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Repeated dose toxicity: inhalation

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chronic toxicity: inhalation
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Report Date:
Reference Type:
Evaluation of nasal tract lesions in derivation of the inhalation reference concentration for hexamethylene diisocyanate
Foureman GL et al.
Bibliographic source:
Inhalation Toxicology 6 (Suppl.): 341-355

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
according to
other: EPA OTS 798.3320 (Combined Chronic Toxicity / Carcinogenicity)
GLP compliance:

Test material

Test material form:

Test animals

Fischer 344
Details on test animals and environmental conditions:
- Source: Charles River Laboratories (Kingston, New York))
- Age at study initiation: approx. 7 weeks
- Mean weight at study initiation: males: 161-176 g; females: 121-124 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 8 days

- Temperature (°C): 20.5-23.3
- Humidity (%): 35-55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 1

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: unchanged (no vehicle)
Details on inhalation exposure:
The chambers used in this study were Hazelton-2000s. These chambers were constructed of stainless steel with clear plastic windows. Each chamber had an approximate volume of two cubic meters. The chambers were equipped with stainless steel, wire mesh cages for individual housing of animals. The cage-racks were fitted with removable feed troughs and an automatic watering system. The air supplied to the chambers passed through an activated-charcoal trap and a HEPA filter before it was conditioned.
Chamber airflow was measured using a calibrated orifice plate and an electro-mechanical transducer. The airflow through each chamber was monitored continuously and recorded at 5-6 minute intervals by a MACSYM 2 (Analog Devices Inc., MA) computer system. The airflow set-point for each chamber was 700 Lpm. Thus, the air turnover rate was set at approximately 21 chamber volumes per hour.
The air exhausted from each exposure chamber was filtered through an activated-charcoal trap and a HEPA filter before it was vented outside
the building.
The thermocouples and the relative humidity sensors (Model SSP128B) were manufactured by HoneyweIl (Minneapolis. Minnesota). Static pressure was measured using Magnahelic gauges. The desired ranges for chamber temperature and relative humidity were 20 to 24°C and 40 to 60 %. respectively. The static pressure set-point was -0.5 inch of water relative to atmospheric pressure. The temperature and relative humidity data were recorded at 5-6 minute intervals by the MACSYM 2 computer system. Static pressure was manually recorded.
HDI vapor was generated by passing filtered, dry air through liquid HDI in a glass bubbler. During vapor generation the bubbler containing HDI was immersed in a constant temperature water bath (Thermomix 1441E, Braun-Melsungen). The water bath temperature and the airflow through the bubbIer were monitored and recorded at hourly intervals during the six-hour exposures.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Samples from the chambers were collected near the animal's breathing zone at a rate of 1 Lpm using two midget impingers connected in series. An acetonitrile solution (10 mL) of N-4-nitrobenzyl-N-n-propylamine (nitro reagent) in the impingers was used to trap and derivatize the HDI to an UV-absorbing compound. All midget impinger samples were analyzed by high performance liquid chromatography.
Duration of treatment / exposure:
2 years
Frequency of treatment:
6 hours/day; 5 days/week
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 0.005, 0.025, 0.175 ppm (0, 0.035, 0.175, 1.23 mg/m3)
nominal conc.
Doses / Concentrations:
0, 0.005, 0.025, 0.164 ppm (0, 0.035, 0.175, 1.15 mg/m3)
analytical conc.
No. of animals per sex per dose:
60 (main groups) and 10 (satellite groups for sacrifice after one year)
Control animals:
yes, sham-exposed
Details on study design:
A 90-day inhalation toxicity study (Shiotsuka, 1988) was conducted in Fischer 344 rats using analytical concentrations of 0.01, 0.04 and 0.14 ppm of HDI. There were no compound-related changes in body weight in this subchronic study. Compound-related clinical signs of toxicity were limited to ocular irritation and it was observed at all three HDI exposure levels. Somatic lesions (determined by microscopic examination of tissues) of the anterior nasal cavity were observed at all three concentrations. Based on this information the three concentrations selected for the chronic study were 0.005, 0.025 and 0.125 ppm. The highest concentration of 0.125 ppm was increased to 0.175 ppm on study-day 127 and maintained at that level until
termination of the study. This change was instituted because overt signs of toxicity were not observed with 0.125 ppm. Since the rats were predominantly exposed to 0.175 ppm, the high concentration group's target concentration will be referred to as being 0.175 ppm.
The exposure was conducted under dynamic conditions, the air control animals were sham-exposed (conditioned room air) under comparable conditions.
Positive control:


Observations and examinations performed and frequency:
All animals in the study were observed for clinical signs of toxicity and mortality twice on exposure days (prior to the onset of exposure and at approximately one hour post-exposure) and once daily on weekends and holidays.
A pre-exposure ophthalmic examination was conducted on ten rats/sex/exposure level. Prior to scheduled sacrifice, the eyes were examined for all survivors in the satellite groups and the main study groups. The indirect method which utilized a condensing lens between the rat's eyes and the ophthalmoscope was used. The examination was performed in a semi-darkened room. The eyes of each rat were checked for a pupillary reflex using the light reflected from the viewing mirror of the ophthalmoscope. The conjunctiva, cornea, and iris of both eyes were also examined using the ophthalmoscope. Two or three drops of a mydriatic were applied to each eye to dilate the pupil. After pupillary dilatation, the lenses, vitreous humors, retinae and optic discs of each rat were examined with the ophthalmoscope.
All rats were weighed weekly for the first 13 weeks of the study. They were then weighed on weeks 15, 17, 19, 21, 23, 27, 31, 34, 35, 39, 43, 51, 55, 59, 63, 67, 71, 75 and 83. Weekly weighings were resumed for weeks 86 through 105.
At 3-, 6-, 12- and 18-month intervals and prior to sacrifice, hematology, clinical biochemistry, and urinalysis parameters were evaluated on generally 20 rats/sex/level from the main study groups. The same rats were used throughout the study for all determinations to the extent possible. The rats in the bleeding group that died or were sacrificed during the study were replaced so that generally 20 rats/sex/level were available. In addition, blood smears were prepared from rats sacrificed at termination.
Sacrifice and pathology:
Rats in the main study groups were selected for sacrifice based on a randomized listing (SAS software [1]) of animals by test group and sex. All animals sacrificed in extremis or at study termination were killed by carbon dioxide asphyxiation.
All study animals were necropsied following death. The necropsy consisted of a systematic gross examination of each animal's general physical condition, body orifices and cavities, and external and internal tissues (40+ organs/tissues). All necropsy findings were recorded manually and were entered into a computer database. The tissues were fixed in 10 percent buffered formalin. The lungs were removed in toto, weighed and perfused intratracheally with 10 percent buffered formalin. The heads were removed and the nasal and paranasal passages were flushed with 10 percent buffered
At necropsy, terminal body weights and organ weights were recorded for the following tissues: liver, lungs, kidneys, adrenals, heart, spleen, gonads, and brain. In addition, relative organ weights (organ weight: body weight ratio x 100) were calculated. Only data from animals sacrificed after one year and after two years of exposure were evaluated.
Tissues collected at necropsy were processed routinely and stained with hematoxylin and eosin and were examined by a veterinary pathologist. Recuts and special stains were requested as deemed necessary, and microscopic observations were entered into a computerized database for summation and report generation. Emphasis was placed on the standardization of trimming procedures of the nasal cavity in this inhalation study. Following removal of the mandible, tongue and associated structures, coronal nasal sections were made from the following areas:
Level I : Vestibule (anterior incisor)
Level II : Posterior to incisor teeth
Level III : Prepapilla (midpoint between incisors and incisive papilla)
Level IV : Incisive papilla
Level V : First palatal ridge
Level VI : Second palatal ridge
Level VII : First molar teeth (second molar teeth in satellite animals)
Morphometric determination of nasal mucosal thickness (measured by an ocular micrometer) was performed on the first 30 rats/sex/exposure level at the first and second nasal cavity examination levels. In level I the mid-septal mucosa thickness was measured unilaterally at the greatest width of epithelium. In level II there were single measurements taken of dorsal turbinate epithelium, ventral turbinate epithelium, lateral wall epithelium and septal epithelium at the point of maximum epithelial width. Lesions in sections from the first and second palatal ridges were coded as First Palatal Ridge and those in the section from the first or second molar teeth were coded as First Molar.
Statistical analysis of continuous data was first evaluated by Analysis of Variance (ANOVA) test, followed by Duncan's Multiple Range Test for between-group comparisons. Frequency data were visually examined for trends that could indicate a compound-related effect. When such trends were apparent, the data was further evaluated using a Chi-Square procedure, followed by pairwise comparison with the control group using one-tailed Fischer's Exact Test on data showing significant differences by the Chi-Square analysis. A p-value less than or equal to 0.05 was considered statistically significant. Any reference to significance in the text of this report presumes statistical significance. References to biologie or toxicologic significance are explicitly identified as such.

Results and discussion

Results of examinations

Details on results:
0.005 ppm: no effect on mortality rate; ocular irritation (includes lacrimation); no effects on body weights, on clinical chemistry, hematology, urinalyses, gross pathology and no significant effects on organ weights; histopathological lesions in the nasal cavity; no associated exposure-related lesionswere observed in the trachea, larynx or nasal lacrimal duct (for details see the remark field);
0.025 ppm: no effect on mortality rate; ocular irritation (includes lacrimation); no effects on body weights, on clinical chemistry, hematology, urinalyses, gross pathology and no significant effects on organ weights; histopathological lesions in the nasal cavity and lungs; no associated exposure-related lesions were observed in the trachea, larynx or nasal lacrimal duct (for details see the remark field);
0.175 ppm: no effect on mortality rate; transient ocular irritation in males; no lesions of the eye detected by ophthalmoscopic examination; slight body weight decrease in females during the second year of exposure; hematologic effects in females (associated with slight anemia); no effects on clinical chemistry, urinalyses, gross pathology and no significant effects on organ weights; histopathological lesions in the nasal cavity and lungs; no associated exposure-related lesions were observed in the trachea, larynx or nasal lacrimal duct (for details see the remark field).

Effect levels

Dose descriptor:
Effect level:
0.005 ppm (analytical)
Basis for effect level:
other: histopathological findings related to respiratory tract irritation

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Nasal cavity:
Level I: There was increased incidence of epithelial hyperplasia or thickening with hyperkeratosis and erosion at 0.175 ppm. Septal 

epithelial thickness did not statistically differ between control and exposed males. In females, hyperkeratosis of the epithelium was

increased in all exposed groups while epithelial hyperplasia was more frequent in 0.005 ppm and 0.025 ppm exposed groups. There

was a statistically significant difference in septal epithelial thickness between control and 0.005 ppm and 0.025 ppm (but not

0.175 ppm)  exposed females in the nasal vestibule.
Level II: There was prominent, minmal to mild, hyperkeratosis and erosion in both sexes exposed to 0.175 ppm of HDI. In 0.175

ppm males, the erosion was often more severe leading to ulceration. Females receiving 0.025 ppm also demonstrated increased

hyperkeratosis. Squamous metaplasia generally affecting the turbinate tips, septum, and lateral walls was extensive in 0.175 ppm rats 

while a combination of epithelial hyperplasia/metaplasia or mucus secretory cell hyperplasia was most prevalent in 0.005 ppm and 

0.025 ppm rats of both sexes. Inflammation was observed in 0.025 ppm and 0.175 ppm male and female rats an was slightly more

severe in the 0.175 ppm exposure groups. Thickness of the epithelium (regardless of morphologic type) covering the nasal septum, 

dorsal and ventral turbinates and lateral walls was statistically different from controls at all exposure levels in females and at the 

0.025 ppm and 0.175 ppm levels in males.
Level III: There was decreased squamous metaplasia (compared hyperplasia/metaplasia in 0.175 ppm rats. Epithelial hyperplasia 

without metaplastic change was notably increased in 0.025 ppm and 0.175 ppm males and 0.025 ppm females. Hyperkeratosis was

present in some 0.175 ppm rats of both sexes. Mucus secretory cell hyperplasia was increased at all exposure levels in both sexes. 

At this third section, hyaline droplet degeneration of epithelium along the dorsal septum and dorsal meatus was prominent in 0.005

ppm and 0.025 ppm females as wll as 0.025 ppm males. Non-specific inflammation was observed in groups exposed to 0.025 ppm

and 0.175 ppm. Epithelial erosion or ulceration was present in a few animals particularly at 0.1175 ppm.
Level IV: Hyaline droplet degeneration was seen in both sexes with increased incidence in all exposed groups as compared to 

controls, but this change was more prevalent and was graded more extensively in groups exposed to 0.025 ppm. There was notable 

olfactory epithelium degeneration in 0.175 ppm males and females with narrowing or atrophy and occasional focal erosion or 

ulceration. Epithelial hyperplasia of 0.025 ppm females and mucus secretroy cell hyperplasia of all exposure groups in males and 

females were present. Inflammation was observed in 0.025 ppm abd 0.175 ppm males and females.
Level V/VI: In controls, there was considerable minimal to mild background levels of epithelial cell mucus and hyaline droplet, 

degeneration particularly along the nasal turbinate scrolls, adjacent to the septum and pharyngeal duct. There was increased amounts

of muscus and hyaline material after exposure to all concentration of HDI as compared with controls. Epithelial hyperplasia, usually along the septum, was seen in 0.025 ppm females while dorsalseptal erosion, often associated with metaplastic change to a squamous

epithelium, was seen in 0.175 ppm males. Degeneration of the olfactory epithelium was prominent in the 0.175 ppm exposure group

of both sexes. Inflammation was observed in 0.025 ppm and 0.175 ppm males and females.
Level VII: Epithelial changes were similar to those of the previous two sections and only prominent changes were noted in addition to

those observations. Thus the obersevations were less frequent and principal lesions consisted of degeneration of the dorsal olfactory

epithelium of the ethmoid turbinates in 0.175 ppm males and hyaline droplet degereration in 0.005 ppm females.
There were generally minimal to mild, focal to multifocal lesions coded as epithelialization (alveolar lining cell proliferation), interstitial

pneumonia (septal thickening, alveolar cellular content and increased alveolar lining cell prominence), or alveolar macrophage 

accumulation (histiocyte cells in alveolar space). When considered individually or combined there was an exposure-related incidence

of these lesions in rats of both sexes exposed to 0.025 ppm or 0.175 ppm of HDI.


No statistically significant terminal body weight differences between control and exposed rats of either sex in the satellite group.

Non-neoplastic or neoplastic lesions were similar but less developed than those in terminal sacrifice animals; there was no early

dose-related onset of neoplastic gross tissue changes; lesions were restricted to histopathologic alteration of nasal mucosa; after one 

year 0.005 ppm is considered to be a NOEL since the changes observed occurred only in one sex, were qualitatively similar to those

seen in controls and did not show any concentration-dependent increase in degree.

Table 1: Incidence of chronic inflammation in the nasal tract tissues of male rats exposed to HDI over 2 years (according to Foureman et al., 1994)

   I n c i d e n c e (%)
 HDI (ppm)  0  0.005  0.025  0.175
 Level of the nasal tract  
 I 27 42  43  52 
 II 35  35 75 98
 III 10  13 23 65
 IV 20  18 27 55
 V 18  20 32 63
 VI 10 0 0

Table 2: Mean severity scores of chronic inflammation in the nasal tract tissues of male rats exposed to HDI over 2 years (according to Foureman et al., 1994)

   M e a n S e v e r i t y S c o r e
 HDI (ppm)  0  0.005  0.025  0.175
 Level of the nasal tract  
 I 1.1 1.1  1.1  1.2 
 II 1.6 1.7 1.4 2.7
 III 1.2  1.4 1.3 1.4
 IV 1.8  1.4 1.3 1.4
 V 1.3  1.3 1.4 1.4
 VI ---  1.3 --- ---

Table 3: Temporal progression of nasal tract lesions in male rats exposed to HDI over 2 years (according to Foureman et al., 1994)

             I n c i d e n c e (%) w i t h l e s i o n s e v e r i t y s c o r e s (in parentheses)  
 HDI (ppm)  0  0.005  0.025  0.175
 Lesion and Level  Year        

 Mucus hyperplasia

Prepapilla (III)

 1 30 (1.0)  30 (1.0)  60 (1.0) 
   2  13 (1.5) 27 (1.2)  47 (1.4) 63 (1.4) 

Squamous metaplasia

Postincisor (II)

 1 0  0 0 90 (3.4)
   2 7 (2.8)  15 (2.0) 10 (2.2) 93 (3.5)

Olfactory epithelium degeneration

Palatal ridge (V) 

 1  0 10 (1.0) 10 (1.0)
   2 0  2 (1.0) 12 (1.6) 92 (2.3) 

Applicant's summary and conclusion

Executive summary:

In a combined chronic toxicity and oncogenicity study in rats according to OECD TG 453 1,6-hexamethylene diisocyanate (HDI) revealed no effects on mortality rate, organ weights, clinical biochemistry and urinalysis after 2-year inhalation with vapour concentrations up to and including 0.175 ppm (nominal conc.). Exposure-related clinical signs of toxicity were observed only at 0.175 ppm in males and consisted of transiently irritated eyes. There were no exposure-related lesions of the eye detected by ophthalmoscopic examination. A small but consistent decrease in body weight of females exposed to 0.175 ppm was observed particularly during the second year of exposure. Exposure-related hematologic effects were observed at 0.175 ppm in females (slight anemia). There were no compound-related gross lesions. The compound-related histopathologic changes were limited to the nasal cavity and lungs. Lung lesions were noted as epithelialization, interstitial pneumonia or alveolar macrophage accumulation in both sexes in the 0.025 and 0.175 ppm exposure groups. Changes in the nasal cavity were observed in both sexes at 0.005 ppm and above (except males of the lowest dose group) and characterized by a non-specific epithelial tissue reaction to irritation at all exposure concentrations. There were no compound-related neoplastic lesions in any organ/tissue examined. The highest concentration of 0.175 ppm is regarded as a Maximum Tolerated Dose (MTD) because of the following combination of effects: slight decrease in body weight (females approx. 5 %); slight anemia in females; prominent erosion and ulceration in the nasal cavity of both sexes; and degeneration of the olfactory epithelium of both sexes. The lowest concentration of 0.005 ppm is considered to be a NOEC after one year of exposure since the changes observed occurred only in one sex, were qualitatively similar to those seen in controls and did not show any concentration-dependent increase in degree. After two years of exposure to the lowest concentration (0.005 ppm = nominal and analytical conc.), indications of a protective response to non-specific irritation was observed (NOAEC).

Analysis of the results from the principal study revealed that compound-related effects were limited to histopathology in the nasal passages. Although some lesions were noted in the nasal tract of animals from all exposure groups, Foureman et al. (1994) concluded that the olfactory epithelial degeneration should be considered as the significant effect in this study, with a NOAEC of 0.005 ppm and a LOAEL of 0.025 ppm, because it followed a concentration-response relationship for both incidence and severity. The data for this lesion show its absence at the lowest concentration with parallel increases in both incidence and severity at the two highest concentrations. For the other lesions, including chronic inflammation, mucus cell hyperplasia, epithelial hyperplasia, hyaline droplet degeneration, and squamous metaplasia no concordance in incidence and severity was found. In response to an irritant, the character of lesions in the nasal tract such as squamous metaplasia, mucus cell hyperplasia, and hyaline droplet formation appears to be more adaptive than adverse..