Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
available as unpublished report, no restrictions, fully adequate for assessment
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: OECD Guideline 412 (Repeated Dose Inhalation Toxicity: 28/14-Day)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Celitement
- Chemical name: Calcium hydrosilicate
- Physical state: White powder
- pH-value: 10.5
- Expiration date of the lot/batch: 12/2015
- Stability under test conditions: Hydration reaction with water
- Storage condition of test material: Room temperature protected from light, in original container in evacuated desiccator

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories
- Age at study initiation: Females 10 weeks, Males 11 weeks
- Weight at study initiation: Males: 358 gram, females: 201 gram
- Diet: Cereal-based (closed formula) rodent diet (VRF1 (FG)) obtained from SDS Special Diets Services, Witham, England, ad libitum.
- Water: Domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC), ad libitum.

HOUSING
Animals were housed in macrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany)) and strips of paper (Enviro-dri, obtained through Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment.
- After allocation, the animals were housed four rats to a cage main study separated by sex.
- For mating, one male and one female were housed together.
- Mated females were housed individually in macrolon cages, which were placed in another cage rack. The location of the mated females in the new cage rackswas determined by the date of mating and by the animal number (within each lot the mated females were housed in the order of animal number).
- During exposure periods, the rats were individually housed in the exposure unit. Immediately after each exposure, the animals were returned to their home cages.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
EXPOSURE EQUIPMENT:
The animals were exposed to the test atmosphere in nose-only inhalation units consisting of a cylindrical, polypropylene (Control group; P. Groenendijk Kunststoffen BV) or stainless steel column (Otgher groups; a modification of the design of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom), surrounded by a transparent cylinder. The columns had a volume of 48.2 (Control group) or 55.6 litres (Other groups) and consisted of a top assembly with the entrance of the unit, a mixing chamber, two rodent tube sections, and at the bottom the base assembly with the exhaust port. Each rodent tube section had 20 ports for animal exposure. Empty ports were used for test atmosphere sampling, and measurement of temperature, relative humidity, oxygen and carbon dioxide. The remaining ports were closed. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column. Male and female rats of each group were placed in alternating order. Animals were rotated once a week with respect to their position in the column. Habituation to the restraint in the animal holders was not performed because in the experience of the lab habituation does not help to reduce possible stress (Staal et al., 2012). In the experience of the lab, the animal's body does not exactly fit in the animal holder which
always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. The units were illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the units was at least 1 litre/min per animal. The air entering the units was controlled at 22 ± 3˚C and the relative humidity was maintained between 30 and 70%.

GENERATION OF THE TEST ATMOSPHERE:
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. The test atmospheres were generated using a turntable dust feeder (Reist and Taylor, 2000) and an eductor (Fox Valve Development Corp., Dover, NJ, USA; Cheng et al., 1989). The compartment of the dust feeder containing the test substance was flushed with a stream of compressed dry air (about 5 L/min) to avoid humidification and hence coagulation of the test substance. The test substance was aerosolized in the eductor, which was supplied with a flow (controlled using a reducing valve) of humidified compressed air. The eductors were calibrated by measuring the total air flow at a range of driving air pressures encompassing the driving pressures used during the study. The driving air pressure was used to monitor the total air flow. The resulting aerosol was led to the top inlet of the exposure units. From there, the aerosol was directed downward and led to the noses of the animals. At the bottom of the unit, the test atmosphere was exhausted. The exposure chamber for the control animals (group 1) was supplied with a stream of humidified air only, which was controlled using a rotameter. The animals were placed in the exposure unit after stabilization of the test atmospheres. Test atmosphere generation and animal exposure were performed in an illuminated laboratory at room temperature.

ACTUAL CONCENTRATION:
The actual concentration of the test substance in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere at 4.6 Ln/min through fiber glass filters (Sartorius, 13400-47). During the study, samples of 276.0 (Low dose group), 115.0 (Mid dose group), or 23.0 (High dose group) Ln were obtained. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test substance present on the filter, by the volume of the sample taken. Samples were taken at least three times per day for each exposure condition. Since the test substance was described to be slightly hygroscopic, it was investigated whether filter weights had to be corrected for possible water uptake. To this end, the weight change of gravimetric filters – loaded by sampling from the test atmospheres at each target concentration – was determined after drying of the filters for at least 24 hours using ambient air, and using dry air. In addition, the weight change of filters was also determined after direct application of the test substance (instead of sampling for an atmosphere) and subsequent drying using dry air. Since drying did not significantly affect the weight of the loaded filters (the change in mass of the test substance on the filters was <5% in all drying scenarios), it was concluded that correction for hygroscopy was not necessary.

TIME TO ATTAIN CHAMBER EQUILIBRATION (T95):
The concentration C in a perfectly stirred test atmosphere in a chamber with volume V (L) and flow F (L/min) increases according to C = C∞ * (1 – e-(F*T/V)), in which T (min) is the time and C∞ is the steady state concentration. The time it takes to reach 95% of the steady state concentration (T95) was calculated from the above formula as: T95 = (-V * Ln 0.05) / F = 3V/F.

NOMINAL CONCENTRATION :
The nominal concentration was calculated from the daily consumption of test substance (by weight), the duration of test atmosphere generation, and the average air flow. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration).

PARTICLE SIZE MEASUREMENT
Particle size distribution measurements were carried out using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure concentration. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated.

TOTAL AIR FLOW:
The air flow of the low, mid, and high dose groups was monitored using the driving air pressure of the eductors. During the study, air pressure was measured using pressure transducers (Midas type 1002, JUMO, Weesp, the Netherlands) and recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). Air flow of the control group was measured every hour by recording the readings of the rotameter.

TEMPERATURE, RELATIVE HUMIDITY:
The temperature and relative humidity of the test atmospheres were recorded hourly during exposure using an RH/T device (TESTO type 0636 9735 probe with 635-1 read-out unit, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany) or a CAN transmitter with temperature and relative humidity probe (G.Lufft Mess- und Regeltechnik GmbH).

OXYGEN AND CARBON DIOXIDE CONCENTRATION:
The oxygen (Oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) concentrations in the animals’ breathing zone were measured once at the beginning of the study.
Details on mating procedure:
- Each female was caged with one male from the same group.
- Subsequently, animals were caged together (after daily exposure) until mating occurred or 1 week had elapsed.
- Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm.
- The day on which sperm was detected in the vaginal smear was considered gestation day (GD) 0.
- Upon evidence of copulation the females were caged individually for the birth and rearing of their pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ACTUAL CONCENTRATION:
The average actual concentrations (± standard deviation) of CELITEMENT in the test atmospheres as determined by gravimetric analysis were 10.3 (± 0.7), 50.3 (± 2.9) and 205.8 (± 17.4) mg/m3 for the low-, mid-, and high-concentration groups, respectively. These concentrations were close to the respective target
concentrations of 10, 50 and 200 mg/m3.

TIME TO ATTAIN CHAMBER EQUILIBRATION
The time to reach 95% of the steady state concentration (T95), based on chamber volume and average air flow was about 1.3 minutes for the low-, and high-concentration group, and about 1.4 minutes for the mid-concentration group. The animals were placed in the exposure units at least 6 minutes after the start of test atmosphere generation.

NOMINAL CONCENTRATION AND GENERATION EFFICIENCY
The average nominal concentrations (± standard deviation), based on test material consumption, air flow and duration of test atmosphere generation, were 42.8 (± 7.4), 131.1 (± 10.4) and 321.3 (± 52.8) mg/m3 for the low-, mid-, and highconcentration groups, respectively. The corresponding generation efficiencies were 25%, 39% and 65%, respectively.

PARTICLE SIZE MEASUREMENT
The average (± standard deviation) mass median aerodynamic diameter (MMAD) of the low-, mid-, and high-concentration test atmospheres were 2.98 (± 0.32), 2.43 (± 0.20) and 2.57 (± 0.24) μm, with corresponding average geometric standard deviations (± standard deviation) of 2.45 (± 0.15), 2.72 (± 0.31) and 2.7 (± 0.25), respectively. Thus, average particle size was within the range of 1 – 3 μm MMAD with a gsd in the range of 1.5 – 3.0, as recommended by OECD guideline 412.

TOTAL AIR FLOW:
The average chamber air flows (± standard deviation) were 129.1 (± 0.3), 120.7 (± 0.2), and 128.4 (± 0.4) L/min for the low-, mid-, and high-concentration groups, respectively. The average chamber airflow for the control group was 35.9 (± 0.3) L/min .

TEMPERATURE, RELATIVE HUMIDITY:
The temperature and relative humidity in the exposure chambers were within their respective target ranges of 22 ± 3˚C and 30-70% during the entire exposure period. The average temperature (± standard deviation) was 22.1 (± 0.6), 21.9 (± 0.3), 21.6 (± 0.4), and 21.5 (± 0.4)°C for the control, low-, mid-, and high-concentration groups, respectively. The average relative humidity (± standard deviation) was 34.8 (± 2.5), 44.1 (± 1.3), 46.1 (± 1.4), and 44.7 (± 2.6) % for the control, low-, mid-, and high-concentration groups, respectively.

OXYGEN AND CARBON DIOXIDE CONCENTRATION:
The oxygen and carbon dioxide concentrations during exposure were determined on the first day of exposure. Oxygen concentrations were 20.3, 20.6, 20.5 and 20.5% (v/v), and carbon dioxide concentrations were 0.572, 0.195, 0.222 and 0.196% (v/v) for the control, low-, mid-, and high-concentration groups, respectively. Oxygen and carbon dioxide were not measured at the end of the main study. However, given the high air flow through the exposure chambers (>> 1 litre/min per animal) and the results of the measurements at the start of the exposure period, it was considered inconceivable that the limits of >19% oxygen and <1% carbon dioxide – as described in OECD guideline 412 – were exceeded at any moment during the study.
Duration of treatment / exposure:
- Exposure took place in male and female rats during a premating period of 2 weeks
- Male and female rats were exposed daily during the mating period
- Female animals were exposed daily from mating (i.e. the finding of a sperm positive vaginal smear; gestation day 0) up to (and including) gestation day 19. Females that appeared to be not pregnant were exposed daily during the same period. Male rats were exposed daily until sacrifice (16 days after the start of the mating period).
Frequency of treatment:
Daily during mating and post-mating period
5 days/week during pre-mating period
Doses / concentrations
Remarks:
Doses / Concentrations:
10.3, 50.3, 205.8 mg/3.
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were selected on the basis of a 14-day range finding study in which groups of 5 male and 5 female rats were exposed to target concentrations of 0, 40, 200 or 1000 mg/m3. Effects in this range finding study at 1000 mg/m3 comprised the death of one rat, clinical signs, reduced body weights and food consumption, increased weights of the lungs, heart and adrenals, and macroscopic changes in the lungs, while at 200 mg/m3 increased lung and heart weights were still observed in females.

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The animals were also observed about halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness (observation of other abnormalities was limited due to the animals’ stay in restraining tubes). All animals were also thoroughly checked again after exposure. In weekends and on public holidays only one check per day was carried out (if there was no exposure). All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

BODY WEIGHT:
- The body weight of each animal was recorded at least once during the acclimatization period and at initiation of exposure (day 0). Subsequently, males were weighed weekly. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 1, 7, 14 and 20 during presumed gestation, and on day 0 and 4 of lactation. Non-mated females were weighed once per week after the mating period.
- In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption was measured per cage over the same periods the body weights were measured, except during the mating period. The results were expressed in g per animal per day and g per kg body weight per day.

NEUROBEHAVIOURAL TESTING (detailed clinical examinations, FOB and motor activity):
- In addition to the above daily general clinical observations, detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly. In the week prior to sacrifice, the detailed clinical observations were included in the Functional Observation Battery in the animals concerned.
- FOB and motor activity testing were performed in 5 animals/sex/group shortly prior to sacrifice of the male and female rats. These determinations were performed on male animals with the lowest identification number in each cage, and on females with a litter.

HAEMATOLOGY:
- On day 10 of the premating period, 5 rats/sex/group (those with the lowest identification numbers in each cage) were fasted overnight (water was freely available) and blood was taken by orbita punction whilst under CO2/O2 anaesthesia. EDTA was used as anticoagulant.
- In each sample the following determinations were carried out: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell counts, prothrombin time, thrombocyte count, mean corpuscular volume (MCV; calculated), mean corpuscular haemoglobin (MCH; calculated), mean corpuscular haemoglobin concentration (MCHC; calculated).

CLINICAL CHEMISTRY:
- On day 10 of the premating period, 5 rats/sex/group (the same as used for haematology) were fasted overnight (water was freely available) and blood was taken by orbita punction whilst under CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation.
- The following measurements were made in the plasma: Alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (PO4).
Sperm parameters (parental animals):
Parameters examined in parental animals: testis weight, epididymis weight.
Litter observations:
PARTURITION AND LITTER EVALUATION:
- At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition and any difficulties occurring during parturition were recorded.
- To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

LITTER SIZE, SEXES AND WEIGHT:
- The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0 and 4 of lactation.
- The pups were individually weighed on days 0 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.

CLINICAL SIGNS:
- Any signs or abnormal behaviour of pups were recorded on day 0 and 4 of lactation.
- At necropsy of the dams, at day 4 (or day 6 for female 83) of lactation, pups were examined externally for gross abnormalities
Postmortem examinations (parental animals):
SACRIFICE, GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS:
- Animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes
- Male animals were sacrificed after their last exposure (a total of 25 exposure days).
- Female animals were sacrificed four days after delivery, except for female 83 (high-concentration group) that was killed six days after delivery.
- Three females from the controls group, 3 from the low-concentration group, 1 from the mid-concentration group and two from the high-concentration exposure group that were not pregnant and one female from the high-concentration exposure group that failed to mate were sacrificed 24 days after the last mating date.
- A necropsy was also performed on female from the high-concentration group that was found dead during the mating period.
Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which was preserved in Bouin's fixative: ovaries (after counting the corpora lutea), uterus (after counting of the implantation sites), testes, epididymides, seminal vesicles, prostate, all gross lesions. The testes and epididymides were weighed (paired organs together) as soon as possible after dissection to avoid drying.
In addition the following organs of five animals/sex/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected) were preserved: adrenals, bone marrow (femur), brain (including sections of cerebrum, cerebellum, medulla/pons), heart, small and large intestines (including Peyer’s patches), kidneys, liver, lungs with trachea and larynx, lymph nodes (axillary, tracheobronchial (mediastinal), cervical), nose, peripheral nerve (sciatic), spinal cord (cervical, mid-thoracic, and lumbar), spleen, stomach, thymus, thyroid, urinary bladder.
- Adrenals, brain, heart, kidneys, liver, lungs with trachea and larynx, spleen and thymus, were weighed (paired organs together) as soon as possible after dissection to avoid drying.

MICROSCOPIC EXAMINATION:
- Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin.
- Microscopic examination was performed on the preserved organs of all animals of the control (group 1) and high concentration group (group 4) (except for the nose which would have been examined only if gross changes had been observed).
- The larynx was examined at three levels (one level to include the base of the epiglottis), the trachea at three levels (including a longitudinal section through the carina of the bifurcation), and each lung lobe at one level. Because treatmentrelated changes were noted in the lungs of animals of the high-concentration group, the lungs were also examined microscopically in rats of the intermediateconcentration groups. Microscopic examination was also performed in the intermediate-concentration groups on the testes, epididymides, seminal vesicles and prostate of males that failed to sire and on the ovaries and uterus of females that were not-pregnant. Furthermore, organs showing gross lesions of animals of all groups were examined microscopically.
Postmortem examinations (offspring):
At necropsy of the dams, at day 4 of lactation, pups were examined externally for gross abnormalities and killed by appropriate techniques. Pups were stored in a freezer (≤-18°C) for possible skeletal analyses. In consultation with the sponsor, the pups were discarded after finalisation of the report.
Statistics:
The resulting data were analysed using the methods mentioned below. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**).
- Continuous data; decision tree for continuous data
- Dichotomous data; decision tree for dichotomous data
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis nonparametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated
measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Incidences of histopathological changes: Fisher’s exact probability test.
Reproductive indices:
FERTILITY AND REPRODUCTIVE PERFORMANCE:
- number of females mated (= placed with males)
- number of males mated (= placed with females)
- number of females inseminated
- number of males with pregnant females
- number of pregnant females (confirmed by the presence of implantation sites at necropsy)
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- litter size (mean number of pups delivered)
- number of implantation sites
- number of corpora lutea
Offspring viability indices:
F1 VIABILITY INDICES
- number of live pups at day 0 or 4
- number of pups lost at day 4
- number of litters lost entirely
- number of (male/female) pups at day 0 or 4

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

SURVIVAL AND CLINICAL SIGNS:
- One female (high-concentration group) was found dead in the feed tin in its home cage in the morning during the mating period. This rat, probably got stuck in the feed tin. It did not show clinical signs prior to its death. Also macroscopic examination did not reveal any abnormalities. Therefore the death of this rat was not ascribed to treatment. All other rats survived until scheduled necropsy.
- Pre-exposure observations did not reveal treatment-related signs during premating, mating, post-mating/ gestation or lactation. In addition, there were no treatment-related clinical abnormalities half way exposure during the animal’s stay in the restraining tubes or directly after exposure.

NEUROBEHAVIOURAL TESTING:
- The results of the neurobehavioural observations and motor activity assessment in rats did not indicate any neurotoxic potential of the test substance.

BODY WEIGHTS:
- There were no statistically significant differences in body weights during the premating period, in males in the post-mating period, or in dams during the gestation period and the lactation period.

FOOD CONSUMPTION:
- There were no treatment-related or statistically significant differences in food consumption expressed per animal during the premating period, in males in the post-mating period, or in dams during the gestation period and the lactation period. If expressed per kg body weight an incidental increase in food intake in males of the high-concentration group was statistically significant during day 7-14 of the premating period. This was considered a chance finding because there were no differences in food intake per kg body weight in the post mating period in males, nor in females at any stage.

HAEMATOLOGY:
- Thrombocytes were statistically significantly decreased in males of the high-concentration group. This finding may be a chance finding because it was not observed in females and was still within the range of recent historical control data. There were no other changes in red blood cell variables.
- A statistically significant decrease in absolute basophils counts was noted in males of the mid- and high-concentration group but the percentage distribution of differential white blood cells was not significantly affected and the values in the treatment groups were within the range of recent historical control data. Therefore the later changes were ascribed to relatively high control values rather that to treatment.

CLINICAL CHEMISTRY:
- Total protein and albumin concentration were statistically significantly decreased in males of the high-concentration group. These changes were not clearly within the range of recent historical control data. There were no other changes in clinical chemistry variables.

ORGAN WEIGHTS:
- The absolute and relative weights of the lungs were statistically significantly increased in males and females of the high-concentration group.
- The absolute and relative weights of the adrenals were statistically significantly increased in females of the high-concentration group.
- There were no significant changes in the weight of the male reproductive organs or any other organ examined.

MACROSCOPIC EXAMINATION:
- Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were unremarkable and normal findings in rats of this strain and age.

MICROSCOPIC EXAMINATION:
- Microscopic analysis revealed bronchiolitis in the lungs of all animals examined in the mid- and high-concentration group.
- None of the animals in the control group and the low-concentration group showed bronchiolitis. In addition, 9 out of 10 high-concentration animals showed accumulation of alveolar macrophages whereas this finding occurred only incidentally in the other groups, including the controls.
- The other histopathological findings were not considered to be treatment related. They represent common findings in rats of this strain and age and were about equally distributed among the groups or occurred in one or a few animals only.
-A statistically significantly decreased incidence of erythrophagocytosis in the treacheobronchial lymph node in high-concentration males was considered a chance finding.

MICROSCOPIC EXAMINATION OF REPRODUCTIVE ORGANS of non-pregnant females or of males that failed to sire:
- Microscopic examination on the testes, epididymides, seminal vesicles and prostate of males that failed to sire and on the ovaries and uterus of females that were not pregnant did not reveal any treatment-related effect.

FERTILITY AND REPRODUCTIVE PERFORMANCE:
- Fertility was not affected by the treatment. Three females in the control group, three females in the low-concentration group, one female in the mid-concentration group and two females in the high-concentration group were mated but appeared to be not pregnant. One female in the high-concentration group was not mated. Another female in the high-concentration group was found dead on mating day 3. This resulted in 9, 9, 11 and 8 pregnant females in the control group, low-, mid- and high-concentration group, respectively.
- There were no relevant differences in pre-coital time, mating index, male or female fertility index between the groups
- No treatment related effects were found in the mean number of implantation sites or corpora lutea between the groups.
- The reproductive performance was not affected by the treatment. In each group the duration of gestation was comparable and the gestation index was 100% (all pregnant females delivered.
- There were no dose-related differences in prenatal loss. There were no stillborn pups; live birth index was 100% and perinatal loss 0% in all groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Local
Effect level:
10.3 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects noted in the lungs
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
50.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The absolute and relative weights of the adrenals were statistically significantly increased in females of the high-concentration group.
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
>= 205.8 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed.

Results: F1 generation

Details on results (F1)

LITTER DATA AND SEX:
- The number of pups born and the sex ratio were comparable in the various groups.
- The viability index (pups surviving 4 days) was not affected by the treatment.
- The viability index was 100% in the control, mid- and high-concentration group and, due to one missing pup, 98.8% in the low-concentration group.

PUP OBSERVATIONS:
- There were no treatment-related signs in pups during the lactation period.

PUP WEIGHT:
- There were no noticeable or statistically significant differences in mean pup weight between the test groups and the controls on day 0 or day 4 of lactation

MACROSCOPIC OBSERVATIONS OF PUPS THAT DIED DURING LACTATION:
- None of the pups was found dead during lactation, and hence no macroscopic examination was conducted. In the low-concentration group one pup was missing on day 4. Macroscopic findings were not recorded in this missing pup

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 205.8 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the test conditions of this study, the NOAEL for parental effects was placed at 10.3 and 50.3 mg/m3 for local and systemic effects, respectively. Because fertility parameters, reproductive performance and development were not affected by the test substance, the NOAEL for reproduction and developmental effects was placed at ≥ 205.8 mg/m3 (the highest concentration tested).
Executive summary:

In a GLP compliant combined repeated dose inhalation toxicity study and reproduction/developmental toxicity screening test performed according to OECD guideline 422 and 412, male and female Wistar rats were exposed to the test substance. The test substance was administered by inhalation (nose only, during 6 hours per exposure day) to groups of 12 male and 12 female rats during a premating period of 2 weeks and during mating and gestation op to GD19. Male animals were sacrificed after a total of 25 exposure days. Dams and pups were sacrificed four days after delivery. The test substance was administered at target concentrations of 0 (clean air only), 10, 50 or 200 mg/m3. The average actual concentrations of the test substance in the test atmospheres, as determined by gravimetric analysis, were close to the respective target concentrations, namely 10.3, 50.3 and 205.8 mg/m3. One female rat of the high-concentration group was found dead during the mating period. The death of this rat was not ascribed to treatment. Pre-exposure and postexposure observations did not reveal treatment-related clinical signs. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. There were no treatment-related differences in body weights or food consumption during the premating period, during the post-mating period in males, or in dams during the gestation period and the lactation period. Haematology and clinical chemistry was conducted in 5 rats/sex/group at the end of the premating period. Total protein and albumin concentrations were decreased in males of the high-concentration group. Lung weights were increased in the high-concentration group in both sexes. Adrenals weights were increased in the high-concentration group in females. Microscopic examination revealed bronchiolitis in the lungs in the mid- and high-concentration group, and accumulation of alveolar macrophages in the high-concentration group. There were no effects of the test substance on fertility and reproductive performance, litter data or pup observations in any group. Based on the effects noted in the lungs, the local NOAEL for parental effects was placed at the low-concentration of 10.3 mg/m3. The systemic NOAEL was placed at 50.3 mg/m3 based on adrenal effects. Because fertility parameters, reproductive performance and development were not affected by the test substance, the NOAEL for reproduction and developmental effects was placed at ≥ 205.8 mg/m3 (the highest concentration tested).