Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
02 July 2012 to 04 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See the read-across report attached in Section 13.
Cross-reference
Reason / purpose:
other: read-across target
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: solid
- Appearance: pink powder
- Storage condition of test material: ambient / dark / under inert gas

Test animals

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

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

IN-LIFE DATES:
From: 02 July 2012
To: 04 March 2013

Administration / exposure

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

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

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

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

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

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

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The substrate collection plates (34 mm stainless steel) and back up filter (34 mm Westech) were weighed before and after sampling to determine the total amount of test and/or vehicle aerosol collected in each particle size range.
After weighing, the substrate collection plates and back up filters of were retained in amber glass jars and stored in a refrigerator set to maintain 4 °C.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at the testing facility.
Duration of treatment / exposure:
F0 animals were dosed for 10 weeks prior to mating; for F0 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F0 females were dosed throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning; for F1 males, this treatment continued until the day prior to termination (a total of ca. 17 weeks). F1 females were dosed throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.
Frequency of treatment:
Daily (ca. 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of gestation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca. 1 hour per day
From Day 3-4 of lactation: ca. 2 hours per day
From Days 5-20 of lactation until prior to termination (ca. Day 21 of lactation): ca. 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.
Doses / concentrationsopen allclose all
Dose / conc.:
5 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 6 µg/L air
analytical conc. F1 generation: 4 µg/L air
Dose / conc.:
10 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 15 µg/L air
analytical conc. F1 generation: 10 µg/L air
Dose / conc.:
20 other: µg/L air (target conc.)
Remarks:
analytical conc. F0 generation: 25 µg/L air
analytical conc. F1 generation: 17 µg/L air
No. of animals per sex per dose:
- F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected for use based on results from a preliminary reproduction study in rats. In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values (TLVs) proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.

Examinations

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

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

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

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

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

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

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

- Sexual Maturation in F1 Animals
Commencing at 28 days of age, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day. Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

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

LITTER OBSERVATIONS
- The number of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined from Day 1 onwards for the presence of milk in the stomach and for any externally visible abnormalities daily. The pups were weighed en masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day 21 all pups were weighed individually.
- Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Prior to Day 14 of lactation, any externally abnormal decedent pup was preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.
Sacrifice and pathology:
SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

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

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

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

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

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

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

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

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

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

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

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there were 2/28 males noted as having wheezing respiration. Animal 422 had this sign recorded on only one day (Day 14 of the study) and Animal 424 had wheezing recorded on 5 occasions (Days 91 and 112-115 of the study). Animal 333 (Group 3F) had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over ca. Days 83-90 of the study. Due to the signs dosing for the animal was stopped for a few days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals, these signs were considered to be incidental.
Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
- F1 animals
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- F0 animals
Animal 138 (Group 1F) was killed prematurely on Day 97 of the study. The animal was sacrificed at the time of parturition as the animal had difficulty giving birth and there was a pup protruding from the vagina (the animal gave birth to one live pup). The uterus also contained live foetuses and one late death. Animal 330 (Group 3F) was killed prematurely on Day 94 of the study. The animal had a prolonged parturition and had given birth to 3 live pups. One dead foetus was found in the right uterine horn at necropsy. There were no abnormalities detected at histological evaluation.
Animals 228 (Group 2M) and 236 (Group 2F) were killed prematurely on Day 85 and Day 83, respectively due to clinical signs. The male animal had shavings stained red, a cold body, reduced activity, rolling gait, staggering and weight loss. Necropsy findings for this animal included yellow froth filled duodenum, ileum and jejunum, pale foci on kidney, pale foci left lung lobe, enlargement of adrenal gland, small thymus, urinary bladder adhesions. Histological findings included a mild ulcer in the larynx. The female had partially closed eyes, dilated pupils, tremors, unkempt coat, walking on tip toes, irregular respiration, staggering and subdued. Necropsy findings included pale extremities and fluid accumulation in both horns of the uterus (the animal was sacrificed prior to having a clear indication of mating). There were no abnormalities detected at histological evaluation.
There was no treatment related pattern to these deaths and these were not positively attributed to treatment.
- F1 animals
Animal 521 (Group 1M), animal 717 (Group 3M), animal 748 (Group 3F), Animal 752 (Group 3F) and animal 816 (Group 4M) were killed prematurely. However, none of these premature deaths were considered to be related to treatment but were considered to be due to accidental injury.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 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 and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- F0 animals
At target 20 μg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls. At target 20 μg/L, there was a transient reduction in food consumption in the females on commencement of treatment compared with the controls; however, consumption for the remainder of the pre-mating period was similar to the controls. Slight intergroup differences in the group mean food consumption in the males at target 5 and 10 μg/L were not attributed to treatment. Slight intergroup differences in group mean food consumption throughout gestation and lactation were not attributed to treatment.
- F1 animals
At target 20 μg/L, there was a slight reduction in group mean food consumption in the males over Days 40-68 of the study; these reductions achieved statistical significance. Slight intergroup differences in group mean food consumption at target 5 and 10 μg/L were not attributed to treatment. Group mean food consumption in the females prior to mating and throughout gestation and lactation were comparable to the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In all treated groups of the F0 generation, the levels of test material in the blood increased significantly on commencement of dosing in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups, which did not indicate any obvious accumulation over the dosing period.
In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test material through the mother’s milk during lactation resulted in an increased exposure to the test material in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values, indicating an increased exposure throughout the dosing period.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 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 pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related findings observed in the reproductive tract in the F0 or F1 generations.
Histological findings were confined to the respiratory tract. Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

- F0 animals
In the larynx there was a broadly dosage-related minimal to moderate squamous metaplasia with minimal to moderate submucosal inflammation. Minimal to marked ulceration of the laryngeal epithelium was associated with the squamous metaplasia in several animals from all treated groups. Occasional incidences of mineralisation, intraluminal necrotic debris or intra-epithelial pustules were seen in some of the treated animals.
In the lungs the principal test material related change was seen in centroacinar regions where there was minimal or mild inflammation and focal or diffuse minimal or mild bronchoalveolar hyperplasia. This latter finding was considered reactive. Minimal or mild goblet cell hyperplasia in the bronchial or bronchiolar epithelium was present in animals exposed to target 20 μg/L together with occasional incidences of degeneration and/or squamous metaplasia of the bronchiolar epithelium. Minimal inflammatory findings (inflammatory cell foci and perivascular inflammatory cell infiltration) were also present with a greater incidence in animals exposed to the test material than in controls.
In the nasal cavity, minimal or mild goblet cell hyperplasia and minimal to moderate eosinophilic globules in the olfactory epithelium were observed in all the male treated groups and in females exposed to target 10 or 20 μg/L. At all dose levels, there was a greater incidence of minimal or mild submucosal inflammatory cell infiltration compared to controls.
In males, inflammation of the nasolacrimal duct and squamous metaplasia of the ductal epithelium was seen in most animals exposed to target 10 or 20 μg/L. In addition to these changes, incidences of minimal or mild focal degeneration of the olfactory, respiratory or transitional epithelia, minimal or mild atrophy of the olfactory epithelium, ulceration and focal squamous metaplasia were observed, mainly in animals exposed to target 10 or 20 μg/L, but occasionally in animals at target 5 μg/L. Deposits of crystalline material, presumed to be test material, was seen in the nasolacrimal ducts of a few animals in the treated groups.
Minimal goblet cell hyperplasia was observed in the pharynx of most males exposed to target 20 μg/L and there were occasional incidences of minimal or mild focal epithelial degeneration, focal inflammation and focal squamous metaplasia in males exposed to target 10 or 20 μg/L.
In the trachea, minimal or mild focal squamous metaplasia and inflammation at the carina and minimal or mild focal epithelial degeneration at sites other than the carina were observed at all dose levels.
Other microscopic findings observed were considered incidental, or of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
- F1 animals
Crystals were occasionally observed in the nasal cavity and in the pharynx from animals exposed to target 10 or 20 μg/L. They consisted of small amounts of needle-shaped crystals either deposited on the olfactory epithelium in the nasal cavity, or free in the lumen of the pharynx. These crystals were considered to result from deposition of test material in some parts of the respiratory tract.
Squamous metaplasia in the nasal cavity was observed mainly in the nasolacrimal duct and to a lesser extent in the transitional and respiratory epithelia.
In the trachea, findings such as epithelial degeneration, squamous metaplasia and submucosal inflammation were observed predominantly in the carina.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
The stages of the oestrus cycles and their mean duration were similar in all groups for both generations.

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

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

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.

LITTER SIZE AND PUP MORTALITY
- F0 generation, F1 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 20 μg/L, there was an increase in the number of animals losing more than 2 pups at birth (total pups born/no. of implantation sites). However, the mean birth index (%) was well within the background range and these increases were considered to be incidental.
- F1 generation, F2 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 10 and 20 μg/L, pup survival (no. losing >3 pups) over Days 0-4 of lactation was slightly lower than the controls. However, the number of animals losing the entire litter was comparable with controls and the remaining animals generally lost 4 pups. In addition, there was no clear dose related response to these reductions and these were considered not to be an effect of treatment.

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

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

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

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

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

Effect levels

Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: F0 Blood Analysis Results

F0 Males

Time-point

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

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

6

Prior to mating

6

13

23

27

Prior to Necropsy

6

19

27

29

F0 Females

Time-point

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

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

7

Prior to mating

6

16

28

39

Prior to Necropsy

7

16

24

33

Control animals were exposed via the food and water.

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

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

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

Table 2: F1 Blood Analysis Results

F1 Males

Time-point

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

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

12

16

16

17

Prior to mating

6

9

13

19

Prior to Necropsy

6

9

14

21

F1 Females

Time-point

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

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

13

12

15

15

Prior to mating

6

10

16

23

Prior to Necropsy

7

10

16

21

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

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

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

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

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

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

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

-7

212

212

207

204

123

128

130

124

0

253

255

246

245

152

156

156

151

7

285

287

275

267*

176

179

178

172

14

314

316

304

288***

195

201

198

191

21

337

343

330

305***

212

218

220

213

28

347

354

339

315***

216

223

229

221

35

366

371

360

338**

232

241

246

237

42

375

382

373

350*

240

252

256

247

49

388

402

384

363*

249

262*

266**

256

56

401

415

397

374*

257

268

271

260

63

419

430

410

389**

262

272

274

265

70

428

441

422

395**

265

276

278

267

77

434

445

433

402**

-

-

-

-

84

442

457

441

414*

-

-

-

-

91

448

465

447

417*

-

-

-

-

98

455

473

453

427*

-

-

-

-

105

463

483

459

436*

-

-

-

-

112

468

485

462

435*

-

-

-

-

119

458

488

478

451

-

-

-

-

Change 0 - 70

-

-

-

-

113

120

122

116

Change 0 - 112

215

231

215

190*

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

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

Day 0 = first day of treatment

 

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

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

269

271

281

266

7

294

297

306

290

14

326

327

334

318

20

379

377

388

366

Weight Gain² (% of control)

110 (-)

106 (96)

107 (97)

100 (91)

Day of Lactation³

1

323

273

282

271

7

321

323

326

314

14

344

345

349

336

21

329

330

337

331

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

 

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

Day

Dose Group (µg/L)

Males

Females

0

5

10

20

0

5

10

20

0

59

62

60

63

56

58

58

60

5

110

121*

98*

84***

76

79

77

76

12

123

135

126

123

106

110

110

106

19

166

174

167

164

135

137

139

135

26

206

221

215

206

157

161

163

157

33

246

263

254

241

178

180

183

177

40

277

295

285

268

193

197

197

193

47

302

323*

311

291

206

209

211

207

54

319

341*

327

308

216

217

219

214

61

340

364*

344

324

226

225

226

224

68

346

369

351

332

231

231

232

228

75

353

376

358

341

-

-

-

-

82

365

390*

374

256

-

-

-

-

89

374

399

385

365

-

-

-

-

96

376

404*

390

369

-

-

-

-

103

384

415*

399

380

-

-

-

-

110

382

418**

402

384

-

-

-

-

Change 0 - 68

-

-

-

-

175

172

174

168

Change 0 - 110

322

356**

343

322

-

-

-

-

*Significantly different from Group 1: p<0.05

**Significantly different from Group 1: p<0.01

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

Day 0 = first day of treatment

 

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

 

Dose Group (µg/L)

0

5

10

20

Day of Gestation¹

0

232

230

229

229

7

259

257

258

253

14

289

287

289

281

20

339

337

339

329

Weight Gain² (% of control)

107 (-)

107 (100)

110 (103)

100 (93)

Day of Lactation³

1

243

243

237

240

7

290

287

282

280

14

321

317

307

307

21

315

311

304

304

¹Pregnant animals only

²Weeks 1 to 20

³Animals rearing young to Day 21 only

Applicant's summary and conclusion

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

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

Male and female Sprague-Dawley rats were exposed to the test material via the inhalation route at target concentrations of 0, 5, 10 and 20 µg/L. F0 animals were randomised into 4 test groups, each containing 28 males and 28 females. These animals were dosed with the test material for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

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

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

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

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

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

There was no effect of treatment on the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation. No test material-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations.

Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L, in the nasal cavity, pharynx, larynx and lung in all dose groups of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

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

In conclusion, under the conditions of this study, a No Observed Effect Level (NOEL) for adult effects was not established due to effects on the respiratory tract. However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of the test material.

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