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Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- modified LLNA (IMDS): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done to discriminate the irritating potential from the sensitizing potential of the test substance.
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Strain: Hsd Win: NMRI (SPF-bred)
- Sex: female
- Source: Harlan Winkelmann GmbH, 33176 Borchen, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: 28-33 g
- Housing: During the study period the animals were single-housed in Makrolon type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 7 days

- Temperature (°C): 22 +/- 2° C
- Humidity (%): 40%-70%
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12
methyl ethyl ketone
vehicle, 2%, 10% or 50%
No. of animals per dose:
Details on study design:
The formulated test item was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days. The volume administered was 25µl/ear. The concentrations were based on the experiences with the test system and the properties of the test substance.

Autopsies - The animals were anaesthetized by inhalation of CO2 and sacrificed one day after the last application. The appropriate organs were then removed. Lymphatic organs were transferred into sterile physiologic saline solution.
Weight and cell count determinations - The weights of the lymph nodes were determined. . The cell counts per ml were determined. The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle treated animals are set to 1.00 (+/- standard deviation).
Ear Swelling - On day 0 and day 3 of the study the ear swelling of the animals was measured, and mean ear swelling was calculated.
Ear weight - On day 4 of the study the ear weight of the sacrificed animals was measured using a punch to take of a piece of every ear with a diameter of 8 mm.
Body weights - The body weights of the animals were recorded initiating the study and at the end of the study.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5% . Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.
Positive control results:
A recently conducted local lymph node assay with alpha hexyl cinnamic aldehyde showed a clear sensitising potential.
Remarks on result:
other: see Remark
Cell counts were dose-dependently increased in all dose groups. The "positive level", which is 1.4 for the cell count index, has been exceeded in all dose groups. Cell count index (test item concentration): 1.00 (0 %) / 3.58 (2 %) / 3.82 (10 %) / 4.69 (50 %).
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labeling

Compared to vehicle treated animals there was a clear dose dependent increase in weights of the draining lymph nodes:

weight index 1.00 (0%) / 2.55 (2%) / 2.92 (10%) / 3.59 (50%).

The "positive level" of ear swelling which is 2 x 10exp-2 mm increase, i.e. about 10% of the control values, has been exceeded in all dose groups:

ear swelling day 1 = 17.42 (0 %) / 17.67 (2 %) / 17.25 (10 %) / 17.08 (50 %); day 4 = 16.83 (0 %) / 20.33 (2 %) / 22.42 (10 %) / 25.25 (50 %); Index day 4 = 1.00 (0 %) / 1.21 (2 %) / 1.33 (10 %) / 1.50 (50 %).

A significant increase compared to vehicle treated animals regarding ear weights was also detected in all dose groups: ear weight day 4 = 10.79 (0 %) / 12.92 (2 %) / 14.05 (10 %) / 17.98 (50 %); Index day 4 = 1.00 (0 %) / 1.20 (2 %) / 1.30 (10 %) / 1.67 (50 %).

An increase in ear swelling and ear weights would point to an acute irritating (inflammatory) response. However, such an irritating property is also combined with a strong skin sensitizing potential of a test compound.

Executive summary:

A modified Local Lymph Node Assay (IMDS; OECD TG 429) was performed on 6 female NMRI mice/group with epicutaneously applied test substance concentrations of 0% (vehicle control), 2%, 10% and 50%. Compared to vehicle treated animals there was a clear increase in weights of the draining lymph nodes and in the cell counts at all dose groups. Additionally an increase in ear swelling and ear weight was detected; this would point to an acute (inflammatory response). However, such an irritating property can also be combined with a strong sensitising potential of the test substance.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A modified Local Lymph Node Assay (IMDS; OECD TG 429) was performed on 6 female NMRI mice/group with epicutaneously applied test substance concentrations of 0% (vehicle control), 2%, 10% and 50%. Compared to vehicle treated animals there was a clear increase in weights and cell counts of the draining lymph nodes at all dose groups. Additionally an increase in ear swelling and ear weight was detected; this would point to an acute (inflammatory response). However, such an irritating property can also be combined with a strong sensitising potential of the test substance. For HDI oligomers, iminooxadiazindione a sensitising potential was concluded.

Justification for selection of skin sensitisation endpoint:
Only one study available

Respiratory sensitisation

Endpoint conclusion
Additional information:

For assessment of respiratory sensitisation a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer). The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances reveal good correlation.

 Toxicological endpoint  HDI oligomers, isocyanurate type  HDI oligomers, iminooxadiazindione type
 Acute oral toxicity  > 2000 mg/kg  > 2000 mg/kg
 Acute inhalation toxicity (pulmonary irritant study) NOAEL 3 mg/m³   NOAEL 2.1 mg/m³
 Skin Irritation/Corrosion  slight irritation/no classification required  slight irritation/no classification required
 Eye Irritation/Corrosion  very slight irritation/no classification required  very slight irritation/no classification required
 Skin Sensitisation  classification required  classification required
 Bacterial Mutagenicity (Ames)  negative  negative

With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008; attached to this endpoint summary).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and the data on respiratory sensitisation of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

See below discussion of respiratory sensitisation of the read-across substance HDI oligomers, isocyanurate type:

"Specific, internationally harmonised test procedures for studies to assess the respiratory sensitisation potential of low- or high-molecular weight compounds do not yet exist. Accordingly, the animal studies on respiratory sensitisation available for HDI oligomers, isocyanurate type, although of high quality, have to be seen as investigative studies. Therefore, the toxicological endpoint respiratory sensitisation is evaluated based on a weight of evidence approach, taking into account the results of five animal studies and, with higher weight, human experience.

At first the conduction and result of the animal studies is summarised here:

Three of the studies were using guinea pigs as test species. The principle of these studies was similar as described by Karol (see for example Karol et al., Fundam. Appl. Toxicol. 5, 1985, 459-472). In one of these studies groups of guinea pigs were exposed on 5 consecutive days for 3 hours daily to different concentrations of the aerosolised test substance (0 (vehicle control), 3, 16, 49, and 261 mg/m³; MMAD approx. 1.5 µm, GSD approx. 1.4). After a treatment free period of at least 2 weeks a challenge experiment with the aerosolised test substance at approx. 97 mg/m³ (MMAD approx. 1.5, GSD approx. 1.5) was performed. After one further exposure-free week a re-challenge with a synthetic protein-conjugate of the test substance at 27 mg/m³ (MMAD 3.7 µm, GSD 2.1; approx. 10 mol HDI isocyanurate/mol guinea pig albumin) was conducted. Investigated parameters included lung function measurements during and after challenge in order to detect immediate or delayed effects. Additionally blood was sampled before each challenge and investigated for specific IgG1-antibodies, and extensive lung histopathology was performed after study termination.

In this study lung function tests revealed no evidence of a pulmonary hypersensitivity. No conclusive immediate or delayed change could be detected neither after challenge with the test substance-aerosol nor after conjugate-challenge. Animals of the 261 mg/m³ group showed slight increases in lung weights. Histopathology revealed at this concentration inflammatory changes in the lung; these were seen as causally related to the primary irritation potential of the substance. No evidence of recruitment of eosinophilic granulocytes in the airway walls was observed. A concentration-related increase of specific test substance related IgG1 antibody counts demonstrated that the test substance-aerosol was of adequate respirability for the animals. Overall, the study result shows with respect to the conditions of this test, that the substance has no respiratory sensitising potential after repeated inhalation exposure to the respiratory tract.

The negative result is confirmed in a second study with a similar testing protocol. Here, inhalative induction on 5 consecutive days for 3 hours daily to the aerosolised test substance at an average concentration of 76 mg/m³ was followed by only one inhalation challenge (average conc. 0.7 mg/m³) after a 2-week treatment free period. Again, lung function measurements revealed no indications for respiratory sensitisation.

A third study used a simplified testing protocol. In this study guinea pigs were intradermally injected thrice with a 30 % test substance formulation before performing the challenge experiment with the test substance-aerosol at approx. 85 mg/m³. Also here, no evidence for a respiratory sensitisation potential was seen.

Further evidence for a negative respiratory sensitisation potential gives a modified subacute inhalation study on 10 male rats/dose groups; 10 additional animals were used for a 7-week recovery period. In this study investigations focused on the asthmatogenic potential of the substance and including lung lavage endpoints, blood gas analysis, lung function tests and in depth lung histopathology. Indications for an asthmatogenic potential were not seen, since no infiltration of eosinophilic granulocytes and no indications for a bronchoalveolitis or airway hyperreactivity was found.

More recently, the respiratory sensitisation potential was investigated in the Brown Norway rat (for a discussion on advantages and disadvantages of the different species in resp. sensitisation studies see Boverhof, Toxicology and Applied Pharmacology, 226, 2008, 1 -13; for further reference see Pauluhn, Inhalation Toxicology, 27, 191-206, 2015; Pauluhn, Toxicological Sciences, 104, 2, 2008, 320-331; Pauluhn, Experimental and Toxicologic Pathology 56, 2005, 203 -234; Pauluhn, Inhalation Toxicology 17, 2005, 67 -78). In this study groups of each 16 male Brown Norway rats were twice epicutaneously induced with the monomeric hexamethylene diisocyanate (HDI; CAS 822-06-0) alone or with the mixture of HDI and HDI oligomers, isocyanurate type. Then a treatment free period of two weeks was followed by four inhalative challenge exposures (at intervals of 2 weeks each) either to HDI alone (8 animals/group) or to the mixture of HDI and HDI oligomers, isocyanurate type (8 animals/group). Lung function measurements (immediate and delayed) and examination of bronchoalveolar lavage endpoints serve in that study as indicatorsfor a respiratory sensitisation potential.

As result, lung function measurements did not provide evidence of immediate or delayed changes in breathing patterns. The measurement of inflammatory endpoints in bronchoalveolar lavage fluid (BAL) revealed statistically significant changes in rats sensitized with HDI oligomers, isocyanurate type, and challenged to the aerosol mixture of HDI and HDI oligomers, isocyanurate type. All remaining groups were essentially indistinguishable. Overall, this demonstrated that the monomeric HDI, which is known as upper respiratory tract irritant (anterior-posterior gradient with most prominent effects in the anterior part of the nasal cavity; e.g.Shiotsuka Inhalation Toxicol 18, 659-665, 2006; Pauluhn, Inhalation Toxicology 27, 191-206, 2015) did not gain access to the lower respiratory tract to any appreciable extent while the aerosol of the mixture elicited pulmonary irritation. As explanation for the significant BAL-changes solely observed in the group challenged to the aerosolised mixture of HDI and HDI oligomers, isocyanurate type, a shuttle effect was proposed, that enables the monomeric HDI-vapour to gain access into the pulmonary tract. It was concluded that for BAL-parameter obtained under such testing conditions it cannot be distinguished between acute pulmonary irritation and an allergic outcome (Pauluhn, Inhalation Toxicology 27, 191-206, 2015). Overall, the study has to be judged as inconclusive. Further investigations on the Brown Norway rat model may gain a better understanding on the adequate study design.

The available human data were discussed in the following:

Diisocyanates in general are known respiratory sensitisers in humans. Some publications on case reports or cohort studies, all related to occupational exposure, indicate that HDI-derived homopolymers were a risk for occupational asthma, especially when looking at the earlier publications, when protective measures were not comparable to nowadays standards.

Looking particularly at HDI oligomers, isocyanurate type, conclusive published data are not available. Moreover, only few of the publications give the diisocyanate specification in sufficient detail at all. The workplaces described in the publications can always be characterised as mixed exposure workplaces to e.g. isocyanates, polyalcohols, and solvents. Many of the publications lack also of reliable information of the respective airborne concentrations, since they mainly were initiated as retrospective medical surveys or they rely on incidental cases. It is furthermore important to note, that most of the studies does not address to what extent the residual monomeric HDI content play a role in asthma development. Since 1,6-hexamethylene diisocyanate (CAS 822-06-0, HDI) is a well-known respiratory sensitiser, a clear assignment of the causative agent for asthma is difficult, when residual amount of HDI is present. Recent investigations revealed that the volatile HDI, which itself is an upper respiratory tract irritant, can be “shuttled” with aerosolised HDI-polyisocyanate into the lower respiratory tract (Pauluhn, Inhal Toxicol, 27, 191-206, 2015) and by that might have an impact in asthma development.

The situation is aggravated by the fact that diisocyanates have a potential to cause sensitising as well as irritant effects (Baur, J Occup Med Toxicol 8, 15, 1-8, 2013), and it remains unclear to what extent each may contribute in the development of asthma. Occupational asthma covers both sensitizer-induced and irritant-induced asthma, the latter including e.g. reactive airway dysfunction syndrome (Tarlo et al., Chest 134, 3, 1S-41S, 2008). The classification-criteria in GHS lay down that a substance should be classified as respiratory sensitiser if evidence for a “specific” respiratory hypersensivity exits, however, it is stated that immunological mechanism do not have to be demonstrated. ECHA exemplifies in its Guidance on the Application of the CLP Criteria (ECHA-15-G-05-EN): “The mechanisms by which substances induce symptoms of asthma are not yet fully known. For preventative measures, these substances are considered respiratory sensitisers. However, if on the basis of the evidence, it can be demonstrated that these substances induce symptoms of asthma by irritation only in people with bronchial hyper reactivity, they should not be considered respiratory sensitisers.”

For all these reasons discussed, the available publications have to be thoroughly evaluated for an assessment of this endpoint. Therefore, an overview of the so far available publications dealing with the respiratory sensitisation potential of HDI oligomers, isocyanurate type in the broadest sense is given in the following:

Case reports:

 First Author  Year of publication  Reference  Substance description  Result
Bieler 2011  Occup Med 61, 440-442, 2011  “HDI-containing hardener (70-80 % HDI based aliphatic polyisocyanate and 0.1 - 0.5 % hexamethylene-1,6-diisocyanate monomers)”  Case report of an acute life-threatening extrinsic allergic alveolitis (EAA) of a woman following exposure to a hardener as described on the left. Workplace investigations revealed that she used no respiratory protection and wore no lab coat, but short latex gloves, and that air renewal rate was only 1.5 times/h. Immunological investigations conducted 5 months after the acute EAA revealed specific IgG to HDI and MDI. Concluding, the substance is not described in sufficient detail and a mixed exposure to various chemicals is indicated (normally she was exposed to acrylic paints); the case cannot directly be associated with HDI oligomers, isocyanurate type.
 Seldén  2007  Int J Occup Med Environ Health 20, 3, 287-290, 2007  Varnish that contained "pigments and solvents, whereas the curing agent was based on 60-70 % HDI oligomers (mainly isocyanurate and biuret), 0.1-1% HDI monomer and solvents"; Desmodur N3300 and N100 used as HPLC-standards. Case report of an acute asthma attack of a self-employed man one hour following spray-painting a bathtub. The diagnosis was irritant-induced occupational asthma, probably caused by massive isocyanate exposure. No characteristics of a sensitization or alveolitis were shown; the case was considered to be an example of reactive airways dysfunction syndrome (RADS), caused by particularly high polyisocyanate exposure. Concluding, the substance is not described in sufficient detail and a mixed exposure to various chemicals is indicated (degreasing and cleaning of the tub with a phosphoric acid/paraformaldehyde formula, a styrene-based filler for surface defects, followed by grinding and acetone cleansing before spray-painting the isocyanate-based varnish); the case cannot directly be associated with HDI oligomers, isocyanurate type. 
Redlich   1997  Scand J Work Environ Health 23, 227-231, 1997 Spray paints "containing HDI monomer and several HDI oligomers (prepolymers)"  Case report of a self-employed autobody shop worker who was diagnosed with isocyanate asthma by history, methacholine challenge, and workplace challenge (he spray-painted a car without respiratory protection for 40 min.). Furthermore airway biopsies demonstrated inflammatory changes typical for asthma, including increased airway eosinophils and T-cells. Immunohistochemical staining with specific anti-HDI antibodies demonstrated the presence of HDI adducts in human bronchial biopsies. Concluding, the substance is not described in sufficient detail and a mixed exposure to various chemicals is indicated; the case cannot directly be associated with HDI oligomers, isocyanurate type. 
 Diller  1988  Letter, dated 1988-10-25, sent to Prof. Dr. med. Diller, Bayer AG. Paints, in one case specified as isocyanate-containing DuPont Imron paint, hexamethylene isocyanate.  Two cases described in a letter: 2 workers were occupationally exposed to isocyanate-containing paints and developed severe asthma of which they died from.Only few details given; the substance is not described in sufficient detail and a mixed exposure to various chemicals is indicated; the case cannot directly be associated with HDI oligomers, isocyanurate type. 
 Innocenti  1986  Med Lav 77, 2, 191-194, 1986  “using acrylic paint containing an HDI-based polyisocyanate as activator. The paint contained a residual 0.3 % of free monomer” Case report of a man who worked occasionally as a car painter. A bronchial provocation test with HDI was performed decanting 100 mL or an acrylic hardener containing polyisocyanate and 0.5 % of free HDI monomer from one beaker to another for 10 min. (atmospheric concentrations not known). A late asthmatic reaction occurring 10 h after exposure and improving after 72 h was observed. Concluding, the substance is not described in sufficient detail and a mixed exposure to various chemicals is indicated; the case cannot directly be associated with HDI oligomers, isocyanurate type. 
 Malo  1983  J Allergy Clin Immunol 72, 4, 413-419, 1983  “polyisocyanate activator, which was a mixture of aliphatic ester (27 %), ether ester (21 %), xylene (14 %), toluene (6 %), and polymeric HDI (7 %)” Case report of a man working as a foreman in a garage where painting was done. He developed episodes of dyspnea, wheezing, and fever on working days. Specific inhalation challenge with the activator containing HDI for 5 min. (maximum air concentration of isocyanate = 0.02 ppm) resulted in both an alveolar (suggested by symptomatology, fever, inspiratory crackles, and high leukocyte count) and bronchial reaction (nonallergic bronchial hyperexcitability evidenced by lung function measurements). Concluding, the substance is not described in sufficient detail and a mixed exposure to various chemicals is indicated; the case cannot directly be associated with HDI oligomers, isocyanurate type. 

Exposure surveys

 First Author  Year of publication  Reference  Background/Type of working area  Result
 Stocks  2015  Occup Med, aop, 2015  National programme (UK) for motor vehicle repair workers (SWORD), includes biomonitoring via urine samples. “two-pack spray paints used in body shops, particularly in the motor vehicle repair (MVR) industry, commonly contain the organic aliphatic diisocyanates, 1,6-hexamethylene diisocyanate (HDI) and isophorone diisocyanate”  Key points of the study include: "Isocyanate exposure was a common cause of asthma in the UK motor vehicle repair industry and workers potentially exposed to 1,6-hexamethylene diisocyanate can be screened for urinary hexamethylenediamine, a biomarker of exposure.” “A declining trend in the number of workers with detectable urinary hexamethylenediamine levels measured during screening temporally coincided with a declining trend in incidence of asthma in the motor vehicle repair industry reported to the Surveillance of Work-related and Occupational Respiratory Disease occupational respiratory disease surveillance scheme from 2006 to 2014.” Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.
 Hathaway  2014  J Occup Environ Med 56, 1, 52-57, 2014 “Seventy-three employees from two plants, manufacturing or producing aliphatic diisocyanates, were surveyed using a detailed respiratory history questionnaire with additional questions on accidental skin and inhalation exposures”   Follow up publication of Cassidy et al., J Occup Environ Med 52, 10, 988-994, 2010.Consistent with this previous study, no cases of occupational asthma were identified from exposure to 1,6-hexamethylene diisocyanate, isophorone diisocyanate, methylene bis(4-cyclohexyl isocyanate), or their polyisocyanates even though many employees reported detection of odours (93%) or skin exposures (53%). The results of this study suggest that the use of careful manufacturing procedures seems sufficient to protect against the development of occupational asthma. “Infrequent and very brief accidental exposures to levels sometimes above 5 ppb (TLV) did not seem to be associated with the development of occupational asthma.”
 Castano  2013  J Occup Environ Med 55, 8, 954-959, 2013  Nine male subjects “were referred for investigation of possible isocyanate-induced OA by SIC (specific inhalation challenge), and they also complained of work-related nasal symptoms.” “Eight subjects worked in autobody repair and car painting and one in aircraft painting.”“The isocyanates most commonly used are hexamethylene diisocyanate (HDI), toluene diisocyanate (TDI), methylene diphenyl diisocyanate (MDI), and isophorone diisocyanate. A common setting for exposure to these chemicals is in autobody repair shops where HDI-containing paints are used as a spray paint hardener.” For challenge experiment exposure to HDI (monomer) and MDI was conducted.  “The 4-minute isocyanate SIC induced a nonsignificant fall in nasal volume and no increase in the VAS score. The 120-minute isocyanate SIC induced a significant fall innasal volume at 15, 30, and 60minutes postchallenge thatwas associated with a significant increase in the VAS score at 15 and 30 minutes postchallenge.Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.
 Yucesoy  2012  Toxicol Sci 129, 1, 166-173, 2012  “The main study population consisted of 353 Caucasian French-Canadians from among a larger sample of 410 diisocyanate (HDI, MDI, and TDI)-exposed workers.”Workers were allocated to one of three groups: SIC (specific inhalation challenge) confirmed diisocyanate asthma (DA) workers, symptomatic workers with negative SIC, asymptomatic exposed workers. “this case-control study reports that the SOD2 rs4880, EPHX1 2740171, and GSTP1 rs1695 variants are significantly associated with DA supporting the hypothesis that genetic variability within antioxidant defense systems contributes to the pathogenesis of this disease. After adjustment for confounding variables, variants of GSTM1, GSTT1, GSTP1, EPHX1, and GSTM3 genes also showed significant positive or negative associations with DA.” Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development. 
 Cassidy  2010  J Occup Environ Med 52, 10, 988-994, 2010  Retrospective study of “Employees from plants manufacturing or producing 1,6-HDI monomer and/or HDI polyisocyanate”, including annual pulmonary function tests and data from medical evaluations.  “No significantly accelerated annual decline in force expiratory volume after 1 second in the HDI exposure group compared to the matched control group was observed. No cases of adult onset asthma, beyond those present at time of hire, and no cases of occupational asthma were identified.”“This study provides support for the current American Conference of Governmental Industrial Hygienists threshold limit value time-weighted average of 5 ppb.”
 Dragos  2009  Occup Environ Med 66, 227-234, 2009  “Prospective study in 385 apprentice car painters during their 18 months of training.” The programme includes 300 hours exclusively for painting with diisocyanate paints. “Hexamethylene diisocyanate (HDI) is mainly used as a spray-paint hardener in automobile body shops and in aircraft manufacturing.”  The conclusions drawn from the authors are 1. In the cohort, a small proportion of participants show increases in HDI-specific IgG and IgE after few months of exposure. 2. Increases in specific IgG and IgG4 appear to have a protective effect on the incidence of work-related lower and upper respiratory symptoms, respectively. 3. Assessment of specific antibodies to isocyanates may help identify subjects at risk of developing symptoms. Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.
 Pronk  2007  Am J Respir Crit Care Med 176, 11, 1090-1097, 2007  “The prevalence of respiratory symptoms and sensitization was assessed in 581 workers in the spray-painting industry.” ”Spray painters, who are exposed to HDI oligomer mixtures, are among the occupational groups with the highest incidence of occupational asthma in industrialized counties…”For serologic analysis HDI oligomer–HSA conjugates were prepared with Desmodur N3300 and Desmodur N100.  “Respiratory symptoms were more prevalent in exposed workers than among comparison office workers. …The results provide evidence of exposure–response relationships for both work-related and non–work-related respiratory symptoms and specific sensitization in a population exposed to oligomers of HDI. Specific IgE was found in only a minority of symptomatic individuals. Specific IgG seems to be merely an indicator of exposure.” Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.
 Glindmeyer 2004   Am J Ind Med 46, 104-111, 2004  “A cross-sectional study of 240 painters spraying polyurethane enamels was undertaken at four aircraft maintenance plants.”“.., paint aerosol is a heterogeneous material presenting a mixed exposure material. Exposure standards or guidelines apply to certain specific paint aerosol components, such as specific solvents, pigments, or other additives. The most commonly used isocyanate in polyurethane paint formulations is HDI, and more specifically its trimeric oligomers, HDI-biuret and HDI-isocyanurate.”  “These results suggest important respiratory effects from exposures to spray paint aerosols at levels generally in compliance with existing standards for otherwise unregulated particulates and for the isocyanate component of the paint.” Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.
 Hathaway  1999  J Occup Environ Med 41, 378-383, 1999  “The study presented here compares pulmonary function change over time between workers exposed to HDI and a control group. In this study worker exposure occurs during the production of HDI biuret and HDI trimer. There is no significant inhalation exposure to the HDI biuret or HDI trimer products since these compounds have low volatility and are not handled in a manner that could generate mist or aerosols.”  As a result the pulmonary function tests reveal virtually identical values of FCV (forced vital capacity) and FEV 1 (forced expiratory volume in 1 second) for these workers and a matched control group. “Exposures to HDI were measured during this period. The time-weighted average exposure to HDI during work not requiring respiratory protection in the unit (approximatly 2 hours per day) was 0.5 parts per billion. The average daily high peak exposure was 2.9 parts per billion. Exposure to these levels appear to pose no risk of accelerated decline in pulmonary function.”
 Cullen  1996  Occup Med 46, 3, 197-204, 1996  A cross-sectional survey was conducted of 23 (about one in five) autobody shops in the New Haven area, including 102 workers (office workers, shop floor workers in different exposure categories, dedicated painters).“Different brands of paint containing aliphatic isocyanates were in use. The paint systems were two-compound paints prepared in the autobody shop; a polyol with pigments and solvents had to be mixed with polymeric isocyanates in a solvent.” HDI and partially polymerised HDI derivatives are mentioned in the text as major hazards.  …, “the survey suggests that there is a high prevalence of airway symptoms among workers in autobody shops, at least in part due to work-related asthma. Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.
 Vandenplas  1993  J Allergy Clin Immunol 91, 4, 850-861, 1993  “Twenty workers, who were referred consecutively for possible OA that resulted from exposure to spray paints underwent inhalation challenges with the monomer and prepolymers of HDI on separate days with pure HDI monomer and the commercial formulation of HDI prepolymers to which they had been exposed at work.”“These commercial hardeners are made of a mixture of prepolymers (30% to 60%) and the monomer (< 1%).” In challenge experiment the publication specifies for one subject (No 13) that he had been exposed to a paint hardener that contained HDI and IPDI in the form of both monomers and prepolymers. Concentrations of HDI prepolymers in challenge experiment were not measured directly due to technical reasons; no data on particle size measurement (if any) given in publication.  The respective publication is a follow-up of Vandenplas et al., Am Rev Respir Dis 145, 582-587, 1992: The data of eleven of the subjects were already reported in this previous publication.Specific inhalation challenges elicited a positive asthmatic reaction in 10 of the 20 subjects. Among these subjects, four had positive bronchial reactions (two early, one late, and one dual) to both the monomer and the prepolymers. Four other subjects had asthmatic reactions (two early, one late, and one dual) after exposure to the prepolymers but not after exposure to the monomer. According to the authors “these observations show that, although they are nonvolatile, the prepolymers of HDI can induce OA…”According to the authors this study shows that prepolymers of HDI can induce OA (occupational asthma), but deficiencies of the study (limited number of subjects, doubtful validity of determination of test atmosphere concentration for HDI prepolymers, inconsistent pattern of asthmatic reaction following challenge for the four “positives” (two early, one late, and one dual), hyperreactivity to baseline metacholine challenge) limits the assessment of the asthmatic reactions observed. Concluding, based on this study HDI oligomers, isocyanurate type cannot unambiguously be identified as the causative agent for asthma development and the underlying mechanism (allergic versus irritant hyperreactivity).
 Grammer  1988  J Allergy Clin Immunol 82, 627-633, 1988  “We have prospectively evaluated 150 workers exposed to hexamethylene diisocyanate (HDI) and its trimer (THDI) during an 18-month period.”“The study population consisted of workers in a factory that spray-paint truck cabs with paints that contain HDI and its trimer THDI.”  “There were no instances of immunologically induced disease among the 21 % workers in this sample with antibody; … The antibody was generally low-level IgG that may be a sensitive indicator to detect exposure to certain reactive chemicals. The level of antibody was not different among job classes of between smokers and nonsmokers. Moreover, there was no correlation between antibody level and exposure duration in these workers whose exposure levels are all well below NIOSH recommendations.
 Welinder  1988  Clin Allergy 19, 85-93, 1988  “A group of thirty car painters exposed to vapours and aerosols of paint containing prepolymer and monomer of HDI was investigated.”“The paint was applied to the cars by spraying in special chambers with an air exchange rate of more than 20 times per hour. The paints were of a two-component type with isocyanate as the curing agents. The isocyanates were based on one or two oligomers of HDI (Desmodur N-100 (DN) or N-3300;...with about 0.5 % monomer HDI.”Exposure levels were not investigated in this study; exposure levels of < 5 -89 μg HDI/m³ air and 200 -8500 μg/m³ referred to measurements in similar facilities. It was stated in the publication that all but two of the workers used respiratory protection devices, but only one half were effectively protected by respirators (visors) supplied with air from a compressor; the others used mainly gas-masks with charcoal filters, without protection against respirable particles.  “Thirteen subjects (43 %) had suffered symptoms of rhinitis and/or conjunctivitis, three (10 %) had attacks of cough, and seven (23 %) had attacks of dyspnoea and/or chest tightness. Three (10 %) had chronic bronchitis. In total, 10 (33 %) had suffered symptoms from the bronchi. There was no association between exposure time or degree of protection, and symptoms. Eight subjects (27 %) of the subjects were atopics. They did not report more symptoms than non-atopics.Ten subjects who had complained of symptoms from the bronchi underwent further medical examination. Two of those had a clear clinical asthma. One of those had suffered asthma since childhood. ….The other developed asthma of late type during the time he had worked as a car painter, and it was associated with isocyanate exposure. Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.
 Séguin  1987  J Occup Med 29, 340-344, 1987 “The prevalence of occupational asthma was assessed in four paint shops of a large airplane assembly plant where 51 employees were exposed to different types of isocyanate, …. The airplane assembly plant used paints containing different types of isocyanates, including TDI, MDI, HDI, and PPI (polymethylene polyphenylisocyanate), the latter two being the most commonly used.”   “The diagnosis of occupational asthma was confirmed in six subjects (…) through specific inhalation challenges in the laboratory to a paint system component containing PPI. Thus, the prevalence of occupational asthma was 11.8 % in these paint shops….”Concluding, based on this study HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development.

In summary, the available animal studies and human data, although assessed in a weight of evidence approach, give an inconclusive picture. Based on this data HDI oligomers, isocyanurate type cannot be identified as the causative agent for asthma development. It is believed that the content of monomeric HDI (CAS 822-06-0) in the products is responsible for the respective respiratory hyperreactivity cases. In this context of interest is that the monomeric HDI-vapour, which is an upper tract respiratory irritant, can be shuttled by the aerosol into the lower respiratory tract when sprayed in the presence of a prepolymer-aerosol (Pauluhn, Inhal Toxicol, 27, 191-206, 2015) and by that might cause an asthmatic response. Monomeric HDI (CAS 822-06-0) is already legally classified for respiratory sensitisation.

Besides, the underlying mechanism (allergic versus irritant hyperreactivity) is not clear. Further studies may help to clarify the open questions, but since there is currently no regulatory accepted model, this issue cannot be solved in the short-term."

Justification for selection of respiratory sensitisation endpoint:
Specific, internationally harmonised test procedures for experiments to assess the respiratory sensitisation potential of low- or high-molecular weight compounds do not yet exist. Accordingly the available studies of the read-across substance HDI oligomers, isocyanurate type for respiratory sensitisation, although of high quality, have to be seen as investigative studies. Therefore this toxicological endpoint is evaluated based on a weight of evidence approach, taking into account the results of five animal studies and additionally human experience. However, data are inconclusive concerning respiratory sensitisation.

Justification for classification or non-classification

Respiratory Sensitisation

According to Regulation (EC) No 1272/2008, Annex I no classification is currently warranted for Respiratory Sensitisation. This is since the available data give no conclusive picture. Further studies would be necessary for a solid assessment of this toxicological endpoint, but since there is currently no regulatory accepted model, this issue cannot be solved in the short-term.


Skin Sensitisation

According to Regulation (EC) No 1272/2008, Annex I, the substance has to be classified as Skin Sensitising Cat.1 (H317: May cause an allergic skin reaction).

Regulation (EU) No 286/2011 amending Regulation (EC) No 1272/2008 states that, where data are sufficient, a refined evaluation allows the allocation of skin sensitizer into sub-category 1A (strong sensitizers) or sub-category 1B (other skin sensitizers). Where data are not sufficient a classification as Skin Sensitisation Category 1 without sub-categorisation applies.

HDI derived homopolymers (in short HDI homopolymers), like HDI oligomerisation products, uretdione type, isocyanurate type, iminooxadiazindione type and biuret type (all CAS no 28182 -81 -2), that are composed solely by different oligomerisation products of 1,6-hexamethylene diisocyanate monomer, were considered to have a similar skin sensitisation potential. This is based on structural analogy, on similar physico-chemical properties (vapour pressure, viscosity, hydrolytically unstable, reactive with nucleophiles) and on results of in vivo skin sensitisation assays of the substances. Consequently, all of these substances should be allocated to the same category/sub-category for skin-sensitisation.


Here in brief is the rationale for the categorization of HDI homopolymers for skin sensitisation:

With regard to sub-categorisation, studies of HDI homopolymers give an inconsistent picture. From the majority of studies it could be seen, that application of the formal criteria for sub-categorisation according to Regulation (EU) No 286/2011 leads to category 1B, but some studies does also point to a strong sensitisation potential (category 1A), and in some cases a discrimination between sub-categories 1A and 1B is not possible based on the test results.

The classification criteria of Regulation (EU) No 286/2011 cover human as well as animal data. For HDI homopolymers conclusive human data on skin sensitisation are not available (Abschlussbericht zum Forschungsvorhaben FP 272, IVDK, Goettingen, September 2011), which could be partly ascribed to the instability of the test preparations (Frick et. al., Contact Dermatitis 51, 73-78, 2004). Few publications point to human experience with positive patch test reactions indicating skin sensitization (Aalto-Korte et. al., Contact Dermatitis 63, 357-363, 2010), but this seems not to be a very frequent observation.

For HDI monomer, the precursor of HDI homopolymer, no sub-categorisation is currently concluded, since limited data on potency and inconsistent human and animal data does not allow a clear discrimination. Taking into account the database on HDI monomer a sub-categorisation of HDI homopolymers as 1A (strong sensitizer) based on worst case conclusion from the animal data with (some) HDI homopolymers seems not to be adequate and proportionate, since the less reactive HDI homopolymers are not assumed to be the more potent skin sensitizers than the respective monomer. Indeed, scientific evidence exists for some time that chemical reactivity or, more precisely, protein reactivity is linked to the potency of skin sensitisation (Lepoittevin et. al., Allergic Contact Dermatitis: The Molecular Basis. Springer, Berlin, 1998).

Overall, in a weight of evidence approach based on limited data on human experiences and inconsistent animal data as well as based on a comparison of the available data with HDI monomer it is concluded that the available data for HDI homopolymers currently do not allow a solid assessment of the potency. Therefore according to Regulation (EU) No 286/2011, (“Skin sensitisers shall be classified in Category 1 where data are not sufficient for sub-categorisation") HDI homopolymers, and also HDI oligomers, iminooxadiazindione type, should be currently classified in Category 1, without further sub-categorization. 


A full and detailed justification concerning the classification of these HDI homopolymers is available and attached to this endpoint summary.