Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 and 8 March 2017 to 6 and 7 June 2017 (males); 9 and 10 March 2017 to 8 and 9 June 2017 (females)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
7 September 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Flue dust, portland cement
EC Number:
270-659-9
EC Name:
Flue dust, portland cement
Cas Number:
68475-76-3
Molecular formula:
It is a UVCB.
IUPAC Name:
tricalcium dipotassium silanedione carbonate sulfate silicate
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material:01-2016
- Expiration date of the batch: 31 December 2018
- Purity: 01-2016
- Supplier: Sponsor (Cimeurope S.A.R.L)
- Appearance: grey/beige powder
- Expiration date: 31 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15-25°C)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
JUSTIFICATION FOR THE SPECIES/STRAIN SELECTION
For this study rats were chosen as test system, because this animal species is normally used in toxicity studies of this type and is accepted by the relevant authorities. The Wistar rat strain was used because it is routinely used at the test facility for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: about 7 weeks
- Weight at study initiation: mean male 237 g and mean female 165 g for female
- Fasting period before study: overnight
- Housing: Makrolon cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and wooden block (ABEDD, Vienna, Austria). Animals were housed in groups of five of the same sex.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England)
- Water: ad libitum, domestic tap-water
- Acclimation period: 13-16 days

DETAILS OF FOOD AND WATER QUALITY
- Diet: Each batch of diet is analyzed by the supplier for nutrients and contaminants. The results were in line with the expected values.
- Water: Water was suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) supplied by N.V. Vitens. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyzes water samples taken on the premises of the test facility for a limited number of variables. The parameters met the requirements of the Dutch Drinking Water Act

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
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1.8 - <= 1.89 µm
Remarks on MMAD:
The average (± standard deviation) mass median aerodynamic diameter (MMAD) of the particles in the low-, mid- and high-concentration test atmospheres were 1.30 (± 0.16), 1.85 (± 0.04) and 1.89 (± 0.02) μm, with average geometric standard deviations of the distribution of particle sizes of 1.80 (± 0.31), 1.98 (± 0.09) and 2.16 (± 0.12), respectively.
Details on inhalation exposure:
EXPOSURE EQUIPMENT
The animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated laboratory room different from the room where the animals were housed. Animals of groups 2, 3 and 4 were exposed in inhalation chambers consisting of a cylindrical stainless steel column, surrounded by a transparent cylinder (a modification of the design of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom). The column had a volume of 55.6 L and consisted of a top assembly with the entrance of the unit, a mixing section, 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. Negative control animals (group 1) were exposed to clean air in a polypropylene nose-only inhalation chamber with a volume of 48.2 L manufactured by P. Groenendijk Kunststoffen BV, which was very similar in construction to the stainless steel chambers described above. Empty ports were used for test atmosphere sampling (for analysis of the actual concentration and particle size) and measurement of oxygen, carbon dioxide, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats were placed in alternating order. Animals were rotated with respect to their position in the column on a weekly basis. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Habituation to the restraint in the animal holders was performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012). In our experience, the animal’s body does not exactly fit in the animal holder which always results in some leakage from high to 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 animals’ thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the unit was at least 1 liter/min for each rat. The air temperature and relative humidity in the unit were maintained at 22 ± 3˚C and between 30 and 70%, respectively.

GENERATION OF TEST ATMOSHPERE
The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. The test atmosphere was 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 material was flushed with a stream of dry compressed air (about 2.5-3.5 L/min) to avoid humidification of the test material. The test material was aerosolized in the eductor, which was supplied with a flow (controlled using a reducing valve) of dry compressed air. The resulting aerosol was led through a glass cyclone, which was used to remove the largest particles from the aerosol, and was subsequently introduced at the top inlet of the exposure chamber. The eductors were calibrated by measuring the total air flow at a range of driving air pressures of the eductors encompassing the driving pressures used during the study. The driving air pressure was used to monitor the total flow. A bypass stream of humidified compressed air (measured by mass view meter; Bronkhorst Hi Tec, Ruurlo, The Netherlands) was added at the top of the exposure chamber to ensure a relative humidity above 30%. The resulting test atmosphere 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 negative control animals (group 1) was supplied with a stream of humidified compressed air only, which was controlled by a reducing valve and measured by mass view meter (Bronkhorst Hi Tec). 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.

TIME TO ATTAIN CHAMBER EQUILIBRATION (T95)
The time to reach 95% of the steady state concentration (T95) was calculated as: 3V/F. This follows from the formula C = C∞ (1 – e^-(FT/V)), describing the increase in concentration C in a perfectly stirred chamber with volume V [L] and flow F [L/min], where T [min] is the time and C∞ is the steady state concentration.

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

PARTICLE SIZE MEASUREMENT
Particle size distribution measurements were performed using a 10-stage cascade impactor (2110k cascade impactor, 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 condition. The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were calculated (Lee, 1972).

TOTAL AIR FLOW, TEMPERATURE, RELATIVE HUMIDITY, OXYGEN AND CARBON DIOXIDE CONCENTRATION
The chamber air flow, temperature and relative humidity of the test atmosphere were recorded at least hourly during exposure. Air flow was measured by recording the readings of the mass view meters and driving air pressure of the eductors. The temperature and relative humidity were measured using an RH/T device (TESTO type 0636 9735 probe with 635-1 read-out unit, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany). 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 were measured twice for each group - during the first week and near the end of the exposure period - at a moment when all animals of a given group were placed in the exposure chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of the test material in the atmosphere was measured at least three times during exposure on each exposure day by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) samples of test atmosphere through fibre glass filters (Sartorius 13400-47, Ø 47 mm) at a flow of 4.6 Ln/min. During the range finding study, samples with a volume of 124.2, 48.0 or 9.2 Ln were taken for groups 2, 3 and 4, respectively. During the main study, samples of 400.0, 125.0 or 100.0 Ln were taken for groups 2, 3 and 4, respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere, and then weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken.
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/m³ air (nominal)
Dose / conc.:
20 mg/m³ air (nominal)
Dose / conc.:
60 mg/m³ air (nominal)
Dose / conc.:
5.09 mg/m³ air (analytical)
Dose / conc.:
21.3 mg/m³ air (analytical)
Dose / conc.:
61 mg/m³ air (analytical)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations to be tested in the sub-chronic study were selected on the basis of a 14-day range finding study in which groups of five male and five female Wistar rats were exposed to target concentrations of 40, 200 and 1000 mg/m3 for 6 hours/day, 5 days/week. Exposure at 40 mg/m3 resulted in limited changes in BAL parameters indicative of cellular damage and slightly increased neutrophil numbers in BAL fluid, accompanied by microscopic changes in the larynx characterized by minimal to mild epithelial hyperplasia/metaplasia and focal inflammatory changes. Exposure at 200 mg/m3 resulted in more substantial changes in BAL parameters, increased lung weights, reduced food intake and growth in males during part of the study, and histopathological changes in the nose (epithelial ulceration, inflammation and/or degeneration), larynx (inflammation and squamous hyperplasia/metaplasia) and lungs (enlarged macrophages, collagenization/fibrosis and epithelialization at the bronchoalveolar junctions). Exposure at 1000 mg/m3 resulted in clinical abnormalities indicating respiratory difficulties; other findings were comparable in nature, but generally more severe than in animals exposed at 200 mg/m3.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS AND DETAILED 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 hindered due to the animals’ stay in restraining tubes. All animals were thoroughly checked again in the afternoon.

BODY WEIGHT:
The body weight of each animal was recorded once before the start of the exposure period: on day -4 or -5 for males, and on day -6 or -7 for females. During the exposure period, the animals were weighed just before exposure on the first day (day 0), and twice a week thereafter. Since no exposure-related effects were seen during the first 4 weeks of the study, body weight measurements were thereafter continued on a once a week basis for the remainder of the study duration. The animals were also weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios. In addition, the two animals which did not survive until scheduled sacrifice were weighed at intercurrent death.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

OPHTHALMOSCOPIC EXAMINATION:
Observations were made prior to the start of the exposure in all animals (day -8 to -5) and towards the end of the exposure period in animals of the control and high-concentration groups (day -83 to -86). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

HAEMATOLOGY:
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell count (lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time (PT), thrombocyte count (platelet count), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ration albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate
Sacrifice and pathology:
SACRIFICE AND GROSS PATHOLOGY
Surviving animals of the main groups were sacrificed at the end of the exposure period in such a sequence that the average time of sacrifice was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. A thorough necropsy was also performed on the animals that were found dead or were killed in moribund condition.

ORGAN WEIGHTS
At sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight:
- adrenals
- heart
- kidneys
- liver
- lungs (left lobe only, because the right lobes were lavaged, After weighing, the left lung lobe was infused with the fixative)
- ovaries
- spleen
- testes
- thymus
- uterus

BRONCHOALVEOLAR LAVAGE (BAL)
The lungs of all animals were lavaged according to a standardized method. In short: the right half of the lungs (after binding off the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 mL saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 mL saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations. Samples of bronchoalveolar lavage fluid were discarded after analysis.
- Biochemical determinations in BAL fluid: The volume of the supernatant was determined. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N- acetylglucosaminidase (NAG), and gamma-glutamyltransferase (GGT) were determined.
- Cellular determinations in BAL fluid: Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grünwald Giemsa. The differential cell counts were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).

HISTOPATHOLOGY
For histopathological examination, samples of the following tissues and organs of all animals were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The left lung lobes (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.

- Tissues and organs: adrenals, aorta, axillary lymph nodes, brain (a), caecum, colon, epididymis, eyes (with optic nerve), exorbital lachrymal glands, femur with joint, heart, kidneys, liver, lungs (b)/trachea (c)/larynx (d), mammary glands (females), cervical lymph nodes, nasopharyngeal tissue (e)(with teeth), nerve peripheral (sciatic nerve), oesophagus, olfactory bulb, ovaries, pancreas, para thyroids, pharynx, parotid salivary glands, pituitary gland, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, and jejunum), spinal cord (f), spleen, sternum with bone marrow, stomach, sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all gross lesions
(a) Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum). (b) Lungs: The left lung lobe was examined microscopically at three levels. (c) Trachea: Three levels were examined microscopically (including a longitudinal section through the carina of the bifurcation). (d) Larynx: Three levels (one including the base of the epiglottis) were examined microscopically. (e) Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [nose associated lymphoid tissue, NALT]). (f) Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar). (g)Stomach: Non-glandular and glandular parts were examined microscopically.

- Slide preparation: Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the low- and mid-concentration groups (groups 2 and 3) were not processed. The noses of the animals of groups 2 and 3 were decalcified and embedded in paraffin concurrently with the noses of the animals of groups 1 (control) and 4 (high concentration).

- Histopathological examination: All preserved tissues of all animals of the control and high-concentration groups and of the animals which were found dead or were sacrificed in moribund condition, were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the intermediate concentration groups were examined microscopically. Since exposure-related changes were observed in the nasal tissues (levels 5 and 6), larynx, tracheobronchial lymph nodes and the left lung lobe of animals of the high-concentration group, histopathological examination of these tissues was extended to animals of the intermediate concentration groups.
Statistics:
- Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ with automatic data transformation. ay 0 body weight data were used as covariate in the analysis of the post-treatment data unless removed during data pre-processing.
- Pre-treatment body weight, organ weight, haematology, clinical chemistry, and bronchoalveolar lavage data: ‘Generalized Anova/Ancova Test’ with automatic data transformation method.
- Food consumption (main study): Dunnett’s multiple comparison test. No statistics was applied on food intake in the range finding study (only one cage per sex).
- Incidences of histopathological changes: Fisher’s exact probability test.
- Arithmetic means and standard deviations were determined.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wound) were observed in a few males of the control and low concentration group; these are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two animals did not survive until scheduled sacrifice. In both cases, the mortality was unrelated to the exposure to the test material. A female of the high-concentration group was found dead in the restraining tube during exposure on day 46. The animal was found with a twisted neck. Probably, it had room to manoeuver in the tube, turned its head and got stuck (a rare observation during nose-only exposure). A female of the low-concentration group was humanely sacrificed on day 67, because it was suffering from an othematoma (a hematoma in the external ear) which was not expected to heal and was probably causing substantial distress given the swelling and the restraint in the restraining tube required for nose-only exposure.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no exposure-related changes in body weight up to the highest concentration tested.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination did not reveal any exposure-related abnormalities.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis of haematology parameters revealed the following statistically significant differences between animals exposed to the test material and unexposed controls:
- Slightly decreased prothrombin time in females of the high-concentration group. This finding was not accompanied by similar changes in males, or by changes in any of the other red blood cell and coagulation parameters examined. Since an increase (rather than a decrease) in prothrombin time is commonly associated with adverse changes, this minor change was considered not to be toxicologically relevant and probably unrelated to treatment.
- Increased absolute numbers of neutrophils in males of the high-concentration group; relative neutrophil numbers were also slightly higher (and as a consequence, the percentage of lymphocytes was slightly lower) than controls, but the difference did not reach the level of statistical significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Analysis of clinical chemistry parameters revealed the following statistically significant differences between animals exposed to the test material and unexposed controls:
- Increased plasma concentration of cholesterol in females of the high-concentration group. Since no changes were observed in related parameters (or in males) and there were no indications for an impaired liver function, no toxicological relevance was attached to this incidental finding.
- Decreased albumin/globulin ratio in females of the mid-concentration group. In the absence of a concentration-response relationship and any changes in plasma protein or albumin levels (from which the A/G ratio is calculated), this was considered to be a chance finding.
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):
Organ weight data showed the following statistically significant differences between animals exposed the test material and controls:
- Increased absolute and relative lung weight in animals of the mid- and high-concentration group. Relative lung weight was also increased in males of the low-concentration group.
- Decreased absolute weight of the ovaries in females of the high-concentration group. In the absence of any significant changes in relative ovary weight or any corroborative changes in e.g. histopathology, no toxicological relevance was attached to this finding.
- Increased relative weight of the heart in females of the mid-concentration group, which was considered to be chance finding since a concentration-response relationship was not observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy, no exposure-related macroscopic changes were observed. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Microscopic evaluation revealed exposure-related histopathological changes in the nose, larynx, lungs and tracheobronchial lymph nodes.
- The changes in the nose were predominantly present in the caudal parts. In level 5 and level 6, the olfactory epithelium showed the presence of eosinophilic droplets. This was observed in level 5 in 16/20 high-concentration animals, 13/20 mid-concentration animals and 1/20 low concentration animals. In level 6, this was observed in 18/20 high-concentration animals, 17/20 mid-concentration animals and 3/20 low-concentration animals. In level 4, the eosinophilic droplets were seen in 2/20 high concentration animals only.
- The changes in the larynx were predominantly present at the epiglottis and comprised squamous metaplasia of the lining epithelium. The changes in the lungs comprised the presence of enlarged macrophages containing dark particles (presumably the test material), observed in all animals of the mid- and high concentration group and in 7/20 animals of the low-concentration group. Similar macrophages were also observed in the draining tracheobronchial lymph nodes in 12/20 high-concentration animals, 16/20 mid-concentration animals and 2/20 low-concentration animals.
- Further, minimal (multi)focal epithelialization at the bronchoalveolar junctions was observed in 3/20 high-concentration animals.
- The other organs and tissues did not reveal any exposure-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure.
- The other organs and tissues did not reveal any exposure-related histopathological changes.

The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Analysis of bronchoalveolar lavage (BAL) parameters revealed the following differences between animals exposed to the test material and unexposed controls:
- Concentration-dependent increase in all biochemical parameters in all exposed groups, which reached the level of statistical significance for ALP at all concentrations tested; LDH and GGT were significantly increased in animals of the mid- and high-concentration group, NAG in males of high-concentration group, and total protein levels were statistically significantly higher than controls in males of the mid- and high-concentration group. Note that total protein seemed decreased in females of the high-concentration group, but this was an artefact caused by an outlier in the control group; total protein content actually increased with increasing dose levels, similar to the changes observed in males.
- Concentration-related increase in the absolute number of neutrophils in BAL fluid of all groups of animals exposed to the test material. In addition, lymphocyte numbers were slightly increased in females of the mid- and high-, and in males of the high-concentration group; macrophage numbers were elevated in females of the high-concentration group.
- Total cell numbers were statistically significantly increased in females of the mid- and highconcentration group and the percentage distribution of white blood cells in BAL fluid shifted from ~100% macrophages (which is normally observed in healthy animals) towards an increased relative contribution of neutrophils, which reached statistical significance in males of the mid- and high-concentration and in females of all exposed groups. In addition, the percentage of lymphocytes was slightly increased in males of the mid- and highconcentration group.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
>= 61 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
5.09 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
other: bronchoalveolar lavage

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

TIME TO ATTAIN CHAMBER EQUIBRILATION (T95)

The time to reach 95% of the steady state concentration (T95), based on chamber volume and average air flow was calculated to be about 1.9 minutes for group 2, and about 4.2 minutes for groups 3 and 4. 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) – calculated from the test material consumption, the air flow and the duration of test atmosphere generation – were 55.0 (± 9.8), 123 (± 15) and 193 (± 25) mg/m3for the low-, mid- and high-concentration groups, indicating generation efficiencies of 10%, 18% and 33%, respectively, which – especially for groups 2 and 3 – are rather low. These low efficiencies were probably related to the use of cyclones to remove the larger particles from the aerosol, causing relatively high losses of test material.

TOTAL AIR FLOW, TEMPERATURE, RELATIVE HUMIDITY, OXYGEN AND CARBON DIOXIDE CONCENTRATION

The average total air flows (± standard deviation) were 49.6 (± 0.2), 85.7 (± 0.3), 39.3 (± 0.2) and 39.9 (± 0.2) L/min for the control, low-, mid- and high-concentration groups, respectively. The temperature and relative humidity in the exposure chambers were within the respective target ranges of 22±3°C and 30-70% during the entire exposure period. The average temperature (± standard deviation) was 21.7 (± 0.3), 21.8 (± 0.3), 21.8 (± 0.3) and 21.6 (± 0.3)˚C for the control, low-, mid- and high-concentration groups, respectively. The average relative humidity during exposure was 44.7 (± 1.2), 36.1 (± 1.0), 42.1 (± 0.7) and 41.2 (± 1.6) % for the control, low-, mid- and high-concentration groups, respectively.The oxygen concentration during exposure was in the range of 20.2 – 20.5% (v/v) and the carbon dioxide concentration was in the range of 0.268 – 0.625% (v/v), which was well within the limits of >19% oxygen and <1% carbon dioxide described in the OECD.

Applicant's summary and conclusion

Conclusions:
The NOAEC for systemic toxicity was determined to be at least 61.0 mg/m3.
The LOAEC for local toxicity was determined to be 5.09 mg/m3.
Executive summary:

The toxicity of the test substance upon repeated exposure by inhalation was investigated in a GLP compliant sub-chronic (90-day) study, performed in accordance with OECD guideline 413, in Wistar rats (Triskelion, 2017). Four groups of 10 male and 10 female rats each were exposed (nose-only) to target concentrations of 0 (control), 5, 20 or 60 mg/m3 for 6 h/d, 5 d/week over a 90-day period, with a total number of 65 exposure days. Animals were sacrificed on the day after the last exposure. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were macroscopically examined at necropsy, the right lung lobes of half of the animals of each group (5 males, 5 females) were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobe together with the full respiratory tract and a large number of organs and tissues were examined microscopically. The concentrations to be tested in the sub-chronic study were selected on the basis of a 14-day range finding study in which groups of 5 male and 5 female Wistar rats were exposed to target concentrations of 40, 200 and 1000 mg/m3 for 6 h/d, 5 d/w. Exposure at 40 mg/m3 resulted in limited changes in bronchoalveolar lavage (BAL) parameters indicative of cellular damage and slightly increased neutrophil numbers in BAL fluid, accompanied by microscopic changes in the larynx characterized by minimal to mild epithelial hyperplasia/metaplasia and focal inflammatory changes. Exposure at 200 mg/m3 resulted in more substantial changes in BAL parameters, increased lung weights, reduced food intake and growth in males during part of the study, and histopathological changes in the nose (epithelial ulceration, inflammation and/or degeneration), larynx (inflammation and squamous hyperplasia/metaplasia) and lungs (enlarged macrophages, collagenization/fibrosis and epithelialization at the bronchoalveolar junctions). Exposure at 1000 mg/m3 resulted in clinical abnormalities indicating respiratory difficulties; other findings were comparable in nature, but generally more severe than in animals exposed at 200 mg/m3. In the sub-chronic main study, the target concentrations of the test substance were accurately achieved as demonstrated by the results of gravimetric analysis of test atmosphere samples. The overall average (± standard deviation) actual concentrations were 5.09 (± 0.79), 21.3 (± 1.9) and 61.0 (± 4.1) mg/m3 for the low-, mid- and high-concentration groups, respectively. The average Mass Median Aerodynamic Diameter (MMAD) of the particles in the test atmosphere was 1.30 with a geometric standard deviation (GSD) of the size distribution of 1.80, 1.85 (GSD of 1.98) and 1.89μm (GSD of 2.16) for the low-, mid- and high-concentration groups, respectively. Exposure-related mortality did not occur during the study. A female animal of the high concentration group was found dead with a twisted neck in the restraining tube during exposure; a female animal of the low-concentration group was humanely sacrificed, because it was suffering from an othematoma, which was probably causing substantial distress given the swelling and the restraint required for nose-only exposure. Clinical and ophthalmoscopic observations revealed no exposure-related abnormalities. Growth and food consumption were not adversely affected by the exposure and were comparable across the groups. Analysis of hematology parameters revealed an increase in neutrophil numbers in blood of male animals of the high-concentration group, which may be related to the inflammatory changes in the lower airways (described below). Exposure to the test material did not result in any toxicologically relevant changes in clinical chemistry parameters. Analysis of BAL parameters revealed concentration-related elevations in all of the biochemical markers for cellular damage investigated (ALP, GGT, LDH, NAG, and total protein), with statistical significance generally being reached in the mid- and high concentration groups. At the low-concentration, although average results for all markers were above control level, statistical significance was only reached for ALP. The changes in biochemical markers were accompanied by a concentration-related increase in neutrophil numbers in BAL fluid, observed at all exposure concentrations. In addition, lymphocyte numbers were statistically significantly increased in females of the mid- and high-, and in males of the high concentration group; and macrophage numbers were increased in females of the high concentration group. Consequently, the percentage distribution of white blood cells in BALF shifted from ~100% macrophages – which is normally observed in healthy animals – towards an increased contribution of primarily neutrophils. Organ weight data showed an increase in absolute and relative (to body weight) weight of the lungs in males and females of the mid- and high-concentration group. Relative lung weight was also increased in males of the low-concentration group. Macroscopic examination at scheduled termination revealed no exposure-related gross pathology. Microscopic examination revealed histopathological changes in the nose, larynx, lungs and tracheobronchial lymph nodes. The changes in the nose were characterized by the presence of eosinophilic droplets in the olfactory epithelium in the causal parts of the nasal tissues, primarily observed in animals of the mid- and high-concentration, and in single animals of the low-concentration group. In the absence of any other histopathological changes in the nose, the toxicological relevance of this finding – which may be induced by low-grade irritation, but is also found as a spontaneous finding in older rodents – was considered to be limited. The histopathological changes in the larynx were characterized by focal squamous metaplasia of the epithelium, predominantly observed at the epiglottis. This lesion was found in only a few animals (the incidence was not statistically significantly higher than in controls) and in females a clear concentration-effect relationship was lacking, but – given the findings in the range finding study – a relation with the exposure could not be completely excluded. The changes in the lungs comprised the presence of enlarged macrophages containing dark particles, observed in all animals of the mid- and high-concentration group and in 7/20 animals of the low-concentration group. Similar macrophages were also observed in the draining tracheobronchial lymph nodes (in 2/20, 16/20 and 12/20 animals of the low-, mid- and high- concentration group, respectively), indicating a physiological response to clear the particulate material from the lungs. In addition, minimal (multi)focal epithelialization at the bronchoalveolar junctions was observed in the lungs of 3/20 animals of the high-concentration group, which – in view of the results of the range finding study – was considered to be related to the exposure to the test material. Under the conditions of this study, inhalation exposure to the test substance up to 61.0 mg/m3 (actual concentration) did not result in systemic toxicity. Based on this result, the No- Observed-Adverse-Effect Concentration (NOAEC) for systemic toxicity was at least 61.0 mg/m3. As toxicologically relevant local effects were noted at all concentrations tested, although of minimal severity at the low concentration, a NOAEC for local toxicity could not be established. The lowest concentration tested in this study, 5.09 mg/m3 (actual concentration), was a Lowest-Observed-Adverse-Effect Concentration (LOAEC) for local toxicity upon sub-chronic inhalation exposure to the test substance.