2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June to Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 39 (2009)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

EC Number:

247-953-0

EC Name:

2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Cas Number:

26747-90-0

Molecular formula:

C18H12N4O4

IUPAC Name:

bis(3-isocyanato-4-methylphenyl)-1,3-diazetidine-2,4-dione

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2015-12-08

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Nederland, AD Horst, Netherlands
- Strain: HsdCpb:Wu (SPF)
- Age at study initiation: approx. 2 months
- Weight at study initiation: at the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 9-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: dry conditioned air

Mass median aerodynamic diameter (MMAD):

>= 2 - <= 3 µm

Geometric standard deviation (GSD):

2

Details on inhalation exposure:

- MODE OF EXPOSURE:
Animals were exposed to the aerosolized test item in polycarbonate exposure restrainers. Restrainers were chosen that accommodated the animals' size. These restrainers were designed so that the rat's tail remained outside the restrainer, thus restrained-induced hyperthermia can be avoided. This type of exposure principle is comparable with a directed-flow exposure design (Moss and Asgharian, 1994) and is preferable to whole-body exposure on scientific (Pauluhn, 1984; 1988) and technical reasons (rapid attainment of steady-state concentrations, no technical problems with regard to test atmosphere inhomogeneities, better capabilities to control all inhalation chamber parameters, easier cleaning of exhaust air, and lower consumption of test item). Moreover, contamination of the hair-coat can largely be avoided and confounding effects as a result of uptake of test item by non-inhalation routes are minimized. The chambers used are commercially available (TSE, DE-61348 Bad Homburg) and the performance as well as their validation has been published (Pauluhn, 1984; Pauluhn, 1994; Pauluhn and Thiel, 2007).

- DESCRIPTION OF APPARATUS:
Dry conditioned air was used to aerosolize the test substance as described below. The test atmosphere was then forced through openings in the inner concentric cylinder of the chamber, directly towards the rats' breathing zone. This directed-flow arrangement minimizes re-breathing of exhaled test atmosphere. Each inhalation chamber segment is suitable to accommodate 20 rats at the perimeter location. All air flows were monitored and adjusted continuously by means of calibrated and computer controlled mass-flow-controllers. A digitally controlled calibration flow meter was used to monitor the accuracy of mass-flow-controller. As
demonstrated in Table 9, the ratio between supply and exhaust air was selected so that 90% of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Aerosol scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that
the total exhaust air flow rate was adjusted on-line and maintained at the specified 90%. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers. The pressure difference between the inner inhalation chamber and the exposure zone was 0.02 em H20 (Pauluhn, 1994). The exposure system was accommodated in an adequately ventilated enclosure. Temperature and humidity are measured by the
Data Acquisition and Control System using calibrated sensors. The sensors were located in the inhalation chamber.

- INHALATION CHAMBER:
The aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter.= 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). To be able to perform all measurements required to define exposure in a manner that is similar to the exposure of rats, 'two segment' chambers were used in all groups. Details of this nose-only exposure system, including its validation, have been published previously (Pauluhn, 1994; Pauluhn and Thiel, 2007).

- INHALATION CHAMBER EQUILIBRIUM CONCENTRATION:
The test atmosphere generation conditions provide an adequate number of air exchanges per hour [30 L/min x 60 min / (3.8 L/chamber) = 237, continuous generation of test atmosphere]. Based on OECD-GD39 the equilibrium concentration (t95) can be calculated as folIows:
t95 (mln) = 3x (chamber volume/chamber airflow)
Under the test conditions used a chamber equilibrium is attained in less than one minute of exposure. The test atmosphere can by no means be diluted by bias-air-flows.

- CONDITIONING THE COMPRESSED AIR:
Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a BEKO RA 55 compressed air dryer. Adequate control devices were employed to control supply pressure.

- AIR FLOWS:
During the exposure period air flows were monitored continuously by flow meters and, if necessary, readjusted to the conditions required. Measured air-flows were calibrated with precision flow-meters and/or specialized flow-calibration devices (Bios DryCal Defender 510; SMG Interlink, USA) and TSI Mass Flow meter 4043 (TSI Incorporated, USA) and were checked for correct performance at regular intervals.

- TREATMENT OF EXHAUST AIR:
The exhaust air was purified via cotton-wool, activated char coal filter, and HEPA filters. These filters were disposed of by Bayer AG.

- INHALATION CHAMBER TEMPERATURE AND HUMIDITY:
Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using FTF11 sensors (ELKA ELEKTRONIK, Lüdenscheid, Germany). The position of the probe was at the exposure location of rats. Measurements were performed in the exhaust air. Temperature and humidity data are integrated for 30-seconds and displayed accordingly. The humidity sensors are calibrated using saturated salt solutions according to Greenspan (1977) and Pauluhn (1994) in a two-point calibration at 33% (MgCI2) and at 75% (NaCI) relative humidity. The calibration of the temperature sensors is also checked at two temperatures using reference thermometers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- ANALYSIS OF TEST ATMOSPHERES:
Nominal concentration: The nominal concentration was not calculated from the ratio of the total quantity of test item consumed during the exposure period and the total throughput of air through the inhalation chamber as this would have required a dismantling of the dust generator.
Total mass concentration: The test substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Gottingen, Germany; postsampling conditioning period of 15 min at room temperature). This method was used to define the actual concentration.
Sampling: Chamber samples were taken in the vicinity of the breathing zone. The number of samples taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values. If technically feasible, per exposure day three samples were collected from the concentration of 11.1 mg/m3, two samples at 1.2 and 3.1 mg/m3 , and one sample at 0.28 mg/m3. The actual concentrations reported refer to mg/m3 test item (gravimetric concentrations).

- STABILITY OF TEST ATMOSPHERES:
The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Bedford, UK) and at 0.3 mg/m3 the R&P Team 1400a (amibent particulate monitor; Thermo Fisher Scientific Inc., USA). Samples were taken continuously from the vicinity of the breathing zone. This chamber monitoring allows for an overall survey of toxicologically relevant technical parameters (inlet and exhaust flows as well as atmosphere homogeneity, temporal stability, and generation performance). Interruptions in exposure (e.g. resulting from obstruction of nozzles or other technical mishaps) are recorded and, if applicable, a commensurate interval is added to the exposure duration for compensation.

- CHARACTERIZATION OF AERODYNAMIC PARTICLE-SIZE DISTRIBUTION:
Samples for the analysis of the particle-size distribution were also taken in the vicinity of the breathing zone. The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. Each impactor stages was covered with an aluminum foil which were subjected to gravimetric analysis. Gravimetric analyses were made using a digital balance.

The parameters characterizing the particle-size distribution were calculated according to the following procedure:
Mass Median Aerodynamic Diameter (MMAD): Construct a 'Cumulative Percent Found - Less Than Stated Particle Size' table, calculate the total mass of test substance collected in the cascade impactar. Start with the test substance collected on the stage that captures the smallest particle-size fraction, and divide this mass of the test substance by the total mass found above. Multiply this quotient by 100 to convert to percent. Enter this percent opposite the effective cut-off diameter of the stage above it in the impactor stack. Repeat this step for each of the remaining stages in ascending order. For each stage, add the percentage of mass found to the percentage of mass of the stages below it. Plot the percentage of mass less than the stated size versus particle size in a probability scale against a log particle-size scale, and draw a straight line best fitting the plotted points. A weighted least square regression analysis may be used to achieve the best fit. Note the particle size at which the line crosses the 50% mark. This is the estimated Mass Median Aerodynamic Diameter (MMAD).

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.3 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

1 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

3 mg/m³ air (nominal)

Doses / concentrations 4

Dose / conc.:

10 mg/m³ air (nominal)

Doses / concentrations 5

Dose / conc.:

0.28 mg/m³ air (analytical)

Doses / concentrations 6

Dose / conc.:

1.2 mg/m³ air (analytical)

Doses / concentrations 7

Dose / conc.:

3.1 mg/m³ air (analytical)

Doses / concentrations 8

Dose / conc.:

11.1 mg/m³ air (analytical)

No. of animals per sex per dose:

5 animals per sex per dose (main groups);
5 animals per sex for control and high dose group (satellite groups for 4-week recovery);
6 males per dose (satellite groups for bronchoalveolar lavage at the end of the 4-week exposure period)

Control animals:

yes, sham-exposed

Details on study design:

- DOSE SELECTION RATIONALE:
Based on the results of a subacute pilot inhalation study in rats (Kopf, 2015) the concentrations for the subacute (4-week) inhalation toxicity study in rats were chosen. Wistar rats were nose-only exposed for 6 hours per day on five consecutive days to mean actual concentrations of 9, 39 and 155 mg/m3. Animals exposed to air under otherwise identically circumstances served as negative controls. Five male and female rats per group were allowed to recover during a 2-week postexposure period. The exposure took place in directed-flow nose-only inhalation chambers. The conventional endpoints (clinical observations, body temperature, body weights, gross necropsy, and reflex measurement) were evaluated. At each sacrifice, the weights of the lungs were determined. Mortality did not occur. Clinical symptoms (e.g. irregular breathing, labored breathing,
tachypnea, bradypnea, piloerection, high-legged gait) and significant hypothermia were observed during the course of the study at 39 mg/m3 and above. Comparisons between the control animals and the exposure groups revealed significantly decreased absolute body weights as well as incremental body weight gain during the exposure period in rats exposed to 155 mg/m3 test item. Significantly increased lung weights were found at 9 mg/m3 and above at the end of the exposure period and at 155 mg/m3 at the end of the recovery phase. Furthermore findings associated to the respiratory tract were seen during necropsy (e.g. less collapsed, light-colored, white hepatoid area and/or exalted areas in the lungs, strongly enlarged lung-associated lymph nodes, dilated heart ventricles dilated). In summary, there is evidence of portal-of-entry toxicity at concentrations tested in this study since clinical respiratory symptoms match well with concentration-dependent increased lung weights and the respiratory tract-associated macroscopic findings observed during necropsy. There is a trend of recovery after the 1-week exposure period. Taking all findings into account, a no observed effect level (NOEL) could not be established due to macroscopic lung-associated findings, significantly increased lung weights and enlarged lung-associated lymph nodes at the low concentration (9 mg/m3). From the above mentioned results of the subacute pilot inhalation study, the target concentrations of 0.3, 1.0, 3.0 and 10 mg/m3 were chosen for the subacute (4-week) inhalation toxicity study with the purpose to demonstrate concentration-dependent effects and to generate a NOAEL.

- POST-EXPOSURE RECOVERY PERIOD:
Five male and female rats each of the control and high dose groups were examined during a post-exposure recovery period of 4 weeks.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

- BODY WEIGHTS:
Body weights of all animals were measured on a twice per week basis during the exposure period and once weekly during the exposure-free recovery period.

- FOOD AND WATER CONSUMPTION:
Food and water consumption were determined on a per week basis.

- CLINICAL OBSERVATIONS:
The appearance and behavior of each rat was examined carefully at least twice on exposure days (before and after exposure) and once a day during the exposure-free weekends and recovery period. Each rat was first observed in its home cage and then individually examined. If considered applicable due to unequivocal signs, in nose-only exposed rats observations were also made during exposure. Assessments from restrainers were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, severe respiratory signs, hemorrhage). Observations were made and recorded systematically; individual records were maintained for each animal, if applicable. Cage side observations included, but were not limited to, changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as well as somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. Since these signs can only be assessed adequately in their home cages, no specific assessment was performed during exposure while animals were restrained.

- FUNCTIONAL OBSERVATION BATTERY (FOB): The first five core rats of each group were used for functional observation battery investigation on day relative 23. Each rat was firstly observed in the home cage and then individually examined. The following reflexes were evaluated: visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds {finger snapping) and touch (back). Measurements of grip strength were measured qualitatively but defined as semi quantitative.

- CLINICAL PATHOLOGY AND HEMATOLOGY:
Blood samples (non fasted) for hematology, coagulation and serum chemistry parameters were collected from all core animals (end of the exposure period) and recovery toxicology animals (end of the recovery period) during sacrifice by cardiacpuncture. The tubes contained EDTA di-potassium salt as an anticoagulant for blood samples collected for hematology. The tubes used for serum chemistry determinations will not contain any anticoagulant, but may contain a serum separator gel. Citrate solution (micro tubes) was used as the anticoagulant for coagulation parameters.
1. Hematology: Hematrocit, Hemoglobin, Leukocytes, Erythrocytes, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Thrombocyte count, Reticulocytes, Leukocyte differential count (Lymphocytes, Granulocytes, Segmented neutrophils, Eosinophilic neutrophils, Basophils, Monocytes, Plasma cells, miscellaneous abnormal cell types), Fibrinogen, PTT, TPZ (Quick sec).
2. Clinical Pathology: Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Glutamate dehydrogenase (GLDH), y-Glutamylaminotransferase (y-GT), Lactate dehydrogenase (LDH), Alkaline phosphatase (APh), Albumin, Bilirubin (total), Calcium, Chloride, Cholesterol, Creatinine kinase, Creatinine, Magnesium, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea.

- OPHTHALMIC EXAMINATION:
Prior to the first exposure all animals {excluded lavage animals) and towars the end of the dosing phase control and high dose animals were examined. The mydriaticum Stulln® was used for pupil dilation. Examinations included an examination of the anterior segment of the eye, the posterior segment of the eye and adnexal structures. Structures examined in the anterior segment of the eye included the cornea, sclera, iris, pupil, lens, aqueous, and anterior chamber. Structures examined in the posterior segment of the eye included the vitreous body, retina and lense. Examination of adnexal structures included conjunctiva, eyelids and eyelashes. Data were collected on-line using a validated computerized system {as provided by Pristima® system).

Sacrifice and pathology:

-ORGAN WEIGHTS:
The following organs were weighed at necropsy after exsanguination: Adrenal glands, Brain, Heart, Kidneys, Liver, Lung (incl. trachea), Lung associated lymph nodes (posterior mediastinal lymph nodes of the hilus region), Ovaries, Spleen, Testes, Thymus.
No organ weight data were collected from animals found dead. Paired organs were weighed together.

- NECROPSY:
All surviving rats were sacrificed at the end of the exposure and post-exposure observation period using sodium pentobarbital as anaesthetic and complete exsanguination by heart puncture (Narcoren®; at least 120 mg/kg body weight, intraperitoneal injection). All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

- HISTOPATHOLOGY:
The following organs/tissues were collected and fixed in 10 % neutral buffered formalin or Davidson's solution: Adrenals, aorta, bone and bone marrow section (sternum), brain (cerebrum, cerebellum, pons/medulla), epididymides, esophagus, eyes with optic nerve, eyelids, extraorbital lacrimal glands, femur with knee joint, Harderian glands, head with nasal cavity, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidneys including pelvis, lacrimal glands, larynx, liver, lungs and main bronchi (all lobes), lymph nodes (lung associated, mandibular, mesenterics, popliteal, mediastinal), mammary gland, muscle (biceps femoris), ovaries with oviducts, pancreas, pharynx, pituitary gland, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulation glands), skin (flank, nose region and facial area), spinal cord (cervical, thoracal, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, ureters, urinary bladder, uterus with cervix, vagina, Zymbal glands and tissues with macroscopic findings.
Histopathology was performed on all organs/tissue shown above at least in the control and high dose groups. The tissues of the respiratory tract were examined in all groups, including those of the recovery groups. Other groups (and/or tissues) were evaluated at the discretion of the clinical pathologist only if warranted by specific changes.

Other examinations:

- RECTAL TEMPERATURE:
First five core animals of each group were subjected to body temperature measurements. The rectal temperatures were measured directly after cessation of
exposure (approximately within 1/2 hour after the end of exposure) using a digital thermometer with a rectal probe for rats on days rel. 0, 8 and 22.

- BRONCHOALVEOLAR LAVAGE (BAL):
Samples of bronchoalveolar lavage fluid were collected from the lungs of rats (six male rats/group) at the end of the exposure period (one day after the last exposure). In BAL-fluid (BALF), several indicators of pulmonary damage were assessed:
Body Weight, Lung weight (absolute), Lung weight (relative), Recovery of lavage fluid, Total cell count in BAL, Mean cellular diameter, Mean cellular volume, Lactate dehydrogenase, Protein, Phospholipids in BALF, gamma-Glutamyltransferase, Number of cells counted per cytospot, Alveolar macrophages, Polymorphonuclear cells, Lymphocytes, Eosinophils, Foamy, Alveolar macrophages with RBC, Alveolar macrophages with particulates, Red Blood Cells (RBC) Score, Alveolar macrophages (count), Polymorphonuclear cells (count), Lymphocytes (count), Eosinophils (count), Foamy alveolar macrophages (count), Alveolar macrophages with RBC and/or PM (count), Adjusted alveolar macrophages, Alveolar macrophages with particulates of AM, BAL-volume

Statistics:

- IN-LIFE DATA: In-life data and organ weights (excluded clinical observation and reflexes) were done on- or offline by using the Pristima® System (Xybion Medical System Corporation, New Jersey,USA). For clinical pathology data the LORENZO LabCentre System (iSOFT Laboratory System, iSOFT Health GmbH, Germany) was used. The gross pathological findings were entered online into the PATHDATA computer system using the laboratory specific macro menu. Histopathological findings were entered online into the PATHDATA computer program version 6.2.c2.

- BODY WEIGHTS, FOOD AND WATER CONSUMPTION, RECTAL TEMPERATURES, CLINICAL PATHOLOGY AND ORGAN WEIGHTS EVALUATION: Data were statistically evaluated using the online Pristima® System. For continuous variables, the statistical test procedure was based on prior knowledge of the respective variable derived from previous studies. For normally distributed variables with equal variances across treatment groups Dunnett's tests were performed. Heteroscedastic normally distributed variables were analyzed using appropriately adjusted Dunnett's tests, using Satterthwaite adjustments for the degrees of freedom and taking the different variances within the groups into account. For log-normally distributed variables, Dunnett's tests were performed after log transformation of the original values. If experience with historical data indicated that the assumptions for parametric analyses are violated, Bonferroni-adjusted Mann-Whitney U-tests were employed in the analyses. For small sample sizes, the exact version of this test was used.

- BAL DATA: Means and SD were statistically evaluated using the ANOVA procedure (vide infra).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Comparisons between the control and the exposure groups did not reveal any significantly concentration-dependent differences in body weights during the exposure period. Nonetheless slight tendency of decreased body weights occurred in female rats at 11.1 mg/m3 when compared to the air control group during the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Lung weights were significantly increased at 3.1 mg/m3 and above. Furthermore lung-associated lymph node (LALN) weights were significantly increased in females at 11.1 mg/m3.
.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lung-associated lymph nodes (LALN) were increased in size in males at 11.1 mg/m3 as well as in females starting at 3.1 mg/m3 at the end of the exposure period. Additionally, some minor gross findings were detected in individual animals at the end of the exposure period and after the end of the recovery period which are reflected to be not test item-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the exposure period, minimal to slight hypercellularity of the bronchoalveolar region occurred in all rats at 3.1 mg/m3 and concentrations above. In two male animals exposed to 1.2 mg/m3 hypercellularity was found with minimal degree. This hypercellularity consists of inflammatory infiltrates, thickened airway epithelium, alveolar macrophages and focal septal thickening. Furthermore enlarged and/or foamy macrophages, partly with brownish cytoplasm, were seen in all rats at 11.1 mg/m3 . In lung-associated lymph nodes, an increased cellularity of the paracortex (grading minimal or slight) occurred in males and females at 3.1 mg/m3 and above. Furthermore cellularity of the paracortex was minimally increased in one rat exposed to 1.2 mg/m3.

At the end of the recovery period, hypercellularity was not seen in animals exposed to 11.1 mg/m3. Enlarged and/or foamy macrophages were detected in the lungs of rats exposed to 11.1 mg/m3. In lung-associated lymph-nodes, increased cellularity of the paracortex was still detectable in rats exposed to 11.1 mg/m3. Additionally, minimal epitheloid cell accumulations occurred in all animals at 11.1 mg/m3.

From a histopathological point of view first biological effects were seen starting at 1.2 mg/m3. Nonetheless due to incidence (2 rats out of 1 0) and severity (minimal) of the hypercellularity and the absence of any significantly inflammatory and cytotoxic effects in the bronchoalveolar lavage at 1.2 mg/m3 this concentration is considered to be not adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Bronchoalveolar lavage (BAL) findings:
At 3.1 mg/m3 the following relevant BAL parameters were significantly changed: alveolar macrophages, neutrophils, lymphocytes and phospholipids. Furthermore at this exposure concentration lactate dehydrogenase, protein, gamma-Glutamyltransferase (GGT) and foamy macrophages were increased but do not reach statistically significance. These findings are interpreted to be indicative of inflammatory and/or cytotoxic effects and thus clearly adverse.

Animals exposed to 11.1 mg/m3 revealed significantly changed parameters of toxicological relevance in the bronchoalveolar lavage fluid (e.g. lung weights, total
cell counts, lactate dehydrogenase, protein, GGT, neutrophils, alveolar macrophages) demonstrating cytotoxicity.

Significantly increased mean cellular diameter and mean cellular volume seen in bronchoalveolar lavage investigation at 0.3 and 1.2 mg/m3 are considered to be not adverse. Increase in cellular diameter and volume are reflected to be physiologically processes due to initiated cellular clearance in the lungs, mainly resulting due to phagocytosis of particles.

- Body temperature findings:
No test item induced concentration-dependent effects were found in body temperature.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

1.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.1 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Executive summary:

In a subacute inhalation toxicity study according to OECD TG 412 and OECD GD 39 five male and five female Wistar rats per dose group were nose-only exposed for 4 weeks (6 hours /day, 5 days/week) to a solid aerosol of Metalink U. The mean actual concentrations (gravimetric) were 0.28, 1.2, 3.1 and 11.1 mg/m3. Rats exposed under otherwise identical test conditions to dry air served as concurrent control group. Additional 5 rats/sex/group (control and high level exposure groups) were allowed to recover during a 4-week post-exposure period. Additional 6 male rats/group were subjected to bronchoalveolar lavage at the end of the 4-week exposure period. The characteristics of the test atmospheres were in compliance with the recommendations of OECD TG 412. The mean Mass Median Aerodynamic Diameters (MMAD) were in the range of 2 - 3 µm within the exposure groups (Geometric Standard Deviation: 1.9 - 2.1 (cascade impactor)). Mortality did not occur in this study. There were no adverse findings seen in clinical observations, ophthalmology, functional observation battery and clinical pathology up to 11.1 mg/m3 . No test item induced concentration-dependent effects were found in food and water consumption as well as in body temperature.

Comparisons between the control and the exposure groups did not reveal any significant concentration-dependent differences in body weights during the exposure period. Nonetheless a slight tendency of decreased body weights occurred in female rats at 11.1 mg/m3 when compared to the air control group during the recovery period.

Lung weights were significantly increased at 3.1 mg/m3 and above. Furthermore lung-associated lymph nodes (LALN) weights were significantly increased in females at 11.1 mg/m3 . Further changes in organ weights are reflected to be not test item associated.

Significantly increased mean cellular diameter and mean cellular volume seen in bronchoalveolar lavage investigation at 0.3 and 1.2 mg/m3 are considered to be not adverse. Increase in cellular diameter and volume are reflected to be physiologically processes due to initiated cellular clearance in the lungs as consequence of internalized particles in the respiratory tract, mainly resulting due to phagocytosis of particles. Absolute lung weights were significantly increased at 3.1 mg/m3 and above. At 3.1 mg/m3 the following relevant parameters were significantly changed: alveolar macrophages, neutrophils, lymphocytes and phospholipids. Furthermore at this exposure concentration lactate dehydrogenase, protein, gamma-Giutamyltransferase (GGT) and foamy macrophages were increased but did not reach statistically significance. Animals exposed to 11.1 mg/m3 revealed significantly changed parameters of toxicological relevance in the bronchoalveolar lavage fluid (e.g. lung weights, total cell counts, lactate dehydrogenase, protein, GGT, neutrophils, alveolar macrophages) were demonstrating clearly evidence of cytotoxicity.

Macroscopically, lung-associated lymph nodes (LALN) were increased in size in males at 11.1 mg/m3 as well as in females starting at 3.1 mg/m3 at the end of the exposure period. Additionally, some minor gross findings were detected in individual animals at the end of the exposure period and after the end of the recovery period which are reflected to be not test item-related.

At the end of the exposure period, minimal to slight hypercellularity of the bronchoalveolar region occurred in all rats at 3.1 mg/m3 and concentrations above. In two male animals exposed to 1.2 mg/m3 hypercellularity was found with minimal degree. This hypercellularity consists of inflammatory infiltrates, thickened airway epithelium, alveolar macrophages and focal septal thickening. Furthermore enlarged and/or foamy macrophages, partly with brownish cytoplasm, were seen in all rats at 11.1 mg/m3 . In lung-associated lymph nodes, an increased cellularity of the paracortex (grading minimal or slight) occurred in males and females at 3.1 mg/m3 and above. Furthermore cellularity of the paracortex was minimally increased in one rat exposed to 1.2 mg/m3. At the end of the recovery period, hypercellularity was not seen in animals exposed to 11.1 mg/m3 . Enlarged and/or foamy macrophages were detected in the lungs of rats exposed to 11.1 mg/m3 . In lung-associated lymph-nodes, increased cellularity of the paracortex was still detectable in rats exposed to 11.1 mg/m3 . Additionally, minimal epitheloid cell accumulations occurred in all animals at 11.1 mg/m3. From a histopathological point of view, first biological effects were seen starting at 1.2 mg/m3 . Nonetheless due to incidence (2 rats out of 10) and severity (minimal) of the hypercellularity and the absence of any significantly inflammatory and cytotoxic effects in the bronchoalveolar lavage at 1.2 mg/m3 , this concentration is considered to be not adverse.

In summary, there is evidence of portal-of-entry toxicity in rats after repeated inhalation {4 weeks) of Metalink U. Clearly adverse findings were observed at 3.1 mg/m3 . These adverse findings were significantly increased lung weights in correlation with toxicologically relevant changes in bronchoalveolar lavage parameters (PMN, L YM, LDH, Protein, GGT). Furthermore these lung findings match well with histopathological observations of minimal to slight hypercellularity of the broncho-alveolar region in all rats at 3.1 mg/m3 and concentrations above. Macroscopically, lung-associated lymph nodes were increased in size in females at 3.1 mg/m3.

Since lung weights were not significantly increased and furthermore neither inflammatory nor cytotoxic findings were seen in the bronchoalveolar lavage at 1.2 mg/m3 , the histopathological finding of minimal hypercellularity at 1.2 mg/m3 in 2 out of 10 rats is considered to be not adverse.

Taking all information into account, in this subacute inhalation toxicity study the No-Observed-Adverse-Effect-Concentration (NOAEC) is considered to be 1.2 mg/m3 .