Reaction mass of 2-ethylhexyl (6-isocyanatohexyl)-carbamate and bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

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:

Jan-March 2017 (in life phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2009)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

EC Number:

247-735-5

EC Name:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

Cas Number:

26488-60-8

Molecular formula:

C16H30N2O3

IUPAC Name:

6-Isocyanatohexylamino 3-ethylheptanoate

Constituent 2

Reference substance name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

EC Number:

278-583-8

EC Name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Cas Number:

76977-79-2

Molecular formula:

C24H48N2O4

IUPAC Name:

6-(2-Ethylhexyloxycarbonylamino)hexylamino 3-ethylheptanoate

impurity 1

Reference substance name:

6-[3-(6-Isocyanatohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexylamino 3-ethylheptanoate

Molecular formula:

C24H42N4O5

IUPAC Name:

6-[3-(6-Isocyanatohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexylamino 3-ethylheptanoate

impurity 2

Reference substance name:

Hexamethylene diisocyanate

EC Number:

212-485-8

EC Name:

Hexamethylene diisocyanate

Cas Number:

822-06-0

Molecular formula:

C8H12N2O2

IUPAC Name:

1,6-diisocyanatohexane

impurity 3

Reference substance name:

Unknown impurities

Molecular formula:

not applicable

IUPAC Name:

Unknown impurities

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, strain Crl:(Wi)WU BR (SPF), animals of this strain have been used at the test institute in toxicological studies for years. Historical data on their physiology, diseases and spontaneous alterations are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Animals were certified to be about 6 weeks at reception and 2 months old at the commencement of study (first exposure day). Hence they fulfil the criterion for young adult Wistar rats.
- Weight at study initiation: At the study start the variation of individual weights did essentially not exceed ±20 per cent of the group means.
- Housing: Singly in conventional Makrolon® Type IIIH cages with gnawing sticks. The legal requirements for housing experimental animals (Directive 2010/63/EU) were followed. A radio program was playing during the light period.
- Diet and water: ad libitum
- Acclimation period: at least 9-14 days

DETAILS OF FOOD AND WATER QUALITY: given in report

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40 - 60 %
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.35 - <= 1.67 µm

Geometric standard deviation (GSD):

1.86

Remarks on MMAD:

The Mass Median Aerodynamic Diameters (MMAD) were in the range of 1.35 – 1.67 µm within the exposure groups (Geometric Standard Deviation: 1.79 – 1.91).

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed under dynamic directed-flow nose-only exposure conditions.
- Exposure apparatus: Dry conditioned air was used to aerosolize the test substance. 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.
Each segment of the aluminium inhalation chamber had 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. Previous validation data indicated that this inhalation system provided a high degree of spatial and temporal uniformity of test atmosphere. Details of this nose-only exposure system, including its validation, have been published previously (1994, 55-62; Pauluhn and Thiel, J. Appl. Toxicol. 27, 2007, 160-167).
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 cm H2O.
- Method of holding animals in test chamber: 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. 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, Journal of Applied Toxicology 13, 1994, 55-62; Pauluhn and Thiel, J. Appl. Toxicol. 27, 2007, 160-167).
- Method of conditioning 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.
- System of generating particulates/aerosols: A modified Schlick binary nozzle was used in order to attain a high and temporally stable concentration of exposure atmospheres. Using a Thermostat (Julabo Water Bath, JULABO GmbH, 77960 Seelbach, Germany), the nozzle temperature was continuously maintained at 25 °C. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was approximately 600 kPa. The test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump). Under dynamic conditions the test substance was atomized into the glass baffle (pre-separator) of the inhalation chamber. Different air dilution cascades were applied to achieve the adequate aerosol concentration for the different exposure groups. Animals exposed to dry conditioned air under otherwise identically conditions were used as negative controls.
- 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; INTERLINK MARKETING CORP dba, SMG/INTERLINK, TX 75023, USA) and TSI Mass Flow meter 4043 (TSI Incorporated, USA) and were checked for correct performance at regular intervals.
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [30 L/min x 60 min/ (7.6 L/ chamber) = 237, continuous generation of test atmosphere]. Thus, under the test conditions used, the chamber equilibrium is attained in less than one minute of exposure. The test atmosphere can by no means be diluted by bias-air-flows.
- Temperature, humidity: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using HC2-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, Germany). The position of the probe was at the exposure location of rats (Figure 1 - Figure 3). Measurements were performed in the exhaust air.
- Treatment of exhaust air: 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 %.

TEST ATMOSPHERE
- Samples taken from breathing zone: yes
- Brief description of analytical method used: The test item concentration was determined by gravimetric analysis using glass fibre filters (filter: glass-fibre filter, Sartorius, Göttingen, Germany; digital balance). This method was used to define the actual concentration. The relative proportion of constituents prone to evaporate was determined: Since the test item did not reveal any volatile fractions, no drying procedure prior filter weighing was applied.
- The particle-size distribution was analysed using a BERNER critical orifice cascade impactor. The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust real-time aerosol photometer (Casella, Bedford, UK).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three samples per exposure day were collected from the 25 mg/m³ exposure chamber. Due to low aerosol concentrations in the breathing zone at 1 and 5 mg/m³ only 1-2 analyses were sampled per day.
The test item concentration was determined by gravimetric analysis using glass fibre filters (filter: glass-fibre filter, Sartorius, Göttingen, Germany; digital balance). This method was used to define the actual concentration of the test item.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: mg/m³ (target conc.)

Remarks:

analytical conc. 0.9 mg/m³

Doses / concentrations 2

Dose / conc.:

5 other: mg/m³ (target conc.)

Remarks:

analytical conc. 5.2 mg/m³

Doses / concentrations 3

Dose / conc.:

25 other: mg/m³ (target conc.)

Remarks:

analytical conc. 25.4 mg/m³

No. of animals per sex per dose:

5; further 5 males and females in control and high dose groups were used for a reversibility study; further 12 males in control and high dose groups were used for the investigation of broncho-alveolar lavage (BAL), six of them at the end of exposure and six of them at the end of the 4-week recovery period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The concentrations for this subacute inhalation toxicity study were based on a 1-week pilot study (T102849-4; the procedures of OECD GD No. 39 (2009) and OECD TG 412 (2009) were followed). In that study Wistar rats were nose-only exposed for 6 hours per day on five consecutive days for 1 week to mean actual concentrations of 0 (air control), 7, 22 and 61 mg/m³ (gravimetric concentrations from actual breathing zone). Five male and female rats per group were allowed to recover during a 2-week post exposure period. The result can be summarized as follows: The NOAEL was considered 7 mg/m³. Evidence of compensatory clearance mechanisms and first physiological effects were seen in the lung at this concentration.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: The appearance and behaviour 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. Observations were made and recorded systematically; individual records were maintained for each animal, if applicable.

DETAILED CLINICAL OBSERVATIONS: Yes, see above

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: The individual body weights were recorded from animals at least once during the quarantine/acclimation period. Additionally body weights were determined during the exposure period prior to inhalation exposure twice weekly (Mondays and Fridays). During the post exposure period body weights (reversibility groups) were determined once a week (Mondays). Individual weights were also recorded directly prior to scheduled necropsies.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was measured weekly (Mondays) in all animals of core groups (5/sex and dose) and reversibility groups (5/sex, control and high dose group)

WATER CONSUMPTION: Yes, water consumption was measured in all animals of core groups (5/sex and dose) and reversibility groups (5/sex, control and high dose group)
- Time schedule for examinations: weekly (on Mondays)

OPHTHALMOSCOPIC EXAMINATION: Yes, ophthalmoscopic examinations were conducted prior the first exposure and towards the end of the dosing phase (on day 24 of the dosing phase) in all animals of core groups (5/sex and dose) and reversibility groups (5/sex, control and high dose group), furthermore at the end of the recovery period (day 24 of the recovery phase) reversibility groups (5/sex, control and high dose group).
- Time schedule for examinations: prior the first exposure and towards the end of the dosing phase, for animals of recovery groups also at the end of the recovery period

HAEMATOLOGY: Yes, blood samples for haematology, coagulation and serum chemistry parameters were collected from all animals (non-fasted) of core groups (5/sex and dose) at the end of the exposure period) and from animals of reversibility groups (5/sex, control and high dose group) at the end of the recovery period).
- Time schedule for collection of blood: each during sacrifice
- Animals fasted: Yes
- Parameters examined: Erythrocyte count, Haemoglobin concentration, Haematocrit, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Reticulocyte count, Erythrocyte morphology, Fibrinogen, PTT, Prothrombin time, Leukocyte count, Differential blood count, Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Thrombocyte count/Platelets count, Large unstained cells/atypical leucocytes.

CLINICAL CHEMISTRY: Yes, blood samples for haematology, coagulation and serum chemistry parameters were collected from all animals (non-fasted) of core groups (5/sex and dose) at the end of the exposure period) and from animals of reversibility groups (5/sex, control and high dose group) at the end of the recovery period).
- Time schedule for collection of blood: each during sacrifice
- Animals fasted: Yes
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Glutamate dehydrogenase, Lactate dehydrogenase, Gamma glutamyl transferase, Creatine kinase, Triglycerides, Cholesterol, Creatinine, Urea nitrogen, Total bilirubin, Total protein, Albumin, Chloride, Calcium, Inorganic phosphate, Potassium, Sodium, Magnesium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, a FOB was conducted in all animals of groups (5/sex and dose)
- Time schedule for examinations: Day 23 (the first exposure day is defined as Day 0)
- Battery of functions tested: 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 behavioural changes stimulated by sounds (finger snapping) and touch (back). Measurements of grip strength were measured qualitatively but defined as semi quantitative.

RECTAL TEMPERATURES: The rectal temperatures in all core animals (5/sex and dose) were measured directly after cessation of exposure (approximately within ½ hour after the end of exposure) using a digital thermometer with a rectal probe for rats on days 0, 8 and 22 of the dosing phase (the first exposure day is defined as Day 0).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals of core groups (5/sex and dose), reversibility groups (5/sex, control and high dose group) and animals assigned to BAL examinations were given a gross-pathological examination.
The following organs were weighed at necropsy of animals of core groups (5/sex and dose) and reversibility groups (5/sex, control and high dose group): Adrenal glands, Brain, Heart, Kidneys, Liver, Lung (plus remnant of trachea), Thymus, Lung associated lymph nodes (LALN) [posterior mediastinal lymph nodes of the hilus region], Spleen, Ovaries, Testes.

HISTOPATHOLOGY: Yes, histopathology was performed
For all animals of core groups (5/sex and dose) and reversibility groups (5/sex, control and high dose group) the following organs/tissues were histopathologically examined: nasal cavities (4 levels: L-I to L-IV) / according to Young, Fundam. Appl. Toxicol. 1: 309-312, 1981), pharynx, larynx, trachea, lung, lung associated lymph nodes (hilus)
For all core animals in the control and high dose groups the following organs/tissues were additionally histopathologically examined: Gross findings, Adrenal glands, Aorta, Bone and bone marrow section – sternum, Brain (cerebrum, cerebellum, pons/medulla), Epididymides, Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve, Eyelids, Femur with knee joint, Heart, Head with nasal cavities (Young, 1981), Harderian glands, Intestine large (cecum, colon, rectum), Intestine small (duodenum, ileum, jejunum), Kidneys including pelvis, Lacrimal glands, Larynx (Kaufmann et al., Exp. Toxicol. Pathol. 61(6): 591-603, 2009), Liver, Lungs (all lobes), Lymph nodes (mandibular, mesenteric, iliac, lung associated lymph nodes), Skin - mammary region, Ovaries incl. oviducts, Pancreas, Parathyroid gland(s), Pharynx, Pituitary gland, Prostate gland, Salivary glands (submandibular, sublingual, Parotid glands), Sciatic nerve, Seminal vesicles with coagulation glands, Skeletal muscle, Skin (back-, mammary-, muzzle- region), Spinal Cord (cervical, thoracic, lumbar), Spleen, Sternum, Stomach, Testes, Thymus, Thyroid gland, Tongue, Trachea, Urinary bladder, Uterus (plus cervix), Ureters, Vagina.

Other examinations:

BRONCHOALVEOLAR LAVAGE: Yes, male animals of all exposure groups were assigned to BAL examinations (6 males/dose, each by end of exposure and by end of reversibility period)
After complete exsanguination, the excised wet lungs were weighed and following ligation of the trachea, the lung lobes were lavaged.
Parameters examined in BAL: Total cell counts, Cell diameter, Cell volume, Cytodifferentiation, Lactate dehydrogenase, Total protein, g-Glutamyltranspeptidase, Phospholipids.

Statistics:

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) and general variability (standard deviation, minimum and maximum) as appropriate.
Body weights, Rectal temperatures, and Clinical Pathology evaluation: 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. Statistical tests were not performed for groups, which were smaller than 3. All statistical tests were performed using standard procedures within the Pristima® System.
Organ Weights: If appropriate, detailed statistical calculations were made using DUNNETT’s test procedures.
Analysis of variance (ANOVA BCTIC): This parametric method checks for normal distribution of data by comparing the median and mean. This program (FORTRAN source code) was originally obtained from BCTIC and is used as means to evaluate off-line data. The ANOVA BCTIC procedure is applied in the GLP validated and released computer program ANOVA.exe (in-house created).
BAL data: Means and SD were determined and statistically compared using the ANOVA procedure.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical symptoms were observed in the control animals as well as in rats exposed to the test item up to 25 mg/m³.
Rectal temperature: There were no test item induced adverse findings seen in rectal temperature.

Mortality:

no mortality observed

Description (incidence):

Mortality did not occur in any exposure group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly decreased cumulative body weight gain was found in male animals starting at 5 mg/m³ when compared to controls whereas absolute body weight was significantly decreased at 25 mg/m³ in males on day 4 only.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item induced adverse findings seen in food consumption.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was significantly increased at 25 mg/m³ when compared to the control groups.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item induced adverse findings seen in ophthalmology.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item induced adverse findings seen in haematology.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item induced adverse findings seen in clinical biochemistry.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative lung weights were significantly increased at 25 mg/m³ at the end of the exposure period showing recovery at the end of the recovery period.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item induced adverse findings seen at necropsy.

Neuropathological findings:

no effects observed

Description (incidence and severity):

A battery of reflex measurements performed on Day 23 revealed no differences between animals exposed to the test item compared to animals in control groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the nasal cavity, minimal to slight focal to multifocal epithelial alterations were observed starting at 5 mg/m³ in females and 25 mg/m³ in males accompanied by an inflammatory infiltration, predominantly consisting of mononuclear cells and interspersed single eosinophils. After the recovery period, this finding was not observed anymore. Corresponding to the nasal findings, minimal to slight epithelial alteration was observed in the pharynx with tendency to recover. In the nasal cavity, an increase in extent and severity of intracellular eosinophilic globules up to slight degree within the olfactory epithelium of the nasal conchae was noticed at 25 mg/m³. After the recovery period, incidence and severity of eosinophilic globules increased minimally in test item exposed animals and in controls. Starting at 1 mg/m³, the larynx of male and female rats revealed a slight to marked squamous metaplasia with cornification and hyperplasia of basal cells, which was exposure-dependent and diffusely distributed at level 1 and 2. The finding was associated with an underlying slight to moderate inflammation, predominantly consisting of mononuclear cells. Findings at the larynx recovered partly after the recovery period. Starting at 1 mg/m³ in females and at 5 mg/m³ in males, a minimal epithelial alteration at the bifurcation was observed in the tracheas mostly accompanied by an inflammatory infiltrate of minimal degree. After the recovery period, no differences were visible between control and test item exposed animals. In the lung, starting at 1 mg/m³, minimal to slight chronic inflammation was observed at the bronchiolo-alveolar junction (consisting of hypercellularity, hyperplasia/thickening and infiltrate with mononuclear cells). Epithelial alterations of minimal degree were seen at branching sites of larger bronchi starting at 1 mg/m³, showing a distinct recovery after the recovery period. The bronchus-associated lymphatic tissue revealed a minimal to slight lymphoid hyperplasia starting at 1 mg/m³ that recovered partly after the recovery period. Lung-associated lymph nodes showed minimal to slight lymphoid hyperplasia in both sexes starting at 5 mg/m³. In addition, an increase of minimal to moderate histiocytosis was observed in females starting at 1 mg/m³ and in males starting at 5 mg/m³. At the end of the post-exposure observation period, lymphoid hyperplasia and histiocytosis were reduced in degree and/or incidence. Instead, an increase of minimal macrophage accumulation was noted in the lung-associated lymph nodes of exposed animals, that was not observed directly after exposure period.
All other microscopic findings were incidental and spontaneous in nature and could not be attributed to the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL analysis: After the exposure period, percentage of lymphocytes and absolute number of lymphocytes were significantly increased starting at 1 mg/m³. Percentage of alveolar macrophages and adjusted alveolar macrophages (including alveolar macrophages with red blood cells and particulates) were significantly decreased starting at 5 mg/m³. Gamma-glutamyltransferase, percentage of polymorphonuclear cells (PMN) and absolute PMN count were significantly increased at 25 mg/m³. After the 4 week recovery period, no statistically significantly changed parameters were found when compared to controls in the bronchoalveolar lavage demonstrating recovery.

Details on results:

In summary, there is evidence of portal-of-entry related inflammatory processes that are considered to be adverse, already starting at 1 mg/m³. Those effects are chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung in combination with statistically increased lymphocytes in the bronchoalveolar lavage as well as the diffuse squamous metaplasia and inflammation of the laryngeal mucosa at 1 mg/m³ and above. At 5 mg/m³ and above inflammatory processes were more pronounced and affected other locations as nasal cavity and pharynx. Therefore, a NOAEL was not established in this study.

Effect levels

1

Dose descriptor:

NOAEL

Remarks on result:

other: A NOAEL could not be established in the study.

2

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/m³ air

System:

other: respiratory system

Organ:

other: respiratory tract

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Executive summary:

A repeated exposure 4-week subacute inhalation study according to OECD TG 412 with the liquid aerosol of the test item was conducted in young adult male and female Wistar rats. In that study the animals were nose-only exposed for 6 hours per day on five consecutive days for 4 weeks to concentrations of 0 (air control), 1, 5, and 25 mg/m³ (gravimetric conc. 0.9, 5.2 and 25.4 mg/m³). Five male and female rats of the control group and the high concentration group were allowed to recover during a 4-week post exposure period. Additionally 6 male animals of each exposure group were allowed to recover during a 4-week post exposure group and were subsequently used for bronchoalveolar lavage investigation.

The test atmospheres were in compliance with the OECD TG 412 (MMAD 1.35 – 1.67 µm; GSD: 1.79 – 1.91) and thus of good respirability for rats.

Mortality did not occur in any exposure group and clinical symptoms were not seen up to the highest concentration of 25 mg/m³. There were no test item induced adverse findings seen in functional observation battery, rectal temperature, food consumption, ophthalmology, clinical pathology, or necropsy. Water consumption was significantly increased at 25 mg/m³ when compared to the control groups. Significantly decreased cumulative body weight gain was found in male animals starting at 5 mg/m³ when compared to controls whereas absolute body weight was significantly decreased at 25 mg/m³ in males on day 4 only. Absolute and relative lung weights were significantly increased at 25 mg/m³ at the end of the exposure period showing recovery at the end of the recovery period.

Histopathology revealed no treatment-related findings in organs or tissues except the respiratory tract. With respect to the respiratory tract, histopathology together with BAL analysis showed evidence of portal-of-entry related inflammatory processes that are considered to be adverse, already starting at 1 mg/m³. Those effects are chronic inflammatory reaction seen at the bronchiolo-alveolar junction of the lung in combination with statistically increased lymphocytes in the bronchoalveolar lavage as well as a diffuse squamous metaplasia and inflammation of the laryngeal mucosa at 1 mg/m³ and above. At 5 mg/m³ and above inflammatory processes were more pronounced and affected other locations as nasal cavity and pharynx. Therefore, a NOAEL could not be established in this study. All other microscopic findings were incidental and spontaneous in nature and could not be attributed to the test item.