotoxicity inhalation study on the analogue substance MnCl2 exist. The study performed according to OECD guideline 426, groups of Wistar Han female rats received manganese chloride by inhalation during gestation (days 6 to 19) and lactation (until post-natal day 20) (unpublished study report 12, 2016). The target concentrations were 5, 15 and 25 μg/L. Variations for the aerosol concentrations were noted and considered by the authors to be due to hygroscopic properties of the test item. In this context, the authors applied a correction factor to the gravimetric concentration to take into account water adsorption. Therefore, some uncertainties remain on the level of the real exposure reported in this study. Particle size measurements indicated that test aerosol is respirable to rats (MMAD less than 2 μm).

Breast milk measurement showed that the level of manganese chloride increased with increased administrated concentration which confirm the exposure of manganese via the milk. Blood measurement showed that the manganese concentration was below the lowest calibration solution in the control and low dose group (except two animals), around 1 μg/L in the mid dose group and between 1 – 2 μg/L in the high dose group.

One dam was killed in extremis on day 15 post coitum in the highest tested group. This dam showed breathing noises and labored breathing (highest grade) and was in a weakened condition. At the highest dose level, up to 70% of the dams showed breathing noises during the gestation period, with a frequency decreasing toward the end of the period. Mean food consumption was dose-dependently decreased from day 6 to 11 post-coitum at the two highest concentrations (-7 and -27% respectively compared to the control group) and recovered thereafter. There was no effect during lactation. Mean body weight gain was reduced at the two highest concentrations. In particular, a body weight loss up to 3% was reported within the first 5 days after treatment start in the highest tested group. Statistical significance was reached until day 17 post-coitum at the highest concentration and until day 12 post-coitum at the middle concentration. There was no effect during lactation. There was no effect on reproduction and breeding data or at macroscopical examination. The study report concluded at a NOAEC of 25 μg/L considering that reversibility was observed.

In pups, there was no treatment-related effect at external examination at first litter check, no treatment-related mortality and clinical signs. There were some variations of food consumption which reached statistical significance in males of the highest tested group; without evidence of a dose-response relationship. There were some decreases in body weight and body weight gain, only observed from day 22 post-partum to day 69 post-partum in all treated groups, without dose-dependence. No effect on developmental indices (pinna unfolding, incisor eruption, onset of coat development and opening of eyes) or on sexual maturation was reported. There was no functional/behavioural endpoints assessed during pre-weaning in contrast to OECD guideline recommendations. In particular, no complete functional observational battery (FOB) was possible for pups between days 5 to 11 post-partum due to the immature condition of the pups at this age. Instead a detailed clinical observation was performed without showing any treatment-related effect. The FOB on days 22, 35, 45 and 60 post-partum did not indicate any treatment-related effect. Punctual statistically significant effects (such as increased body temperature, grip strength, landing foot splay) were reported without linear curve. Locomotor activity and its development, assessed from day 13 until 60 post-partum, were not affected by manganese chloride. Learning and memory was demonstrated in all groups. The mean amplitude of startle response, time to maximum amplitude and habituation were not affected by treatment. There was no effect on brain (weight and macroscopical findings) on day 11, 22 or 63 post partum. No findings were noted on the length of the cerebellum and forebrain of all examined groups on day 11, 22 and 63 post-partum, except on day 63 post-partum where a slight decrease of length of cerebellum in males at the low dose and in females at the highest dose and also a increase of forebrain length in females in the mid dose were reported. However, it should be noted that many of the sections available for morphometric analysis and microscopic pathology were deemed non-assessable because of processing or sectioning artefacts. This decreases the confidence in the absence of findings reported in the brain. There was no findings in other organs assessed at macroscopical and microscopical examination on day 11, 22 and 63 post-partum. Overall, the developmental NOAEC was set at 25 μg/L.In conclusion, no effect on neurodevelopment is reported with manganese.

Additional information

A large body of published literature report some developmental effects upon the exposure to manganese in humans and rhodents. These studies are not guideline studies and have confounders. However, due to the large number of them a weight of evidence is considered.

Although the available database on guidelines studies for this endpoint ( on the analogue substance) is robust and report no neurodevelopmental effects, some developmental effects are reported on the prenatal developmental study although some of these effects have been seen to be reversible at post natal life.

The study on the registered substance , though of lesser reliability reports significant effects.

Overall, by weight of evidence a Repro 2 for development is proposed as self classification for KMnO4

Mode of Action Analysis / Human Relevance Framework

A large body of published literature report some developmental effects upon the exposure to manganese in humans and rhodents. These studies are not guideline studies and have confounders. However, due to the large number of them a weight of evidence is considered.

Although the available database on guidelines studies for this endpoint ( on the analogue substance) is robust and report no neurodevelopmental effects, some developmental effects are reported on the prenatal developmental study although some of these effects have been seen to be reversible at post natal life.

The study on the registered substance , though of lesser reliability reports significant effects.

Overall, by weight of evidence a Repro 2 for development is proposed as self classification for KMnO4 - also approved by the RAC and supported by ANSES CoRAP evaluation report.