4,4'-methylenedicyclohexyl diisocyanate

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept 2006 - Feb 2008 (2nd peer-review histopathology)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1981)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: about 2 months
- Weight at study initiation: at the study start the variation of individual weights did essentially not exceed +/- 10 % of the mean
- Housing: singly in Makrolon Type IIIh cages (according to Spiegel & Goennert, Zschr. Versuchstierkunde 1, 38, 1961 and Meister, Zschr. Versuchstierkunde 7, 144-153, 1965)
- Diet and Water: ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-70 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 0.9 µm

Geometric standard deviation (GSD):

2

Remarks on MMAD:

MMAD range: 0.89-0.92 µm (geometric standard deviation approx. 2); highly respirable to rats

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were nose-only exposed to the aerosolized test article in restrainers made of Plexiglas. The type of exposure is comparable with a directed-flow exposure design (Moss and Asgharian, Respiratory Drug Delivery IV, 1994, 197-201).
- Exposure apparatus: Chambers used are commercially available (TSE, Bad Homburg, Germany) and the performance as well as their validation has been published (e.g. Pauluhn, Journal of Applied Toxicology, 14, 55-62, 1994). Each segment of 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 I). Dry conditioned air was used to evaporate 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 was 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. 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. Gas 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 cm H20.
- Generation of aerosol: Atmospheres of the test substance were generated under dynamic conditions using a digitally controlled Havard PHD 2000 pump and a binary nozzle. For nebulization, conditioned (dry, oil free) compressed air (15 I/min, dispersion pressure approximately 600 kPa) was used. In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/baffle system was used. The targeted concentrations were achieved by using air extraction/substitution dilution cascades. By using dilution cascades the targeted concentrations could be achieved without changing the generation principle of atmospheres.
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (> 200 x, continuos generation of test atmosphere). Under such test conditions used chamber equilibrium is attained in less than one minute of exposure (t99% = 4.6 x chamber volume/chamber airflow). The ratio between the air supplied and exhausted was chosen so that approximately 90% of the supplied air is removed via the exhaust system.
- 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 VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Exhaust air treatment: The exhaust air was purified via cotton woll and HEPA filters.
- Temperature and humidity measurements were performed by the computerized Data Acquisition and Control System using FTF11-Sensoren (Fa. Elka Electronic, Lüdenscheid, Germany).

TEST ATMOSPHERE
- The integrity end stability of the aerosol generation and exposure system was monitored using a Microdust Pro (Casella) digital real-time aerosol photometer.
- Samples taken from breathing zone: yes
- Brief description of analytical method used: HPLC and gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
HPLC-method: Nitro-reagent-treated glass fibres were exposed to the test atmosphere. Nitro-reagent react with isocyanates to a stable derivative which was quantified by HPLC.
- Particle size distribution: The particle-size distribution was analyzed using a BERNER-Type Aeras low-pressure critical orifice cascade impactor. > 95 % of the particle mass had an aerodynamic diameter - MMAD (Mass median aerodynamic diameter): The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was 0.91 µm at 0.5 mg/m³, 0.92 µm at 3.0 mg/m³ and 0.89 µm at 18 mg/m³ (geometric standard deviation (GSD) 1.93-1.98).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test-substance concentration in the atmospheres was characterised by gravimetric analysis and via HPLC. In each case separate samples were taken from the breathing zone. Both methods show in good correlation the actual concentrations in the different dose level (gravimetric vs. HPLC value): low conc. 0.47 / 0.42 mg/m³, medium conc. 3.0 / 2.73 mg/m³, high conc. 18.0 / 16.64 mg/m³.

Duration of treatment / exposure:

Thirteen consecutive weeks, additionally animals of the control and high-level groups (10/sex and group) were allowed to recover over an exposure-free time period of approximately 4 weeks.

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.5 mg/m³ air

Remarks:

target conc.; gravimetric conc.: 0.47 mg/m³

Dose / conc.:

3 mg/m³ air

Remarks:

target conc. and gravimetric conc.

Dose / conc.:

18 mg/m³ air

Remarks:

target conc. and gravimetric conc.

No. of animals per sex per dose:

10, additionally 10 animals at 0 mg/m³ (control) and 18 mg/m³ (high concentration) for recovery-groups

Control animals:

other: air control

Details on study design:

- Dose selection rationale: This study was designed on the basis of previous acute inhalation study (see chapter Acute toxicity: inhalation, study of 1995) and two subacute inhalation studies in rats (exposure 5 x 6-hr/day on five consecutive days; exposure 5 x 6-hr/day on five consecutive days for 4 weeks) (see chapter Repeated dose toxicity: inhalation). Based on this data it appeared that the substance aerosol in a concentration of approx. 40 mg/m³ may cause undue respiratory tract irritation in a repeated exposure 13-week inhalation study. Therefore in this repeated inhalation exposure study, the following target concentrations were selected: 0.5, 3 and 18 mg/m³. With regard to respiratory tract irritation 18 mg/m³ was estimated to be slightly irritant which may not cause undue stress to experimental animals during the course of the 3-months study. Based on histology findings from the 4-week study, 3 mg/m³ was considered to be a concentration likely to cause minimum effects upon subchronic exposure, whilst 18 mg/m³ were considered likely to cause unequivocal morphological changes within the respiratory tract, however, without causing severe acute irritation. Accordingly, in regard to the anticipated most sensitive endpoint (clinical evidence of respiratory tract irritation) 0.5 mg/m³ were anticipated to be the no-observed-adverse-effect concentration (NOAEC) and 18 mg/m³ the FEL (frank effect level).
Due to the very low vapor pressure of this diisocyanate the vapor phase was considered to be negligible.

Observations and examinations performed and frequency:

BODY WEIGHT: were measured before exposure on a twice per week basis and during the exposure-free recovery period once per week.

FOOD AND WATER CONSUMPTION: determined once per week.

OBSERVATIONS AND DETERMINATIONS: The appearance and behavior of each rat was examined at least twice on exposure days (before and after exposure) and once a day during the exposure-free recovery period. Cage side observations icluded at least 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.
During the course of study, additional clinical observations which took into account the pattern of examination consistent with a Functional Observational Battery. Furthermore reflexes were tested, based on teh recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257) and Moser et.al. (Fundamental and Applied Tox. 11, 1988, 189-206). Rectal (colonic) temperatures were measured directly after cessation of exposure.

HAEMATOLOGY/CLINICAL CHEMISTRY. General clinical chemistry tests were performed at the end of the 3 months exposure period on all main group animals. The blood required for the glucose determinatin was obtained during the last exposure week. Hematology, Clinical Pathology and Urinanalysis was performed.

OPHTHALMOSCOPIC EXAMINATION: Eye examinations were performed on all animals prior to the first exposure and towards the end of the exposure period.

Sacrifice and pathology:

GROSS PATHOLOGY: The following organs were weighed: Adrenals, brain, heart, kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus
The following tissues were collected: Adrenals, aorta, bone and bone marrow section (femur), brain (cerebrum, cerebellum, pons/medulla), epididymides, esophagus, eyes, eyelids, extraorbital lacrimal glands, femur, heart, harderian glands, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidneys, lachrymal glands, larynx, liver, lungs, lymph nodes (mandibular, bronchial/hilus, and mesenteric), mammary gland, nasal turbinates, optical nerves, ovaries, oviducts, pancreas, pharynx, pituitary gland, prostate gland, salivary gland, sciatic nerve, skeletal muscle, skin (flank and facial areas), spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus with uterine cervix, vagina, tissues with macroscopic findings.

HISTOPATHOLOGY: Histopathology was performed on all tissue specimens shown above at least in the control and high-level exposure groups.

Statistics:

STATISTICAL METHODS: Dunnett Test, Adjusted Welch Test, Kruskal-Wallis Test followed by Adjusted U Test.

Clinical signs:

no effects observed

Description (incidence and severity):

Rats tolerated the exposures without any substance-induced clinical effects. There was no evidence of a conclusive, toxically significant effect on body (rectal) temperatures at any exposure concentration.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no toxically consistent effect on body weights up to and including the 18 mg/m³ group and statistical significant changes appeared to be related to an increase rather than a decrease in body weights (vs. air control). Accordingly, as far as significant changes were observed they are considered to be of no pathodiagnostic relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was a tendency of reduced food consumption in the air control group. The isolated statistical significances are considered to be of no toxicological significance as they indicate increased rather than decreased food consumption.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There was no consistently affected water consumption throughout the exposure period considered to be of toxicological significance. The increased water consumption relative to the air exposure group is not considered to be of any pathognostic relevance.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmology performed did not reveal any conclusive evidence of test substance-induced changes in the dioptric media or in the fundus.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no concentration-dependent changes in any group. Therefore, the statistical significances are considered to be of no pathodiagnostic significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no concentration-dependent changes in any group. Therefore, the statistical significances are considered to be of no pathodiagnostic significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no effects considered to be of pathodiagnostic relevance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The examination of reflexes made within the first and last exposure week did not reveal any differences between the groups.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

With the exception of the significantly increased lung weights (absolute and relative) in male rats of the 18 mg/m³ exposure group, there were no other significant changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross pathological examination of the rats that were sacrificed at the end of the exposure period did not reveal evidence of any treatment-related organ damage.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The subchronic exposure resulted in a concentration-dependent epithelial alteration characterized by squamous epithelial metaplasia and inflammatory changes in the larynx at 0.5 mg/m³ and above. Inflammatory lesions were detected in the anterior nasal cavities and the nasopharynx with typical anterior-posterior gradient of injury. The minimal to slight lesions observed at the level of the bronchiolo-alveolar airways were restricted to few rats exposed at 18 mg/m³. With the exception of the laryngeal findings recorded in the control and 18 mg/m³ groups no clear differences of the 18 mg/m³ and control groups were observed after a four week exposure -free recovery period. Based on conclusion expressed in the critical review from Osimitz et. al. (Toxicology and Applied Pharmacology, 225, 2007, 229-237) grade 1 and 2 laryngeal findings of the kind observed in this study the changes in the larynx at 0.5 mg/m³ are not necessarily adverse, as the kind and extent of change is commonly observed and considered to be a relative rat-specific adaptive response to irritant substances.

Histopathological findings: neoplastic:

not specified

Details on results:

The histopathological evaluation of the nasal cavity and the pharynx revealed epithelial changes which gained statistical significance at 18 mg/m³. The lesions at these locations were characterized by focal inflammatory infiltrates, goblet cell hyperplasia, epithelial alterations and/or epithelial metaplasia. Minimal epithelial alterations, indicating a beginning squamous metaplasia, were seen in the distal airways whereas in the tracheal and bronchial airways (and bifurcartions) of the groups exposed to the test substance and of the air control were indistinguishable. A concentration-independent increase of a minimal laryngeal focal infiltration of inflammatory cells and a minimal to slight squamous metaplasia occurred at 0.5 mg/m³ and above. Apart from some isolated findings observed at 18 mg/m³ this group was essentially indistinguishable from the respective control group after a four week recovery period. Accordingly, clear evidence of reversibility existed. Also in the larynx a clear tendency of reversibility of epithelial metaplasia was apparent.

Collectively, the histopathology findings demonstrate unequivocal respiratory tract effects at 18 mg/m³ which corresponded with the elevated lung weights observed at this group. All findings are suggestive of a direct local irritant mechanism at the location of initial deposition of aerosol. The intensity of lesions showed an anteriorposterior gradient which is typical for water soluble, chemically reactive agents deposited in this region of the respiratory tract. The laryngeal squamous epithelial metaplasia is considered to be a rather rat-specific response. In the rat, the layout of the upper respiratory structures is tandem such that the air could travel in nearly linear fashion from the nose to the bifurcation of the trachea. The linear arrangement of the upper airways allows the larynx of rats to lie close to the posterior edge of the oral and nasal cavities. In the condition, the epiglottis lies against the soft palate. The apposition of the epiglottis to the soft palate in the resting condition isolates the oral cavity from the respiratory airways and makes the rat an obligatory nose breather (virtually no oropharynx). Thus, the direction of flow in rats is almost linear whereas in humans it is rectangular (for more details see DeSesso, Quality Assurance Good Practise, Regulation, and Law 2:213-231, 1993). Commonly, metaplastic changes observed at this location are considered to be causally related to adaptive (differentiation of cells to become more resistant) rather than frank adverse effects.

This study demonstrates that the test substance was tolerated without any systemic adverse effects or clinical findings suggestive of respiratory tract irritation at any exposure level. The absolute lung weights and the lung weight to body weight/brain weight ratios were significantly increased in the mildly more susceptible male rats. With regard to histopathological changes, all lesions observed were related to portal-of-entry, local irritant effects (nasal passages, pharynx, and larynx), i.e. changes that occurred at anatomical structures, to some extent rat specific and favouring focal deposition of irritant particulates. No changes of pathodiagnostic relevance occurred at 3.0 mg/m³, except the laryngeal findings. Taking all findings into account, 3.0 mg/m³ constitutes a no-observed-adverse-effect level (NOAEL) for effects related to respiratory tract irritation. Based on the borderline laryngeal findings observed at 0.5 mg/m³ this exposure concentration did not constitute a no-observed- effect-level (NOEL); however, as the kind and extent of change is commonly observed and considered to be a relative rat-specific adaptive response to irritant substances.

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Histopathological findings related to respiratory tract irritation at next higher concentration (18 mg/m³).

Critical effects observed:

yes

Lowest effective dose / conc.:

18 mg/m³ air

System:

other: respiratory system

Organ:

other: respiratory tract: local irritant effect

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Executive summary:

A subchronic 13 -week inhalation toxicity study according to OECD TG 413 was conducted with 4, 4'-methylenedicyclohexyl diisocyanate. In this study 10 male and 10 female rats per group were nose-only exposed to the aerosolized substance. For all exposure groups (conditioned air, 0.5, 3, 18 mg/m³; target concentrations) the MMAD was in the range of ~ 0.9 µm (GSD ~2) and thus highly respirable for rats. Additional animals (10/group) of control and high dose group were allowed to recover during an exposure-free period of approx. 4 weeks.

The study demonstrated that the test substance was tolerated without any systemic effects or clinical findings at any exposure level. There was no evidence of hematological effects. Clinical pathology and urinanalysis were unobtrusive. There were no statistically significant or conclusive changes in absolute or relative organ weights with the exception of significantly elevated lung weights in the male rats at 18 mg/m³. With regard to histopathological changes, all lesions observed were related to portal-of-entry, local irritant effects (nasal passages, pharynx, and larynx), i.e., changes that occured at anatomical structures, to some extent rat specific and favoring focal deposition of irritant particulates. No changes of pathodiagnostic relevance occured at 3.0 mg/m³, except laryngeal findings, which were regarded as rat specific adaptive response based on a critical review from Osimitz et. al. (Toxicology and Applied Pharmacology: 225, 229-237, 2007). Therefore 3.0 mg/m³ was determined to be a NOAEL for the respitratory irritant effects.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept 2006 - Feb 2008 (2nd peer-review histopathology)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1981)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: about 2 months
- Weight at study initiation: at the study start the variation of individual weights did essentially not exceed +/- 10 % of the mean
- Housing: singly in Makrolon Type IIIh cages (according to Spiegel & Goennert, Zschr. Versuchstierkunde 1, 38, 1961 and Meister, Zschr. Versuchstierkunde 7, 144-153, 1965)
- Diet and Water: ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-70 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 0.9 µm

Geometric standard deviation (GSD):

2

Remarks on MMAD:

MMAD range: 0.89-0.92 µm (geometric standard deviation approx. 2); highly respirable to rats

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were nose-only exposed to the aerosolized test article in restrainers made of Plexiglas. The type of exposure is comparable with a directed-flow exposure design (Moss and Asgharian, Respiratory Drug Delivery IV, 1994, 197-201).
- Exposure apparatus: Chambers used are commercially available (TSE, Bad Homburg, Germany) and the performance as well as their validation has been published (e.g. Pauluhn, Journal of Applied Toxicology, 14, 55-62, 1994). Each segment of 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 I). Dry conditioned air was used to evaporate 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 was 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. 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. Gas 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 cm H20.
- Generation of aerosol: Atmospheres of the test substance were generated under dynamic conditions using a digitally controlled Havard PHD 2000 pump and a binary nozzle. For nebulization, conditioned (dry, oil free) compressed air (15 I/min, dispersion pressure approximately 600 kPa) was used. In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/baffle system was used. The targeted concentrations were achieved by using air extraction/substitution dilution cascades. By using dilution cascades the targeted concentrations could be achieved without changing the generation principle of atmospheres.
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (> 200 x, continuos generation of test atmosphere). Under such test conditions used chamber equilibrium is attained in less than one minute of exposure (t99% = 4.6 x chamber volume/chamber airflow). The ratio between the air supplied and exhausted was chosen so that approximately 90% of the supplied air is removed via the exhaust system.
- 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 VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Exhaust air treatment: The exhaust air was purified via cotton woll and HEPA filters.
- Temperature and humidity measurements were performed by the computerized Data Acquisition and Control System using FTF11-Sensoren (Fa. Elka Electronic, Lüdenscheid, Germany).

TEST ATMOSPHERE
- The integrity end stability of the aerosol generation and exposure system was monitored using a Microdust Pro (Casella) digital real-time aerosol photometer.
- Samples taken from breathing zone: yes
- Brief description of analytical method used: HPLC and gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
HPLC-method: Nitro-reagent-treated glass fibres were exposed to the test atmosphere. Nitro-reagent react with isocyanates to a stable derivative which was quantified by HPLC.
- Particle size distribution: The particle-size distribution was analyzed using a BERNER-Type Aeras low-pressure critical orifice cascade impactor. > 95 % of the particle mass had an aerodynamic diameter - MMAD (Mass median aerodynamic diameter): The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was 0.91 µm at 0.5 mg/m³, 0.92 µm at 3.0 mg/m³ and 0.89 µm at 18 mg/m³ (geometric standard deviation (GSD) 1.93-1.98).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test-substance concentration in the atmospheres was characterised by gravimetric analysis and via HPLC. In each case separate samples were taken from the breathing zone. Both methods show in good correlation the actual concentrations in the different dose level (gravimetric vs. HPLC value): low conc. 0.47 / 0.42 mg/m³, medium conc. 3.0 / 2.73 mg/m³, high conc. 18.0 / 16.64 mg/m³.

Duration of treatment / exposure:

Thirteen consecutive weeks, additionally animals of the control and high-level groups (10/sex and group) were allowed to recover over an exposure-free time period of approximately 4 weeks.

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.5 mg/m³ air

Remarks:

target conc.; gravimetric conc.: 0.47 mg/m³

Dose / conc.:

3 mg/m³ air

Remarks:

target conc. and gravimetric conc.

Dose / conc.:

18 mg/m³ air

Remarks:

target conc. and gravimetric conc.

No. of animals per sex per dose:

10, additionally 10 animals at 0 mg/m³ (control) and 18 mg/m³ (high concentration) for recovery-groups

Control animals:

other: air control

Details on study design:

- Dose selection rationale: This study was designed on the basis of previous acute inhalation study (see chapter Acute toxicity: inhalation, study of 1995) and two subacute inhalation studies in rats (exposure 5 x 6-hr/day on five consecutive days; exposure 5 x 6-hr/day on five consecutive days for 4 weeks) (see chapter Repeated dose toxicity: inhalation). Based on this data it appeared that the substance aerosol in a concentration of approx. 40 mg/m³ may cause undue respiratory tract irritation in a repeated exposure 13-week inhalation study. Therefore in this repeated inhalation exposure study, the following target concentrations were selected: 0.5, 3 and 18 mg/m³. With regard to respiratory tract irritation 18 mg/m³ was estimated to be slightly irritant which may not cause undue stress to experimental animals during the course of the 3-months study. Based on histology findings from the 4-week study, 3 mg/m³ was considered to be a concentration likely to cause minimum effects upon subchronic exposure, whilst 18 mg/m³ were considered likely to cause unequivocal morphological changes within the respiratory tract, however, without causing severe acute irritation. Accordingly, in regard to the anticipated most sensitive endpoint (clinical evidence of respiratory tract irritation) 0.5 mg/m³ were anticipated to be the no-observed-adverse-effect concentration (NOAEC) and 18 mg/m³ the FEL (frank effect level).
Due to the very low vapor pressure of this diisocyanate the vapor phase was considered to be negligible.

Observations and examinations performed and frequency:

BODY WEIGHT: were measured before exposure on a twice per week basis and during the exposure-free recovery period once per week.

FOOD AND WATER CONSUMPTION: determined once per week.

OBSERVATIONS AND DETERMINATIONS: The appearance and behavior of each rat was examined at least twice on exposure days (before and after exposure) and once a day during the exposure-free recovery period. Cage side observations icluded at least 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.
During the course of study, additional clinical observations which took into account the pattern of examination consistent with a Functional Observational Battery. Furthermore reflexes were tested, based on teh recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257) and Moser et.al. (Fundamental and Applied Tox. 11, 1988, 189-206). Rectal (colonic) temperatures were measured directly after cessation of exposure.

HAEMATOLOGY/CLINICAL CHEMISTRY. General clinical chemistry tests were performed at the end of the 3 months exposure period on all main group animals. The blood required for the glucose determinatin was obtained during the last exposure week. Hematology, Clinical Pathology and Urinanalysis was performed.

OPHTHALMOSCOPIC EXAMINATION: Eye examinations were performed on all animals prior to the first exposure and towards the end of the exposure period.

Sacrifice and pathology:

GROSS PATHOLOGY: The following organs were weighed: Adrenals, brain, heart, kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus
The following tissues were collected: Adrenals, aorta, bone and bone marrow section (femur), brain (cerebrum, cerebellum, pons/medulla), epididymides, esophagus, eyes, eyelids, extraorbital lacrimal glands, femur, heart, harderian glands, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidneys, lachrymal glands, larynx, liver, lungs, lymph nodes (mandibular, bronchial/hilus, and mesenteric), mammary gland, nasal turbinates, optical nerves, ovaries, oviducts, pancreas, pharynx, pituitary gland, prostate gland, salivary gland, sciatic nerve, skeletal muscle, skin (flank and facial areas), spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus with uterine cervix, vagina, tissues with macroscopic findings.

HISTOPATHOLOGY: Histopathology was performed on all tissue specimens shown above at least in the control and high-level exposure groups.

Statistics:

STATISTICAL METHODS: Dunnett Test, Adjusted Welch Test, Kruskal-Wallis Test followed by Adjusted U Test.

Clinical signs:

no effects observed

Description (incidence and severity):

Rats tolerated the exposures without any substance-induced clinical effects. There was no evidence of a conclusive, toxically significant effect on body (rectal) temperatures at any exposure concentration.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no toxically consistent effect on body weights up to and including the 18 mg/m³ group and statistical significant changes appeared to be related to an increase rather than a decrease in body weights (vs. air control). Accordingly, as far as significant changes were observed they are considered to be of no pathodiagnostic relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was a tendency of reduced food consumption in the air control group. The isolated statistical significances are considered to be of no toxicological significance as they indicate increased rather than decreased food consumption.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There was no consistently affected water consumption throughout the exposure period considered to be of toxicological significance. The increased water consumption relative to the air exposure group is not considered to be of any pathognostic relevance.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmology performed did not reveal any conclusive evidence of test substance-induced changes in the dioptric media or in the fundus.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no concentration-dependent changes in any group. Therefore, the statistical significances are considered to be of no pathodiagnostic significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no concentration-dependent changes in any group. Therefore, the statistical significances are considered to be of no pathodiagnostic significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no effects considered to be of pathodiagnostic relevance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The examination of reflexes made within the first and last exposure week did not reveal any differences between the groups.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

With the exception of the significantly increased lung weights (absolute and relative) in male rats of the 18 mg/m³ exposure group, there were no other significant changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross pathological examination of the rats that were sacrificed at the end of the exposure period did not reveal evidence of any treatment-related organ damage.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The subchronic exposure resulted in a concentration-dependent epithelial alteration characterized by squamous epithelial metaplasia and inflammatory changes in the larynx at 0.5 mg/m³ and above. Inflammatory lesions were detected in the anterior nasal cavities and the nasopharynx with typical anterior-posterior gradient of injury. The minimal to slight lesions observed at the level of the bronchiolo-alveolar airways were restricted to few rats exposed at 18 mg/m³. With the exception of the laryngeal findings recorded in the control and 18 mg/m³ groups no clear differences of the 18 mg/m³ and control groups were observed after a four week exposure -free recovery period. Based on conclusion expressed in the critical review from Osimitz et. al. (Toxicology and Applied Pharmacology, 225, 2007, 229-237) grade 1 and 2 laryngeal findings of the kind observed in this study the changes in the larynx at 0.5 mg/m³ are not necessarily adverse, as the kind and extent of change is commonly observed and considered to be a relative rat-specific adaptive response to irritant substances.

Histopathological findings: neoplastic:

not specified

Details on results:

The histopathological evaluation of the nasal cavity and the pharynx revealed epithelial changes which gained statistical significance at 18 mg/m³. The lesions at these locations were characterized by focal inflammatory infiltrates, goblet cell hyperplasia, epithelial alterations and/or epithelial metaplasia. Minimal epithelial alterations, indicating a beginning squamous metaplasia, were seen in the distal airways whereas in the tracheal and bronchial airways (and bifurcartions) of the groups exposed to the test substance and of the air control were indistinguishable. A concentration-independent increase of a minimal laryngeal focal infiltration of inflammatory cells and a minimal to slight squamous metaplasia occurred at 0.5 mg/m³ and above. Apart from some isolated findings observed at 18 mg/m³ this group was essentially indistinguishable from the respective control group after a four week recovery period. Accordingly, clear evidence of reversibility existed. Also in the larynx a clear tendency of reversibility of epithelial metaplasia was apparent.

Collectively, the histopathology findings demonstrate unequivocal respiratory tract effects at 18 mg/m³ which corresponded with the elevated lung weights observed at this group. All findings are suggestive of a direct local irritant mechanism at the location of initial deposition of aerosol. The intensity of lesions showed an anteriorposterior gradient which is typical for water soluble, chemically reactive agents deposited in this region of the respiratory tract. The laryngeal squamous epithelial metaplasia is considered to be a rather rat-specific response. In the rat, the layout of the upper respiratory structures is tandem such that the air could travel in nearly linear fashion from the nose to the bifurcation of the trachea. The linear arrangement of the upper airways allows the larynx of rats to lie close to the posterior edge of the oral and nasal cavities. In the condition, the epiglottis lies against the soft palate. The apposition of the epiglottis to the soft palate in the resting condition isolates the oral cavity from the respiratory airways and makes the rat an obligatory nose breather (virtually no oropharynx). Thus, the direction of flow in rats is almost linear whereas in humans it is rectangular (for more details see DeSesso, Quality Assurance Good Practise, Regulation, and Law 2:213-231, 1993). Commonly, metaplastic changes observed at this location are considered to be causally related to adaptive (differentiation of cells to become more resistant) rather than frank adverse effects.

This study demonstrates that the test substance was tolerated without any systemic adverse effects or clinical findings suggestive of respiratory tract irritation at any exposure level. The absolute lung weights and the lung weight to body weight/brain weight ratios were significantly increased in the mildly more susceptible male rats. With regard to histopathological changes, all lesions observed were related to portal-of-entry, local irritant effects (nasal passages, pharynx, and larynx), i.e. changes that occurred at anatomical structures, to some extent rat specific and favouring focal deposition of irritant particulates. No changes of pathodiagnostic relevance occurred at 3.0 mg/m³, except the laryngeal findings. Taking all findings into account, 3.0 mg/m³ constitutes a no-observed-adverse-effect level (NOAEL) for effects related to respiratory tract irritation. Based on the borderline laryngeal findings observed at 0.5 mg/m³ this exposure concentration did not constitute a no-observed- effect-level (NOEL); however, as the kind and extent of change is commonly observed and considered to be a relative rat-specific adaptive response to irritant substances.

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Histopathological findings related to respiratory tract irritation at next higher concentration (18 mg/m³).

Critical effects observed:

yes

Lowest effective dose / conc.:

18 mg/m³ air

System:

other: respiratory system

Organ:

other: respiratory tract: local irritant effect

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Executive summary:

A subchronic 13 -week inhalation toxicity study according to OECD TG 413 was conducted with 4, 4'-methylenedicyclohexyl diisocyanate. In this study 10 male and 10 female rats per group were nose-only exposed to the aerosolized substance. For all exposure groups (conditioned air, 0.5, 3, 18 mg/m³; target concentrations) the MMAD was in the range of ~ 0.9 µm (GSD ~2) and thus highly respirable for rats. Additional animals (10/group) of control and high dose group were allowed to recover during an exposure-free period of approx. 4 weeks.

The study demonstrated that the test substance was tolerated without any systemic effects or clinical findings at any exposure level. There was no evidence of hematological effects. Clinical pathology and urinanalysis were unobtrusive. There were no statistically significant or conclusive changes in absolute or relative organ weights with the exception of significantly elevated lung weights in the male rats at 18 mg/m³. With regard to histopathological changes, all lesions observed were related to portal-of-entry, local irritant effects (nasal passages, pharynx, and larynx), i.e., changes that occured at anatomical structures, to some extent rat specific and favoring focal deposition of irritant particulates. No changes of pathodiagnostic relevance occured at 3.0 mg/m³, except laryngeal findings, which were regarded as rat specific adaptive response based on a critical review from Osimitz et. al. (Toxicology and Applied Pharmacology: 225, 229-237, 2007). Therefore 3.0 mg/m³ was determined to be a NOAEL for the respitratory irritant effects.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

4,4´-Methylenedicyclohexyl diisocyanate has been assessed in the OECD HPV programme, 2005.

Cited from SIAR of SIAM 20 (Paris, April 19 -22, 2005): "No results from repeated-dose toxicity tests are available for the oral and dermal route of exposure. A subacute inhalation study (1, 6 and 36 mg/m³; 6 hours/day on five days/week for 4 weeks) with rats according to OECD TG 412 indicates the respiratory tract to be the target organ of respirable 4,4´-methylenedicyclohexyl diisocyanate aerosol. The reported NOAEL for effects governed by respiratory tract irritation is 1 mg/m³; the LOAEL is 6 mg/m³ (i.e. histopathological changes in nasal passages, larynx and bronchi). The results of the reproduction/developmental toxicity screening test (OECD TG 421) correspond to the results of the subacute study."

Not included in the OECD assessment was a more recent subchronic (13 -week) inhalation toxicity study conducted according to OECD TG 413 with 4,4'-methylenedicyclohexyl diisocyanate. In this study 10 male and 10 female rats per group were nose-only exposed to conc. of 0.5, 3, and 18 mg/m³ of the aerosolized substance. For all exposure groups the MMAD was in the range of ~ 0.9 µm (GSD ~2) and thus highly respirable for rats. Additional animals (10/group) of control and high dose group were allowed to recover during an exposure-free period of approx. 4 weeks.

The result of this study corresponds well to the results of the previous studies: It could be demonstrated that the test substance was tolerated without any systemic effects or clinical findings at any exposure level. There was no statistically significant or conclusive changes in absolute or relative organ weights with the exception of significantly elevated lung weights in the male rats exposed at 18 mg/m³. With regard to histopathological changes, all lesions observed were related to portal-of-entry, local irritant effects (nasal passages, pharynx, and larynx), i.e., changes that occurred at anatomical structures, to some extent rat specific and favouring focal deposition of irritant particulates. 3.0 mg/m³ was determined to be a NOAEL for the respiratory irritant effects in this study.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex VI, not classified for repeated dose toxicity. Classification as STOT repeated is not justified, due to lack of cumulative toxicity. In the repeated dose toxicity studies no systemic effects were observed at all, and the observed local effects from studies with different exposure periods lead to very similar LOAECs and NOAECs, thus no evidence is given for a major time-dependent change of the response (NOAECs OECD 412 vs. OECD 413 vs. OECD 421/general toxicity: 1 - 3 - 1 mg/m³, LOAECs OECD 412 vs. OECD 413 vs. OECD 421/general toxicity: 6 - 18 - 6 mg/m³.)