Reaction mass of 2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol and 6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

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:

October 2015 to June 2016

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:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

EC Number:

253-281-9

EC Name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

Cas Number:

36938-15-5

Molecular formula:

C14H30N4O4

IUPAC Name:

2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol

Constituent 2

Reference substance name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Molecular formula:

C14H30N4O4

IUPAC Name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Test material form:

solid: bulk

Details on test material:

- Batch no.: EGGE 2806-1
- Currenta sample no.: 2187

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: certified for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: certified by analysis

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test item was melted in a warming cupboard at 110°C, over a time period of 24 hours. Subsequently the test item was filled in a warm beaker glass and was then transferred into an Erlenmeyer flask including warm water. During this process the solution was stirred.
- Final dilution of a dissolved solid, stock liquid or gel: For aerosolization, the test item was dissolved in deionized Lewatit© water (70% v/v test item in water stock solution; further dilution was done to achieve the respective target concentrations) to decrease viscosity.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain: Crl:(Wi)WU BR (SPF),
- Age at study initiation: 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:

other: liquid aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: Lewatit© water

Mass median aerodynamic diameter (MMAD):

>= 1.21 - <= 1.58 µm

Remarks on MMAD:

Geometric Standard Deviation: 2.07– 2.64

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 solution 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.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 other: mg/m³ (target conc.)

Doses / concentrations 2

Dose / conc.:

0 mg/m³ air (analytical)

Doses / concentrations 3

Dose / conc.:

80 other: mg/m³ (target con.)

Doses / concentrations 4

Dose / conc.:

84 mg/m³ air (analytical)

Doses / concentrations 5

Dose / conc.:

400 other: mg/m³ (target conc.)

Doses / concentrations 6

Dose / conc.:

447 mg/m³ air (analytical)

Doses / concentrations 7

Dose / conc.:

2 000 other: mg/m³ (target conc.)

Doses / concentrations 8

Dose / conc.:

2 033 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, concurrent vehicle

Details on study design:

- DOSE SELECTION RATIONALE:
The study was based on the results of the subacute (5x6h) inhalation toxicity study in rats followed by a postexposure period of 2 weeks (T102193-6) with a liquid aerosol of the test item (0, 122, 680 and 2911 mg/m³ air (analytical) . The solid test item was solved in Lewatit© water. (for further details see 7.5.2 endpoint study record: rel 1-sup, subacute pilot rat (5 days), Kopf 2016a
- POST-EXPOSURE RECOVERY PERIOD:
Five male and female rats 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.

- 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).

NEUROBEHAVIOURAL EXAMINATION (FOB): Yes
- Time schedule for examinations: on day relative 23
- Dose groups that were examined: 0, 80, 400 2000 mg/m³ (both sexes)
- Battery of functions tested:
Visual placing response
Grip strength (vertical)
Grip strength (horizontal)
Tonus
Cornea reflex
Light reflex
Pinna reflex
Startle reflex / sound
Startle reflex / touch
Tail-pinch response
Righting response (open field)
Righting response (drop method)

Sacrifice and pathology:

-ORGAN WEIGHTS:
The following organs were weighted at necropsy after exsanguination: Adrenal glands, Brain, Heart, Kidneys, Liver, Lung (incl. trachea), Ovaries, Spleen, Testes, Thymus.
No organ weight data were collected from animals found dead. Paired organs were weighted 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:
The rectal (colonic) temperatures were measured at several time points shortly after cessation of exposure (within 1/2 hour of cessation of exposure) using a digital rectal probe (H. Sachs, March, Germany). Five rats/main group/sex were examined after the first exposure, midterm and the end of the exposure period.

- 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 were assessed:
TCC = Total cell count
MCD = Mean cellular diameter
MCV = Mean cellular volume
LDH = Lactate dehydrogenase
PROT = Protein
PLIPf = Phospholipids
GGT = gamma-Glutamyltransferase
AM = Alveolar macrophages
PMN = Polymorphonuclear cells
LYM = Lymphocytes
EOS = Eosinophils
RBC = Red Blood Cells

Statistics:

- IN-LIFE DATA: Statistical tests on body weights and weight gain as well as on absolute organ weights or relative log1O-transformed organ weights are analyzed using the Dunnett Exact Homogeneous Test. Food/water intake per animal and day are calculated and analyzed by the adjusted Mann-Whitney U-tests. Terminal body weights (TWS) serve as covariate for calculation of the organ-to-body-weight ratio (percentage). Likewise, the Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log10-transformation or the Bonferroni/Mann-Whitney U-test are used for the statistical analysis of clinical pathology parameters. Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit. This included measures of general tendency (mean and median (median not given for food and water intake)) and general variability (standard deviation, minimum and maximum) as appropriate. 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 analysed 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.

- RECTAL TEMPERATURE, BRONCHOALVEOLAR LAVAGE: Data were statistically evaluated using the ANOVA procedure.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Red encrusted noses were seen at low incidences in all concentration groups including vehicle controls.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly increased body weights and cumulative body weight gain in males at 84 mg/m³ and above. Body weights and cumulative body weight gain in female rats were significantly increased at 2033 mg/m³ only.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intake was significantly increased in male rats at 84 mg/m³ and above. Isolated significantly increased food intake was found in female rats exposed to 84 mg/m³ on day 28.

Food efficiency:

not specified

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

effects observed, non-treatment-related

Description (incidence and severity):

Isolated not clearly concentration-dependent significantly increased intake of water was seen in male and female rats. These findings are considered to be not of toxicological relevance.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examinations did not reveal any test item related findings in both sexes up to and including the highest concentration of 2033 mg/m³.

Haematological findings:

no effects observed

Description (incidence and severity):

No test item induced concentration-dependent findings of toxicological relevance were seen in clinical pathological parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item induced concentration-dependent findings of toxicological relevance were seen in clinical pathological parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

A battery of reflex measurements was made on day relative 23. Differences between animals exposed to the test item compared to the control group did not occur

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant significantly changed relative organ weights were found in both sexes up to and including 2033 mg/m³ when compared to control animals. Differences in absolute organ weights are considered to reflect that treated animals revealed higher absolute body weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the end of the exposure period as well as after the end of the post-exposure period some minor gross findings were detected in individual animals without any relation to the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

After the end of the exposure period as well as after the additional 4-week post-exposure period, in the organs/tissues evaluated histopathologically no findings were seen which have to be assessed as related to the test compound. All findings seen during histopathological evaluation are known from controls of previous inhalation studies and/or regarded to be of spontaneous nature.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

After the end of the exposure period as well as after the additional 4-week post-exposure period, in the organs/tissues evaluated histopathologically no findings were seen which have to be assessed as related to the test compound. All findings seen during histopathological evaluation are known from controls of previous inhalation studies and/or regarded to be of spontaneous nature.

Other effects:

no effects observed

Description (incidence and severity):

rectal temperatures: No statistically significantly changed body temperatures in both sexes up to and including the highest concentration of 2033 mg/m³.

Details on results:

Bronchoalveolar lavage fluid (BALF):

The average recovery of bronchoalveolar lavage fluid (BALF) was approximately 85-89 % of the instilled volume and was similar amongst all groups.
Mean cellular diameter (MCD) and mean cellular volume (MCV) were dose-dependent increased. An increase in these two parameters is considered to be the consequence of physiological clearance mechanism. Due to phagocytosis of particles/substances the MCD and MCV of macrophages usually increase.
The percentage of foamy macrophages was obviously increased at 2033 mg/m³ whereas relevant reduction of the percentage of alveolar macrophages was observed at that concentration. These findings match well with the increased mean cellular diameter and mean cellular volume since these parameters clearly demonstrate compensatory clearance mechanisms within the respiratory tract due to phagocytosis.
Gamma-Glutamyltransferase (GGT) levels were significantly increased at 2033 mg/m³. Lactate dehydrogenase (LDH) in the BALF was minimally increased at 2033 mg/m³, not reaching statistically significance when compared to the control group.

Taking all results of the bronchoalveolar lavage investigation into account, first relevant biological effects were seen starting at 2033 mg/m³. Nonetheless due to the absence of any significantly inflammatory (e.g. increase in inflammatory cells as neutrophils, lymphocytes or eosinophils) and cytotoxic effects (LDH is not reaching statistically significance) in the bronchoalveolar lavage at 2033 mg/m³, these above mentioned biological effects are considered to be of compensatory nature due to clearance mechanism and not reflected to be adverse.

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 033 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on all parameters examined

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of Mortality and Morbidity

Sex	Analytical concentration (gravimetric) (mg/m3)	Toxicological results	Onset and duration of signs	Onset and duration of mortality
male	0 (Control)	0 / 0 / 16	1d	---
	84	0 / 5 / 11	0d, 1d	---
	447	0 / 4 / 11	0d -2d, 8d	---
	2033	0 / 5/ 16	0d, 1d, 7d, 14d	---
female	0 (Control)	0 / 5/ 10	0d, 1d	---
	84	0 / 1/ 5	0d, 1d	---
	447	0 / 0/ 5	---	---
	2033	0 / 2/ 10	7d, 14d	---

Toxicological results:

number of dead animals / number of animals with signs after cessation of exposure / number of animals exposed

Applicant's summary and conclusion

Executive summary:

In a subacute inhalation toxicity study (OECD TG 412) 5 male and 5 female Wistar rats per dose group were nose-only exposed for 4 weeks (6 hours /day, 5 days/week) to a liquid aerosol of the test item which was dissolved in deionized water to decrease viscosity. The mean actual concentrations (gravimetric) were 84, 447 and 2033 mg/m³. Rats exposed under otherwise identical test conditions to deionized water served as negative control group. Additional 5 rats/sex/satellite 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 OECD test guideline No. 412. The Mass Median Aerodynamic Diameters (MMAD) were in the range of 1.21 – 1.58 µm within the exposure groups (Geometric Standard Deviation: 2.07– 2.64). Such an aerosol is highly inhalable for rats.

Mortality did not occur in any exposure group. Red encrusted noses were seen at low incidences in all concentration groups including vehicle controls. Body temperatures, reflex measurements and ophthalmology were indistinguishable in rats exposed up to and including 2033 mg/m³ when compared to vehicle control animals. No adverse findings in clinical pathology, necropsy and histopathology were observed in rats exposed up to and including the highest concentration of 2033 mg/m³. Furthermore no relevant significantly changed relative organ weights (% of TBW) were found up to and including 2033 mg/m³ when compared to control animals. Comparisons between the control and the exposure groups revealed significantly increased body weights and cumulative body weight gain in males at 84 mg/m³ and above. Body weights and cumulative body weight gain in female rats were significantly increased at 2033 mg/m³ only. Food intake was significantly increased in male rats at 84 mg/m³ and above. Isolated significantly increased food intake was found in female rats exposed to 84 mg/m³ on day 28. Isolated not clearly concentration-dependent significantly increased intake of water was seen in male and female rats. These findings are considered to be not of toxicological relevance.

The results of the bronchoalveolar lavage investigation pointed to first relevant biological effects, most likely associated to physiologically clearance processes, at 2033 mg/m³. Neither toxicologically relevant increases in inflammatory cells as polymorphnuclear cells and lymphocytes in the bronchoalveolar lavage fluid (BALF) nor histopathological lung findings were observed up to and including this concentration. Thus, these above mentioned biological effects seen during the course of the study are considered to be of compensatory nature and not considered to be adverse. Taking all findings into account, the no observed adverse effect level (NOAEL) is considered to be 2033 mg/m³ for respiratory tract responses and extrapulmonary toxicity. Nonetheless clear evidence of compensatory clearance mechanisms and first physiological effects were seen in the lung at this concentration.