Pentane, 1,5-diisocyanato-

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:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study According to ECHA Practical Guide 6 the maximum score for read across is rel. 2

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION
See document attached to section "13.2 Other assessment reports"

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

(1981)

Qualifier:

according to guideline

Guideline:

other: EPA OTS 798.3320 (Combined Chronic Toxicity / Carcinogenicity)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: not applicable (vapour study)

Details on inhalation exposure:

ANIMAL EXPOSURE CHAMBER:
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:
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.
TREATMENT OF CHAMBER EXHAUST:
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.
CHAMBER TEMPERATURE / RELATIVE HUMIDITY / STATIC PRESSURE:
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.
GENERATION OF HDI ATMOSPHERE:
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:

yes

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

Remarks:

Doses / Concentrations: 0, 0.035, 0.175, and 1.23 mg/m³ (0, 0.005, 0.025, 0.175 ppm)
Basis: target conc.

Remarks:

Doses / Concentrations: 0, 0.035, 0.175, and 1.15 mg/m³ (0, 0.005, 0.025, 0.164 ppm)
Basis: analytical conc.

No. of animals per sex per dose:

60 (for main study) and 10 (animals of satellite groups for sacrifice after one year)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 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.

- EXPERIMENTAL DESIGN:
The exposure was conducted under dynamic conditions, the air control animals were sham-exposed (conditioned room air) under comparable conditions.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
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.

BODY WEIGHTS:
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.

FOOD AND WATER CONSUMPTION: No data

OPHTHALMOLOGY:
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.

CLINICAL PATHOLOGY:
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.

Hematology determinations included: erythrocyte morphology, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, reticulocyte count, total erythrocyte count, total and differential leukocyte count.

Clinical biochemistry determinations included: albumin, calcium, chloride, creatine phosphokinase, creatinine, gamma glutamyl-transpeptidase, globulin, glucose, phosphorus, potassium, serum alkaline phosphatase, serum lactic dehydrogenase, serum alanine aminotransferase, serum aspartate aminotransferase, sodium, total cholesterol, total protein, total bilirubin, urea nitrogen.

Urinalysis determinations included: bilirubin, glucose, ketones, microscopic sediment exam., occult blood, pH, protein, specific gravity, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

ANIMAL SACRIFICE:
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.

GROSS PATHOLOGY:
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
formalin.

ORGAN WEIGHTS AND TERMINAL BODY WEIGHTS:
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.

HISTOPATHOLOGY:
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.

Statistics:

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.

Details on results:

0.005 ppm: no effect on mortality rate; no ocular irritation (includes lacrimation); no effects on body weights, clinical chemistry, hematology, urinalyses, or 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 "any other information on results..").
0.025 ppm: no effect on mortality rate; no ocular irritation (includes lacrimation); no effects on body weights, on clinical chemistry, hematology, urinalyses, or 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 "any other information on results..").
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, or 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 "any other information on results..").

Key result

Dose descriptor:

NOAEL

Effect level:

0.035 other: mg/m³ (eq. to 0.005 ppm)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.175 other: mg/m³ (eq. to 0.025 ppm)

System:

respiratory system: upper respiratory tract

Organ:

lungs

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

HISTOPATHOLOGIC LESIONS:

Nasal cavity

The principal target organ in this study was the nasal cavity (upper tract respiratory irritant effects). The results of the coronal nasal sections level I to VII were given in the following.

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, minimal 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 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 and 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 to the previous more anterior section) with increased combined 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 well 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 in a few animals particularly at 0.1175 ppm.

Level IV: Hyaline droplet degeneration was seen in both sexes 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 characterized by destruction of the epithelial architecture often with narrowing or atrophy and occasional focal erosion or ulceration. Epithelial hyperplasia of 0.025 ppm females and mucus secretory cell hyperplasia of all exposure groups in males and females were present. Inflammation was observed in 0.025 ppm and 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 an increased amount of mucus 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 dorsal septal erosion, often associated with metaplastic changes 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 observations 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 degeneration in 0.005 ppm females.

After one year of exposure, an adaptive nasal epithelial response, including mucus secretory cell and epithelial hyperplasia, was observed at the lowest exposure concentration. At the mid and high exposure concentrations, a progression from this response was observed and accompanied by hyaline droplet degeneration, hyperkeratosis, indicators of chronic inflammation and olfactory epithelial damage.

After two years a progression in the adaptive response at the low exposure concentration occurred and it was characterized by hyperplasia/metaplasia and hyaline droplet degeneration. Hyaline droplet degeneration is a morphologic indicator of reversible cellular change and was enhanced by exposure to HDI. At the mid and high exposure concentrations, a progression of the lesions noted in the satellite groups were observed after two years of exposure.

Lungs

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) after two years of exposure. 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.

SATELLITE OBSERVATIONS:

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 terminal sacrifice animals; there was 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	0	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	0	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	0	10 (1.0)	10 (1.0)
	2	0	2 (1.0)	12 (1.6)	92 (2.3)

Executive summary:

In a combined chronic toxicity and oncogenicity study in rats according to OECD TG 453 1,6-hexamethylene diisocyanate (CAS 822 -06 -0) revealed no effects on mortality rate, organ weights, clinical biochemistry and urinalysis after 2 years of 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 NOAEL 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.

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 NOAEL 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study According to ECHA Practical Guide 6 the maximum score for read across is rel. 2

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION
See document attached to section "13.2 Other assessment reports"

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

(1981)

Qualifier:

according to guideline

Guideline:

other: EPA OTS 798.3320 (Combined Chronic Toxicity / Carcinogenicity)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: not applicable (vapour study)

Details on inhalation exposure:

ANIMAL EXPOSURE CHAMBER:
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:
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.
TREATMENT OF CHAMBER EXHAUST:
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.
CHAMBER TEMPERATURE / RELATIVE HUMIDITY / STATIC PRESSURE:
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.
GENERATION OF HDI ATMOSPHERE:
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:

yes

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

Remarks:

Doses / Concentrations: 0, 0.035, 0.175, and 1.23 mg/m³ (0, 0.005, 0.025, 0.175 ppm)
Basis: target conc.

Remarks:

Doses / Concentrations: 0, 0.035, 0.175, and 1.15 mg/m³ (0, 0.005, 0.025, 0.164 ppm)
Basis: analytical conc.

No. of animals per sex per dose:

60 (for main study) and 10 (animals of satellite groups for sacrifice after one year)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 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.

- EXPERIMENTAL DESIGN:
The exposure was conducted under dynamic conditions, the air control animals were sham-exposed (conditioned room air) under comparable conditions.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
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.

BODY WEIGHTS:
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.

FOOD AND WATER CONSUMPTION: No data

OPHTHALMOLOGY:
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.

CLINICAL PATHOLOGY:
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.

Hematology determinations included: erythrocyte morphology, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, reticulocyte count, total erythrocyte count, total and differential leukocyte count.

Clinical biochemistry determinations included: albumin, calcium, chloride, creatine phosphokinase, creatinine, gamma glutamyl-transpeptidase, globulin, glucose, phosphorus, potassium, serum alkaline phosphatase, serum lactic dehydrogenase, serum alanine aminotransferase, serum aspartate aminotransferase, sodium, total cholesterol, total protein, total bilirubin, urea nitrogen.

Urinalysis determinations included: bilirubin, glucose, ketones, microscopic sediment exam., occult blood, pH, protein, specific gravity, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

ANIMAL SACRIFICE:
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.

GROSS PATHOLOGY:
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
formalin.

ORGAN WEIGHTS AND TERMINAL BODY WEIGHTS:
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.

HISTOPATHOLOGY:
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.

Statistics:

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.

Details on results:

0.005 ppm: no effect on mortality rate; no ocular irritation (includes lacrimation); no effects on body weights, clinical chemistry, hematology, urinalyses, or 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 "any other information on results..").
0.025 ppm: no effect on mortality rate; no ocular irritation (includes lacrimation); no effects on body weights, on clinical chemistry, hematology, urinalyses, or 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 "any other information on results..").
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, or 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 "any other information on results..").

Key result

Dose descriptor:

NOAEL

Effect level:

0.035 other: mg/m³ (eq. to 0.005 ppm)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.175 other: mg/m³ (eq. to 0.025 ppm)

System:

respiratory system: upper respiratory tract

Organ:

lungs

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

HISTOPATHOLOGIC LESIONS:

Nasal cavity

The principal target organ in this study was the nasal cavity (upper tract respiratory irritant effects). The results of the coronal nasal sections level I to VII were given in the following.

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, minimal 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 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 and 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 to the previous more anterior section) with increased combined 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 well 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 in a few animals particularly at 0.1175 ppm.

Level IV: Hyaline droplet degeneration was seen in both sexes 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 characterized by destruction of the epithelial architecture often with narrowing or atrophy and occasional focal erosion or ulceration. Epithelial hyperplasia of 0.025 ppm females and mucus secretory cell hyperplasia of all exposure groups in males and females were present. Inflammation was observed in 0.025 ppm and 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 an increased amount of mucus 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 dorsal septal erosion, often associated with metaplastic changes 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 observations 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 degeneration in 0.005 ppm females.

After one year of exposure, an adaptive nasal epithelial response, including mucus secretory cell and epithelial hyperplasia, was observed at the lowest exposure concentration. At the mid and high exposure concentrations, a progression from this response was observed and accompanied by hyaline droplet degeneration, hyperkeratosis, indicators of chronic inflammation and olfactory epithelial damage.

After two years a progression in the adaptive response at the low exposure concentration occurred and it was characterized by hyperplasia/metaplasia and hyaline droplet degeneration. Hyaline droplet degeneration is a morphologic indicator of reversible cellular change and was enhanced by exposure to HDI. At the mid and high exposure concentrations, a progression of the lesions noted in the satellite groups were observed after two years of exposure.

Lungs

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) after two years of exposure. 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.

SATELLITE OBSERVATIONS:

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 terminal sacrifice animals; there was 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	0	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	0	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	0	10 (1.0)	10 (1.0)
	2	0	2 (1.0)	12 (1.6)	92 (2.3)

Executive summary:

In a combined chronic toxicity and oncogenicity study in rats according to OECD TG 453 1,6-hexamethylene diisocyanate (CAS 822 -06 -0) revealed no effects on mortality rate, organ weights, clinical biochemistry and urinalysis after 2 years of 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 NOAEL 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.

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 NOAEL 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.035 mg/m³

Study duration:

chronic

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

A 4 -week vapour inhalation toxicity study is available for the substance. This study was conducted recently according to OECD TG 412 and OECD GD 39 to fulfil the REACH Annex VIII requirements. In this study rats were nose-only exposed for 6 hours/day on 5 days/week to mean actual concentrations of 0, 0.1, 0.6, and 2.2 mg/m³. For investigation of reversibility additional five rats per sex and dose group were allowed to recover during a 4-week postexposure period. Furthermore, bronchoalveolar lavage examinations, conducted at the end of the exposure and recovery period, used additional six male rats per group.

The results seen during the course of the study were indicative for a portal-of-entry toxicity, since relevant findings were associated to the upper respiratory tract; no findings indicating systemic toxicity were seen.

Effects were observed in the highest dose group (2.2 mg/m³) on water and food consumption (at the beginning of the exposure period) and on absolute body weights (in male rats on day 4). Significant reduction in relative body weights occurred discontinuously at 0.6 mg/m³ and above.

Only rats exposed to 2.2 mg/m³ revealed clinical signs. These symptoms were clearly associated to the respiratory tract. Assessment of bronchoalveolar lavage parameters indicate no relevant findings associated to lung injury or lung irritation. Significant reduction of body temperature was measured at 2.2 mg/m³ in males (day 0) and females (day 8). It is well known that reduction of the body temperature can be provoked by substances that cause irritation in the upper respiratory tract.

Histopathological examination revealed test item-induced findings at 0.6 mg/m³ and above at the end of the exposure period. There was epithelial atrophy/degeneration in the nasal cavity of all rats from the high concentration group (2.2 mg/m³) as well as minimal epithelial alteration in female rats from the mid concentration group (0.6 mg/m³). Inflammatory infiltrates were detected in the nasal cavity associated to the mentioned epithelial damage. No substance-induced findings were seen in larynx, trachea, lung and lung-associated lymph nodes. Extrapulmonary organs were indistinguishable. At the end of the 4 week post-exposure period, complete recovery of induced findings was detected in the posterior levels of the nasal cavity. In the anterior levels, epithelial atrophy/degeneration was still detected in rats from the high concentration group with clearly reduced severity.

Concluding, the NOAEC in this study was 0.1 mg/m³ due to epithelial alteration and inflammatory infiltrates in the nasal cavity at 0.6 mg/m³ (LOAEC) at the end of the exposure period.

The results of a 1 -week pilot study confirmed the results qualitatively and quantitatively. In this pilot study additionally four females of each main group were kept overnight in whole body plethysmographs for measurement of delayed lung function. A slight trend, if any, of changes in respiratory rate, respiratory minute volume and PenH was measured at the highest dose group (2.4 mg/m³) when compared to controls.

Based on these results a read across to 1,6 -diisocyanatohexane (HDI, CAS 822 -06 -0) can be justified. This is discussed in a separate document attached to the dossier. Based on the read across the repeated inhalation studies available for HDI can be used to fulfil the requirements of the REACH Annexes IX and X.

For HDI the most relevant evaluation of repeated dose toxicity comes from a 2-year chronic toxicity and oncogenicity study in rats according to OECD TG 453 (Shiotsuka 1989/2010, Foureman 1994). In this study the animals were whole-body exposed to 0, 0.035, 0.175, and 1.15 mg/m³ (0, 0.005, 0.025, and 0.164 ppm) of HDI vapour. The exposure regimen was 6 hours/day, 5 days/week for one year (chronic toxicity assessment) or two years (toxicity and oncogenicity assessment).

Test substance-related effects in this study were transient ocular irritation in males, small but consistent decrease in body weight of females (particularly during the second year of exposure) and slight anaemia in females at 1.15 mg/m³. Histopathologically, substance-related changes were limited to non-neoplastic effects in the nasal cavity and the lungs.

Changes in the nasal cavity were observed in both sexes at 0.035 mg/m³ and above (except males of the lowest dose group) and characterized by non-specific epithelial tissue reactions to irritation at all exposure concentrations (i.e. hyperkeratosis, hyperplasia of the squamous epithelium, chronic active inflammation, squamous metaplasia, mucus secretory cell or goblet cell hyperplasia, hyaline droplet degeneration and minimal degeneration of the olfactory epithelium). Lung lesions were noted as epithelialization, interstitial pneumonia or alveolar macrophage accumulation in both sexes at 0.175 mg/m³ and above after two years of exposure. No evidence of substance-related oncogenicity was found. The highest concentration of 1.15 mg/m³ is regarded as Maximum Tolerated Dose based on a slight decrease in body weight and slight anaemia of females and microscopic changes in the nasal cavity of both sexes. The lowest concentration of 0.035 mg/m³ 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 (0.035 mg/m³) indications of a protective response to the non-specific irritation was observed (NOAEC).

Analysis of the results from the principal study revealed that substance-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 concluded that the olfactory epithelial degeneration should be considered as the significant effect in this study, with a NOAEC of 0.035 mg/m³ and a LOAEC of 0.175 mg/m³, 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.

The further studies of HDI (the 21-day inhalation study of Sangha, 1984, and the 90-day inhalation study of Shiotsuka, 1988) confirm the result qualitatively and quantitatively and were also used to justify the read across.

Justification for classification or non-classification

According to of Regulation (EC) No 1272/2008, Annex I, no classification is warranted for repeated dose toxicity.

A classification with STOT-RE is not justified due to lack of cumulative toxicity: For the read across-substance HDI the NOAECs, and moreover the type of toxicological effects, do not differ between a chronic (2 -year) and a subacute (3-week) study (in both cases 0.035 mg/m³; LOAEC 0.175 and 0.12 mg/m³, respectively; no NOAEC available for the subchronic study). This is also indicated when comparing the subacute study of PDI with its 1-week range finder study; both reveal the same NOAEC of 0.1 mg/m³ (next higher concentration tested/LOAEC 0.6 mg/m³).This is in line with the ECHA Guidance on the Application of the CLP Criteria (2009), which notes on page 365: Where the same target organ toxicity of similar severity is observed after single and repeated exposure to a similar dose, it may be concluded that the toxicity is essentially an acute (i.e. single exposure) effect with no accumulation or exacerbation of the toxicity with repeated exposure. In such a case classification with STOT-SE only would be appropriate.