Trimanganese tetraoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

WoE Approach

Carter et al. (1980) – Oral Route

The blood values of rats that were maintained on a normal-Fe diet while receiving dietary test material differed little from those of the control animals through 100 days of age, whereas animals maintained on a low-Fe diet and exposed to test material during the prenatal and postnatal period developed microcytic anaemia. Overall, young Fe-deficient animals treated during both the prenatal and postnatal period were consistently more haematologically sensitive to test material treatment than animals that received sufficient amounts of dietary Fe.

 

Rehnberg et al. (1980) – Oral Route

The study demonstrates that manganese via Mn3O4 oral exposure can be absorbed in neonates, and can accumulate in tissues. However, the mortality after day 12 in the top dose is not consistent with effects seen in later studies at the same dose level. It is believed that the mortalities are likely to be a consequence of insufficient iron in the diet of the high dose group, rather than a direct toxic effect of Mn3O4, and this is demonstrated in later studies (e.g. Laskey et al. 1985). The effect on long-term development cannot be judged from this study, and it is known that natural homeostatic mechanisms begin to reduce the amount of Mn absorbed fairly early, as seen in this study after 18 days exposure.

 

Rehnberg et al. (1982) – Oral Route

Mn3O4 exposure in neonates results in increase tissue concentrations. Neonate Fe-deficient animals are much more vulnerable to the effects of Mn3O4 exposure than neonate Fe-normal animals, and this can result in increased mortality due to Fe deficiency rather than any direct action of Mn3O4.

Gray LE and Laskey JW (1980) – Oral Route

No effect was seen on growth and general appearance of Mn3O4-treated animals. No effect on body weight, liver or kidney weight following Mn3O4 treatment. The only effects seen were reduced activity level of treated animals after 73 days and retarded growth of testes and accessory sex organs.

 

Read-Across: Elbetieha et al. (2001) – Oral Route

Findings from the study indicate that long-term ingestion of the read-across material MnCl2 may cause some adverse effects on both fertility and reproduction. However, the dosage levels of Mn employed in this investigation are relatively high and it is unlikely that Mn will be present at levels at high as this in the general, or occupational, environment.

 

Read-Across: Jardine (2013) – Inhalation Route

Under the conditions of the study the No Observed Effect Level (NOEL) for reproductive toxicity for the read-across material MnCl2, was determined to be 20 µg/L.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2 July 2012 to 4 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese dichloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2.

Reason / purpose for cross-reference:

other: Target substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (F0) 6 - 8 weeks
- Weight at study initiation: (F0) Males: 155 - 298 g; Females: 130 - 194 g
- Housing: Animals were initially housed 2 per cage by sex in polycarbonate cages measuring approximately 61 x 43.5 x 24 cm with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a stainless steel grid insert measuring approximately 48 x 37.5 x 25 cm. After mating, the males were rehoused with their original cage-mates in solid bottomed cages. Mated females were transferred to individual solid bottomed cages (approximately 58.6 x 42.5 x 21 cm). White paper tissues were supplied as nesting material from Day 20 of gestation. Females with litters were retained in this cage type until termination after weaning. F1 animals retained after weaning were housed 2 per cage in cages measuring approximately 61 x 43.5 x 24 cm, as described above. The F1 animals then followed the same caging regime as described for the F0 animals.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: F0 animals were acclimatised for 13 days before the commencement of dosing. For at least 7 days prior to commencement of dosing the animals were conditioned to the restraint procedures used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing period of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 26°C
- Humidity (%): 33 - 69%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose only

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test aerosols were generated using a Wright Dust Feed generator device. Exposure of the animals to the test material, or vehicle, was achieved utilising a modular nose only stainless steel flow past inhalation chamber.

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

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

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

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


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

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.

Details on mating procedure:

A few days prior to the initiation of mating, the males were separated into individual grid bottomed cages. Pairings were on a 1 male to 1 female basis. Animals were paired in numerical order within the groups. Each female was transferred to the cage of its appropriate co-group male near the end of the work day, where it remained until mating had occurred or 14 days had elapsed. Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrous observed in each lavage recorded. The presence of sperm in such a lavage and/or a copulatory plug in situ was designated as Day 0 of gestation. If the number of males in a group was reduced by mortality, mating was on a 1 male to 2 female basis.
The time taken for each female to show a positive mating sign was evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at Charles River, Edinburgh under Study No. 428133 (Method No. 2813). Full details of the analytical methodology are contained within that report.

Duration of treatment / exposure:

F0 animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation. For F0 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation. For F1 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).

Frequency of treatment:

Daily (ca 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of lactation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca 1 hour per day
From Day 3-4 of lactation: ca 2 hours per day
From Days 5-20 of lactation until prior to termination (ca Day 21 of lactation): ca 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.

Details on study schedule:

- Selection and Weaning of F1 Animals
From each group, at least 24 males and 24 females were selected for post-weaning assessments. The selected pup(s) were the median’th weight pup(s) of that sex in the litter on Day 21 of lactation. These pups were removed from their mother on Day 21 of lactation, individually identified and housed in a new cage. Pups that were not selected for post-weaning assessments remained with their mother until sacrifice.

Dose / conc.:

5 other: µg/L

Remarks:

Nominal conc.

Dose / conc.:

10 other: µg/L

Remarks:

Nominal conc.

Dose / conc.:

20 other: µg/L

Remarks:

Nominal conc.

Dose / conc.:

6 other: µg/L

Remarks:

Analytical conc. F0 generation

Dose / conc.:

15 other: µg/L

Remarks:

Analytical conc. F0 generation

Dose / conc.:

25 other: µg/L

Remarks:

Analytical conc. F0 generation

Dose / conc.:

4 other: µg/L

Remarks:

Analytical conc. F1 generation

Dose / conc.:

10 other: µg/L

Remarks:

Analytical conc. F1 generation

Dose / conc.:

17 other: µg/L

Remarks:

Analytical conc. F1 generation

No. of animals per sex per dose:

- F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected for use based on results from a preliminary reproduction study in rats (Charles River Study 495849). In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values (TLVs) proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.

Positive control:

na

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- All animals were checked for early each morning and as late as possible each day for viability. Furthermore, all animals were examined for reaction to treatment daily during the course of dosing on the study. The onset, intensity and duration of any signs were recorded.

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

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

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

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

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

- Seuxal Maturation
Commencing at 28 days of age, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the
body weight on that day. Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

Oestrous cyclicity (parental animals):

Over a 2 week period prior to the initiation of mating, vaginal lavages were taken early each morning and the stages of oestrous observed were recorded.

Sperm parameters (parental animals):

The tip of the cauda epididymis was placed in Medium 199 containing 0.2% BSA and HEPES. The sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was examined using a Hamilton Thorne sperm motility analyser; sufficient replicates to provide 200 motile sperm were assessed (except where it was obvious that motility was compromised for that animal).
The remaining portion of the cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From a sample of the sperm suspension described above, a sperm smear was prepared and stained with eosin. From the Control and High dose animals, two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce.
One testis wase decapsulated and homogenized. The homogenate may have been sonicated to remove tissue debris etc, as required. The number of homogenisation resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

Litter observations:

The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined from Day 1 onwards for the presence of milk in the stomach and for any externally visible abnormalities daily. The pups were weighed en masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day 21 all pups were weighed individually.
Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Prior to Day 14 of lactation, any externally abnormal decedent pup was preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.

Postmortem examinations (parental animals):

SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

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

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

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

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

HISTOPATHOLOGY
Histological examination was conducted on all adults in the Control and High dose groups of the F0 and F1 generation and a selection of the premature decedents. After a review of the data, histological examination of the respiratory tract tissues of the Control and High dose animals, it was considered appropriate to conduct histopathology on the respiratory tract of all adult animals of the F0 and F1 generation.
The following tissues were processed for microscopic evaluation: adrenal glands, larynx, left testis, left epididymis, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, prostate, pituitary gland, seminal vesicles and coagulating glands, trachea (anterior and posterior), uterus (with oviducts and cervix), vagina.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.

Postmortem examinations (offspring):

SACRIFICE / GROSS NECROPSY
Pups that were not selected for post-weaning assessments were killed at the same time as their mother.
Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

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

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

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

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

HISTOPATHOLOGY
Histological examination was conducted on the brain, spleen and thymus of Control and High dose F1 and F2 weanlings (the selected weanlings at necropsy). A single H&E section was cut, stained and evaluated.

Statistics:

Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Reproductive indices:

For each group the following were calculated:

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

Fertility Index (female) = Number pregnant / Number paired

Gestation Index = Number bearing live pups / Number pregnant

Offspring viability indices:

For each litter and group the following were calculated:

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

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

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

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

Overall Survival Index = Number of pups live on Day 21 of lactation / Total number born (live and dead)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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 jejenum, 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 751 (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 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 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.
- 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 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.

REPRODUCTIVE FUNCTION: ESTROUS 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 ofthe 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.

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.

Dose descriptor:

NOEL

Effect level:

20 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effectswere observed

Remarks on result:

other: Generation: F0 and F1

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

LITER 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.

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 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.

Dose descriptor:

other: Not specified

Generation:

F1

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

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

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 timepoint. At terminal necropsy, these values were 383% and 371% for males and females.

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 timepoint. At terminal necropsy, these values were 350% and 243% for males and females.

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 timepoint. At terminal necropsy, these values were 217% and 129% for males and females.

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 timepoint. At terminal necropsy, these values were 250% and 200% for males and females.

At target 10 μg/L, manganese levels prior to mating were 112% higher than controls in males and 133% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 167% and 129% for males and females.

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

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.

Conclusions:

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

Executive summary:

The reproductive toxicity of the test material was investigated in a two generation study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800.

F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

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

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

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

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

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

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

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

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

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

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

In accordance with Section 1, Annex XI of the REACH regulation, reproductive toxicity testing (EOGRTS) or further developmental toxicity testing (OECD 414) in a second species (rabbit), via the oral route, does not appear scientifically necessary. The principal route of human exposure in the workplace (as regulated by REACH, as opposed to essential dietary intakes regulated elsewhere) is by inhalation. Occupational exposures for inhalation of manganese compounds including oxides are regulated by workplace TLVs or similar measure. The SCOEL-IOELV is 0.2 mg/m3. A rat inhalation multi-generation study of a soluble Mn salt (MnCl2) shows a NOAEL at 20 mg/m3. An oral developmental toxicity in the rat of (insoluble, so less bioavailable) Mn3O4 showed a NOAEL at 250 mg/kg bw/day, significantly above the IOELV. Further oral testing of Mn3O4 will therefore test not only by an inappropriate route, but at dose levels that are far in excess of the current IOELV. It is inconceivable that additional animal data could be used to set a higher reference dose, than the IOELV based on human experience in the workplace. Any further animal testing of Mn3O4 therefore cannot provide any additional useful information for regulatory risk assessment. It is also considered particularly unjustified on animal welfare grounds considering the high number of animals that would be required. Further studies cannot be used for regulatory purposes; the registrant notes that animal studies should not be conducted solely for the purpose of classification. A more detailed justification is provided in the report “Revised Justification for Omission of reproductive and developmental toxicity studies with Mn3O4", as attached.

Reproductive effects observed:

not specified

Reference 3

Endpoint:

toxicity to reproduction

Remarks:

other: haematological effects of chronic Mn3O4 exposure over two generations

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not conducted to GLP, standard observations not made. Focus is on haematology and effect of Mn in rats maintained on low-Fe diets.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The haematological effects of chronic Mn3O4 exposure during both prenatal and postnatal periods was determined. Data was obtained from the parental to F2 generations. Effects of exposure to Mn3O4 during Fe deficiency were determined by placing half of the animals of a low Fe diet and the other half on a normal diet. Areas of biological assessment included growth and development, haematology, tissue accumulation and distribution, and reproduction.

STUDY 1: Rats (known to be at gestation day 1) were randomly assigned to 8 treatment groups. Treatment began on gestational day 2 and was continued in the F1 generation through 244 d of age. Food consumption was evaluated and blood parameters were recorded on days 24, 40, 60, 100 and 224 of age.

STUDY 2: Rats (known to be at gestation day 1) received the normal-Fe diet during gestation. When the pups reached 10 days of age, all litters were randomly separated into 4 treatment groups and dosing was initiated. Treatment continued until tail blood samples were obtained and the animals were killed at 40 days of age. Haematological measurements were recorded.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altamont, NY, USA
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

not specified

Details on exposure:

DIET PREPARATION
Treatment diets were prepared biweekly by adding test material to obtain the desired Mn concentrations in the diet. Basal diet initially contained 50 ppm Mn as manganese sulphate; the remainder of additive was as test material.
Experimental diets were stored in a cool room (10°C) and were used within 2 - 4 weeks to minimise loss of nutritional quality.

Mean particle diameter:
STUDY 1: 0.62 µm (range 0.1 - 2.8 µm), 80% of particles evaluated were <1 µm in diameter.
STUDY 2: 1.2 µm (range: 0.1-5.6 µm), 45% of total particles were <1µm in diameter.

Details on mating procedure:

STUDY 1: Animals were received at day 1 of gestation.
STUDY 2: Animals were received at day 1 of gestation.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

STUDY 1: treatment continued from day 2 of gestation to day 224 of age (F1 generation)
Study 2: treatment continued through pregnancy to day 40 of age (F1 generation).

Frequency of treatment:

Daily (in diet)

Dose / conc.:

400 other: ppm Mn

Remarks:

Nominal in diet

Dose / conc.:

1 100 other: ppm Mn

Remarks:

Nominal in diet

Dose / conc.:

3 550 other: ppm Mn

Remarks:

Nominal in diet

No. of animals per sex per dose:

No data

Control animals:

yes, plain diet

Statistics:

All data were analysed by a computer-based statistical package containing analysis of variance (ANOVA), Duncan's multiple range test, and correlation procedures. Data were analysed for homogeneity of variance.

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Dose descriptor:

other: Not specified

Generation:

F1

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Study 1:

Red Blood Cell count, Haematocrit and Mean cell volume values of animals maintained on normal-Fe diets differed significantly from animals maintained on low-Fe diets.

- Mortality: mortality rate was increased (>50%) in animals at the highest dose group (low-Fe), from birth to 40 d of age. Consequently all remaining animals in this group were killed at day 40. All blood values were significantly lower than the control animals on the low-Fe diet.

- Blood parameters: haematocrit values in the low-Fe groups was depressed from 24 to 100 days of age. There were no evident Mn dose responses.

A strong correlation between dietary Fe levels and certain serum constituents was noted at both 100 and 200 days of age.

- Bodyweights and food consumption: a significant dose-related decrease in bodyweight was seen at days 52 and 88. Animals receiving the low-Fe diet consumed more food than the controls. This was not significant.

Study 2:

No significant Mn -related effects.

Conclusions:

The blood values of rats that were maintained on a normal-Fe diet while receiving dietary test material differed little from those of the control animals through 100 days of age, whereas animals maintained on a low-Fe diet and exposed to test material during the prenatal and postnatal period developed microcytic anemia. Overall, young Fe-deficient animals treated during both the prenatal and postnatal period were consistently more haematologically sensitive to test material treatment than animals that received sufficient amounts of dietary Fe.

Executive summary:

Haematological values were studied in rats after chronic exposure to test material. Data were obtained at selected ages from the P0 through the F2 generation. Effects of exposure to test material during Fe-deficiency were determined by placing half of the animals on a low-Fe diet (20 mg/kg) while the other half were maintained on a normal-Fe diet (240 mg/kg). Animals treated with test material and maintained on a normal-Fe diet showed little variation from controls through 100 days of age. However, animals (24 - 100 days of age) maintained on a low-Fe diet and receiving Mn treatment during the prenatal and postnatal periods developed microcytic anemia. Overall, young Fe-deficient animals treated during both the prenatal and postnatal period were consistently more haematologically sensitive to test material treatment than animals that received sufficient amounts of dietary Fe.

Reference 4

Endpoint:

fertility, other

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A well documented study with a clearly defined scope and with sufficiently detailed material and methods. However, there is no statement on impurity profile of the test material. Furthermore, since the study was conducted with manganese chloride tetrahydrate, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2.

Reason / purpose for cross-reference:

other: Read-across target

Qualifier:

no guideline followed

Principles of method if other than guideline:

EXPERIMENT 1: Fertility was estimated in male mice exposed to MnCl2 via drinking water for 12 weeks. Water consumption and body weights were measured daily. Following exposure each male was placed in an individual cage with 2 virgin untreated females of the same strain, for 10 days after which males were removed and females were sacrificed 10 days later by cervical dislocation. The number of pregnant females, implantation sites, viable foetuses and resorptions were recorded.

EXPERIMENT 2: Fertility was estimated in adult female mice exposed to MnCl2 via drinking water for 12 weeks via drinking water. Body weight, water consumption and general appearance were recorded daily. Treated females were divided into groups of 3 animals each and housed with a sexually mature untreated male of proven fertility for 10 days. 10 days following the removal of untreated males, the mice were sacrificed by cervical dislocation. The following measurements were recorded: number of pregnant females, number of implantations, number of viable foetuses and number of resorptions.

EXPERIMENT 3: Female mice exposed to MnCl2 via drinking water were sacrificed after 12 weeks of exposure and body weight and weights of paired ovaries and uteri were recorded.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 50 days old
- Weight at study initiation: approximately 28 g
- Diet: ad libitum
- Water: ad libitum
The mouse diet had a manganese content of 60 mg//kg chow and the tap water contained 0.3 mg/L. The manganese intakes that are reported in the study are based on the manganese chloride added to the drinking water only and do not account for the background levels in food and water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1 °C
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test material was dissolved in tap water at the required test concentrations.

Details on mating procedure:

Experiment 1:
Following exposure each male was placed in an individual cage with 2 virgin untreated females of the same strain. They were left together for 10 days during which two estrous cycles should have elapsed.

Experiment 2:
Treated females were divided into groups of 3 animals each and housed with a sexually mature untreated male of proven fertility for 10 days. During this period, two estrous cycles should have elapsed.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

daily (in water)

Remarks:

Doses / Concentrations:
0, 1000, 2000, 4000 and 8000 mg/L
Basis:
nominal in water

Remarks:

Doses / Concentrations:
0, 108, 172, 352, 707 mg/kg bw
Basis:
actual ingested
(males)

Remarks:

Doses / Concentrations:
0, 100, 188, 359, 635 mg/kg bw
Basis:
actual ingested
(females)

No. of animals per sex per dose:

- Experiment 1
14 males and 28 females per dose

- Experiment 2
15 females per dose group

- Experiment 3
8 females in the control group, 12 females in each of the treatment groups

Control animals:

yes

Details on study design:

- Dose selection rationale: these concentrations were chosen according to previous studies.

Positive control:

na

Parental animals: Observations and examinations:

- Experiment 1:
Bodyweights and water consumption of the treated males were recorded daily.

- Experiment 2:
Bodyweight, water consumption and general appearance of treated females were recorded daily.

- Experiment 3:
Bodyweight was recorded.

Oestrous cyclicity (parental animals):

na

Sperm parameters (parental animals):

na

Postmortem examinations (parental animals):

Experiment 1:
10 days after the end of the 10 day mating period, the males were removed from mating cages and the females were sacrificed by cervical dislocation under light ether anaesthesia. The number of pregnant females, number of implantation sites, number of viable foetuses and the number of resorptions were recorded.

Experiment 2:
10 days after the end of the 10 day mating period, the males were removed from mating cages and the females were sacrificed by cervical dislocation under light ether anaesthesia. The number of pregnant females, number of implantations, number of viable foetuses and number of resorptions were recorded.

Experiment 3:
At the end of the 12 week exposure period the weights of paired ovaries and uteri were recorded.

Statistics:

Data were expressed as means ± S.D. Differences between control and test material exposed groups were analysed using Fisher’s exact test, (two-tail) and Student’s t-test. The P values of <0.05 were considered statistically significant.

Clinical signs:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: See "Details on results" for information

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

EFFECT ON WATER CONSUMPTION: In experiment 1, ingestion of test material by adult male mice significantly decreased the average daily water consumption per animal as compared to the controls. Similar results was observed with the female mice in experiment 2.

BODYWEIGHT: In both experiments there were no significant effects of the test material on body weight gain.

EFFECTS OF TEST MATERIAL ON FERTILITY OF MALE MICE:
Fertility was significantly reduced in males exposed to test material at 8000 mg/L. The number of implantation sites, viable foetuses and the total number of resorptions in females impregnated by male rats exposed to test material was not statistically different from their control counterparts.

EFFECTS OF TEST MATERIAL ON THE FERTILITY OF FEMALE MICE:
Fertility was not significantly reduced in females exposed to all concentrations of test material. The number of implantation sites and viable foetuses were significantly reduced in pregnant females exposed to 8000 mg/L test material compared to control unexposed pregnant females.

EFFECTS ON BODY AND ORGAN WEIGHTS (EXP. 3): Exposure of female mice to test material at any concentration had no effect on body weight. Ovarian weights were significantly increased in females exposed to 4000 and 8000 mg/L, whereas uterine weights were significantly increased in 1000 mg/L, 2000 mg/L, 4000 mg/L and 8000 mg/L test material exposed females.

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Dose descriptor:

other: Not specified

Generation:

F1

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Table 1: Effect of 12 weeks of ingestion of test material on average daily water consumption in male mice^a

Treatment	Water consumption (mL/day)	Test material from drinking water (mg/kg per day)^b
Control	4.64 ± 0.34	-
1000 mg/L	3.12 ± 0.28*	108.3 ± 6.34
2000 mg/L	2.50 ± 0.22**	172 ± 13.02
4000 mg/L	2.68 ± 0.29**	352 ± 14.91
8000 mg/L	2.72 ± 0.24**	706.5 ± 18.26
^aData are expressed as mean ± SD ^bThe calculations of Mn intake are based soled on the MnCl2added to the drinking water. * P <0.05 significantly different as compared to control value (Student’s t-test). ** P < 0.005 significantly different as compared to control value (Student’s t-test).

 

Table 2: Effect of 12 weeks ingestion of test material on average daily water consumption in female mice

Treatment	Water consumption (mL/day)	Test material from drinking water (mg/kg per day)^a
Control	4.59 ± 0.37	-
1000 mg/L	2.94 ± 0.29*	99.83 ± 8.61
2000 mg/L	2.92 ± 0.27 **	187.54 ± 9.34
4000 mg/L	2.79 ± 0.39**	358.84 ± 14.16
8000 mg/L	2.25 ± 0.18**	634.92 ± 21.52
^aResults are expressed as mean ± SD * P <0.05 significantly different as compared to control value (Student’s t-test). ** P < 0.005 significantly different as compared to control value (Student’s t-test).

 

Table 3: Effect of long term exposure to test material via drinking water on fertility of male mice.

Treatments	No. of males	No. of females	No. of pregnant females (%)	No. of implantations^a	No. of viable foetuses^a	Total no. of resorptions
Control	14	28	26/28 (92)	9.00 ± 2.22	8.76 ± 3.35	7
1000 mg/L	14	28	25/28 (89)	8.73 ± 1.68	8.50 ± 1.74	13
2000 mg/L	14	28	22/28 (78)	8.86 ± 1.75	8.40 ± 2.23	10
4000 mg/L	14	28	20/28 (71)	8.15 ± 1.81	7.60 ± 1.87	11
8000 mg/L	14	28	17/22 (66)*	8.00 ± 1.96	7.70 ± 1.89	6
^aResults are expressed as mean ± SD * P <0.05 significantly different as compared to control value (Fisher’s exact test (two tail)).

 

Table 4: Effect of long-term exposure to test material via drinking water on fertility of female mice

Treatments	No. of females	No. of pregnant females (%)	No. of implantations^a	No. of viable foetuses^a	No. of mice with resorptions (%)	Total no. of resorptions
Control	15	13/15 (86)	9.41 ± 1.68	9.41 ± 1.8	0/13 (0.0)	0
1000 mg/L	15	13/15 (86)	9.08 ± 1.62	9.00 ± 1.68	3/13 (23)	3
2000 mg/L	15	13/15 (86)	8.42 ± 1.92	8.25 ± 2.05	2/13 (15)	2
4000 mg/L	15	9/15 (60)	8.43 ± 2.38	8.28 ± 2.22	1/9 (11)	1
8000 mg/L	15	10/17 (66)	7.80 ± 1.55*	7.60 ± 1.58*	2/10 (20)	2
^aResults are expressed as mean ± SD * P<0.025 significantly different as compared to control value (Student’s t-test).

 

Table 5: Body and organ weights of female mice exposed to test material for 12 weeks via drinking water^a

Details	Treatments
	Control	1000 mg/L	2000 mg/L	4000 mg/L	8000 mg/L
No. of animals	8	12	12	12	12
Body weight (g)	34.6 ± 6.09	32.84 ± 5.12	33.31 ± 3.40	33.21 ± 3.02	33.33 ± 4.53
Ovarian weights (mg/10 gm b.wt)^b	2.12 ± 0.83	2.5 ± 0.74	2.4 ± 0.79	3.50 ± 1.06*	4.70 ± 2.3*
Uterine weights (mg/10 gm b.wt)^b	23.60 ± 8.51	31.80 ± 1.28*	34.50 ± 8.12*	35.50 ± 6.9**	33.7 ± 9.7
^aResults are expressed as mean ± SD ^bRelative weights * P <0.05, significantly different compared to control value (Student’s t-test) ** P <0.005, significantly different compared to control value (Student’s t-test)

 

Conclusions:

Findings from the study indicate that long-term ingestion of the test material may cause some adverse effects on both fertility and reproduction. However, the dosage levels of Mn employed in this investigation are relatively high and it is unlikely that Mn will be present at levels at high as this in the general, or occupational, environment.

Executive summary:

The effect of long-term ingestion of the test material was investigated on fertility of male and female Swiss mice. Adult male or female mice ingested a solution of test material along with drinking water at concentrations of 1000, 2000, 4000 and 8000 mg/L for 12 weeks. Fertility was significantly reduced in male mice exposed to test material solution at a concentration of 8000 mg/L, but not at the other concentrations. There were no treatment-related effects on the number of implantation sites, viable foetuses or the number of resorptions in female rats impregnated by males who had ingested test material. Fertility was not significantly reduced in female mice exposed to test material solution at all concentrations used in this study. However, the numbers of implantations and viable foetuses were significantly reduced in females exposed to test material solution at a concentration of 8000 mg/L. There was no significant effect on the number of resorbed foetuses in females exposed to test material solution compared to their control counterparts. Absolute body weight was not significantly affected in females exposed to test material solutions. However, ovarian weight was significantly increased in females exposed to test material solution at concentrations of 4000 and 8000 mg/L. A significant increase in the uterine weight was also observed at all concentrations used in the study. These results indicate that ingestion of high levels the test material by adult male and female mice causes some adverse effects on fertility and reproduction.

Reference 5

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Exposure length not clearly defined, uncertainty about group size.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice were exposed to Mn3O4 in the diet, from day 15 of lactation. At 30 days of age male pups were weaned and housed in groups of 2 or 3 with littermates. Growth, general appearance and the presence of any gross behavioural abnormalities were noted. Furthermore, locomotor activity was assessed. At necropsy, bodyweights and selected organ weights were recorded.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass., USA
- Age at study initiation: (P): Age unknown - pregnant dams were received at day 12 of gestation.
- Housing: Pregnant dams were housed individually in transparent plastic mouse cages (33 x 20 x 13 cm). At day 30 of age male pups were weaned and housed in groups of two or three with littermates.
- Diet: dams were placed on a casein diet in day 13 of gestation; the diet contained 50 ppm Mn as manganese sulphate

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12:12 light:dark

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The treatment group were fed on a diet containing the basic casein diet (50 ppm MnSO4) with an additional 1050 ppm Mn3O4. Controls were maintained on the casein diet.

Details on mating procedure:

Pregnant mice were received on day 12 of gestation

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Sacrifice was performed on days 54, 73 and 90

Frequency of treatment:

continuous (in diet)

Details on study schedule:

On day 15 of lactation, 26 healthy litters were randomly assigned to one of two treatment regimens (the control group which received plain diet, and the treatment group which received diet with the addition of 1050 ppm as Mn3O4).

Dose / conc.:

1 050 other: ppm Mn3O4

Remarks:

Nominal in diet

No. of animals per sex per dose:

no data

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

No observations reported

Litter observations:

- At day 73 of age, 15 control males and 11 males from the Mn3O4-treated litters were tested during the day for 2-h RLA levels in a residential maze. The residential maze contained 8 photocells. The number of beam interruptions over a 2 hour period was recorded as a measure of locomotor activity.

Postmortem examinations (parental animals):

No observations reported

Postmortem examinations (offspring):

SACRIFICE
- Four nonsibling males from each treatment group were sacrificed at 58 days of age. These animals were subjected to post-mortem examinations (macroscopic and/or microscopic examination) as follows: prepubital glands, seminal vesicles and paired testes were weighed. Sex accessory glands were measured.
- At day 73 of age, 15 control males and 11 males from the Mn3O4-treated litters were subject to behavioural testing. After behavioural testing, 17 control and 12 treated males were sacrficed and their preputial, seminal vesicle, testis and bodyweights were recorded.
- At day 90 of age, 22 control males and 10 males from the Mn3O4-treated litters were sacrificed.
- In addition, the liver and kidney weights were taken in a few cases at 73 and 90 days.

Statistics:

A two-way multivariate analysis of variance (ANOVA) was used to test the hypotheses that age and Mn treatment alter reproductive development.

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Dose descriptor:

other: Not specified

Generation:

F1

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

The growth and general appearance of the Mn-treated animals was normal throughout the experiment. Administration of 1050 ppm Mn did not induce tremors, ataxia, or hind limb paralysis, which are characteristic of severe cases of manganism in humans. However, the activity levels of the Mn-treated males were significantly reduced.

Growth of the reproductive tissues was retarded by Mn administration.

Unlike reproductive development, body, liver, and kidney weights were unaffected by treatment.

Conclusions:

No effect was seen on growth and general appearance of Mn3O4- treated animals. No effect on body weight, liver or kidney weight following Mn3O4 treatment. The only effects seen were reduced activity level of treated animals after 73 days and retarded growth of testes and accessory sex organs.

Executive summary:

Mice were exposed to Mn3O4 in the diet, from day 15 of lactation. At 30 days of age male pups were weaned and housed in groups of 2 or 3 with littermates. Growth, general appearance and the presence of any gross behavioural abnormalities were noted. Furthermore, locomotor activity was assessed. At necropsy, bodyweights and selected organ weights were recorded.

Results of the study demonstrated that chronic dietary administration of test material to the male mouse altered behavioural and reproductive development.

It is not clear from the present study whether the reduction in RLA is causally related to or only correlated with reproductive dysfunction, as both are affected by Mn treatment. It is possible that the reduced locomotor activity seen in the present study resulted directly from the action of Mn on the brain rather than being mediated through a reduction in gonadal hormones.

Although growth of the testes and sex accessory gland was retarded by Mn administration, body and liver weights were unaffected; this indicates a specific effect on the hypothalamic-pituitary-gonadal axis rather than a general retardation of the growth of all organs.

Overall, chronic exposure to test material in the diet at 1050 ppm Mn retarded the sexual development and lowered reactive locomotor activity levels in male mice.

Reference 6

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not to GLP but follows basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Pre-weanling rats (M and F ) were dosed daily by gavage from day 1-day 21 postpartum with particulate Mn3O4 dosed, by gavage, as a suspension in sucrose solution. Animals were sacrificed on days 3, 6, 9, 12, 15, 18 and 21. Brain, liver, kidney and testes were removed, weighed, and analysed for Mn and Fe contents. Red blood cells, white blood cells, haematocrit, mean cell volume, and haemoglobin were measured in all control animals, all group 4 animals, and 21-day old group 2 and 3 animals.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altamont, NY, USA
- Housing: Individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

other: 50% (w/v) sucrose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Mn3O4 particles were suspended in a 50% (w/v) sucrose solution to achieve Mn concentrations of 21 (group 2), 71 (group 3) and 214 (group 4) µg/µL.

DOSING METHODS
Each animal received 1 µL/g bw of dose solution or vehicle.

Details on mating procedure:

48 timed-pregnant rats were used

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

neonates were dosed from day 1 to day 20 after birth (21 day exposure).

Frequency of treatment:

Daily

Details on study schedule:

Pups of the pregnant rats were delivered naturally and each litter was normalised to six pups (3 males and 3 females) on day 1.

Dose / conc.:

21 other: µg/µL

Remarks:

Actual ingested

Dose / conc.:

71 other: µg/µL

Remarks:

Actual ingested

Dose / conc.:

214 other: µg/µL

Remarks:

Actual ingested

No. of animals per sex per dose:

36 male pups / 36 female pups per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

Pregnant females were randomly assigned to 4 groups.

Litter observations:

Haematological examinations were made on the control and high dose groups on days 3, 6, 9, 12, 15, 18 and 21 of age and on groups 2 and 3 animals at day 21 of age.

Postmortem examinations (offspring):

4 females and 4 males (non-littermates) were randomly selected from each dosage group were weighed and sacrificed on day 3, 6, 9, 12, 15, 18 and 21 of treatment. Brain, Liver, Kidney and testes were removed, weighed and analysed from Mn and Fe content. RBC's, WBC's, haematocrit, mean cell volume and haemoglobin were determined in blood samples.

Statistics:

Statistical analyses included paired t-test, analysis of variance (ANOVA), covariance, Duncan's multiple range test, and probit analysis.

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings:

not specified

Dose descriptor:

other: LD50

Generation:

F1

Effect level:

403 other: µg/g.d

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: Probit analysis

Dose descriptor:

other: LD50

Generation:

F1

Effect level:

353 other: µg/g/d

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Probit analysis

Dose descriptor:

other: LD50

Generation:

F1

Effect level:

225 other: µg/g/d

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: Probit analysis

Dose descriptor:

NOEC

Generation:

F1

Effect level:

1 200 other: g/g/d

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: Not specified

Reproductive effects observed:

not specified

BODYWEIGHT: bodyweights of animals receiving 21 µg/g day were not statistically different from those of the controls at any time during the study. Bodyweights of animals exposed to 71 and 214 µg/g day were significantly different to those of the controls from day 3 onwards.

MORTALITY: survival was not affected up to day 12. Significant mortality occurred beyond day 12.

ORGAN WEIGHTS: No treatment-related effects were noted before day 12 of age. Liver weights were significantly depressed in the two highest dose groups at 18 and 21 days of age. Kidneys in the highest dose groups expressed increased weights at day 18 and 21. Brain weights were depressed in the higher dose groupsby day 12 of treatment and remained depressed throughout. Testes weights in the higher dose groups were significantly lower than those of the controls between 15 and 21 days of age.

Mn CONCENTRATIONS: The degree of accumulation was not proportional to the dose. The highest concentrations were present in the liver. Significant increases in Mn concentrations in the testes were seen in the two highest dose groups by day 6.

Liver>brain>kidney>testes at 18 -21 days of age. Tissue concentrations were highest on day 18 and began to drop by 21, despite no change in the dose levels

HAEMATOLOGY: There was a significant dose-related depression in RBC's, HCT and HGB at day 21 of age. In the high dose group this depression was apparent by day 15.

Conclusions:

The study demonstrates that manganese via Mn3O4 oral exposure can be absorbed in neonates, and can accumulate in tissues. However, the mortality after day 12 in the top dose is not consistent with effects seen in later studies at the same dose level. It is believed that the mortalities are likely to be a consequence of insufficient iron in the diet of the high dose group, rather than a direct toxic effect of Mn3O4, and this is demonstrated in later studies (e.g. Laskey et al 1985). The effect on long-term development cannot be judged from this study, and it is known that natural homeostatic mechanisms begin to reduce the amount of Mn absorbed fairly early, as seen in this study after 18 days exposure.

Executive summary:

Mn accumulation was evaluated in selected tissues of preweanling rats dosed daily with test material. Significant findings included a high rate of Mn accumulation in the preweanling rat; a Mn dose-related acceleration of postpartum liver iron depletion; a Mn dose-related depression in red blood cells, haematocrit, haemoglobin, body weight, and survival by 21 days postpartum; and a Mn distribution in tissues with liver > brain > kidney > testes at 18 - 21 days of age.

Reference 7

Endpoint:

toxicity to reproduction

Remarks:

other: Assessment of tissue Mn over two generations

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not to GLP, not a standard reproductive toxicity study. Follows basic scientific principles, basic observations made.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were chronically exposed to Mn3O4 particulate through two generations. Animals were exposed from day 2 of gestation (F1 generation) through 24 days of age (F2 generation). Observations were made of growth, tissue content and distribution of Mn and Fe in animals exposed to a normal Fe diet and low-Fe diets. These observations were made at selected periods and included pregnant and lactating dams (F1 dams), weanlings (F1 and F2), adolescents (F1), adults (F1), and neonates (F2).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altamont, NY, USA)
- Age at study initiation: time-pregnant animals were recieved on day 1 of gestation
- Housing: Individually
- Diet: TD-76476 (240 mg/kg Fe) and TD-76475 (20 mg/kg Fe) obtained from Teklad Test Diets, Madison, USA, ad libitum*
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

* test diest contained a background level of Mn of 50 µg/g as manganese sulphate

Route of administration:

oral: feed

Details on exposure:

Rats were divided into two groups: one maintained on a low-Fe diet (20µg/g), the second maintained on a normal Fe diet (240 µg/g). Each group was then divided into 4 subgroups to receive different amounts of Mn in the diet.

DIET PREPARATION
- Diets were prepared in 10 kg amounts.
- Storage temperature of food: 10°C
-Mn content: before mixing the basal casein diet contained 50 µg/g Mn as manganese sulphate. Particulate Mn3O4 was added to achieve total Mn concentrations.

Details on mating procedure:

Time-pregnant animals were recieved on day 1 of gestation (F1 generation)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Animals were exposed from day 2 of gestation (F1 generation) through 24 days of age (F2 generation)

Frequency of treatment:

Daily in feed

Details on study schedule:

- At term pups were delivered naturally. At day 1 postpartum the F1 litters were normalised to 6 pups (3 male/ 3 female), crossfostering when necessary. Pups were weaned on day 24 postpartum. At 92-94 days of age, F1 females were mated to non-littermate males of the same dose group. Half of the dams delivered naturally and half were delivered by caesarean section . F2 pups that were delivered naturally were normalised to 6 pups (3 male/ 3 female) per litter.

Remarks:

Doses / Concentrations:
0, 400, 1100 or 3550 µg/g
Basis:
nominal in diet

No. of animals per sex per dose:

3 males and 3 females

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

Milk was collected from parental and F1 lactating dams 24 days postpartum. Placenta was collected from F1 term dams.
Blood was collected at 24 and 40 days of age in the F1 generation.

Postmortem examinations (parental animals):

Liver, kidney and brain (cerebrum) were taken at all sacrifice ages from the dams; testes were taken from males. Adrenals, pituitary, and spleen were taken at day 224 of age.
All organs, after dissection, were weighed, placed in plastic vials, and stored at -60°C. Before analysis tissues were thawed, and a portion was removed and analysed for Mn and Fe content by AAS.

Postmortem examinations (offspring):

Liver, kidney and brain (cerebrum) were taken at all sacrifice ages from the F2 neonates; testes were taken from males

Statistics:

Statistical analyses included analysis of variance (ANOVA) and Dunnett's multiple comparison test.

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Dose descriptor:

other: Not specified

Generation:

F1

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

BODYWEIGHT:

Rats chronically exposed to Mn3O4 in a normal-Fe diet showed no change in growth rate. Both male and female animals on the low-Fe diets showed Mn related depressions in bodyweight. Bodyweights of the De-deficient males receiving 1100 µg/g Mn in the diet were depressed 20 -30% through day 100 of age. Bodyweights of females from the same treatment group were depressed 10 -15% through day 60 of age. Bodyweights of both males and females receiving a 400 µg/g Mn, low-Fe diet were depressed 10 -15% through day 60 of age. Beyond day 100 of treatment, no bodyweight deficits were observed in any treatment group.

MORTALITY:

Mortality of animals maintained on the low-Fe diet supplemented with 3550 µg /g Mn was greater than 75% at day 40 of age. A small number of these animals were killed at day 40 of age for tissue samples; all remaning animals in this dose group failed to survive beyond day 50 of age. Blood analysis on these animals indicated severely depressed Fe levels.

Mn TISSUE CONCENTRATIONS:

Liver Mn accumulation in Fe deficiency was significantly increased in term and lactating dams, 40 day old animals, F1 and F2 weanlings and term pups compared to corresponding animals fed the normal Fe diet. In general there was a dose-related increase in liver Mn in both Fe groups. Dose related concentrations of Mn in the kidney were less obvious than the liver. concentrations of Mn in the cerebrum were highest in F1 and F2 weanlings. The lowest brain Mn concentrations were found in the F2 neonates.

Significant dose related increases in testicular Mn were seen only in 24 day old F1 weanlings.

Conclusions:

Mn3O4 exposure in neonates results in increase tissue concentrations. Neonate Fe-deficient animals are much more vulnerable to the effects of Mn3O4 exposure than neonate Fe-normal animals, and this can result in increase mortality due to Fe deficiency rather than any direct action of Mn3O4.

Executive summary:

Rats were chronically exposed to test material through two generations. At specific ages, observations were made of growth, tissue content, and distribution of Mn and Fe as affected by chronic exposure to Mn through an Fe-sufficient diet and an Fe-deficient diet.

During the study, chronic dietary exposure to test material resulted in dose-related increases in tissue Mn accumulation, and concomitant Fe deficiency promoted Mn accumulation. Tissue concentrations of Mn were highest in the weanling, while rats 40 days of age or older, which are protected by biliary excretion and tissue barriers, accumulated minimal amounts of Mn. Although maximal dietary exposure (mg/kg bw/day) occurs between day 24 and 40 of age, all tissues exhibited a decline in Mn concentrations during this period. This indicates that Mn uptake and retention are maximal during the preweanling period. Chronic particulate exposure of test material to animals on a normal Fe diet resulted in significant Mn absorption at levels of 3550 µg/g diet. Animals on low-Fe diet supplemented with 400 and 1100 µg/g Mn had significantly elevated liver Mn concentrations through 224 days of age.

These results indicate that the populations most susceptible to increased Mn accumulation are those absorbing large amounts of Fe, the preweanling, the lactating dam, and the Fe-deficient rat.

Effect on fertility: via oral route

Quality of whole database:

A number of literature papers have been included in the dataset most of which were well reported and performed to sound scientific principles. As such, most were assigned a relibaility score of 2 in line with the criteria of Klimisch. Therefore, the overall quality of the database is considered to be high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

20 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

The study was performed under GLP conditions and in accordance with standardised guidelines; however, since the study was conducted with manganese dichloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2 in line with the criteria of Klimisch (1997).

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

WoE Approach

Carter at al. (1980) – Oral Route

In the first study, reported by Carter (1980), the haematological values were studied in rats after chronic exposure to test material. Data were obtained at selected ages from the P0 through the F2 generation. Effects of exposure to test material during Fe-deficiency were determined by placing half of the animals on a low-Fe diet (20 mg/kg) while the other half were maintained on a normal-Fe diet (240 mg/kg). Animals treated with test material and maintained on a normal-Fe diet showed little variation from controls through 100 days of age. However, animals (24 - 100 days of age) maintained on a low-Fe diet and receiving Mn treatment during the prenatal and postnatal periods developed microcytic anemia. Overall, young Fe-deficient animals treated during both the prenatal and postnatal period were consistently more haematologically sensitive to test material treatment than animals that received sufficient amounts of dietary Fe.

Rehnberg et al. (1980) – Oral Route

In the second study, reported by Rehnberg (1980), Mn accumulation was evaluated in selected tissues of preweanling rats dosed daily with test material. Significant findings included a high rate of Mn accumulation in the preweanling rat; a Mn dose-related acceleration of postpartum liver iron depletion; a Mn dose-related depression in red blood cells, haematocrit, haemoglobin, body weight, and survival by 21 days postpartum; and a Mn distribution in tissues with liver > brain > kidney > testes at 18 - 21 days of age.

Rehnberg et al. (1982) – Oral Route

In the third study, reported by Rehnberg (1982), rats were chronically exposed to test material through two generations. At specific ages, observations were made of growth, tissue content, and distribution of Mn and Fe as affected by chronic exposure to Mn through an Fe-sufficient diet and an Fe-deficient diet. During the study, chronic dietary exposure to test material resulted in dose-related increases in tissue Mn accumulation, and concomitant Fe deficiency promoted Mn accumulation. Tissue concentrations of Mn were highest in the weanling, while rats 40 days of age or older, which are protected by biliary excretion and tissue barriers, accumulated minimal amounts of Mn. Although maximal dietary exposure (mg/kg bw/day) occurs between day 24 and 40 of age, all tissues exhibited a decline in Mn concentrations during this period. This indicates that Mn uptake and retention are maximal during the preweanling period. Chronic particulate exposure of test material to animals on a normal Fe diet resulted in significant Mn absorption at levels of 3550 µg/g diet. Animals on low-Fe diet supplemented with 400 and 1100 µg/g Mn had significantly elevated liver Mn concentrations through 224 days of age. These results indicate that the populations most susceptible to increased Mn accumulation are those absorbing large amounts of Fe, the preweanling, the lactating dam, and the Fe-deficient rat.

Gray LE and Laskey JW (1980) – Oral Route

In the fourth study, reported by Gray (1980), mice were exposed to Mn3O4 in the diet, from day 15 of lactation. At 30 days of age male pups were weaned and housed in groups of 2 or 3 with littermates. Growth, general appearance and the presence of any gross behavioural abnormalities were noted. Furthermore, locomotor activity was assessed. At necropsy, bodyweights and selected organ weights were recorded. Results of the study demonstrated that chronic dietary administration of test material to the male mouse altered behavioural and reproductive development. It is not clear from the present study whether the reduction in RLA is causally related to or only correlated with reproductive dysfunction, as both are affected by Mn treatment. It is possible that the reduced locomotor activity seen in the present study resulted directly from the action of Mn on the brain rather than being mediated through a reduction in gonadal hormones. Although growth of the testes and sex accessory gland was retarded by Mn administration, body and liver weights were unaffected; this indicates a specific effect on the hypothalamic-pituitary-gonadal axis rather than a general retardation of the growth of all organs. Overall, chronic exposure to test material in the diet at 1050 ppm Mn retarded the sexual development and lowered reactive locomotor activity levels in male mice.

Read-Across: Elbetieha et al. (2001) – Oral Route

Data on a more available form of manganese were also considered. In the first such study, reported by Elbetieha (2001), the effect of long-term ingestion of the manganese chloride was investigated on fertility of male and female Swiss mice. Adult male or female mice ingested a solution of test material along with drinking water at concentrations of 1000, 2000, 4000 and 8000 mg/L for 12 weeks. Fertility was significantly reduced in male mice exposed to test material solution at a concentration of 8000 mg/L, but not at the other concentrations. There were no treatment-related effects on the number of implantation sites, viable foetuses or the number of resorptions in female rats impregnated by males who had ingested test material. Fertility was not significantly reduced in female mice exposed to test material solution at all concentrations used in this study. However, the numbers of implantations and viable foetuses were significantly reduced in females exposed to test material solution at a concentration of 8000 mg/L. There was no significant effect on the number of resorbed foetuses in females exposed to test material solution compared to their control counterparts. Absolute body weight was not significantly affected in females exposed to test material solutions. However, ovarian weight was significantly increased in females exposed to test material solution at concentrations of 4000 and 8000 mg/L. A significant increase in the uterine weight was also observed at all concentrations used in the study. These results indicate that ingestion of high levels the test material by adult male and female mice causes some adverse effects on fertility and reproduction.

Read-Across: Jardine (2013) – Inhalation Route

The reproductive toxicity of manganese was investigated in a two generation study which was conducted with manganese dichloride, which represents a more bioavailable form of manganese than the registration substance.

During the study, F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

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

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

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

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

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

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

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

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

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

 

Adaptation for Further Reproductive Toxicity Testing

In accordance with Section 1, Annex XI of the REACH regulation, reproductive toxicity testing (EOGRTS) or further developmental toxicity testing (OECD 414) in a second species (rabbit), via the oral route, does not appear scientifically necessary. 

The principal route of human exposure in the workplace (as regulated by REACH, as opposed to essential dietary intakes regulated elsewhere) is by inhalation. Occupational exposures for inhalation of manganese compounds including oxides are regulated by workplace TLVs or similar measure. The SCOEL-IOELV is 0.2 mg/m³. A rat inhalation multi-generation study of a soluble Mn salt (MnCl2) shows a NOAEL at 20 mg/m³. An oral developmental toxicity in the rat of (insoluble, so less bioavailable) Mn3O4 showed a NOAEL at 250 mg/kg bw/day, significantly above the IOELV. Further oral testing of Mn₃O₄will therefore test not only by an inappropriate route, but at dose levels that are far in excess of the current IOELV. It is inconceivable that additional animal data could be used to set a higher reference dose, than the IOELV based on human experience in the workplace. Any further animal testing of Mn3O4 therefore cannot provide any additional useful information for regulatory risk assessment. It is also considered particularly unjustified on animal welfare grounds considering the high number of animals that would be required. Further studies cannot be used for regulatory purposes; the registrant notes that animal studies should not be conducted solely for the purpose of classification.

Effects on developmental toxicity

Description of key information

Armour (2018)

Under the conditions of the study, the NOAEL for maternal toxicity was 750 mg/kg/day (bodyweight gain and gravid uterine weight) and the NOAEL for embryo-foetal toxicity was concluded to be 250 mg/kg/day due to litter size and mean foetal weights that were slightly reduced. Embryo-foetal survival was considered unaffected by treatment, but foetal development was adversely affected with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs.

Waiver (for testing on second species)

In accordance with Section 1, Annex XI of the REACH regulation, reproductive toxicity testing (EOGRTS) or further developmental toxicity testing (OECD 414) in a second species (rabbit), via the oral route, does not appear scientifically necessary. 

The principal route of human exposure in the workplace (as regulated by REACH, as opposed to essential dietary intakes regulated elsewhere) is by inhalation. Occupational exposures for inhalation of manganese compounds including oxides are regulated by workplace TLVs or similar measure. The SCOEL-IOELV is 0.2 mg/m³. A rat inhalation multi-generation study of a soluble Mn salt (MnCl2) shows a NOAEL at 20 mg/m³. An oral developmental toxicity in the rat of (insoluble, so less bioavailable) Mn3O4 showed a NOAEL at 250 mg/kg bw/day, significantly above the IOELV. Further oral testing of Mn₃O₄will therefore test not only by an inappropriate route, but at dose levels that are far in excess of the current IOELV. It is inconceivable that additional animal data could be used to set a higher reference dose, than the IOELV based on human experience in the workplace. Any further animal testing of Mn3O4 therefore cannot provide any additional useful information for regulatory risk assessment. It is also considered particularly unjustified on animal welfare grounds considering the high number of animals that would be required. Further studies cannot be used for regulatory purposes; the registrant notes that animal studies should not be conducted solely for the purpose of classification.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2016 to 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (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 Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau.

Version / remarks:

November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 70 days of age.
- Weight at study initiation: 220 to 319 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. During acclimatisation up to four animals were housed together. During pairing, the animals were housed as one (stock) male and one female. During gestation one female was housed per cage.
- Diet: SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Food was available ad libitum.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water was available ad libitum.
- Acclimation period: Five days.

ENVIRONMENTAL CONDITIONS
- Temperature: Monitored and maintained within the range of 20 - 24 ºC.
- Humidity: Monitored and maintained within the range of 40 - 70 %.
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 hours light : 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

other: 1.0 % w/v methylcellulose (MC) aqueous solution.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The required amount of test material was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser.
- A series of suspensions/formulations at the required concentrations were prepared by dilution of individual weighings of the test material.
- Frequency of preparation: Weekly
- Storage of formulation: Formulations in the concentration range of 1 to 200 mg/mL are stable at ambient temperature (15 to 25 °C) for up to 1 day; refrigerated (2 to 8 °C) for up to 15 days.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Concentration in vehicle: Constant doses in mg/kg/day, calculated from the most recently recorded scheduled body weight.
- Amount of vehicle: Dose Volume 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analysed to assess the stability and homogeneity of the test material in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 3 of treatment were analysed for achieved concentration of the test material.

Preparation of Calibration Standards
A primary standard solution (1 000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of the test material in extract solvent (20 mL) which was then made to volume (50 mL) with diluent.
Solutions for instrument calibration were prepared by appropriate dilution of the primary standard using diluent to contain the test material at nominal concentrations of 2 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 8 μg/mL and 10 μg/mL.
Calibration solutions were read on the Atomic Absorption Spectrometer, at the beginning of each sample analysis sequence and then standard 3 at an interval of every 10 samples.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of extract solvent (20 mL) and made to volume (200 mL) using diluent. The extract was diluted using diluent, to provide a solution containing the test material at an expected concentration within the range 4 μg/mL to 8 μg/mL. The concentration of the test material in the final solution was quantified by Atomic Absorption Spectrometer.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (1 % w/v methylcellulose) with known amounts of the test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Instrumentation Parameters
Atomic Absorption Spectrometer (AAS): Perkin Elmer, AAnalyst 200
Lamp: Perkin Elmer Manganese Lumina, 6 mA
Fuel: Acetylene, 2.5 L/min
Oxidant: Air 10 L/min
Wavelength: 279.48 nm
Slit width: 1.8/0.6 mm
Integration time: 3.0 seconds
Number of replicates: 3
Read delay: 5 seconds

Calculations
The response of the test material in each calibration standard was measured. Calibration curves were constructed by quadratic (2nd order) regression of the response versus calibration standard concentration. The response observed for the test material in sample and procedural recovery solution was measured. The concentration of the test material was determined using the following equation:

Analysed concentration, mg/mL = (-b + √(b^2 - 4a (c - Y)) / 2a) x (V / W) x (D / 1 000)

Procedural recovery values were determined using the following equation:

Procedural recovery (%) = (Analysed concentration, mg/mL / Fortified concentration, mg/mL) x 100

Where
Y = Mean absorbance response for the test material in test sample
a,b,c = Coefficients derived from quadratic regression of calibration data
V = Dilution volume of sample (mL)
W = Weight of sample (g)
D = Density of sample (g/mL)
Note: A nominal mass of 1 g for sample weight was used when calculating results for procedural recovery samples.

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
- The specificity of the analysis in control samples.
- The linearity of detector response over the calibration standard concentration range.
- The repeatability of the mid-level calibration standard.
- The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 1 mg/mL and 200 mg/mL during the method validation.

Homogeneity in 1 % w/v Methylcellulose Formulations
The homogeneity of the test material in 1 % w/v methylcellulose formulations were assessed at nominal concentrations of 1 mg/mL and 200 mg/mL, during ambient and refrigerated storage.
Freshly prepared specimen formulations (400 mL) were equally sub divided into 4 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient Temperature Storage (15 to 25 °C)
On receipt, the contents of one bottle (Bottle 1) of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) and 1 and 2 hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation. The remainder of the bottle was stored at ambient temperature and after 1 days storage the contents were remixed and sampled as detailed above.

Refrigerated Storage (2 to 8 °C)
The remaining bottles (Bottles 2, 3 and 4) were refrigerated on receipt and on Day 1, Day 8 and Day 15; the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottles were mixed by 20-fold inversion followed by magnetic stirring for 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.

Concentration of Dose Formulations
For first and last occasions, six 1 mL samples (accurately weighed), were taken from the middle of the formulation by Pharmacy personnel. Three samples were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved. The contingency samples for first occasion were analysed due to a suspected instrument error during the original analysis.

RESULTS
Method Validation
The analytical procedure was successfully validated for the test material with respect to the specificity of analysis, the linearity of detector response, repeatability, method accuracy and precision.

The specificity of the AA assay was demonstrated by the absence of a response for the test material in the control sample.
Linearity was confirmed over the nominal concentration range 2 μg/mL to 10 μg/mLwith a coefficient of determination >0.992. The repeatability was <5 % for six replicate readings of a standard solution containing the test material at a nominal concentration of 5 μg/mL. A mean procedural recovery value of 99.8 % (CV=2.44 %, n=5) was obtained for 1 mg/mL and 101.4 % (CV=0.79 %, n=5) was obtained for 200 mg/mL.

Homogeneity of Dose Formulations
The homogeneity of the test material in 1 % w/v methylcellulose formulations were assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 100 mg/mL. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 9 % of the initial time zero value and the coefficient of variation was less than 7.5 %.
Recovery results during the trial remained within ± 7.5 % of the mean recovery found during validation showing the continued accuracy of the method. This is with the exception of 2 recoveries which were excluded in accordance with SOPs.

Concentration of Dose Formulations
The mean concentrations of the test material in test formulations analysed during the study and the deviation of the mean result from the nominal value were assessed. For the last occasion the mean concentrations were within 6 %, confirming the accuracy of formulation. Coefficient of variation values remained within 3 %, confirming the accuracy of analysis. For the first occasion samples the mean concentrations were within 23 % and the coefficient of variation values were within 15 %.

For the last occasion, procedural recovery results remained within ± 7.5 % of the mean recovery found during validation showing the continued accuracy of the method. For the first occasion the procedural recovery results were variable.

Clarification of Study Conduct
The first and last occasion samples were analysed outside of their confirmed stability/homogeneity period. Atomic absorption is not a stability indicating method and in this case only measures the amount of manganese present in a sample. Manganese will not be unstable therefore the fact that the samples were analysed outside of their confirmed stability period had no impact upon the results. The last occasion samples were all within acceptable limits, confirming that the animals were dosed the correct amount of test material, and also showing that no degradation has occurred. The first occasion Group 2 samples were all within acceptable limits. Group 3 and Group 4 had a high % CV and Group 4 also had a high mean concentration. Of the six samples analysed for Groups 3 and 4, three samples had a concentration of >15 % of nominal concentration, with the highest being +40 % of nominal. The procedural recoveries for the first occasion were not within acceptable limits. It is suspected that an analytical error occurred with these samples, although this could not be confirmed. As samples were not corrected for procedural recoveries this had no impact upon the data reported

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of analysis, linearity of detector response, repeatability, method accuracy and precision. The homogeneity was confirmed for the test material in 1 % w/v methylcellulose formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during ambient temperature (15 - 25 ºC) for 1 day and refrigerated storage (2 - 8 °C) for up to 15 days. The mean concentrations of the test material in test formulations analysed for the study were within ± 15 % of nominal concentrations confirming accurate formulation, this is with the exception of the first occasion Group 4.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1 with identified stock males. A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. Day 0 of pregnancy when positive evidence of mating was detected. On the day of positive evidence of mating (Day 0) only females showing at least two copulation plugs were allocated.
- Allocation: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Duration of treatment / exposure:

Day 6 to 19 after mating.

Frequency of treatment:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Duration of test:

13 days.

Dose / conc.:

83 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 83, 250 and 750 mg/kg/day) were selected in conjunction with the Sponsor and were based on the effects of a preliminary embryo-foetal study at this laboratory (Envigo study number JR43JW) which investigated dose levels of 250, 500 or 1 000 mg/kg/day. In that study there were no unscheduled deaths, no signs associated with dosing and no treatment related clinical signs at all dose levels investigated. For females that received 250 or 500 mg/kg/day there were no inter-group differences in body weight performance or food consumption and no findings detected at necropsy of the dams or external examination of the foetuses. Amongst females that received 1 000 mg/kg/day, mean body weight gain and mean food consumption towards the end of gestation was lower than that of control. In addition, the extent of post-implantation loss and late resorptions (with one total litter resorption) was higher than control, the resultant mean number of live young was lower than control and the mean foetal and litter weights were also lower than control for females that received 1 000 mg/kg/day.
The total litter resorption and another litter with a high number of late resorptions at 1 000 mg/kg/day was considered to preclude the use of this dose on this OECD 414 study. Therefore, the high dose level for this study was set at 750 mg/kg/day with low and intermediate dose levels of 83 and 250 mg/kg/day utilising a common ratio of 3 to investigate the dose response of any potential toxicity observed.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes. A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.
Detailed observations were recorded daily at the following times in relation to dose administration:
One to two hours after completion of dosing of all groups.
As late as possible in the working day.
- A detailed physical examination was performed on each animal on Days 0, 5, 12, 18, and 20 after mating to monitor general health.
- Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3, 6-20 after mating.
Group mean weight changes were calculated from the weight changes of individual animals. Weight changes were calculated and plotted graphically with respect to Day 6 of gestation.
Adjusted body weights on Day 20 after mating were calculated from the body weight at termination minus the gravid uterine weight. Body weight change values for the period Day 6-20 were also presented, after being adjusted for the contribution of the gravid uterus.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.
Group mean food consumptions and standard deviations for each period were derived from unrounded cage values.

POST-MORTEM EXAMINATIONS: Yes. A complete necropsy was performed in all cases. All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
- Sacrifice on gestation day #: Animals surviving until the end of the scheduled study period were killed on Day 20 after mating.

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

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

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

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations – Number of live fetuses) / Number of implantations) x 100
All group values and SD (as appropriate) were calculated from the individual litter values.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
For females surviving to Day 20 after mating only, the following was recorded:
- Gravid uterus weight: Yes, including cervix and ovaries.
The following were recorded for all animals (including those prematurely sacrificed, where possible) for each ovary/uterine horn:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Apparently non pregnant animals and for apparently empty uterine horns the number of uterine implantation sites were checked after staining with ammonium sulphide.

Fetal examinations:

Examination of all viable foetuses and placentae dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each foetus was recorded.
Examination of nominally 50 % of foetuses in each litter: Sexed internally and eviscerated.
Fixation: Foetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining foetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed foetuses were subject to free-hand serial sectioning. IMS fixed foetuses were processed and stained with Alizarin Red.
- Soft tissue examinations: Yes: Serial sections were examined for visceral abnormalities.
- Skeletal examinations: Yes: Assessed for skeletal development and abnormalities.

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

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of foetuses are tabulated on an individual basis for affected litters and foetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to foetal weight.
Group incidences of observations on foetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric foetal and litter incidences.
Findings observed were classified, according to severity and incidence, as:
- Major abnormalities: Normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.
- Minor abnormalities: Minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
- Variants: Alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

In the Foetal examinations appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.

Statistics:

Statistical Analysis
The following data types were analysed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed: Group 1 vs 2, 3 and 4

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs seen at physical examinations or at post-dose observation considered related to treatment with the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (3F 49) was found dead on Day 16 of gestation, having had irregular breathing at recording of clinical signs. Macroscopic examination at necropsy revealed trauma to the oesophagus (although a perforation was not detected), adhesions between the lungs, heart and thorax, clear fluid in the thorax and dark lungs and bronchi. This animal was found to be pregnant with 17 foetuses in utero. This death may have been caused by damage to the oesophagus during the dose administration procedure and not a result of treatment with the test material.

One female (3F 48) was killed on Day 17 of gestation for welfare reasons. This animal had shown signs of underactivity, fast breathing, piloerection, pallor, and partially closed eyelids prior to despatch. Macroscopic examination at necropsy revealed a perforated oesophagus (although partially autolysed, water leakage was visible when testing for damage), adhesions between the lungs, heart and thorax and dark lungs and bronchi. This animal was found to be pregnant with 16 foetuses in utero. This death is considered to have been caused by damage to the oesophagus during the dose administration procedure and not a result of treatment with the test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight and bodyweight gain up until Day 18 of gestation were similar to that of the Control for females which received 750 mg/kg/day. Bodyweight gain thereafter from Days 18-20 of gestation was lower than that of the Control for females which received 750 mg/kg/day. This is likely a consequence of the slightly low litter size and mean foetal weights in this group.
Bodyweight and bodyweight gain throughout gestation were similar to that of the Control for females which received 83 or 250 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of females receiving 83, 250 and 750 mg/kg/day was similar to that of Controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Gravid uterine weights were marginally low for animals which received 750 mg/kg/day when compared to the control group. This is likely a consequence of the slightly low mean foetal weights in this group. Adjusted bodyweight values were unaffected by treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test-material related macroscopic abnormalities detected in the adult females at scheduled termination on Day 20 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

The number of dams with abortions was 0.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of both implantations and total live young was slightly lower than that of the control for all groups receiving the test material. The extent of mean pre- and post-implantation loss was higher than that of the control for females which received the test material, although a dose response was not apparent and a review of the individual litter values did not suggest an effect of treatment so no effect of treatment was inferred.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

0 litter losses by total resorption were observed.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Resorption in the groups 1, 2 3 and 4 was as follows: 10, 14, 11 and 14. The summary of resorptions as early/late and total is given in Table 4.

Dead fetuses:

no effects observed

Description (incidence and severity):

No stillbirths were observed.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries were observed.

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Description (incidence and severity):

One female (Group 3 female 48) was killed for welfare reasons on Day 17 of gestation, and one female (Group 3 female 49) was found dead on Day 16 of gestation. One female (Group 4 female 73) was found to be not pregnant at macroscopic examination. 20, 20, 18 and 19 females in Groups 1, 2, 3 and 4, respectively were found to be pregnant with live young on Day 20 of gestation.

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Basis for effect level:

other: No maternal effect at highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day, slightly reduced.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Table 4 details the numbers of live offspring. The percentages of live offspring were 95.9, 92.6, 94.4 and 92.6 for the 0, 83, 250 and 750 mg/kg bw dose groups, respectively.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

For females which received the test material at 750 mg/kg/day, litter weight and overall foetal weight was marginally lower than that of the control as well as if compared to those animals which received 83 and 250 mg/kg/day.
There was no effect of treatment on litter or foetal weights in animals which received the test material at 83 or 250 mg/kg/day.
None of the offspring were reported as being runts.
There was no effect of treatment on placental weights in animals which received the test material.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The mean number of both implantations and total live young was slightly lower than that of the Control for all groups receiving the test material. The extent of mean pre- and post-implantation loss was higher than that of the Control for females which received the test material, although a dose response was not apparent and a review of the individual litter values did not suggest an effect of treatment so no effect of treatment was inferred.

At 750 mg/kg/day there were a number of foetuses with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs. These findings are outside of both concurrent and Historical Control Data (HCD).

At 83 mg/kg/day there was a slightly increased foetal incidence of short 13th rib compared to concurrent control and just outside of HCD but no such effects were seen in the 250 mg/kg/day and the 750 mg/kg/day group. Therefore, this finding was considered incidental due to the lack of dose response.

At 750 mg/kg/day there was an increased incidence of partially undescended lobe of thymus (9 foetuses in 7 litters) compared to concurrent control (4 foetuses in 4 litters) and just outside of HCD (up to 5 foetuses in 4 litters).

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

changes in litter size and weights

other: Skeletal variations

Developmental effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Formulation Analysis

The analytical procedure was successfully validated with respect to specificity of analysis, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity was confirmed for the test material in 1 % w/v methylcellulose formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during ambient temperature (15 - 25 ºC) for 1 day and refrigerated storage (2 - 8 °C) for up to 15 days.

The mean concentrations of the test material in test formulations analysed for the study were within ± 15 % of nominal concentrations confirming accurate formulation, this is with the exception of the first occasion Group 4.

Table 1: Group Mean Body Weight Values (g) During Gestation

Day	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
0	Av	Mean SD N	261 24.5 20	259 18.5 20	264 19.3 20	258 19.9 19
3	Av	Mean SD N	276 24.3 20	276 20.9 20	282 18.8 20	275 20.8 19
6	Av	Mean SD N	289 26.2 20	289 19.0 20	295 19.7 20	287 21.5 19
7	Wi	Mean SD N	292 25.9 20	293 20.1 20	299 19.5 20	289 23.1 19
8	Wi	Mean SD N	295 26.0 20	297 19.4 20	303 19.8 20	294 24.6 19
9	Wi	Mean SD N	300 26.4 20	302 21.0 20	307 18.4 20	298 22.0 19
10	Wi	Mean SD N	304 26.3 20	306 20.3 20	313 19.5 20	302 22.9 19
11	Wi	Mean SD N	310 27.9 20	313 21.6 20	322 20.3 20	309 24.2 19
12	Wi	Mean SD N	315 29.5 20	319 22.5 20	328 21.4 20	316 24.6 19
13	Wi	Mean SD N	322 28.5 20	327 22.9 20	337 22.4 20	323 24.3 19
14	Wi	Mean SD N	330 27.6 20	333 22.9 20	342 22.7 20	329 25.1 19
15	Wi	Mean SD N	338 28.9 20	342 22.8 20	351 23.3 20	336 27.0 19
16	Wi	Mean SD N	350 30.8 20	352 22.8 20	361 22.8 20	347 26.7 19
17	Wi	Mean SD N	362 31.4 20	366 23.9 20	374 26.3 19	359 26.7 19
18	Wi	Mean SD N	377 34.5 20	382 27.0 20	393 29.4 18	375 29.3 19
19	Wi	Mean SD N	394 37.3 20	397 27.9 20	409 31.7 18	388 31.1 19
20	Wi	Mean SD N	421 42.3 20	418 26.0 19	432 31.9 18	409 33.4 19

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi: Treated groups compared with Control using Williams’ test

 

Table 2: Body Weight Change - Group Mean Values (g) During Gestation

Days	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
0 - 3	KW	Mean SD N	15 6.0 20	17 6.3 20	18 5.5 20	17 2.8 19
3 - 6	Av	Mean SD N	13 7.9 20	13 7.4 20	14 5.1 20	12 6.0 19
6 - 7	Wi	Mean SD N	3 7.0 20	3 5.7 20	3 3.7 20	2 4.0 19
7 - 8	Wi	Mean SD N	3 3.7 20	4 4.2 20	4 3.1 20	5 3.1 19
8 - 9	Wi	Mean SD N	5 3.7 20	5 4.4 20	4 3.9 20	4 4.6 19
9 - 10	Wi	Mean SD N	4 5.9 20	4 3.7 20	6 3.4 20	4 3.8 19
10 - 11	Wi	Mean SD N	6 4.6 20	7 5.2 20	9 4.1 20	7 3.9 19
11 - 12	Sh	Mean SD N	5 8.1 20	6 5.8 20	6 4.6 20	7 3.4 19
12 - 13	Sh	Mean SD N	6 9.8 20	7 3.8 20	9 4.4 20	7 5.1 19
13 - 14	Sh	Mean SD N	9 7.1 20	7 4.3 20	5 2.6 20	6 6.0 19
14 - 15	Wi	Mean SD N	8 5.3 20	8 3.9 20	9 3.4 20	7 3.1 19
15 - 16	Sh	Mean SD N	11 6.6 20	10 4.4 20	10 9.1 20	11 3.6 19
16 - 17	Sh	Mean SD N	12 6.4 20	14 4.7 20	13 9.6 19	12 4.8 19
17 - 18	Wi	Mean SD N	15 6.5 20	16 4.9 20	17 6.2 18	16 5.4 19
18 - 19	Wi	Mean SD N	17 5.0 20	15 4.9 20	16 5.4 18	13* 4.6 19
19 - 20	Wi	Mean SD N	27 5.7 20	24 4.4 19	23* 4.4 18	21** 5.5 19
6 - 18	Sh	Mean SD N	88 25.8 20	92 10.3 20	98 15.5 18	88 13.2 19
18 – 20	lWi	Mean SD N	44 9.4 20	39 4.1 19	39 6.1 18	34** 7.0 19
6 - 20	Sh	Mean SD N	132 32.3 20	130 11.3 19	138 18.5 18	122* 18.2 19

KW: Pre-treatment comparison of all groups using Kruskal-Wallis test followed by pairwise Wilcoxon rank sum tests

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

l: Data were log transformed for the statistical analysis

* p < 0.05

** p < 0.01

 

Table 3: Gravid Uterine Weight, Adjusted Body Weight and Adjusted Body Weight Change - Group Mean Values (g) On Day 20 Of Gestation

	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
Body weight on day 6	Av	Mean SD N	289 26.2 20	289 19.0 20	294 17.9 18	287 21.5 19
Terminal body weight on day 20	Wi	Mean SD N	419 41.1 20	420 29.0 20	431 31.9 18	410 33.2 19
Body weight change days 6 - 20	Sh	Mean SD N	130 31.6 20	130 12.5 20	137 18.7 18	123* 18.2 19
Gravid uterine weight	Wi	Mean SD N	95 15.8 20	90 15.5 20	91 20.4 18	86 14.0 19
Adjusted bodyweight day 20	Wi	Mean SD N	324 28.8 20	329 21.0 20	339 21.3 18	324 26.5 19
Adjusted body weight change days 6 - 20	Sh	Mean SD N	35 21.8 20	40 11.6 20	45 9.1 18	36 12.3 19

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p < 0.05

 

Table 4: Litter Data - Group Mean Values on Day 20 of Gestation

		Statistical Test		Dose Group (mg/kg/day)
				0	83	250	750
Corpora Lutea		Du	Mean SD N	18.0 2.63 20	18.8 2.59 20	20.5* 3.01 18	17.9 2.53 19
Implantations		Wi	Mean SD N	17.1 1.99 20	16.2 2.23 20	16.0 3.73 18	16.3 2.14 19
Resorptions	Early	Wc	Mean SD N	0.7   20	1.1   20	0.9   18	1.2   19
	Late	Wc	Mean SD N	0.0   20	0.1   20	0.0   18	0.1   19
	Total	Wc	Mean SD N	0.7   20	1.2   20	0.9   18	1.3*   19
Live young	Male	Wi	Mean SD N	8.7 2.80 20	7.1 2.17 20	6.7 3.01 18	8.5 2.52 19
	Female	Wi	Mean SD N	7.8 2.17 20	7.9 2.43 20	8.4 2.66 18	6.6 2.27 19
	Total	Wi	Mean SD N	16.4 2.19 20	15.0 2.68 20	15.1 3.69 18	15.1 2.25 19
Sex ratio (%M)		Wa	Mean SD N	52.0   20	47.5   20	44.4   18	56.1   19
Implantation Loss (%)	Pre-	Wa	Mean SD N	6.9   20	13.5   20	22.0**   18	9.3   19
	Post-	Wa	Mean SD N	3.9   20	7.4   20	5.5   18	7.8*   19

Du: Treated groups compared with Control using Dunnett’s test

Wi: Treated groups compared with Control using Williams’ test

Wc: Treated groups compared with Control using Wilcoxon rank sum test

Wa: Treated groups compared with Control using Wald’s test

* p < 0.05

** p < 0.01

 

Table 5: Placental, Litter and Foetal Weights - Group Mean Values (g) on Day 20 of Gestation

	Statistical Test		Dose Group (mg/kg/day)
			0	83	250	750
Placental weight	lWi	Mean SD N	0.56 0.040 20	0.58 0.055 20	0.60 0.095 18	0.56 0.073 19
Litter weight	Wi	Mean SD N	58.69 10.993	55.48 11.255 20	56.40 13.907 18	52.56 9.639 19
Litter Size	Wi	Mean SD N	16.40 2.186 20	15.00 2.675 20	15.11 3.692 18	15.05 2.248 19
Male foetal weight	Sh	Mean SD N	3.66 0.375 20	3.81 0.228 20	3.88 0.165 18	3.58 0.274 19
Female foetal weight	Wi	Mean SD N	3.45 0.357	3.57 0.213 20	3.62 0.197 18	3.37 0.281 19
Overall foetal weight	Wi	Mean SD N	3.56 0.366 20	3.69 0.210 20	3.74 0.174 18	3.48 0.261 19

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

l: Data were log transformed for the statistical analysis

 

Table 6: Foetal Examinations – Major Abnormality Findings – Group Incidences

	Foetuses				Litters
	Dose Group (mg/kg/day)
	0	83	250	750	0	83	250	750
Number Examined	328	300	272	286	20	20	18	19
Total Number Affected	6	0	3	40	3	0	3	10
Cervical/Thoracic- Visceral
Retroesophageal aortic arch	1	0	0	0	1	0	0	0
Right sided aortic arch	1	0	0	0	1	0	0	0
Right sided azygos vein/descending aorta	1	0	0	0	1	0	0	0
Muscular ventricular septal defect	1	0	0	0	1	0	0	0
Large atrium	1	0	0	0	1	0	0	0
Diaphragmatic hernia	0	0	1	0	0	0	1	0
Lumbar (and abdominal)/Sacral/Caudal- Visceral
Omphalocele	0	0	1	0	0	0	1	0
Lumbar (and abdominal)/Sacral/Caudal- Appendicular Skeletal
Bent scapula(e)	4	0	1	37	1	0	1	10
Short/thickened humerus	1	0	0	32	1	0	0	8
Bent humerus	4	0	0	6	1	0	0	5
Bent radius/ulna/fibula	1	0	0	18	1	0	0	5

Note: Individual foetuses/litters may occur in more than one category.

 

Table 7: Foetal Examinations – Minor Skeletal Abnormality and Variants Findings – Group Incidences

	Foetuses				Litters
	Dose Group (mg/kg/day)
	0	83	250	750	0	83	250	750
Number Examined	164	148	137	142	20	20	18	19
Minor skeletal abnormalities- Cranial
Fissure(s)	1	0	1	0	1	0	1	0
Interparietal fissure(s)	1	0	0	1	1	0	0	1
Minor skeletal abnormalities- Ribs
Medially thickened / kinked / marked	4	1	3	49	1	1	3	11
Minor skeletal abnormalities- Sternebrae
Misaligned ossification sites	1	1	0	0	1	1	0	0
Minor skeletal abnormalities- Appendicular
Misshapen scapula	0	0	0	1	0	0	0	1
Total Minor skeletal abnormalities
Total affected by one or more	7	2	4	50	4	2	4	12
Rib and vertebral configuration- Cervical rib
Short supernumerary	1	1	3	3	1	1	3	3
Full supernumerary	0	1	0	0	0	1	0	0
Rib and vertebral configuration- 13th Rib
Short with/without costal cartilage	1	5	0	1	1	3	0	1
Rib and vertebral configuration- Number of 14th Ribs
Short supernumerary	9	6	3	4	7	3	2	3
Full supernumerary	0	0	1	0	0	0	1	0
Total	9	6	4	4	7	3	3	3
Pelvic girdle
Unilateral caudal shift	0	1	0	0	0	1	0	0
Delayed/Incomplete ossification/unossified- Cranial
Cranial centres/marked	26	13	19	30	11	7	11	12
Presphenoid	2	0	1	0	1	0	1	0
Hyoid	36	18	22	12	14	9	11	6
Delayed/Incomplete ossification/unossified- Sternebrae
5th and/or 6th	110	104	77	91	20	20	17	19
Other	22	6	8	9	9	6	6	5
Total	111	105	78	93	20	20	17	19
Delayed/Incomplete ossification/unossified- Vertebrae
Cervical	7	2	1	4	4	2	1	3
Thoracic	17	16	16	16	9	11	10	10
Lumbar	2	0	1	1	1	0	1	1
Sacrocaudal	26	13	12	10	8	8	8	6
Caudal	2	1	0	2	2	1	0	2
Delayed/Incomplete ossification/unossified- Ribs
Any/marked	2	0	1	5	1	0	1	3
Delayed/Incomplete ossification/unossified- Appendicular
Pelvic Bones	21	10	13	10	7	7	7	8
Clavicles	0	0	0	1	0	0	0	1
Metacarpals	3	1	1	2	2	1	1	2
Metatarsals	2	1	1	3	2	1	1	3

Note: Individual foetuses/litters may occur in more than one category.

 

Table 8: Foetal Examinations - Minor Visceral Abnormality and Necropsy Findings - Group Incidences

	Foetuses				Litters
	Dose Group (mg/kg/day)
	0	83	250	750	0	83	250	750
Number Examined	164	152	135	144	20	20	18	19
Total Number Affected	28	26	15	23	16	15	10	13
Visceral abnormalities- Brain
Dilated interventricular foramen	0	1	2	0	0	1	1	0
Visceral abnormalities- Eyes
Variation in lens shape	1	0	2	0	1	0	2	0
Visceral abnormalities- Thyroid
Absent lobe	0	1	0	0	0	1	0	0
Visceral abnormalities- Thymus
Partially undescended lobe	4	1	1	9	4	1	1	7
Thymic remnant	1	0	0	0	1	0	0	0
Visceral abnormalities- Oesophagus
Right sided	1	0	0	0	1	0	0	0
Visceral abnormalities- Caudal vena cava
Anomalous confluence with left hepatic vein	1	0	0	0	1	0	0	0
Visceral abnormalities- Diaphragm
Thinning with liver protrusion	0	3	1	3	0	3	1	3
Visceral abnormalities- Liver
Folded posterior caudate lobe	0	2	0	0	0	2	0	0
Small posterior caudate lobe	0	0	0	1	0	0	0	1
Visceral abnormalities- Ureter(s)
Dilated	1	0	0	0	1	0	0	0
Visceral abnormalities- Testis(es)
Undescended	1	1	0	0	1	1	0	0
Malpositioned	1	1	1	1	1	1	1	1
Visceral abnormalities- Umbilical artery
Left	3	1	0	0	3	1	0	0
Haemorrhages -Head
Brain	4	6	5	3	4	4	5	3
Haemorrhages- Neck/Thorax
Thoracic cavity	1	0	0	0	1	0	0	0
Haemorrhages- Abdomen
Abdominal cavity	7	11	5	6	5	6	4	5
Liver lobes	6	1	3	1	4	1	3	1

Note: Individual foetuses/litters may occur in more than one category.

 

Table 9: Control Incidence Major Abnormalities - Crl:CD(SD) Rat

	Study Number
	1		2		3		4		5		6
Number of foetuses/litters examined	317	20	319	20	300	21	304	19	282	20	322	22
Cervical/Thoracic
Diaphragmatic hernia	-	-	-	-	-	-	-	-	-	-	-	-
Lumbar (and abdominal)/sacral/caudal
Omphalocele	-	-	-	-	-	-	-	-	-	-	-	-
Appendicular
Bent scapula(e )	1	1	-	-	-	-	-	-	2	2	-	-
Short/thickened humerus	-	-	-	-	-	-	-	-	-	-	-	-
Bent humerus	-	-	-	-	-	-	-	-	-	-	-	-
Bent radius/ulna/fibula	-	-	-	-	-	-	-	-	-	-	-	-
	Study Number
	7		8		9		10		11
Number of foetuses/litters examined	296	20	310	20	331	21	291	20	305	20
Cervical/Thoracic
Diaphragmatic hernia	-	-	-	-	-	-	-	-	-	-
Lumbar (and abdominal)/sacral/caudal
Omphalocele	-	-	-	-	-	-	-	-	-	-
Appendicular
Bent scapula(e )	-	-	-	-	-	-	-	-	-	-
Short/thickened humerus	-	-	-	-	-	-	-	-	-	-
Bent humerus	-	-	-	-	-	-	-	-	-	-
Bent radius/ulna/fibula	1	1	-	-	-	-	-	-	-	-
	Current Study - Dose Group (mg/kg)
	0		83		250		750
Number of foetuses/litters examined	328	20	300	20	272	18	286	19
Cervical/Thoracic
Diaphragmatic hernia	-	-	-	-	1	1	-	-
Lumbar (and abdominal)/sacral/caudal
Omphalocele	-	-	-	-	1	1	-	-
Appendicular
Bent scapula(e )	4	1	-	-	1	1	37	10
Short/thickened humerus	1	1	-	-	-	-	32	8
Bent humerus	3	1	-	-	-	-	3	3
Bent radius/ulna/fibula	1	1	-	-	-	-	18	5

 

Table 10: Control Incidence Minor Skeletal abnormalities - Crl:CD(SD) Rat

	Study Number
	1		2		3		4		5		6
Number of foetuses/litters examined	160	20	160	20	138	21	152	19	141	20	163	22
Skeletal abnormalities
Ribs - medially thickened/kinked	2	2	-	-	-	-	-	-	1	1	2	1
Appendicular - misshapen scapula	-	-	-	-	-	-	-	-	-	-	-	-
Cervical rib - full supernumerary	-	-	-	-	-	-	-	-	-	-	-	-
13th rib - short with/without costal cartilage	3	3	3	3	1	1	3	2	-	-	-	-
14th rib - full supernumerary	-	-	-	-	-	-	-	-	-	-	-	-
Pelvic girdle - unilateral caudal shift	1	1	-	-	-	-	-	-	-	-	-	-
Delayed/Incomplete ossification/unossified
Ribs - any	1	1	-	-	-	-	-	-	-	-	-	-
Appendicular - clavicle	-	-	-	-	-	-	-	-	-	-	-	-
	Study Number
	7		8		9		10		11
Number of foetuses/litters examined	149	20	153	20	166	21	147	20	153	20
Skeletal abnormalities
Ribs - medially thickened/kinked	2	1	1	1	-	-	-	-	1	1
Appendicular - misshapen scapula	-	-	-	-	-	-	-	-	-	-
Cervical rib - full supernumerary	-	-	-	-	-	-	1	1	-	-
13th rib - short with/without costal cartilage	-	-	1	1	1	1	2	2	2	2
14th rib - full supernumerary	-	-	-	-	-	-	-	-	-	-
Pelvic girdle - unilateral caudal shift	1	1	-	-	-	-	-	-	1	1
Delayed/Incomplete ossification/unossified
Ribs - any	1	1	1	1	-	-	-	-	-	-
Appendicular - clavicle	-	-	-	-	-	-	-	-	-	-
	Current Study - Dose Group (mg/kg)
	0		83		250		750
Number of foetuses/litters examined	164	20	148	20	137	18	142	19
Skeletal abnormalities
Ribs - medially thickened/kinked	4	1	1	1	3	3	49	11
Appendicular - misshapen scapula	-	-	-	-	-	-	1	1
Cervical rib - full supernumerary	-	-	1	1	-	-	-	-
13th rib - short with/without costal cartilage	1	1	5	3	-	-	1	1
14th rib - full supernumerary	-	-	-	-	1	1	-	-
Pelvic girdle - unilateral caudal shift	-	-	1	1	-	-	-	-
Delayed/Incomplete ossification/unossified
Ribs - any	2	1	-	-	1	1	5	3
Appendicular - clavicle	-	-	-	-	-	-	1	1

 

Table 11: Control Incidence Minor Visceral Abnormalities - Crl:CD(SD) Rat

	Study Number
	1		2		3		4		5		6
Number of foetuses/litters examined	157	20	159	20	136	21	152	19	141	20	159	22
Fixed visceral observations
Brain - dilated interventricular foramen	-	-	-	-	-	-	-	-	-	-	-	-
Thyroid - absent	-	-	-	-	-	-	-	-	-	-	-	-
Thymus - partially undescended lobe	4	3	2	2	-	-	1	1	2	2	4	3
Diaphragm - thinning with liver protrusion	1	1	1	1	3	3	-	-	-	-	1	1
Liver - folded posterior caudate lobe	-	-	-	-	1	1	-	-	-	-	-	-
Liver - small posterior caudate lobe	-	-	-	-	-	-	-	-	-	-	-	-
	Study Number
	7		8		9		10		11
Number of foetuses/litters examined	147	20	157	20	165	21	144	20	152	20
Fixed visceral observations
Brain - dilated interventricular foramen	1	1	-	-	1	1	-	-	-	-
Thyroid - absent	-	-	-	-	-	-	-	-	-	-
Thymus - partially undescended lobe	5	4	4	4	2	2	4	4	2	2
Diaphragm - thinning with liver protrusion	-	-	1	1	-	-	3	3	1	1
Liver - folded posterior caudate lobe	-	-	-	-	-	-	-	-	1	1
Liver - small posterior caudate lobe	-	-	-	-	-	-	-	-	-	-
	Current Study - Dose Group (mg/kg)
	0		83		250		750
Number of foetuses/litters examined	164	20	152	20	135	18	144	19
Fixed visceral observations
Brain - dilated interventricular foramen	-	-	1	1	2	1	-	-
Thyroid - absent	-	-	1	1	-	-	-	-
Thymus - partially undescended lobe	4	4	1	1	1	1	9	7
Diaphragm - thinning with liver protrusion	-	-	3	3	1	1	3	3
Liver - folded posterior caudate lobe	-	-	2	2	-	-	-	-
Liver - small posterior caudate lobe	-	-	-	-	-	-	1	1

 

Discussion

In this study, treatment with the test material at 83 or 250 mg/kg/day was well tolerated by pregnant females and there were no effects of treatment on the body weight or food consumption of the parental females, or on placental, litter or foetal weights.

Bodyweight performance of females receiving the test material at 750 mg/kg/day was also unaffected by treatment with the test material up until Day 18 of gestation, however, bodyweight gain between Days 18-20 of gestation was lower than that of the control, and the gravid uterine weight of females which received the test material at 750 mg/kg/day was lower than that of the control. These differences were considered to be as a consequence of the slightly lower litter size and foetal weights in this group, since there was no difference from Control in the bodyweight gain of females on Day 20 of gestation when adjusted for the weight of the gravid uterus. Food consumption was considered unaffected. 

Embryo-foetal survival (as indicated by the extent of pre- and post-implantation losses and the number of total live young) was marginally lower than that of the Control for females which received the test material although there was no dose response and review of the individual litter values did not suggest an effect of treatment.

Foetal pathology examination at 750 mg/kg/day revealed a number of foetuses with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs which are outside of both concurrent and Historical Control Data (HCD). Since these occurred in a high frequency in this group only, these findings are considered related to treatment with test material.

Conclusions:

Under the conditions of the study, the NOAEL for maternal toxicity was 750 mg/kg/day (bodyweight gain and gravid uterine weight) and the NOAEL for embryo-foetal toxicity was concluded to be 250 mg/kg/day due to litter size and mean foetal weights that were slightly reduced. Embryo-foetal survival was considered unaffected by treatment, but foetal development was adversely affected with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs.

Executive summary:

The developmental toxicity of the test material was investigated in accordance with the standardised guidelines OECD 414 under GLP conditions.

The purpose of this study was the assessment of the influence of the test material (an industrial chemical) on embryo-foetal survival and development when administered during the organogenesis and foetal growth phases of pregnancy in the rat.

Three groups of 20 females received the test material at doses of 83, 250 or 750 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1.0 % w/v methylcellulose (MC) aqueous solution at the same volume dose as treated groups and for the same duration. Animals were killed on Day 20 after mating for reproductive assessment and foetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All foetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

The mean concentrations of the test material in test formulations analysed for the study were within ±15 % of nominal concentrations confirming accurate formulation, this is with the exception of the first occasion Group 4.

There were two deaths in the 250 mg/kg/day group but these were considered unrelated to treatment.

There were no signs seen at physical examinations or at post-dose observation considered related to treatment with the test material.

Bodyweight and bodyweight gain throughout gestation were similar to that of the Control for females which received 83 or 250 mg/kg/day.

Bodyweight and bodyweight gain up until Day 18 of gestation were similar to that of the Control for females which received 750 mg/kg/day. Bodyweight gain thereafter from Days 18-20 of gestation was lower than that of the Control for females which received 750 mg/kg/day. This is likely a consequence of the slightly low litter size and mean foetal weights in this group.

Gravid uterine weights were marginally low for animals which received 750 mg/kg/day when compared to the control group. This is likely a consequence of the slightly low mean foetal weights in this group.  Adjusted bodyweight values were unaffected by treatment.

Food consumption of females receiving 83, 250 and 750 mg/kg/day was similar to that of Controls.

There were no test-material related macroscopic abnormalities detected in the adult females at scheduled termination on Day 20 of gestation.

One female (Group 4 female 73) was found to be not pregnant at macroscopic examination. 20, 20, 18 and 19 females in Groups 1, 2, 3 and 4, respectively were found to be pregnant with live young on Day 20 of gestation.

Embryo-foetal survival was considered to be unaffected by treatment.

For females which received the test material at 750 mg/kg/day, litter weight and overall foetal weight was marginally lower than that of the control as well as if compared to those animals which received 83 and 250 mg/kg/day.

There was no effect of treatment on litter or foetal weights in animals which received the test material at 83 or 250 mg/kg/day.

There was no effect of treatment on placental weights in animals which received the test material.

Foetal pathology examination at 750 mg/kg/day revealed there were a number of foetuses with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs. These findings are outside of both concurrent and Historical Control Data (HCD). However, some of these findings have been reported in published literature to be reversible in quantity and severity post-natally but because these occurred in a high frequency in this group only, these findings are considered related to treatment with the test material.

In this study, treatment with the test material at 83 or 250 mg/kg/day was generally well tolerated.

At 750 mg/kg/day, maternal body weight performance was unaffected by treatment with the test material up until Day 18 of gestation, however, bodyweight gain between Days 18-20 of gestation was lower than that of the control, and the gravid uterine weight of females which received the test material at 750 mg/kg/day was lower than that of the control. Litter size and mean foetal weights were slightly reduced. Embryo-foetal survival was considered unaffected by treatment, but foetal development was adversely affected with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs.

Therefore, the No-Observed-Adverse-Effect-Level (NOEL) for maternal toxicity was 750 mg/kg/day and the No-Observed-Adverse-Effect-Level (NOAEL) for embryo-foetal toxicity was concluded to be 250 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A well documented study with a clearly defined scope and with sufficiently detailed materials and methods. However, there is no statement on impurity profile of the test material.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Long-Evans rats were chronically exposed to Mn3O4 (in diet) at 0, 350, 1050 and 3500 ppm from day 1 of gestation up to 224 days of age. Rats were grouped into either low or normal Fe dietary levels. Reproductive development and fertility of the F1 generation were determined.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1:2
- Length of cohabitation: 2 weeks

Duration of treatment / exposure:

Pregnant females were exposed to Mn3O4 in diet from day 2 of pregnancy until weaning of the offspring. Offspring were further exposed and mated at day 90-100. Treatment continued until the end of the study, 224 days.

Frequency of treatment:

Daily

Duration of test:

Up to 224 days of age

No. of animals per sex per dose:

Not reported

Control animals:

yes, plain diet

Dose descriptor:

other: Not specified

Based on:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Abnormalities:

not specified

Developmental effects observed:

not specified

General toxic effects were apparent in young animals at a dietary dose of 3500 ppm Mn and were enhanced by concomitant Fe deficiency. Fertility was reduced in the group exposed to 3500 ppm Mn with a diet containing sufficient Fe. Male reproductive development was delayed by Mn treatment, as measured by testes weight, sperm count, and serum follicle-stimulating hormone and testosterone concentrations.

Conclusions:

No effects on body weight were observed during treatment. Reduced numbers of pregnant animals were observed in the high-dose group, while other reproductive indices/litter parameters were not affected. Testis weights were reduced in the high dose only, but after correction for body weight no difference to control remained. Ovary weights, testosterone, LH, FSH and epididymal sperm count remained unaffected.

Executive summary:

Pregnant females were exposed to Mn3O4 in the diet from day 2 of pregnancy until weaning of the offspring. Offspring were further exposed and mated at day 90-100. Litters were culled to 3 animals/sex. Animals were sacrificed after 24, 40, 60, 100 and 224 days of age for testis weight, sperm count and/or blood hormone analysis. F1 females used for mating were sacrificed at the end of pregnancy for fertility and litter parameters. No effects on body weight were observed during treatment. Reduced numbers of pregnant animals were observed in the high-dose group, while other reproductive indices/litter parameters were not affected. Testis weights were reduced in the high dose only, but after correction for body weight no difference to control remained. Ovary weights, testosterone, LH, FSH and epididymal sperm count remained unaffected.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A non-GLP study conducted to sound scientifici principles with a sufficient level of data to assess the relevant results.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mn accumulation, distribution, and disappearance were evaluated in selected tissues of preweanling rats dosed daily with particulate Mn3O4 for 12 or 27 d postpartum.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altamont N.Y.
- Housing: Individual cages
- Diet: Ad libitum (standard rat chow available to all dams and pups containing background levels of Mn at 50 µg/g Mn)
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hour light cycle

Route of administration:

oral: gavage

Vehicle:

other: 50% sucrose solution

Details on exposure:

VEHICLE
- Concentration in vehicle: Controls (group I) were given 1 µL 50% sucrose (w/v) per gram b.w/d. Groups II and III were administered the 1 µL sucrose suspension (w/v) per gram b.w/d containing 71 µg/µL Mn as Mn3O4.
- Amount of vehicle: 1 µL

DOSE ADMINISTRATION
Pups were weighed daily for dosage determinations.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Timed pregnant rats were used in the study.

Duration of treatment / exposure:

Groups I and III were dosed from day 1 to day 27 from birth, Group II were dosed from day 1 to day 12 from birth.

Frequency of treatment:

Daily

Duration of test:

27 days

Control animals:

yes, concurrent vehicle

Details on study design:

Mothers and pups were maintained on standard laboratory rat Chow containing 50 µg Mn/g. The concentration of Mn in mother’s milk was determined to be 0.2 ±0.5 µg/mL.
Pups were delivered naturally and each litter was adjusted to to six males by cross-fostering if necessary. Dams were removed at postpartum day 24. Controls (group I) were administered with 1 µL 50% sucrose (w/v) per gram of body weight per day by gastric intubation from day 1 through to day 27 after birth. Groups II and III were given the same sucrose suspension supplemented with 71 µg/µL Mn. Group II were dosed from day 1 to 12, whereas group III were dosed up to day 27.

Fetal examinations:

Six nonlittermates from each group were removed, weighed, and quantitatively analysed for Mn. In addition, Fe assays were performed on liver samples by atomic absorption spectrophotometry to determine the effect of postpartum Mn exposure on liver Fe accumulation.

Statistics:

Statistical analyses included paired t-test, analysis of variance, analysis of covariance, and Duncan’s multiple range test.

Dose descriptor:

other: Not specified

Based on:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

not measured/tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Neonatal rats exposed Mn at 71 µg/g bodyweight per day for the first 12 days or 27 days after birth showed no change in growth rate or in liver, kidney, brain, testis, adrenal or pituitary weight from 12 -150 days of age.

Exposure of preweanling rats to Mn for 12 or 27 days postpartum produced elevated Mn concentrations in all of the tissues examined. Removal of test material from the daily intake of animals exposed for 12 days resulted in a rapid decrease in Mn tissue concentrations. Mn in tissues of animals treated for 27 days reached higher concentrations that in those exposed for only 12 days, and the subsequent decrease in Mn concentrations in their tissues was slower.

During treatment with test material, the relative distribution of Mn in tissues shifted considerably from the distribution seen in the control animals, with the relative amount in the cerebrum, hypothalamus, and pituitary increasing significantly. Levels in these organs were 16 -30 times higher than those in control tissues for the same age. the shift in Mn distribution suggests that the liver's capacity to store Mn had been exceeded, with subsequent release of Mn to the systemic circulation. The testes were found to have the lowest Mn accumulation. Conversely, the high and relatively uniform levels of Mn found in the hypothalamus, cerebrum, and pituitary suggest that the blood-brain barrier is ineffective or immature in the preweanling rat. In animals whose dosing was stopped before the establishment of the blood-brain barrier, Mn returned to control levels rapidly. In animals dosed beyond the establishment of this barrier, the return of Mn to control levels was much slower.

Conclusions:

Significant findings included a high rate of Mn absorption and localisation in tissues, especially the cerebrum, hypothalamus and pituitary. In these tissues, the return of Mn concentrations to control levels was much slower when Mn dosing was continued beyond 18-20 days postpartum.

Executive summary:

Mn accumulation, distribution, and disappearance were evaluated in selected tissues of preweanling rats dosed daily with particulate test material for 12 or 27 d postpartum.

Mn was found to accumulate in in all of the tissues examined, and entered the cerebrum, hypothalamus, and pituitary at rates much higher than those seen in adult animals. Removal of Mn from these organs was rapid when dosing ceased at 12 days of age. However, when dosing continued beyond the maturation of tissue barriers and the establishment of a homeostatic mechanism, removal of Mn from the cerebrum, hypothalamus, and pituitary was relatively slow. Since Mn readily enters the brain of the preweanling rat and the residence time is relatively long, it appears that the neonate would be sensitive to any possible effect of the test material.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A key study was performed according to international guidelines and the principles of GLP. In addition, a number of literature papers have been included in the dataset detailing studies which were well reported and performed to sound scientific principles. As such, they were assigned a relibaility score of 2 in line with the criteria of Klimisch. Therefore, the overall quality of the database is considered to be high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Armour (2018)

The developmental toxicity of the test material was investigated in accordance with the standardised guidelines OECD 414 under GLP conditions.

The purpose of this study was the assessment of the influence of the test material (an industrial chemical) on embryo-foetal survival and development when administered during the organogenesis and foetal growth phases of pregnancy in the rat.

Three groups of 20 females received the test material at doses of 83, 250 or 750 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1.0 % w/v methylcellulose (MC) aqueous solution at the same volume dose as treated groups and for the same duration. Animals were killed on Day 20 after mating for reproductive assessment and foetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All foetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

The mean concentrations of the test material in test formulations analysed for the study were within ±15 % of nominal concentrations confirming accurate formulation, this is with the exception of the first occasion Group 4.

There were two deaths in the 250 mg/kg/day group but these were considered unrelated to treatment.

There were no signs seen at physical examinations or at post-dose observation considered related to treatment with the test material.

Bodyweight and bodyweight gain throughout gestation were similar to that of the Control for females which received 83 or 250 mg/kg/day.

Bodyweight and bodyweight gain up until Day 18 of gestation were similar to that of the Control for females which received 750 mg/kg/day. Bodyweight gain thereafter from Days 18-20 of gestation was lower than that of the Control for females which received 750 mg/kg/day. This is likely a consequence of the slightly low litter size and mean foetal weights in this group.

Gravid uterine weights were marginally low for animals which received 750 mg/kg/day when compared to the control group. This is likely a consequence of the slightly low mean foetal weights in this group.  Adjusted bodyweight values were unaffected by treatment.

Food consumption of females receiving 83, 250 and 750 mg/kg/day was similar to that of Controls.

There were no test-material related macroscopic abnormalities detected in the adult females at scheduled termination on Day 20 of gestation.

One female (Group 4 female 73) was found to be not pregnant at macroscopic examination. 20, 20, 18 and 19 females in Groups 1, 2, 3 and 4, respectively were found to be pregnant with live young on Day 20 of gestation.

Embryo-foetal survival was considered to be unaffected by treatment.

For females which received the test material at 750 mg/kg/day, litter weight and overall foetal weight was marginally lower than that of the control as well as if compared to those animals which received 83 and 250 mg/kg/day.

There was no effect of treatment on litter or foetal weights in animals which received the test material at 83 or 250 mg/kg/day.

There was no effect of treatment on placental weights in animals which received the test material.

Foetal pathology examination at 750 mg/kg/day revealed there were a number of foetuses with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs. These findings are outside of both concurrent and Historical Control Data (HCD). However, some of these findings have been reported in published literature to be reversible in quantity and severity post-natally but because these occurred in a high frequency in this group only, these findings are considered related to treatment with the test material.

In this study, treatment with the test material at 83 or 250 mg/kg/day was generally well tolerated.

At 750 mg/kg/day, maternal body weight performance was unaffected by treatment with the test material up until Day 18 of gestation, however, bodyweight gain between Days 18-20 of gestation was lower than that of the control, and the gravid uterine weight of females which received the test material at 750 mg/kg/day was lower than that of the control. Litter size and mean foetal weights were slightly reduced. Embryo-foetal survival was considered unaffected by treatment, but foetal development was adversely affected with bent scapula(e); bent radius/ulna/fibula; short/bent/and thickened humerus with associated medially thickened/kinked/incompletely ossified ribs.

Therefore, the No-Observed- Effect-Level (NOEL) for maternal toxicity was 750 mg/kg/day and the No-Observed-Adverse-Effect-Level (NOAEL) for embryo-foetal toxicity was concluded to be 250 mg/kg/day.

Further review of this study concluded that bent bones of the types reported in this study, especially when observed in isolation, should not be considered malformations. These changes should be considered variations and do not infer any loss of function in the offspring, or a serious or irreversible developmental defect.

 

Laskey et al. (1982)

In a further study, pregnant females were exposed to the test material in the diet from day 2 of pregnancy until weaning of the offspring (Laskey, 1982). Offspring were further exposed and mated at day 90-100. Litters were culled to 3 animals/sex. Animals were sacrificed after 24, 40, 60, 100 and 224 days of age for testis weight, sperm count and/or blood hormone analysis. F1 females used for mating were sacrificed at the end of pregnancy for fertility and litter parameters. No effects on body weight were observed during treatment. Reduced numbers of pregnant animals were observed in the high-dose group, while other reproductive indices/litter parameters were not affected. Testis weights were reduced in the high dose only, but after correction for body weight no difference to control remained. Ovary weights, testosterone, LH, FSH and epididymal sperm count remained unaffected.

 

Rehnberg et al. (1981)

Mn accumulation, distribution, and disappearance were evaluated in selected tissues of pre-weanling rats dosed daily with particulate test material for 12 or 27 d postpartum (Rehnberg, 1981). Mn was found to accumulate in in all of the tissues examined, and entered the cerebrum, hypothalamus, and pituitary at rates much higher than those seen in adult animals. Removal of Mn from these organs was rapid when dosing ceased at 12 days of age. However, when dosing continued beyond the maturation of tissue barriers and the establishment of a homeostatic mechanism, removal of Mn from the cerebrum, hypothalamus, and pituitary was relatively slow. Since Mn readily enters the brain of the pre-weanling rat and the residence time is relatively long, it appears that the neonate would be sensitive to any possible effect of the test material.

Toxicity to reproduction: other studies

Description of key information

Excess oral exposure of test material, to the preweanling rat was found to cause subtle alterations in the reproductive axis and appeared to interfere with the synthesis of T. These effects are not thought to interfere with rodent fertility at chronic dietary levels up to 1100 ppm Mn.

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A well documented study with a clearly defined scope and with sufficiently detailed materials and methods. However, there is no statement on impurity profile of the test material.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Long-Evans rat pups were dosed orally from birth to 21 days with particulate Mn3O4 at 0, 71 or 214 mg Mn/kg body weight/day. Hypothalamic, pituitary or testicular functions were determined by measuring the endogenous or stimulated serum concentration of follicle-stimulating hormone, luteinising hormone, and/or testosterone at 21 or 28 days of age were assessed. Body, testes, and seminal vesicles weight and tissue concentrations of Mn were also evaluated.

GLP compliance:

no

Type of method:

in vivo

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altmont, NY, USA

Route of administration:

oral: gavage

Vehicle:

other: 50 % sucrose solution

Details on exposure:

DOSE ADMINISTRATION
Animals were dosed at the rate of 1 µL/g body weight.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Animals were dosed once a day from day 1 to day 21 in both studies.

Frequency of treatment:

Animals were dosed daily

Duration of test:

21 days

Dose / conc.:

71 other: µg Mn/g bodyweight

Remarks:

Nominal conc. (Study 1)

Dose / conc.:

71 other: µg Mn/g body weights

Remarks:

Nominal conc. (Study 2)

Dose / conc.:

214 other: µg Mn/g body weights

Remarks:

Nominal conc. (Study 2)

No. of animals per sex per dose:

Not reported

Control animals:

yes, concurrent vehicle

Details on study design:

Pregnant rats were received on day 1 of gestation. On day 1 postpartum litters were normalised to 8 pups/litter with at least 1 female per litter.

Statistics:

Data were analysed, where appropriate, by one- or two-way analysis of variance, linear regression, or Duncan's multiple comparison procedures.

Study 1

No Mn-treatment-related effects were seen in bodyweights at 21 of 28 days of age. There was significant depression in testes weight at 21 days in animals receiving 71 µg Mn/g bodyweight, but this difference was not present at day 28. Mn concentrations in the liver and hypothalamus were 150 and 63% higher in the Mn-treated castrated rats when compared to the Mn-treated non-castrated rats.

No Mn-treatment-related effects were seen in the endogenous T, FSH, or LH serum concentrations. No treatment effects were seen in the serum FSH or LH concentrations following castration, in the serum FSH or T concentrations following 2 -hour LH-RH stimulation, or in the 2 -hour hCG-stimulated serum T concentration. Following 7 days of hCG stimulation, however, serum T concentration was significantly lower in the Mn-treated animals. Additionally, the 7 -day hCG treatment resulted in significant and comparable increases in body, testes, and seminal vesicles weight.

Study 2

Significant Mn dose-related depressions in bodyweight were noted at 28 days of age. Testicular growth in animals that received 7 days of hCG stimulation following 214 µg Mn/g bodyweight for 21 days was less that either control animals or animals that received 71 µg Mn/g bodyweight. Mn treatment (214 µg Mn/g bodyweight) resulted in depressed serum T concentration in animals following 7 days hCG stimulation. Neither FSH nor LH serum concentrations showed any Mn-treatment-related differences with any of the assessment procedures.

Conclusions:

Excess oral exposure of test material, to the preweanling rat was found to cause subtle alterations in the reproductive axis and appeared to interfere with the synthesis of T. These effects are not thought to interfere with rodent fertility at chronic dietary levels up to 1100 ppm Mn.

Executive summary:

Rats were dosed orally from birth to 21 days with test material at 0, 71 or 214 µg Mn/g body weight/day. Hypothalamic, pituitary or testicular functions were determined by measuring the endogenous or stimulated serum concentration of follicle-stimulating hormone, luteinising hormone, and/or testosterone at 21 or 28 days of age were assessed. Body, testes, and seminal vesicles weight and tissue concentrations of Mn were also evaluated.

Only slight Mn treatment effects were seen in body and testes weights. No effects were seen either on unstimulated or stimulated FSH or LH serum concentrations. Although no Mn treatment effects were seen on endogenous or 2 hour human chorionic gonadotropin (hCG) stimulate serum T concentrations, there was a reduction in the serum T following 7 days of hCG stimulation. The hypothalamic Mn concentrations in animals with these reproductive effects were three times those where alterations in the dopaminergic pathway have been reported. However, no indication of hypothalamic or pituitary malfunction was found. These results suggest that the site of Mn damage that causes depression of sustained serum T concentrations is in the testicular Leydig cell.

Additional information

Laskey et al. (1985)

Rats were dosed orally from birth to 21 days with test material at 0, 71 or 214 µg Mn/g body weight/day. Hypothalamic, pituitary or testicular functions were determined by measuring the endogenous or stimulated serum concentration of follicle-stimulating hormone, luteinising hormone, and/or testosterone at 21 or 28 days of age were assessed. Body, testes, and seminal vesicles weight and tissue concentrations of Mn were also evaluated.

Only slight Mn treatment effects were seen in body and testes weights. No effects were seen either on unstimulated or stimulated FSH or LH serum concentrations. Although no Mn treatment effects were seen on endogenous or 2 hour human chorionic gonadotropin (hCG) stimulate serum T concentrations, there was a reduction in the serum T following 7 days of hCG stimulation. The hypothalamic Mn concentrations in animals with these reproductive effects were three times those where alterations in the dopaminergic pathway have been reported. However, no indication of hypothalamic or pituitary malfunction was found. These results suggest that the site of Mn damage that causes depression of sustained serum T concentrations is in the testicular Leydig cell.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified for reproduction, Category 2 (d) on the basis of adverse effects on the foetus in the key developmental toxicity study detected at 750 mg/kg bw/day which cannot be attributed to maternal toxicity. The foetal effects are delays in development, and are expected to be fully reversible; they do not represent serious or irreversible toxicity. While a slight decrease in litter size, and slight increase in post-implantation loss are described, the report clearly states that embryofoetal survival was considered to be unaffected by treatment.