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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
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available as unpublished report, no restrictions, fully adequate for assessment

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
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: 7-8 weeks
- Weight at study initiation: Males: 274 gram, females: 185 gram
- Housing: Five animals per cage separated by sex, 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.
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diet Services, Whitham, 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.
- Acclimation period: 9 days

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:
nose only
Vehicle:
air
Details on inhalation 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 twice 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 reducing valve and monitored using a mass view meter (Bronkhorst Hi Tec, Ruurlo, The Netherlands). 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 CELITEMENT 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). Samples of 115.0 (Low dose group), 23.0 (Mid dose group), or 4.6 (High dose group) Ln test atmosphere 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. The air pressure readings were recorded hourly during exposure. Air flow of the control group was measured every hour by recording the readings of the mass view meter.

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 and end of the study
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 40.6 (± 2.1), 205.6 (± 12.9) and 1035 (± 120) mg/m3 for the low, mid and high concentration groups, respectively. These concentrations were close to the respective target concentrations of 40, 200 and 1000 mg/m3.

TIME TO ATTAIN CHAMBER EQUILIBRATION:
The time to reach 95% of the steady state concentration (T95), based on chamber volume and average airflow was calculated to be about 1.3 minutes for the low, mid, and high dose groups. The animals were placed in the exposure unit at least 9 minutes after the start of atmosphere generation.

NOMINAL CONCENTRATION AND GENERATION EFFICIENCY
The average nominal concentrations (± standard deviation) were 141 (± 18), 559 (± 42) and 3239 (± 681) mg/m3 for the low, mid and high concentration groups, respectively. The corresponding generation efficiencies were 29.3%, 37.0% and 33.1%, respectively.

Particle size measurement
Aerodynamic particle size was determined weekly for each exposure condition. Overall, the mass median aerodynamic diameter (MMAD) was in the range of 2.3 – 2.7 μm, and the distribution of particles sizes had a geometric standard deviation (gsd) in the range of 2.2 – 2.7. For the low concentration test atmosphere, the MMAD was 2.68 μm (gsd of 2.35) and 2.25 μm (gsd of 2.58). For the mid concentration test atmosphere, the MMAD was 2.56 μm (gsd of 2.55) and 2.51 μm (gsd of 2.68). For the high concentration test atmosphere, the MMAD was 2.36 μm (gsd of 2.40) and 2.48 μm (gsd of 2.16).

TOTAL AIR FLOW:
The average chamber air flows (± standard deviation) were 131 (± 0), 131 (± 0) and 129 (± 0) L/min for the low, mid and high concentration groups, respectively. The average chamber airflow for the control group was 21.6 (± 0.2) L/min.

TEMPERATURE AND RELATIVE HUMIDITY:
The temperature in the exposure chambers was within the target range of 22 +/- 3°C during the entire exposure period. The average temperature (± standard deviation) was 21.1 (± 0.2), 21.2 (± 0.2), 21.1 (± 0.2) and 21.1 (± 0.3) °C for the control, low, mid and high concentration group, respectively.
The relative humidity in the exposure chambers was within the target range of 30 –70% during the entire exposure period. The average relative humidity (± standard deviation) was 36.3 (± 0.9), 45.6 (± 0.8), 39.6 (± 0.8) and 47.5 (± 1.4) % for the control, low, mid and high concentration group, respectively .

OXYGEN AND CARBON DIOXIDE CONCENTRATION:
The oxygen concentration during exposure was in the range of 20.3 – 20.7 % (v/v) and the carbon dioxide concentration was in the range of 0.074 – 0.585 % (v/v), i.e well within the limits of >19% oxygen and <1% carbon dioxide described in OECD guideline 412.
Duration of treatment / exposure:
14 days (i.e. 10 exposure days in total)
Frequency of treatment:
5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
40.6, 205.6, 1035 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On exposure days, 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. The animals were also thoroughly checked again after exposure. In weekends only one check per day was carried out. All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

BODY WEIGHTS:
The body weight of each animal was recorded three days before the start of exposure, prior to exposure on the first day (day 0) and on day 4, 7, 11 and 14 (the scheduled sacrifice date).

FOOD CONSUMPTION:
Food consumption of the animals was measured per cage by weighing the feeders. The results were expressed in g per animal per day. Food consumption was measured over weekly periods, starting at the start of the exposure period.
Sacrifice and pathology:
PATHOLOGY- GROSS EXAMINATION
At the end of the exposure period, surviving animals were sacrificed in such a sequence that the average time of killing 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 autopsy was also performed on one female of the high-concentration group that was found dead after the first exposure on day 0. The following organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the organ to body weight ratios were calculated: adrenals, heart, kidneys, liver, spleen, testes, lungs with trachea and larynx. These organs and the nose and gross lesions of the animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin).
Statistics:
See "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS
- One female of the high-concentration group was found dead after the first exposure on day 0. All other rats survived until scheduled necropsy on day 14.
- Post-exposure signs were generally confined to rats of the high-concentration group and included hunched posture, dyspnoea and piloerection in males (mainly at the first exposures) and females (at several stages throughout the study), and sniffing, grunting and sneezing in females (after the last exposure on day 13). Occasionally blepharospasm and nasal discharge were noted in rats of the highconcentration group after exposure.
- Pre-exposure signs were generally limited to the high-concentration group. In males occasional nose encrustations were noted; in females dyspnoea, sniffing, grunting, sneezing, hunched posture and/or piloerection were noted, especially at the end of the study.
- No clinical abnormalities were observed half way exposure during the animal’s stay in the restraining tubes.

BODY WEIGHTS:
- Body weights were statistically significantly decreased in males of the high-concentration group from day 4 of the study and in female of this group on day 14. Growth was not affected in the other groups.

FOOD CONSUMPTION:
- In males and females of the high-concentration group, food intake (measured per cage) was lower than in controls. In the low- and mid-concentration groups there were no clear differences with the controls.

ORGAN WEIGHTS:
- The absolute and relative weights of the lungs were statistically significantly increased in males of the high-concentration group and in females of the mid- and high-concentration group.
- The relative weight of the heart was statistically significantly increased in males of the high-concentration group and in females of the mid- and high-concentration group.
- The relative weight of the adrenals was statistically significantly increased in males of the high-concentration group. In females of all test groups the relative adrenal weight tended to be higher than in controls, but there was no clear dose relationship and statistical significance was not obtained (in four or five animals/group). The weights of the liver, kidneys, spleen and testes were not affected by the treatment. The elevated relative testis weight in high-concentration males was ascribed to the well-known inverse correlation between terminal body weight en relative testis weight.

MACROSCOPIC EXAMINATION:
The high-dose female that was found dead after exposure on day 0, showed white deposition in the nose and discoloration of the lung. The probable cause of death was obstruction of the nose.
Macroscopic examination at the end of the exposure period showed red spots or discoloration of the lungs in one mid-concentration female and in one male and two females of the high concentration group. One high-concentration female had incompletely collapsed lungs. The other macroscopic findings were unremarkable.

Effect levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Effects of CELITEMENT 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 occasional macroscopic changes in the lungs. At 200 mg/m3, increased lung and heart weights were still observed in females. At 40 mg/m3, no clear effects of CELITEMENT were detected. However, the weight of the adrenals was significantly increased in males of the high-concentration group and elevated in females of all dose groups. Although the findings in females were not statistically significant and not clearly dose related, it cannot completely be excluded that they represents a treatment-related effect.
Executive summary:

In a GLP compliant repeated dose inhalation toxicity study performed according to OECD guideline 412, male and female Wistar rats were exposed the test substance. The test substance was administered by inhalation (nose only, during 6 hours per exposure day) to groups of 5 male and 5 female rats per dose during a period of 2 weeks. The test substance was administered at target concentrations of 0 (clean air only), 40, 200 or 1000 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 40.6, 205.6 and 1035 mg/3. Besides mortality, clinical signs, body weights and food consumption were reported. At the end of the exposure period, surviving animals were sacrificed for pathological examination. Effects in this 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. At 40 mg/m3, no clear effects of the test substance were detected. However, the weight of the adrenals was significantly increased in males of the high-concentration group and elevated in females of all dose groups. Although the findings in females were not statistically significant and not clearly dose related, it cannot completely be excluded that they represents a treatment-related effect. On the basis these results, the following concentrations were selected for the main study: 0, 10, 50 and 200 mg/m3.