Hexamethylene diisocyanate, oligomerisation product, blocked with 2-butanone oxime

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

22.10.2009

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(E)-(butan-2-ylidene)amino N-(5-{3-[6-(3-{6-[({[(butan-2-yl)amino]oxy}(hydroxy)methyl)amino]hexyl}-5-{5-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]pentyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-5-{5-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]pentyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl}pentyl)carbamate; (E)-(butan-2-ylidene)amino N-[5-(3-{6-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]hexyl}-5-{5-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]pentyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)pentyl]carbamate; (E)-(butan-2-ylidene)amino N-[6-(3-{6-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]carbamate

EC Number:

617-779-3

Cas Number:

85940-94-9

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

(E)-(butan-2-ylidene)amino N-(5-{3-[6-(3-{6-[({[(butan-2-yl)amino]oxy}(hydroxy)methyl)amino]hexyl}-5-{5-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]pentyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-5-{5-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]pentyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl}pentyl)carbamate; (E)-(butan-2-ylidene)amino N-[5-(3-{6-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]hexyl}-5-{5-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]pentyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)pentyl]carbamate; (E)-(butan-2-ylidene)amino N-[6-(3-{6-[({[(E)-(butan-2-ylidene)amino]oxy}carbonyl)amino]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]carbamate

Details on test material:

- Name of test material (as cited in study report): HDI Trimer MEKO blocked
- Physical state: Liquid / colorless, clear
- Analytical purity: 77.3% in acetone

- Lot/batch No.: GCZ 3405-1
- Expiration date of the lot/batch: 2009-01-15

- Stability under test conditions: The stability was guaranteed by the sponsor, and the sponsor holds this responsability.
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rats were selected since this rodent species is recommended in the respective test guidelines.
Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: about 7 weeks
- Weight at study initiation: The weight of the animals used did not exceed +/- 20% of the mean weight of each sex. No more data. (males of the
main group: 210-225g); females of the main group: 165-175g
- Fasting period before study: no data
- Housing: 5 animals by cage
- Diet (e.g. ad libitum): GLP diet
- Water (e.g. ad libitum): Tap water at libitum
- Acclimation period: yes but no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): in the range 20 -24°C
- Humidity (%): in the range 30 - 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): a light/dark rythm of 12 hours

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: acetone

Remarks on MMAD:

MMAD / GSD: See Table 7.5.3b
It is technically not achievable to have GSD abd MMAD in the range of the values provided in the guideline for this test substance.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
- Method of holding animals in test chamber: The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation
chamber.

- Source and rate of air:
- Method of conditioning air:
- System of generating aerosols: The aerosol was generated with compressed air in a mixing stage with conditioned supply air and
passed via the cyclonic separator into the inhalation system

- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination:
The particle size analysis was carried out with a cascade impactor
The calculation of the particle size distribution was carried out in the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (DIN 66141: Darstellung von Korngrößenverteilungen and DIN 66161: Partikelgrößenanalyse, Beuth-Vertrieb GmbH, Berlin und Köln, FRG).

- Treatment of exhaust air:


TEST ATMOSPHERE
- Brief description of analytical method used:
The concentrations of the inhalation atmospheres in test groups were analyzed by gravimetry. This analytical method is judged to be valid because
the polymeric test substance does not possess an appreciable vapor pressure.
A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the aerosol was drawn
through the filter.
The concentration of acetone as solvent was analyzed by a calibrated online total hydrocarbon analyzer.
- Samples taken from breathing zone: yes



VEHICLE (if applicable)

- Composition of vehicle:acetone

- Concentration of test material in vehicle: 77.3% in acetone

- Purity of vehicle: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determination of test groups concentrations:
Gravimetrical measurements
Daily means of the test substance concentration were calculated based on three measured samples per concentration and exposure.
The aerosol concentration in mg/m3 was calculated from the difference between the weight of the preweighed filter and the weight of the filter after
sampling, with reference to the sample volume of the inhalation atmosphere.

Analytical determination of acetone concentration
Daily means of the continuously monitored acetone concentration were calculated per concentration and exposure.
The measurements (total hydrocarbon analyzers) were recorded using line recorders and transferred to an automated measuring system

From the daily mean values of each test substance and solvent concentration, mean concentrations and standard deviations for the entire study
were derived.

Duration of treatment / exposure:

13 weeks / head-nose

Frequency of treatment:

6 hours per day on 5 consecutive days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Basis:
nominal conc.

Doses / concentrations 2

Dose / conc.:

5 mg/L air (nominal)

Remarks:

basis: nominal concentration

Doses / concentrations 3

Dose / conc.:

25 mg/L air (nominal)

Remarks:

basis: nominal concentration

Doses / concentrations 4

Dose / conc.:

150 mg/L air (nominal)

Remarks:

basis: nominal concentration

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Considering the results provided by the 14-day inhalation range finding study (where rats were exposed to 750, 150 and 30 mg/m3) and
considering a NOEC of 30 mg/m3 in this study, the concentrations selected for the 90-day exposure repetead inhalation study are: 150 mg/m3,
concentration which would cause toxic effects, 25 mg/m3, the mid concentration and 5 mg/m3 which could be expected to be the NOEC.

- Rationale for animal assignment: Prior to the preflow period, the animals were distributed according to weight among the individual test groups,
separated by sex.
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

- Recovery groups: Yes, a group of 20 animals (5 rats per sex per group were exposed to either acetone (control group) or to the high target
concentration of 150 mg/m3 for 90 days and observed for potential reversibility for 28 days.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

MORTALITY: Yes
- Time Schedule: Evident signs of toxicity or mortality were examined twice a day on working days, and once a day on Saturdays, Sundays and public holidays.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The clinical condition of the test animals was recorded once during the preflow period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.

During exposure only a group wise examination was possible.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was determined at the start of the preflow, at the start of the exposure period and
then, as a rule, twice a week (on Monday and Friday) as well as one day prior to gross necropsy. As a rule, the animals were weighed at the same time of the day.

FOOD CONSUMPTION:
- Food consumption
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day. The animals were maintained in
social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food
consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the
animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible.

FOOD EFFICIENCY:
- Body weight gain: Food efficiency (group means) could not calculated based upon individual values for body weight and food consumption. Food
consumption and body weight were measured on different study days

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Before the start of the exposure period (day -5/ -6) the eyes of all main-group animals, and at the end of the study (day 90) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an
ophthalmoscope (HEINE Optotechnik, Herrsching, FRG) after administration of a mydriatic.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: sampling was conducted at least 16 hours post exposure
- Anaesthetic used for blood collection: Yes: isoflurane. Animals were exposed to isoflurane only for 1 or 2 minutes, threfore, it is hardly
conceivable, that isoflurane reduced the chronic inflammatory effects of the test compound. (Reference: Protective effects of isoflurane pretreatment in endotoxin-induced lung injury. Reutershan J, Chang D, Hayes JK, Ley K. Anesthesiology. 2006 Mar;104(3):511-7).

- Animals fasted: Yes / No / No data
- How many animals: Hematological examinations were performed in main and recovery group animals.
- Parameters checked were: Leucocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin,
Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes and Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked were: ALT, AST, ALP, GGT, Na, K, Cl, INP, Ca, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB, TRIG, CHOL and Mg.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: RECTAL TEMPERATURE
Measurement of the rectal temperature were carried out in all main-group animals. Using a digital thermometer (Testo) rectal temperatures were
determined prior to exposure on study day -4/-5, during the exposure period on study days 0, 42 and 85. All measurements were performed in the
afternoon (shortly after exposure on exposure days).

Sacrifice and pathology:

The main group and recovery group animals were killed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava. The
animals were then necropsied and assessed by gross pathology.

GROSS PATHOLOGY: Yes
Organ weight
1. Anesthetized animals
2. Lung
3. Liver
4. Kidneys
5. Adrenal glands
6. Testes
7. Uterus
8. Ovaries
9. Thymus
10. Spleen
11. Brain
12. Heart
13. Thyroid glands
14. Epididymides

HISTOPATHOLOGY: Yes, The following organs or tissues were preserved in neutral buffered 4% formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum, colon and rectum
7. Duodenum, jejunum and ileum
8. Epididymides, prostate and seminal vesicle
9. Extraorbital lacrimal glands (Harderian gland included)
10. Eyes with optic nerve
11. Female mammary gland
12. Femur with knee joint
13. Heart
14. Kidneys
15. Larynx
16. Liver
17. Lungs
18. Lymph nodes (mediastinal, mesenteric and axillary lymph nodes)
19. Nose (nasal cavity)
20. Oesophagus
21. Ovaries
22. Pancreas
23. Parathyroid glands
24. Pharynx
25. Pituitary gland
26. Salivary glands (mandibular and sublingual glands)
27. Sciatic nerve
28. Skeletal muscle
29. Skin
30. Spinal cord (cervical, thoracic and lumbar cords)
31. Spleen
32. Sternum with marrow
33. Stomach (forestomach and glandular stomach)
34. Testes
35. Thymus
36. Thyroid glands
37. Trachea
38. Urinary bladder
39. Uterus, oviducts and vagina

Other examinations:

No data

Statistics:

Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the
hypothesis of equal means.
Clinical pathology parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided).If the resulting p-value was equal or
less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal
medians.
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in rats of both sexes of the 150 mg/m3 dose group the total white blood cell (WBC) counts were increased (in females not statistically significant), which was due to an increase of the absolute neutrophil counts. In females of the high dose group also the relative neutrophil counts were statistically significantly increased at the expense of the relative lymphocyte counts. The total white blood cell counts in dosed males as well as the absolute neutrophil counts in dosed rats of both sexes were still increased after 4 weeks of recovery.
The mild increase of the total white blood cell counts and the neutrophil counts in male and female rats after inhalation exposure to 150 mg/m3 test substance was most probably due to a systemic inflammation, which was still present after the 4 weeks recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in males of the 150 mg/m3 dose group the total bilirubin values were higher compared to controls.
The reason for the higher bilirubin values in males of the 150 mg/m3 dose group could not be elucidated. Neither any other parameter in clinical chemistry nor any red blood cell parameter
was altered in these rats. Additionally, in corresponding females no indication of a liver, kidney or red blood cell system damage could be found in clinical pathology. Although a treatment-related reason for the higher bilirubin values in males could not be excluded, this single parameter alteration was not regarded as adverse.

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase of the absolute and relative weight in the lungs of males and females in the 150 mg/m3 group is observed and is regarded to be treatment-related.
The statistically significant weight decreases noted in the spleen (low and mid concentration) and thymus (mid concentration) in female animals is considered to be incidental since no histopathological correlate or dose-dependent relationship was found.
In the lungs, the statistically significant weight increase in males and females exposed to 150 mg/m3 test substance correlated microscopically with a granulomatous inflammation, which was accompanied by lympho-reticular hyperplasia in the bronchial-associated lymphoid tissue (BALT)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The mediastinal lymph nodes were “enlarged” in all males and females of the 150 mg/m3 group and in 7/10 males and 5/10 females of the 25 mg/m3 group. This enlargement is regarded to be treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the nasal cavity, lungs and mediastinal lymph nodes in both male and female animals and in the trachea of males only.

Nasal cavity: Minimal to slight subepithelial lymphoid infiltrates started to be observed in the septum of the nasal cavity (level II and III) of males and females in the 25 mg/m3 group, with increasing incidence in the 150 mg/m3 group. Hyaline droplets were minimally seen in the respiratory and olfactory epithelium in levels II, III and IV in the 25 mg/m3 group and increased in incidence and grading (minimal to moderate) in the 150 mg/m3 group, affecting both males and females.
Trachea: In the epithelium of the trachea, goblet cells started to be observed in males of the 25 mg/m3 group (3/10). They increased in incidence (7/10 males) and severity (minimal to moderate) in the 150 mg/m3 group. Other findings observed in few males of the high concentration group included minimal to slight hyperplasia and inflammation of the respiratory epithelium and minimal granulomas in the carina.
Lungs: In the lungs, a granulomatous inflammation affected all males and females of the 25 mg/m3 and 150 mg/m3 group. In the mid concentration group, the severity was minimal to moderate in males and minimal to slight in females. In the high concentration group the severity increased up to marked in males and moderate in females. The bronchial-associated lymphoid tissue (BALT) showed lympho-reticular hyperplasia with development of granulomas starting from minimal to slight in the mid concentration group (males only) up to moderate (males) or slight (females) in the high concentration group.
Mediastinal lymph nodes: In the mediastinal lymph node, 10/10 males and 9/10 females of the 25 mg/m3 group showed lympho-reticular hyperplasia with development of multifocal granulomas ranging from minimal to moderate. In the 150 mg/m3 group, this finding was marked in all males and slight to marked in females. In few animals the granulomas showed partly (central) necrosis of varying extend.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No deaths were recorded throughout the study.
During the preflow period, the exposure period and the post-exposure observation period the animals showed no clinical signs and findings different from normal.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights and the body weight change of the test substance exposed groups were not statistically significantly different from the control group 0.

FOOD CONSUMPTION
No substance-related changes of food consumption were observed during the whole study period.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainder of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.


HAEMATOLOGY
At the end of the administration period, in rats of both sexes of the 150 mg/m3 dose group the total white blood cell (WBC) counts were increased (in females not statistically significant), which was due to an increase of the absolute neutrophil counts. In females of the high dose group also the relative neutrophil counts were statistically significantly increased at the expense of the relative lymphocyte counts. The total white blood cell counts in dosed males as well as the absolute neutrophil counts in dosed rats of both sexes were still increased after 4 weeks of recovery.
In the differential blood cell counts some other cell fractions were statistically significantly altered in the exposed rats, but these changes were within the historical control ranges and therefore were regarded as not treatment-related.

CLINICAL CHEMISTRY
At the end of the administration period, in males of the 150 mg/m3 dose group the total bilirubin values were higher compared to controls.
The reason for the higher bilirubin values could not be elucided. Neither any other parameter in clinical chemistry nor any red blood cell parameter was altered in these rats. Additionally, in corresponding females, no indication of a liver, kidney or red blood cell system damage could be found in clinical pathology. Although a treatment related reason for the higher bilirubin values in males could not be excluded, this single parameter
alteration was not regarded adverse (ECETOC 2002).

URINALYSIS


NEUROBEHAVIOUR


ORGAN WEIGHTS
Tables 7.5.3c and 7.5.3d
Absolute weight:
The statistically significant increase of the absolute weight in the lungs of males and females in the 150 mg/m3 group is regarded to be treatment-
related. All other mean absolute weight parameters did not show relevant differences compared to the control group and are regarded to be within
the normal range of test animals of that age.
Relative weight:
The statistically significant increase of the relative weight in the lungs of males and females in the 150 mg/m3 group is regarded to be treatment-
related. The statistically significant weight decreases noted in the spleen (low and mid concentration) and thymus (mid concentration) in female
animals is considered to be incidental since no histopathological correlate or dose-dependent relationship was found.
All other mean relative weight parameters of the treated animals did not show relevant differences compared to the control groups.


GROSS PATHOLOGY
The mediastinal lymph nodes were “enlarged” in all males and females of the 150 mg/m3 group and in 7/10 males and 5/10 females of the 25 mg/m3 group. This enlargement is regarded to be treatment-related.
All other gross lesions noted are considered to be incidental or spontaneous in nature and not related to treatment.


HISTOPATHOLOGY: NON-NEOPLASTIC
Table 7.5.3e
Treatment-related findings were observed in the nasal cavity, trachea, lungs, and the mediastinal lymph node of both sexes, with the lungs and mediastinal lymph node showing the most severe changes. Less severe findings were noted in the nasal cavity (males and females) and trachea (males). All of them were present in a dose-dependent manner starting at 25 mg/m3.
Minimal to slight subepithelial lymphoid infiltrates started to be observed in the septum of the nasal cavity (level II and III) of males and females in the 25 mg/m3 group, with increasing incidence in the 150 mg/m3 group. Hyaline droplets were minimally seen in the respiratory and olfactory epithelium in levels II, III and IV in the 25 mg/m3 group and increased in incidence and grading (minimal to moderate) in the 150 mg/m3 group, affecting both males and females.
The slight increase of subepithelial lymphoid infiltrates (level II and III) and the development of hyaline droplets (level II, III, and IV) in the nasal cavity represent findings after a slight
epithelial irritation. However, since the lymphoid infiltrates were of low severity they may be regarded as non-adverse. Moreover, hyaline droplets are considered to represent an adaptive change to treatment; therefore their presence is regarded as non-adverse. At the end of the recovery period, the nasal cavity showed a full recovery for lymphoid infiltrates in females and a partial recovery for hyaline droplets in both males and females.

In the epithelium of the trachea, goblet cells started to be observed in males of the 25 mg/m3 group (3/10). They increased in incidence (7/10 males) and severity (minimal to moderate) in the 150 mg/m3 group. Other findings observed in few males of the high concentration group included minimal to slight hyperplasia and inflammation of the respiratory epithelium. Moreover, minimal granulomas occurred in the carina. The accumulation of goblet cells in the epithelium of the trachea in males was the most common finding and is regarded as an adaptive effect. However, the epithelial hyperplasia, inflammation and granulomas in the carina, although of low grading, are considered to be adverse. After a 4 week treatment-free period, the male trachea showed a full recovery.

In the lungs, a granulomatous inflammation affected all males and females of the 25 mg/m3 and 150 mg/m3 group. In the mid concentration group, the severity was minimal to moderate in males and minimal to slight in females. In the high concentration group the severity increased up to marked in males and moderate in females. The bronchial-associated lymphoid tissue (BALT) showed lympho-reticular hyperplasia with development of granulomas starting from minimal to slight in the mid concentration group (males only) up to moderate (males) or slight (females) in the high concentration group.
In the mediastinal lymph node, 10/10 males and 9/10 females of the 25 mg/m3 group showed lympho-reticular hyperplasia with development of multifocal granulomas ranging from minimal to moderate. In the 150 mg/m3 group, this finding was marked in all males and slight to marked in females. In few animals the granulomas showed partly (central) necrosis of varying extend.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS: RECTAL TEMPERATURE
Rectal temperatures of the treated main-group animals were not different to those of the controls during the whole study period.

RECOVERY GROUP
Tables 7.5.3 f, g and h
Absolute weight:
The statistically significant increase of the absolute weight in the lungs of males in the 150 mg/m3 group is regarded to be treatment-related.
A statistically significant increase of the absolute weight was observed in the liver of females in the 150 mg/m3 group.
All other mean absolute weight parameters did not show relevant differences compared to the control group and are regarded to be within the normal range of test animals of that age.
Relative weight:
The statistically significant increase of the absolute weight in the lungs of males in the 150 mg/m3 group is regarded to be treatment-related.
Other statistically significant changes were noted for the relative weights, such as an increase in the liver weight in females and a decrease in the
testes weight in males. As neither liver nor testes were regarded target organs in the main groups (no weight changes, no histopathomorphological
findings), these weak weight changes are considered incidental and not related to treatment.
All other mean absolute weight parameters did not show relevant differences compared to the control group and are regarded to be within the normal range of test animals of that age.
Gross pathology: At gross pathology, “lobulations” were observed in the lungs (2/5 males, 3/5 females) and the mediastinal lymph nodes were “enlarged” in all male and female animals of the 150 mg/m3 recovery group.
All other gross lesions noted are considered to be incidental or spontaneous in nature and not related to treatment.
Histopathology: In the lungs, all animals showed granulomatous inflammation with a severity ranging from slight to marked in males and slight to
moderate in females. The bronchial-associated lymphoid tissue (BALT) showed lympho-reticular hyperplasia with development of granulomas in 5/5 males (slight to moderate) and 3/5 females (minimal to slight).
In the medistinal lymph nodes, lympho-reticular hyperplasia with development of multifocal granulomas affected 5/5 males (moderate to severe) and 5/5 females (moderate to marked). In few animals the granulomas showed partly (central) necrosis of varying extend.
In the nasal cavity, minimal subepithelial lymphoid infiltrates were observed in the septum in males (level II and III). Hyaline droplets were found in the respiratory or olfactory epithelium in males (levels II, III and IV) and females (level III and IV) with higher incidence in level III.
Compared to control animals, no findings were observed in the trachea in males.

Effect levels

Key result

Dose descriptor:

NOEC

Effect level:

5 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Lowest effective dose / conc.:

25 mg/L air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Table 7.5.3c Absolute weight

	Male animals			Female animals
Group	5 mg/m^3	25 mg/m^3	150 mg/m^3	5 mg/m^3	25 mg/m^3	150 mg/m^3
Lungs	99%	103%	132%**	101%	104%	126%**

*: p <= 0.05

**: p <= 0.01

Table 7.5.3d Relative weight

	Male animals			Female animals
Group	5 mg/m^3	25 mg/m^3	150 mg/m^3	5 mg/m^3	25 mg/m^3	150 mg/m^3
Lung	101%	111%	141%**	104%	106%	130%**
Spleen				91%*	90%**	106%
Thymus				93%	84%*	107%

*: p <= 0.05

**: p <= 0.01

Table 7.5.3e Histopathology

Nasal cavity level II	Male animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Infiltration, lymphoid	0	0	2	8
Droplets, hyaline	0	0	0	3
	Female animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Infiltration, lymphoid	0	0	3	5
Droplets, hyaline	0	0	1	1

Nasal cavity leve III	Male animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Infiltration, lymphoid	0	0	0	7
Droplets, hyaline	0	0	2	6
	Female animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Infiltration, lymphoid	0	0	1	7
Droplets, hyaline	0	0	2	8
Nasal cavity level IV	Male animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Droplets, hyaline	0	0	0	4
	Female animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Droplets, hyaline	0	0	1	7

Trachea	Male animals
Group	0 mg/m^3	5 mg/m^3	25 mg/m^3	150 mg/m^3
Animals examined	10	10	10	10
Gobletcell accumulation	0	0	3	7
Hyperplasia, respiratory epithelium	0	0	0	3
Inflammation	0	0	0	2
Granuloma	0	0	0	2

Lungs	Male animals
Group	0 mg/m^3	5 mg/m^3	25 mg/m^3	150 mg/m^3
Animals examined	10	10	10	10
Inflammation, granulomatous	0	0	10	10
·       Grade 1	0	0	3	0
·       Grade 2	0	0	6	0
·       Grade 3	0	0	1	6
·       Grade 4	0	0	0	4
BALT: hyperplasia, lymphoreticular	0	0	5	10
·       Grade 1	0	0	4	1
·       Grade 2	0	0	1	3
·       Grade 3	0	0	0	6
	Female animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Inflammation, granulomatous	0	0	10	10
·       Grade 1	0	0	4	0
·       Grade 2	0	0	6	4
·       Grade 3	0	0	0	6
BALT: hyperplasia, lymphoreticular	0	0	0	8
·       Grade 1	0	0	0	4
·       Grade 2	0	0	0	4

Mediastinal Lymph node	Male animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Hyperplasia, lympho-reticular with development of granuloma	0	0	10	10
·       Grade 1	0	0	0	0
·       Grade 2	0	0	2	0
·       Grade 3	0	0	8	0
·       Grade 4	0	0	0	10
	Female animals
Group	0 mg/m^3	5mg/m^3	25mg/m^3	150mg/m^3
Animals examined	10	10	10	10
Hyperplasia, lympho-reticular with development of granuloma	0	0	9	10
·       Grade 1	0	0	4	0
·       Grade 2	0	0	3	1
·       Grade 3	0	0	2	3
·       Grade 4	0	0	0	6

Table 7.5.3f Recovery group - Absolute weight

	Male animals	Female animals
Group	150 mg/m^3	150 mg/m^3
Lungs	158%**	-
Liver	-	108%**

**: p <= 0.01

Table 7.5.3g Recovery group - Relative weight

	Male animals	Female animals
Group	150 mg/m^3	150 mg/m^3
Lungs	132%**	-
Liver	-	109%*
Testes	90 %**	-

*: p <= 0.05

**: p <= 0.01

Table 7.5.3h Recovery group - Histopathology - lungs/mediatinal lymph nodes/nasal cavity

Lungs	Male animals		Female animals
Group	0 mg/m^3	150mg/m^3	0mg/m^3	150mg/m^3
Animals examined	5	5	5	5
Inflammation, granulomatous	0	5	0	5
·       Grade 2	0	2	0	3
·       Grade 3	0	2	0	2
·       Grade 4	0	1	0	0
BALT: hyperplasia, lymphoreticular	0	5	0	3
·       Grade 1	0	0	0	1
·       Grade 2	0	1	0	2
·       Grade 3	0	4	0	0

Mediastinal Lymph node	Male animals		Female animals
Group	0 mg/m^3	5mg/m^3	0mg/m^3	150mg/m^3
Animals examined	5	5	5	5
Hyperplasia, lympho-reticular with development of granuloma	0	5	0	5
·       Grade 3	0	1	0	3
·       Grade 4	0	3	0	2
·       Grade 5	0	1	0	0

Nasal cavity	Male animals		Female animals
Group	0 mg/m^3	150mg/m^3	0mg/m^3	150mg/m^3
Animals examined	5	5	5	5
Level II
Infiltration, lymphoid	0	4	0	0
Droplets, hyaline	0	1	0	0
Level III
Infiltration, lymphoid	0	2	0	0
Droplets, hyaline	0	4	0	4
Level IV
Droplets, hyaline	0	2	0	2

Applicant's summary and conclusion

Conclusions:

This study showed that changes specifically induced by HDI Trimer MEKO-blocked were found in the respiratory tract of the rats exposed to
150 and 25 mg/m3 in a concentration-related manner. The location of the damage was essentially limited to the lung periphery. There was no
evidence of damage to any organs except the respiratory organs.
Regarding the significant toxic lesions observed in the respiratory tract confirmed at microscopic examination in the lungs and the mediastinal lymph nodes, regarding also the non-reversibility of effects in the lungs and mediastinal lymph nodes of the highest dose group within a 4-week recovery period and the significant histopathological effects observed in the mid-group dose (25 mg/m3) whereas no effects has been observed for the same group dose (30 mg/m3) in the 14 days inhalation study, it could be presumed that the test substance has the potential to be harmful to human following repeated exposure by inhalation route.
As a consequesnce, HDI Trimer MEKO blocked is classified for repeated exposure by inhalation route as R48/20 according to the Annex VI of the Directive 67/548/EEC and as a STOT RE 2 according the Annex I of the CLP regulation (EC) N°(1272/2008).
Therefore, according to the Annex I of the CLP Regulation (EC) No 1272/2008, it could be concluded that HDI Trimer MEKO blocked is classified as a
STOT-RE 2 and as R48/20 according to the Annex VI of the Directive 67/548/EEC.

Executive summary:

In a subchronic inhalation toxicity study, performed according to the OECD guideline 413, in compliance with GLP, HDI Trimer MEKO-blocked in 77.3% of acetone was administered to 10 Wistar rats per sex and concentrations by nose-head exposure at concentrations of 0, 5, 25 and 150 mg/m3. Moreover a recovery group of with 5 rats per sex per group were exposed to either acetone (control group) or to the high target concentration of 150 mg/m3 for 90 days and observed for potential reversibility for 28 days.

During the exposure period, the target concentrations were maintained as constant and stable as could be provided with liquid areosol generation techniques in the concentration range tested.

No mortality has been induced during the exposure period.

All animals tolerated the treatment without clinical symptoms.

Regarding pathology, treatment-related findings were observed in the nasal cavity, trachea, lungs, and the mediastinal lymph nodes of both sexes, with the lungs and mediastinal lymph nodes showing the most severe changes. Less severe findings were noted in the nasal cavity (males and females) and trachea (males). All of them were present in a dose-dependant manner starting at 25 mg/m3.

In the lungs, the statistically significant weight increase in males and females exposed to 150 mg/m3 test substance correlated microscopically with a granulomatous inflammation, which was accompanied by lympho-reticular hyperplasia in the bronchial-associated lymphoid tissue (BALT). The enlarged mediastinal lymph nodes observed at gross pathology in the 25 mg/m3 and 150 mg/m3 group correlated microscopically with lympho-reticular hyperplasia accompanied by multifocal granulomas. The presence of granulomas in the alveolar parenchyma and regional mediastinal lymph node is considered adverse. No recovery of these findings was noted after a 4-week recovery period.

The slight increase of subepithelial lymphoid infiltrates (level II and III) and the development of hyaline droplets (level II, III, and IV) in the nasal cavity represent findings after a slight epithelial irritation. However, since the lymphoid infiltrates were of low severity they may be regarded as non-adverse. Moreover, hyaline droplets are considered to represent an adaptive change to treatment; therefore their presence is regarded as non-adverse. At the end of the recovery period, the nasal cavity showed a full recovery for lymphoid infiltrates in females and a partial recovery for hyaline droplets in both males and females.

The accumulation of goblet cells in the epithelium of the trachea in males was the most common finding and is regarded as an adaptive effect. However, the epithelial hyperplasia, inflammation and granulomas in the carina, although of low grading, are considered to be adverse. After a 4 week treatment-free period, the male trachea showed a full recovery.

No histopathological effects were seen in any other organ outside the respiratory tract.

In clinical pathology, the mild increase of the total white blood cell counts and the neutrophil counts in male and female rats after inhalation exposure to 150 mg/m3 test substance reflected a secondary systemic response to the serious irritation and inflammation of the lungs, which was still present after the 4 weeks recovery period.

No treatment-related adverse effects has been observed at the lower concentration. Therefore, the NOAEC is 5 mg/m3.

The exposure of rats to HDI Trimer MEKO blocked caused concentration dependent pulmonary lesions as indicated by the increased weights (absolute and relative) of lung of the high concentration group and corresponding histological findings in lungs and mediastinal lymph nodes in the high and intermediate concentration groups. These effects may become systemically effective and caused increased total white blood cell counts, which is triggered by the increased neutrophil counts.

Overall, at the high concentration of 150.0 mg/m3, all these effects (histological changes in lungs and mediastinal lymph nodes, the changes in differential white blood cell count) were not reversible within 4 weeks recovery period. Moreover, less severe, full or partly reversible histological changes were also observed in nasal cavity and trachea

This study showed that changes specifically induced by HDI Trimer MEKO-blocked were found in the respiratory tract of the rats exposed to 150 and 25 mg/m3 in a concentration-related manner. These changes are considered to be local effects and were partly due to the irritant potential of the test substance. There was no evidence of damage to any organs except the respiratory organs.

Regarding the significant toxic lesions observed in the respiratory tract confirmed at microscopic examination in the lungs and the mediastinal lymph nodes, regarding also the non-reversibility of effects in the lungs and mediastinal lymph nodes of the highest dose group within a 4-week recovery period and the significant histopathological effects observed in the mid-group dose (25 mg/m3) whereas no effects has been observed for the same group dose (30 mg/m3) in the 14 days inhalation study, it could be presumed that the test substance has the potential to be harmful to human following repeated exposure by inhalation route. As a consequesnce, HDI Trimer MEKO blocked is classified for repeated exposure by inhalation route as R48/20 according to the Annex VI of the Directive 67/548/EEC and as a STOT RE 2 according the Annex I of the CLP regulation (EC) N°(1272/2008).