Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Poc:DH (SPF-bred)
- Source: Harlan Winkelmann GmbH Laboratory Animal Breeders, Borchen, Germany.
- Age at study initiation: 5 - 7 weeks
- Weight at study initiation: mean weight 319 - 342 g
- Housing: in groups of 2 or 3 in type IV Makrolon cages
- Diet and water: ad libitum
- Acclimation period: at least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- Air changes (per hr): >/= 10
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
intradermal induction: 5 % test substance
epicutaneous induction: 50 % test substance
challenge: 10 % test substance
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
intradermal induction: 5 % test substance
epicutaneous induction: 50 % test substance
challenge: 10 % test substance
No. of animals per dose:
20 test animals/10 controls
Details on study design:
RANGE FINDING TESTS:
Intradermal induction: One guinea pig was given intradermal injections twice, in each case 0.1 mL of the test substance concentrations 0%, 1%, 2.5%, 5%. The injection sites were evaluated after 24 and 48 hours.
Epicutaneous induction: 4 guinea pigs were treated epidermally in each case with 0.5 mL of the test substance concentrations 0%, 25%, 50%, 100% under occlusive conditions for 24 hours. Skin reactions were evaluated 24 and 48 hours after removal of the patch. Evidence of skin irritating properties of the 100% test substance formulation was seen.
Challenge: 5 guinea pigs, that were treated in the same manner as the control animals during the inductions, received epidermally in each case 0.5 mL of the test substance concentrations 0%, 5%, 25%, 40% under occlusive conditions for 24 hours. Skin reactions were evaluated 24 and 48 hours after removal of the patch. Evidence of skin irritating properties of the 25% test substance formulation for challenge was seen.

MAIN STUDY:
Intradermal induction: The dorsal region and the flanks of the guinea pigs were shorn one day prior to the application. Starting behind the nape of the neck, 3 injections each in a row were made on the left and the right side of the spinal column. The 1st and 2 nd injections were made as contiguous as possible and the 2nd and 3rd injections in a distance of about 2 cm. The application volume is 0.1 mL/injection site, the test substance concentration is 5% in paraffin oil (injection site medial/bilateral) and 5% in paraffin oil/FCA 1/1 (injection site caudal/bilateral), respectively. Animals of the control group were treated in the same manner with the corresponding amount of the vehicle.

Epicutaneous induction: Performed one week after the intradermal induction. On the day prior to topical treatment, the test areas were shorn. Hypoallergenic patches (2 x 4 cm), treated with 0.5 mL 50 % test substance formulation (test group) or solely vehicle (control group) were placed between and on the injection sites, covered with aluminium foil and held securely in place on the skin using a self-adhesive tape. At the end of the 48-hour exposure period, remaining substance was removed with sterile physiological saline solution.

Challenge: Performed three weeks after the intradermal induction. The dorsal region and the right flank were shorn one day prior to the challenge. During the challenge hypoallergenic patches loaded with a 10 % test substance formulation was placed on the right flank (caudal, for test substance and control group) and held securely in place with a self-adhesive tape for 24 hours. A patch loaded only with the vehicle (0.5 mL application volume) was placed on the right flank (cranial) as control. At the end of the exposure period, remaining test substance was removed with paraffin oil, and 6 hours later the skin of the animals was shorn in the zone of the challenge area. The skin reactions were assessed 48 and 72 hours after start of the application.

EVALUATION CRITERIA: A substance is interpreted to be sensitizing if by comparison with the control group 30% or more of the test group animals reacted positive.
Positive control substance(s):
yes
Remarks:
, 2-Mercaptobenzothiazole
Positive control results:
Using a 2,5 % formulation of 2-mercaptobenzothiazole for intradermal induction, a 40 % formulation for topical induction and a 40 % formulation for challenge, 60 % of the test animals exhibited dermal reactions. No reddening of the skin was observed on control group animals.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
10 %
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 13.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
10 %
No. with + reactions:
17
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 10 %. No with. + reactions: 17.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Appearance and behaviour of the test substance group were not different from the control group. By the end of the study the body weight development of the treatment group animals was in the same range as that of the control group.

None of the animals in the test group reacted to the control patch moistened with the vehicle. None of the animals in the control group reacted to the test substance formulation or to the vehicle.

48 hours after the beginning of the challenge treatment the test substance formulation led to skin redness (grade 1 or 2) in 13/20 animals in the test group, one of the 13 positive reacting animals showed additionally an oedema grade. 72 hours after the challenge 17/20 animals in the test group showed an erythema grade 1 or 2 and the oedema in the specified animal still persisted. In total, 19/20 animals in the test group (95%) showed skin reaction to the test substance formulation.

Executive summary:

A Skin Sensitisation test (GPMT) was conducted according to OECD TG 406 on female guinea pigs (20 test animals and 10 controls). Based on range finding tests the following concentrations were used in the main test: 5 % for intradermal induction, 50 % for topical induction and 10 % for challenge. As vehicle paraffin oil was used.

48 and 72 hours after the beginning of the challenge treatment 19/20 (95 %) animals in the test group showed skin reaction to the 10% test substance formulation (erythema grade 1 or 2, oedema grade 1 in one animal). None of the animals in the control group showed skin reactions. Thus, under the conditions of the maximization test and with respect to the evaluation criteria the test substance exhibits a skin-sensitisation potential.

 

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

The substance was tested positive on skin sensitisation in a fully reliable Guinea Pig Maximation test conducted according to OECD TG 406 on 20 test animals. After being induced with a 5 % concentration for intradermal induction and a 50 % concentration for epicutaneous induction the challenge experiment at a 10 % test substance concentration revealed 19/20 animals with skin reactions. As none of the animals from the control group reacted, a skin-sensitising potential was concluded for the substance.

This result is confirmed by a Local Lymph Node Assay according to OECD TG 429 tested with concentrations of 10%, 20%, and 50% test substance. No dose-response correlation was obtained in this LLNA, as the stimulation indices determined were 22.16 (10%), 21.94 (25%) and 29.62 (50%).


Justification for selection of skin sensitisation endpoint:
A fully reliable GPMT is selected, which confirms the endpoint conclusion as "sensitising" for the substance. The LLNA key-study (also result "sensitising") was not selected here, as it shows no dose-response correlation, and is thus of no added value.

Respiratory sensitisation

Endpoint conclusion
Additional information:

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 et.al., Toxicology and Applied Pharmacology, 226, 2008, 1 -13; for further reference see Pauluhn, Inhalation Toxicology, 27, 191-206, 2015; Pauluhn et.al., Toxicological Sciences, 104, 2, 2008, 320-331; Pauluhn et.al., Experimental and Toxicologic Pathology 56, 2005, 203 -234; Pauluhn et.al., 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 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. 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, 3.4.2.2.1.1 (“Skin sensitisers shall be classified in Category 1 where data are not sufficient for sub-categorisation") HDI homopolymers, and also HDI oligomers, isocyanurate 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.