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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-02-05 to 2008-02-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Directive 67/302/EEC
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Isophorone Diisocyanate from Bayer MaterialScience AG; purity: 99.8%; Batch No. LL 48/3-55; Sample No. 77 ;

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS
- Strain: Hsd Cpb:WU (SPF) Wistar rats
- Source: Harlan-Winkelmann, Borchen (Germany)
- Age: 2 months at study initiation
- Weight at study initiation:   Group I (0 mg/m3) males 186-213 (mean 200) g   females 137-164 (mean 149) g; Group II (0.05 mg/m3) males 197-225 (mean 210) g   females 143-166 (mean 154) g; Group III (0.2 mg/m3) males 201-227 (mean 211) g   females 146-171 (mean 155) g; Group IV (1.0 mg/m3) males 194-225 (mean 209) g   females 146-169 (mean 154) g

- Number of animals: 10 per sex and dose group for the doses 0.05 mg/m3 and 0.2 mg/m3; 10 (and additionally 10 for satellite groups) per sex and dose group for doses 0 mg/m3 and 1.0 mg/m3
- Housing: single in conventional Makrolon Type IIIh cages
- Diet:. ad libitum, standard fixed-formula diet
- Water: ad libitum, tab water
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity: 40-70 %
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h / 12 h

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: dry conditioned air
Remarks on MMAD:
MMAD / GSD: At the concentrations tested, the liquid re-condensed IPDI particulates cannot be reliably measured with regard to particle size as they would
instantlys re-equilibrate with the vapor phase either due to low pressure or due to dilution sheath air. This means, smaller particles of IPDI are prone
to instant evaporation. The samples for analysis of the particle size distribution were taken in the vicinity of the breathing zone.
Details on inhalation exposure:
ADMINISTRATION / EXPOSURE 
- Type of exposure: directed-flow nose-only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Plexiglas exposure restrainers, commercially available (TSE, Bad Homburg)
- Source and rate of air: dry conditioned air ; air exchanges > 200x/h
- Method of conditioning air: compressed air was supplied by Boge compressors and conditioned automatically by a VIA compressed air dryer
- System of generating particulates/aerosols: under dynamic condtitions the various concentrations of the test substance were generated by usin g a glass bubbler. A metereed flow of nitrogen was bubbled through a fritted disk (dipped into the test substance) was instantly diluted with precon ditioned air. Targeted concentrations were achieved by "pull-push" dilution principles.
- Temperature and humidity in air chamber: 20.7-21.2 °C, 9.1-24 % ; measurements were performed by computerized data acquisition using sensors
- Air flow rate: 30l/min, controlled continuously by calibrated mass flow meters
- Air change rate: > 200/per hour, continuous generation of test atmosphere
- Method of particle size determination: Laser-Velocimeter (TSI), samples are taken in the vicinity of the breathing zone,
- Treatment of exhaust air: purified via cotton-wool/activated charcoal and HEPA filters
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
test atmoshere was determined by HPLC after derivatization of the isocanate functionality, chamber samples were taken in vicinty of breathing zone
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week; 6 hours/day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/m3; 0.05 mg/m3; 0.2 mg/m3; 1.0 mg/m3
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
--; 0.116 mg/m3, 0.462 mg/m3; 1.673 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
--; 0.055 mg/m3; 0.273 mg/m3; 1.106 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10 male and 10 female per dose group for the doses 0.05 mg/m3 and 0.2 mg/m3; 10 (and additionally 10 for satellite groups) per sex and dose group for doses 0 mg/m3 and 1.0 mg/m3
Control animals:
yes
Details on study design:
Rats were assigned to four exposure groups and 10 rats per group/sex were exposed by inhalation to design concentrations of 0 (air control); 0.05; 0.2 and 1 mg/m3 for 13 weeks (target concentrations; 6 hours/day on five days/week). Rats of main groups were sacrified at the end of the 13-week exposure period. Additional animals (10 rats/sex/group) were allocated to the control and high-level exposure groups that served as satellite rats which were subjected to a postexposure observation period of approx. 4 weeks.
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: appearance and behavior at least twice on exposure days (before and after each exposure), once a day on  exposure-free days; additional observations during exposure where  indicated by (e.g. spasms, abnormal movements, severe respiratory signs,  hemorrhage) Cage side observations (e.g. changes on skin/hair coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, sensori-and somatomotor activity and behavior pattern; observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration.
- Mortality: (if applicable) time recorded as precisely as possible  during observations
- Body weight: Mondays and Fridays during exposure period, once per week  during postexposure period - Food and water consumption: each week
- Ophthalmoscopic examination: prior to first exposure and towards end of  exposure period with indirect ophthalmoscope five minutes after 
treatment  with mydriatic: anterior/posterior segment of the eye and adnexal structures
- Haematology and Clinical Chemistry: End of exposure and recovery period :  hematocrit, hemoglobin, leukocytes, erythrocytes,mean corpuscular volume,  mean corpuscul ar hemoglobin concentration, thrombocyte count, reticulocytes, Heinz' bodies, Leukocyze differential count, aspartate  aminotransferase (optimized , ASAT), alanine aminotransferase(optimized, ALAT), glutamate dehydrogenase (GLDH), gamma-Glutamylaminotransferaase,  lactate  dehydrogenas e (LDH), alkaline phosphatase (APh),  albumin, bilirubin, blood glucose, total protein, triglycerides,  cholesterol, creatinine, urea, sodium, potassium,  calcium,  magnesium, phosphate, chloride; prothrombin time ("Hepato quick")
- Urinalysis: for 10 animals per sex per group towards the end of the 3 month study period, 16 hour overnight  sampling during last week of study, s emiquantitative determination of pH,protein, glucose, blood, bilirubin, urobilinogen, ketone bodies, sediment composition and urine osmolarity (qu antitatively)
- Histopathology: 10 animals/sex/gruop one day after their terminal exposure and after approx. 4 weeks of recovery 10 animals/sex of the control group and of the high-level exposure gruop were euthanized. Body weights were recorded and examination of external surface of the body was performed and organs/tissues were collected as described in the protocol.
- Organ weights: Organs weighted at necropsy after exsanguination
- Other:   Rectal temperatures: on weeks 0, 6 and 12   
Reflexes on weeks 0, 6 and 11: visual placing response and grip strenghth  on wire mesh, abdominal muscle tone, corneal and pupillary reflexes,  
pinnal reflex, righting reflex, tail-pinch response, foot splay reflex, startle reflex  (finger snapping and touch on back)

Sacrifice and pathology:
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: not listed separately in test report
- Weights: absolute, relative to body weight and relative to weight of  brain for adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen,  testes, 
thymus
- Microscopic:    All groups including recovery groups: nasal cavity, larynx,  lungs,  pharynx, trachea, lymph nodes lung associated;   
All groups excluding recovery groups: 
adrenal glands, aorta, esophagus,  eyes, eyelids, exorbital lacrimal glands, heart, duodenum, kidneys,  liver, mesenteric lymph nodes, optic nerves,  ovaries, oviducts, skin (mammary region and muzzle), spleen, stomach  (fore- and glandular), testes, thymus, organs 
and tissues with  macroscopic findings; brain, epididymides, femur, Harderian  glands, head, jejunum, ileum, caecum, colon, rectum, remaining inte stine, mandibular lymph nodes, pancreas, pituitary gland, prostate, salivary  glands, sciatic nerve, seminal vesicles, skeletal muscle (tigh), spinal  cor d, sternum, thyroid glands (with parathyroids), tongue, ureters, urinary bladder, uterus (with cervix), vagina ;
Not evaluated: Lymph nodes (popliteal); urethra, Zymbal's glands, physical identifier
Other examinations:
no further information
Statistics:
STATISTICAL METHODS:
- Descriptive analysis: all variables that are not dichotomous
- Others depending on prior experience:   Dunnett test: where approximately normal distribution with equal  variances across treatments 
was anticipated ;   p value adjusted Welch test: where heteroscedasticity appeared to e  more likely;   Kruskal-Wallis test followed by adjusted 
MWW tests (U tests): where the  assumptions for a parametric analysis of variance were questionable; if not otherwise noted, all pair-wise tests are two-sided comparisons.

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:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death: No test substance-induced mortality was observed.

- Clinical signs: All animals tolerated the exposures without any test substance-specific clinical signs

- Body weights: There was no toxicologically consistent effect on body weights in all dose groups. Statitical significant changes appeared to be related to an increase rather than a decrease in body weights (vs. air control). As far as significant changes were observed they are considered to be of
no pathodiagnistic relevance

- Food and water consumption: There was a no consistent evidence of effected food consumption in the exposure groups. The isolated statistical sig nificances are considered to be of no toxicological significance. There was no consistently affected water consumption troughout the exposure peri od considered to be of toxicological significance. The sightly increased water consumption relative to the air control is not considered to be of any pathognostic relevance.

- Ophthalmoscopic examination: No conclusive evidence of substance-induced  changes in the dioptric media or in the fundus.

- Haematology: There were no conclusive concentration-dependent changes in any dose group. Therefore, the statistical significances are considere d to be of no pathodiagnostic significance.

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

- Urinalysis: No effects considered to be of pathodiagnostic relevance.

- Neurobehaviour: Examination of reflexes did not reveal any differences between the  groups.  

- Organ weights: No significant concentration depended changes in organ weights or the organ-to-body weight or organ-to-brain weight ratios 

- Gross pathology: No evidence of any treatment-related organ damage

- Histopathology: Evaluation of nasal cavity and the larynx revealed minimal or slight epithelial changes in the high dose group (1.1 mg/m3). After a f our week recovery period minimal epithelial metaplasia could still be detected; however, clear evidence of recovery existed. Sex differences were not observed.
Concentration (substance)- independend alterations of the testes and in the retina were observed in control rats and in the high dose group

- Other: Determination of the RECTAL TEMPERATURES revealed no evidence of a conclusive, toxicologically significant effect on body (rectal) temperat ures at any exposure concentration used in this study 

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
0.27 mg/m³ air
Sex:
male/female
Basis for effect level:
other: upper respiratory tract irritation
Dose descriptor:
LOAEC
Effect level:
1.1 mg/m³ air
Sex:
male/female
Basis for effect level:
other: histopathological changes in nasal cavity and larynx

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

Conclusions:
This subchronic 13 week inhalation study demonstrates that rats exposed up to 1.1 mg/m3 of the test substance Isophorone Diisocyanate did not display any substance-induced clinical effects, changes in reflexes and body temperature, conclusively affected body weights or food/water consumption. There was no evidence of hematological effects. Clinical pathology and urinanalysis were unobtrusive. There were no statistically significant or conclusive dose-dependend changes in absolute or relative organ weights. The histopathological evaluation of the nasal cavity and the larynx revealed minimal or slight epithelial changes at 1.1 mg/m3. After a four week recovery period minimal epithelial metaplasia could still be detected; however clear evidence of recovery existed. This study demonstrated that the test substance was tolerated without any systemic adverse effects or clinical findings suggestive of respiratory tract irritation at any exposure level. Taking all findings into account, 0.27 mg/m3 constitutes a No-Observed-Adverse-Effect-Concentration (NOAEC).
Executive summary:

A subcronic 13 week inhalation study with Isophorone Diisocyanate has been conducted in young adult Wistar rats. 10 male and 10 female rats per group were exposed (dynamic directed-flow, nose-only) 6 hours/day on five days/week for 13 weeks to following concentrations of the test substance: 0 (air control), 0.05, 0.27 and 1.1 mg/m3. Additional animals of the control and high-level exposure group (10/sex/group) were allowed to recover over an exposure-free time period of approximately 4 weeks. This subchronic 13 week inhalation study demonstrates that rats exposed up to 1.1 mg/m3 of the test substance did not display any substance-induced clinical effects, changes in reflexes and body temperature, conclusively affected body weights or food/water consumption. There was no evidence of hematological effects. Clinical pathology and urinanalysis were unobtrusive. There were no statistically significant or conclusive dose-dependend changes in absolute or relative organ weights. The histopathological evaluation of the nasal cavity and the larynx revealed minimal or slight epithelial changes at 1.1 mg/m3. After a four week recovery period minimal epithelial metaplasia could still be detected; however clear evidence of recovery existed. This study demonstrated that the test substance was tolerated without any systemic adverse effects or clinical findings suggestive of respiratory tract irritation at any exposure level. Taking all findings into account, 0.27 mg/m3 constitutes a No-Observed-Adverse-Effect-Concentration (NOAEC).