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 (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 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- 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. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdWin:NMRI (SPF)
- Source: Harlan Nederland, 5960 AD Horst, The Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 26 - 32 g
- Housing: single housing in conventional Makrolon Type III cages
- Diet: Provimi Kliba SA 3883 maintenance diet for rats and mice, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 (vehicle control), 10, 30, 100 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated once before application in A/OO. The formulation was applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance. For negative and positive control a dose group treated only with the vehicle A/OO and one treated with hexyl cinnamic aldehyde in A/OO, respectively, in the above described manner was used.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling
- ear weight
- body weights
The test is positive if the stimulation index exceeds 1.4 for cell count indices; for ear swelling if the "positive level" of 0.02 mm increase is reached. These levels are exclusively defined for the NMRI outbreed mice used in this study.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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 differentes 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:
After treatment with Alpha Hexyl Cinnamic Aldehyde mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals, which are of statistical significance.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The “positive level”, which is 1.4 for cell counts has been exceeded in all dose groups. This increase is of statistical significance. Cell count index (test item concentration): 1.00 (0 %) / 2.31 (10 %) / 2.45 (30 %) / 2.90 (100 %). Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 10 %.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labeling

This study does point to a non-specific (irritant) and to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell counts of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test substance.

Compared to vehicle treated animals there were clear increases regarding the weights of the draining lymph nodes and the cell counts, which are of statistical significance in all dose groups. The "positive level" of index 1.4 for the cell counts has been exceeded in all dose groups.
(Weight index: 1.0 (0%), 2.07 (10%), 2.41 (30%), 2.18 (100%); Cell count index: 1.00 (0 %) / 2.31 (10 %) / 2.45 (30 %) / 2.90 (100 %).)

The "positive level" of ear swelling which is 2x 10exp-2 mm increase, i.e. about 10 % of the control values, has been exceeded in all dose groups. These changes are of statistical significance.
(Ear swelling: day 1 = 17.17 (0 %) / 17.25 (10 %) / 17.25 (30 %) / 17.75 (100 %); day 4 = 17.50 (0 %) / 24.50 (10 %) / 26.42 (30 %) / 27.17 (100 %); Index day 4 = 1.00 (0 %) / 1.4 (10 %) / 1.51 (30 %) / 1.55 (100 %).)

A significant increase compared to vehicle treated animals regarding ear weights was detected in all dose groups.
(Ear weight = 11.78 (0 %) / 16.92 (10 %) / 18.28 (30 %) / 18.18 (100 %); Index day 4 = 1.00 (0 %), 1.44 (10 %), 1.55 (30 %), 1.54 (100 %)).

Differentiation indices (DI), which is the quotient of the relative lymph node reaction divided by the relative acute skin reaction was < 1 for all concentrations tested, i.e. 0.82, 0.71 and 0.86 (for calculation of DI see: Homey et.al., Toxicol. and Appl. Pharmacol. 153, 1998, 83 -94; Vohr et.al. Arch. Toxicol. 73, 2000, 501 -509). These DI values point to an irritating potential of the test item. However, such an irritant property could also be combined with a skin sensitizing potential of a test compound. Thus, a skin sensitizing property cannot be excluded.

The body weights of the animals were not affected by any treatment.

 

Executive summary:

A modified LLNA (IMDS; OECD TG 429) was performed on 6 female NMRI mice per dose group using test substance of 0 % (vehicle control), 10 %, 30 % and 100 %.

Compared to vehicle treated animals clear increases regarding the weights of the draining lymph nodes and the cell counts were seen. The "positive level" of index 1.4 for the cell counts has been exceeded in all dose groups.

The "positive level" of ear swelling has also been significantly exceeded and a significant increase regarding ear weights was detected. These changes were of statistical significance in all dose groups. An increase in these parameters would point to an acute irritating (inflammatory) response. However, such an irritant property could also be combined with a skin sensitizing potential of a test compound. Thus, a skin sensitizing property cannot be excluded.

Also it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 10%.

Summarizing, the study does point to a non-specific (irritant) and to a specific immunostimulating (sensitizing) potential of the test item.

 

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

A modified LLNA (IMDS; OECD TG 429) was performed on 6 female NMRI mice per dose group using test substance of 0 % (vehicle control), 10 %, 30 % and 100 %.

Compared to vehicle treated animals clear increases regarding the weights of the draining lymph nodes and the cell counts were seen. The "positive level" of index 1.4 for the cell counts has been exceeded in all dose groups.

The "positive level" of ear swelling has also been significantly exceeded and a significant increase regarding ear weights was detected. These changes were of statistical significance in all dose groups. An increase in these parameters would point to an acute irritating (inflammatory) response. However, such an irritant property could also be combined with a skin sensitising potential of a test compound. Thus, a skin sensitising property cannot be excluded.

Also it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 10%.

Summarizing, the study does point to a non-specific (irritant) and to a specific immunostimulating (sensitizing) potential of the test item.

 


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

Respiratory sensitisation

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

For respiratory sensitisation a read across to a substance with a similar chemical composition (HDI oligomerisation product, isocyanurate type, EC No. 931 -274 -8, CAS No 28182 -81 -2) is applied. The read across is based on physicochemical and toxicological similarity of the two substances. Especially a recent comparative pulmonary irritant potency study according to TRGS 430 (Technical Rule for Hazardous Substances 430; published by the German Federal Ministry of Labour and Social Affairs, 2009) confirmed for HDI oligomerisation product, allophanate-type the same toxicological mode of action (irritant portal of entry toxicity) with a similar potency (NOAEC for pulmonary irritation: 3.4 mg/m³ for HDI oligomerisation product, allophanate-type versus 3.3 mg/m³ for HDI oligomerisation product, isocyanurate type). For further justification of the grouping and read-across according to regulation (EC) No 1907/2006, Annex XI, 1.5 see document attached to chapter "Assessment Reports".

The read-across substance (EC No. 931 -274 -8; with residual content of monomeric HDI < 0.2%) revealed no respiratory sensitising potential in two studies with guinea pigs. No specific immediate or delayed effects were observed in a study with respirable aerosolised test substance for induction (5 x 3 hours) and first challenge (both head/nose only exposure), as well as for second challenge with respirable aerosolised test substance-guinea pig-albumin-conjugate. Also no specific immediate or delayed effects were observed in a second study with respirable substance aerosol-challenge (head/nose only exposure), following an induction with 3 consecutive intradermal injections. There are no data available yet regarding the respiratory sensitisation potential in the more sensitive repeated challenge Brown Norway rat bioassay (for reference and discussion see Pauluhn et. al., Experimental and Toxicologic Pathology 56, 2005, 203 -234; Pauluhn et. al., Inhalation Toxicology 17, 2005, 67 -78; Boverhof et. al., Toxicology and Applied Pharmacology 226, 2008, 1 -13). Diisocyanates in general are known respiratory sensitisers in humans. For none of the HDI oligomerisation products respective data are available in humans.


Justification for selection of respiratory sensitisation endpoint:
No study selected since both read-across studies on respiratory sensitisation were assessed.

Justification for classification or non-classification

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

No classification required for respiratory sensitisation according to Regulation (EC) No 1272/2008, Annex I, since the existing animal data indicate no sensitisation potential and no respective data are available for humans.

 

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 sensitiser into sub-category 1A, strong sensitisers, or sub-category 1B, other skin sensitisers. Where data are not sufficient a classification as Skin Sensitisation Category 1 without sub-categorisation applies.

 

HDI oligomerisation product, allophanate type is among the substances that are registered under CAS no 28182-81-2 (CAS name:Hexane, 1,6-diisocyanato-, homopolymer; in short: HDI homopolymers). HDI derived homopolymers, like HDI oligomerisation product, 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. A full and detailed justification concerning the classification of these HDI homopolymers is available and attached to this endpoint summary. Regarding HDI oligomerisation product, allophanate type, although having a slightly different composition (oligomerisation product of HDI and minor amounts of alcohol), allophanate-type HDI oligomerisation product is also member of CAS no 28182-81-2 and should also be allocated to the same category/sub-category for skin sensitization. This is based on the similarity of physico-chemical properties, but also on the assumption and experience that the structural difference resulting from the alcohol will not influence predominantly the skin sensitization potential of the substance.

 

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

Regarding the 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, Göttingen, 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 sensitiser) 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 sensitisers 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 allophanate-type HDI oligomerisation product, should be currently classified in Category 1, without further sub-categorization.