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Description of key information

Key value for chemical safety assessment

Skin sensitisation

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

A modified LLNA was performed according to the OECD test guideline 429 using the cell counting method. In brief, 6 female NMRI mice per testing group were epicutaneously treated for 3 consecutive days on both ears with 25µl of test substance formulations containing 0, 3, 10 or 30% of Hexamethylene diisocyanate, oligomers (biuret) (Bayer, 2006). Measurement of ear swelling was performed in order to discriminate a specific (sensitizing) potential from a non-specific (irritating) potential of the test substance. A significant increase in the weight of the draining auricular lymph nodes and in cell counts was observed. A stimulation index of 1.4 (cell count) was defined to be indicative for sensitizing substances for this method and mice strain. This value was exceeded in all dose groups, indicating a specific immunostimmulating potential of the test substance. Though a significant dose dependent swelling of the ear was observed at day one in all dose groups (>17% of control). This has to be interpreted as clear indication of an acute, unspecific inflammation reaction (irritation) by the high dose levels applied in the assay, even in the absence of visual signs of irritation (edema, erythema). Therefore it can not be excluded, that a significant share of the observed increase in lymph nodes weights and cell counts are due to an unspecific, irritation mediated stimulation. Since the LLNA is only assessing the induction phase of the sensitization reaction no quantification of the challenge reaction is possible. Summarized, a sensitizing potential of the test substance was indicated by the assay, though the quantifiable result (stimulation index) may be over predictive due to the unspecific inflammatory potential of the test substance. The reliability of a dose response assessment is therefore questionable.

 

A guinea pig maximization assay was performed similar to OECD test guideline 406 (Hazelton, 1986). 10 male and 10 female animals were included in each dose group. One intradermal (day 1) and six epicutaneous (days 1, 3, 5, 8, 10, 12, 15) inductions were performed with 0.5ml of a 50% solution of the test substance in paraffin oil. In this assay macroscopically evaluated skin reactions 6h after challenge (edema) were verified histopathologically. This revealed 6 confirmed cases of skin sensitization.

According to the evaluation criteria of the assay the test substance has to be classified as sensitizing, since ≥30% of the test animals exhibit skin reactions in this adjuvant test.

Nevertheless, it should be emphasized that the assay was performed with comparably high induction concentrations (50%) and a very rigid induction regimen (7 instead of 2 topical inductions as required by the guideline).

 

A second guinea pig maximization assay was performed similar to OECD test guideline 406 with 10 male and 10 female animals. Induction was performed intradermally (0.05% test substance) and topically (10% test substance, occlusive for 48h). The tests substance concentration applied at challenge was 2.5%. Edematous skin reactions (moderate to severe) were observed following challenge in all animals sensitized to Hexamethylene diisocyanate, oligomers (biuret). Even though animals of the control groups (vehicle induced) exhibited an elevated score of edema (slight to moderate) this observation has to be interpreted a clear indication for a skin sensitizing potential of the test substance. No histopathological confirmation of macroscopical findings was performed in comparison to the by Hazelton (1986).

 

A published Bühler assay was performed similar to OECD test guideline 406 (Zissu, 1998). In deviation to the OECD guideline which is requesting 3 topical induction steps, 20 female animals were once epicutaneously induced with a 0.5ml of a 50% solution of the test substance in petrolatum. Following a rest period of 14 day challenge was performed with 0.5ml of a 30% solution of the test substance. Skin reactions after challenge were evaluated only once (30h after application) which revels a further deviation from the guideline. Although the report is lacking a detailed description of the test results (e.g. differentiation between edema and erythema), 14/20 animals were reported to exhibit signs of sensitization towards the test substance. 

 

All described assays unequivocally identified a skin sensitizing potential of Hexamethylene diisocyanate, oligomers (biuret). Classification as Skin Sens. 1 (H317: May cause an allergic skin reaction) according to CLP and R43 (May cause sensitisation by skin contact) according to DSD is therefore warranted.

 

Table 1: Overview of the available in vivo sensitisation tests for Hexamethylene diisocyanate, oligomers (biuret) and the respective classifications which would result from applying the criteria indicated in Regulation (EU) No 286/2011.

Animal data

CLP Sub-category

Remark

modified LLNA (Bayer, 2006),

cell count method

EC1.4: exceeded with 3%

cell count index:  

2.17 (3%) - 2.41 (10%) - 3.54 (30%)

1

Unspecific inflammation indicated by significant increase in ear thickness at all dose levels. 

modified GPMT (Hazelton, 1986),

1x intradermal FCA

7x topical induction: 50 %,

sensitization rate: 30 %

1

7 instead of 2 topical inductions as required by the guideline

GPMT (Hazelton, 1983),

intradermal induction conc.: 0.05%,

sensitization rate: 100 %

1A

elevated score of edema (slight to moderate) in control group

Buehler test (Zissu, 1998),

Induction conc.: 50%,

sensitization rate: 70 %

1B

1 instead of 3 topical inductions as required by the guideline.

Human data

CLP Sub-category

 

No reliable data identified

 

 

 

 


Migrated from Short description of key information:
Positive induction assay (LLNA).
Positive guinea pig challenge assays according to OECD test guideline406 (Buehler and Maximiztation assays).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a bioassay with Brown-Norway rats, 2 different HDI homopolymer (biuret) isoforms, varying in the amount of residual HDI monomer (0.1, 2%), were tested (Bayer, 2007). 16 male animals per dose were either exposed epidermally or by inhalation. Epidermal induction was performed on day one (trunk, 100µl of a 25% solution) and day seven (both ears, 50µl of a 12.5% solution each), inhalation induction was performed 5x3h with 50mg/m³ on 5 subsequent days. A single 30min inhalation challenge (50 mg/m³) was performed approx. 21 days following the first induction. Signs of respiratory hypersensitivity were recorded by lung function measurements, determination of inflammatory end point in bronchoalveolar lavage fluid (BALF) and total IgE in serum. One day after specific challenge the animals were subjected to an unspecific methacoline bronchoprovocation challenge. Furthermore lungs and accessory lymph nodes (lung and auricular) were weighed and examined histopathologically.

Besides of investigating the sensitizing potential of the test substance to the respiratory tract, this study was conducted to determine the role of residual monomer content. 

During or following challenge none of the rats sensitized topically or by inhalation exposures to biuret-0.1%HDI or biuret-2%HDI displayed any consistent changes in breathing pattern that may resemble an immediate-onset respiratory response.

Unspecificmethacoline bronchoprovocation challengeonly revealed an alteration of lung function parameters when animals sensitized by inhalation to Biuret-2% HDI were challenged with high MCh concentrations (as indicated by an increase in the enhanced pause (Penh)). Furthermore BALF parameters, lung and lymph node weights, IgE concentrations did not reveal statistically significant variations between the different treatement groups. 

 

Summarized, no validated guideline is available for the assessment of respiratory sensitization in animals. By weight of evidence, and in absence of reliable human data no classification for respiratory sensitization is justified.

For completeness it has to be mentioned, that two appropriate bioassays are available for HDI homopolymer (biuret) of higher oligomeric grade. Since the test substances are not consistent to the registered substance they were not included as supporting evidende. Nevertheless the experimental designs of both assays are representing a sound scientific concept including a powerful induction regimen and single inhalation challenges with concentrations indicating mild signs of respiratory irritation. One assay was performed in guinea pigs (the preferred animal species indicated in 3.4.2.1.3.1 of Commission Regulation (EU) No 286/2011) and revealed no specific pulmonary responses. The other assay was performed in the Brown-Norway rat strain, which is known to be very sensitive for inflammatory responses of the lung. This assay included measurements of immunoglobulin E (IgE) and likewise revealed no indications for a respiratory sensitizing potential of the test substance.


Migrated from Short description of key information:
In animal studies asessing the respiratory sensitizing potential in guinea pigs or Brown Norway rats no effects indicative for an induction or elicitation of respiratory sensitizatiion were observed (e.g. antibody induction, immedeate or delayed-type effects on breathing parameters).

Justification for classification or non-classification

Dangerous substance Directive (67/548/EEC)

The available experimental test data are considered reliable and suitable for the purpose of classification. Based on the criteria for classification of sensitisation of Directive 67/548/EEC, as amended for the 28thtime in Directive 2001/59/EC, classification for skin sensitisation is warranted: "R43". Classification for respiratory sensitisation is not warranted.

Regarding subcategorization according to the 2ndATP to the CLP Regulation (286/2011/EC) the available studies on skin sensitization display heterogeneous results. An overview of the available in vivo sensitisation tests for Hexamethylene diisocyanate, oligomers (biuret) and the respective classifications which would result from applying the criteria indicated in Regulation (EG) No 286/2011 is presented in the End Point Summary of this chapter (table 1).

Apparently no consistent pattern is distinguishable by comparing the subcategories which would result from the single assays. Subcategorization would either result in 1A (GPMT, Hazelton, 1983) or 1B (Buehler, Zissu, 1998) or the study design is not suitable for a clear discrimination of subcategories based on the formal criteria of Regulation (EG) No 286/2011 (modified LLNA, Bayer 2006; modified GPMT, Hazelton, 1986). Furthermore the quality of some of the studies does not fully comply with current standards e.g. insufficient documentation, no details on test substance purity. The observation of heterogenous test result dependent on test type and test performance is consistent with other HDI homopolymers (for details see Classification of HDI homopolymers for skin sensitisation according to Regulation (EU) No 286/2011, 2012).

Conclusive human data on skin sensitisation are not available for Hexamethylene diisocyanate, oligomers (biuret). However very few publications point to human experience with positive patch test reactions (Aalto-Korte et. al., Contact Dermatitis 63, 357-363, 2010), though this seems not to be a very frequent observation.

Summarized, based on a weight of evidence on limited data on human experience and inconsistent animal data, the available data for Hexamethylene diisocyanate, oligomers (biuret) does currently do not allow a solid assessment of the skin sensitization potency according to Regulation (EG) No 286/2011. Based on 3.4.2.2.1.1 (“Skin sensitisers shall be classified in Category 1 where data are not sufficient for sub-categorisation") Hexamethylene diisocyanate, oligomers (biuret) should be currently classified in Category 1, without further sub-categorisation.

For a complete discussion on classification for skin sensitisation of HDI homopolymers, including Hexamethylene diisocyanate, oligomers (biuret), see document attached to this endpoint summary.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the 2ndtime in Directive EC 286/2011, classification for skin sensitisation is warranted: Category 1. Classification for respiratory sensitisation is not warranted.

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