1,3-diethyl-2,4-diisocyanato-5-methylbenzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Assessment of Preferential T-Helper 1 or T-Helper 2 Induction by Low Molecular Weight Compounds Using the Local Lymph Node Assay in Conjunction with RT-PCR and ELISA for Interferon-g and Interleukin-4

Author:

Van de Briel, R.J., De Jong, W.H., Spiekstra, S.W., Van Dijk, M., Fluitman, A., Garssen, J. and Van Loveren, H.

Year:

2000

Bibliographic source:

Toxicology and Applied Pharmacology 162, 77–85 (2000)

Materials and methods

Principles of method if other than guideline:

The aim of the study was to establish whether the LLNA was sufficient to not only identify allergens but also mark them as either a contact or a respiratory allergen. To this end, IFN-g and IL-4 mRNA expression as well as the production of these cytokines was measured at various time points after primary sensitization with DNCB and TMA. In a second study, production of IFN-g and IL-4 was measured after primary sensitization with DNCB and TMA, as well as the respiratory sensitizers toluene-2,4-diisocyanate (TDI) and phthalic anhydride (PA).

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Toluene diisocyanate

IUPAC Name:

Toluene diisocyanate

Details on test material:

- Name of test material (as cited in study report): toluene-2,4-diisocyanate (TDI; 99.8% purity),

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: obtained from own breeding colony, or from Harlan/CPB, Zeist, The Netherlands
- Age at study initiation: 6–8 weeks
- Diet (e.g. ad libitum): ground standard laboratory chow (RMH-B, Hope Farms, Woerden, The Netherlands) ad libitum
- Water (e.g. ad libitum): ad libitum

- Other: The breeding colony of the animals was pre-screened/monitored for endogenous pathogenic viruses and bacteria and was found negative.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0.75% (w/v) for TDI

No. of animals per dose:

4-8 animals (differing depending on the type of experiment conducted)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
For three consecutive days, 25 µl of 1% DNCB, 12.5% TMA, and AOO (first study) or 1% DNCB, 10% TMA, 0.75% TDI, 25% PA, and AOO (second study) were applied to the dorsum of both ears. At various days after the last application, the local (auricular) lymph nodes (LN) were excised and weighed. For RT-PCR, they were snap-frozen in liquid nitrogen and stored at 270°C. For the lymphocyte proliferation test and ELISA, local LN cell suspensions were made.

Statistics:

Statistical analysis was performed using the two-tailed Student’s t test.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: no data available

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: no data available

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Criteria used for interpretation of results: EU-GHS

Conclusions:

The publication is based on experiments according to the local lymph node assay, and was conducted to not only identify allergens but also mark them as either a contact or a respiratory allergen and is considered to be of high quality (reliability Klimisch 2). The validity criteria of the test system are fulfilled. The test material did induce sensitisation and should be classified accordingly.

Executive summary:

Vandebriel and co-workers investigated whether the LLNA was sufficient to not only identify allergens but also mark them as either a contact or a respiratory allergen (Vandebriel et al., 2000). It has been shown that contact allergens preferentially induce a T-helper 1 (TH1) response, whereas respiratory allergens preferentially induce a T-helper 2 (TH2) response. These responses can be discriminated on the basis of cytokine production, such as IFN-g, which is produced by TH1 cells, and IL-4, which is produced by TH2 cells. To this end, IFN-g and IL-4 mRNA expression as well as the production of these cytokines was measured at various time points after primary sensitisation with DNCB and TMA. In a second study, production of IFN-g and IL-4 was measured after primary sensitization with DNCB and TMA, as well as the respiratory sensitizers toluene-2,4-diisocyanate (TDI) and phthalic anhydride (PA); these two were assayed 7 days after the first application. Topical application of DNCB and TMA for three consecutive days does not induce IFN-g mRNA expression relative to the vehicle control, and similarly induces IL-4 mRNA expression. While application of DNCB and PA results in a similar proliferative response, PA induces 16-fold more IL-4 (and fourfold less IFN-g). Furthermore, while application of TMA and TDI results in a 1.8-fold larger proliferative response compared to DNCB, 15- to 30-fold more IL-4 is produced (and similar amounts of IFN-g). Thus, TMA, TDI, and PA can be discriminated from DNCB on the basis of IL-4 production within the LLNA. IFN-g production was similar for DNCB, TMA, and TDI, and fourfold lower for PA, while IL-4 production was similar for TMA, TDI, and PA, and 24-fold lower for DNCB.

In summary, both studies showed induction of IL-4 production by respiratory allergens, with little or no induction by the contact allergen, holding promise for the possibility of identifying allergens (based on lymphocyte proliferation) and respiratory allergens within the LLNA by measuring IL-4 production 7 days after the first application.