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
Remarks:
in vivo
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD Toolbox 2.2, is a harmonized system for OSAR application, where the OECD principles are met.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: other guideline: OECD Guideline 406 (Skin sensitisation);EU Method B.6 (Skin sensitisation)
Deviations:
no
GLP compliance:
not specified
Type of study:
other: the prediction is based on 11 data points from 5 chemicals
Test material information:
Composition 1
Species:
other: Mouse; guinea pig





The prediction was based on dataset comprised from the following descriptors: "Skin sensitisation"
Estimation method: Taking highest mode value from the 5 nearest neighbours
Domain logical expression:Result: Out of Domain

("a" and ("b" and "c" and "d" and "e" ) )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Diisocyanates by US-EPA New Chemical Categories

Domain logical expression index: "b"

Parametric boundary:The target chemical should have a value of log Kow which is >= 3.19

Domain logical expression index: "c"

Parametric boundary:The target chemical should have a value of log Kow which is <= 6.12

Domain logical expression index: "d"

Parametric boundary:The target chemical should have a value of Similarity which is >= 0.413

Domain logical expression index: "e"

Parametric boundary:The target chemical should have a value of Similarity which is <= 0.703

Interpretation of results:
other: Positive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The substance is predicted to be sensitising.
Executive summary:

The chemical 2,4,6-triisopropyl-m-phenylene diisocyanate (CAS 2162 -73 -4) was evaluated by the QSAR OECD Toolbox software for its skin sensitisation potential. The prediction was based on the measured values of chemicals assigned into the category.

The target chemical was classified as "Diisocyanates" by "US EPA New Chemical Categories". Therefore primarily analogues chemicals were searched by this profiling method. 21 chemicals were assigned into the category. The target chemical was predicted "positive" based on the experimental data of the nearest neighbours. Thereafter, the chemicals in the category were scanned for the differences relevant for the investigated endpoint. All chemicals in the category exhibited the same mechanism of binding to proteins by "Iso(thyo)cyanateprotein acyltransfer" mechanism, therefore the chemicals were not subcategorized according to this property. The chemicals in the category possess different organic functional groups but this subcategorization method destroys the category. Some of category members showed a quite high level of structural similarity. Taking only maximal values of skin sensitisation potential from available data points the chemical 1,1 methylenebis (4-isocyanatocyclohexane) (CAS 5124-30-1) appeared different by test conditions. This chemical was negative in the Guinea Pig Maximization Test while in the Buehler test it was positive. It was the only difference of this chemical to the others and by the reason that the guinea pig maximization test was the single test among 11 available tests on 5 category members the chemical was removed as an outlier.

The target chemical falls out of applicability domain because of logKow. However, the similarity of category members regarding protein binding mechanism because of the isocyanate functional group is crucial for exerting their sensitization activity and considered to be meaningful to accept prediction.

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

Skin sensitisation:

The potential of the substance toluene diisocyanate was investigated in a guinea pig maximisation test in guinea pigs by Karol, et al. (1981). The test substance was solved in olive oil and applied to skin epi- and percutaneous for topical induction and epicutaneous open or via injection for challenge purposes. Topical induction was conducted in group I with a total dose of 50 µL 1% TDI, in group II with a total dose of 50 µL 10% TDI, in group III with a total dose of 50 µL 25% TDI, in group IV with a total dose of 50 µL 100% TDI and in group V with a total dose of 100 µL 100% TDI (applied as two 25 µL applications on day 1 and two 25 µL applications to new dorsal sites on Day 3). Intradermal induction was conducted with injections of 50 µL 100% TDI into each of two dorsal sites.

Topical challenge was performed with the application of 25 µL 0.1% TDI onto clean depilated dorsal sites. These concentrations of TDI did not cause dermal irritation in control guinea pigs. Skin sensitivity (contact sensitivity) to TDI was apparent by the seventh day following exposure. After 14 days, animals were additionally evaluated for TDI sensitivity by serologic analysis and by bronchial provocation challenge. Antibodies to TDI were detected using passive cutaneous anaphylaxis, double diffusion in gel, and radiolabeled antigen binding (Farr) assays.

In the study reported here, single dermal exposures to TDI produced strong contact sensitivity in guinea pigs. Dermal sensitivity was highly specific for TDI; no cross-reaction was observed upon challenge of animals with HDI. The single dermal exposure to TDI also resulted in prolonged antibody production. The magnitude of the antibody response in the current study was dependent upon the concentration of TDI used for sensitisation. Fifty microliters of 10% TDI was sufficient to stimulate antibody production which was still demonstrable 3 months later. Antibodies were specific for TDI and showed little, if any, cross-reactivity with hexamethylene diisocyanate (HDI), diphenylmethane diisocyanate (MDI), or dicyclohexylmethane diisocyanate (hydrogenated MDI).

Dermal exposure to 100% TDI resulted in pulmonary sensitivity in a percentage of animals. Although all animals produced cytophilic antibodies to TDI as a result of dermal TDI exposure, lung reactions were observed in only some of the animals. Respiratory hypersensitivity to TDI could be demonstrated by inhalation challenge with 0.005 ppm TDI, or aerosols of either TDI -protein conjugates or conjugates of p-tolyl isocyanate with protein. Bronchial challenge with 0.005 ppm TDI vapour elicited respiratory reactions in 2 of 12 animals. However, inhalation challenge with a TDI-protein conjugate elicited reactions in 4 of 12 animals, and challenge with a TMI-protein conjugate elicited respiratory reactions in 5 of the 12 animals. All the respiratory reactions were "immediate" and occurred either during challenge or within 30 min following challenge. In challenges using isocyanate-protein conjugates, hapten-specificity of the pulmonary responses was demonstrated by the failure of aerosolised proteins to evoke pulmonary reactions in any of the animals. TDI-specific pulmonary reactions were elicited more effectively when the TDI (or TMI) was inhaled as a hapten-protein conjugate rather than as TDI vapour. This occurrence may be related to several factors such as solubility, hapten concentration, or size of hapten.

This report of immunologic sensitivity resulting from dermal exposure of animals has importance for setting an appropriate TLV for TDI. Repeated inhalation of TDI as well as dermal contact with TDI each resulted in pulmonary sensitisation to TDI. In the case of dermal contact, 1 drop (50 µL) 100%TDI applied to the skin in the absence of any adjuvant caused antibody production in 100% of animals and pulmonary sensitisation in approximately 30 -40 % of animals.

The potential of toluene diisocyanate to induce skin sensitivity was investigated in different mouse strains via the local lymph node assay to assess the impact of choice of mouse strain (Woolhiser et al., 2000). The objective of these studies was to begin to assess the response of chemical sensitizers in the LLNA across six strains of female mice (C57BL:6, SJL:J, BALB:c, B6C3F1, DBA:2 and CBA). Draining lymph node proliferation was evaluated following exposures to three concentrations of a-hexylcinnamaldehyde (HCA), a moderate contact sensitizer which is one of the chemicals recommended by the OECD as a positive control for the LLNA. In addition, the strong contact sensitizer 2,4-dinitrofluorobenzene and the potent IgE-mediated sensitizer toluene diisocyanate were evaluated at single, moderate concentrations as positive controls for T cell mediated and IgE-mediated responses. The six murine strains demonstrated varying levels of lymph node proliferation following exposure to three chemical sensitizers. These studies suggest that the specific combination of strain and antigen may be more important than a strain’s Th1:Th2 predominance.

DBA:2, B6C3F1, BALB:c and CBA mice had essentially equal levels of lymph node proliferation following exposure to the three chemicals. While C57BL:6 mice gave similar results as CBA mice following DNFB and HCA administration, the LLNA response to TDI was considerably lower. SJL mice provided low stimulation indexes (SI) values for all three chemicals evaluated. Regardless of the level of LLNA response, all six mouse strains identified the sensitisation potential of HCA, TDI or DNFB. Based on these studies, DBA:2, B6C3F1 and BALB:c mice are good choices for continued evaluation as additional mouse strains for use in the LLNA.

Although C57BL:6 mice are reported to be predisposed to Th1 immune responses (Särnstrand et al., 1999) and as such had the lowest response to the IgE inducing chemical TDI, SJL mice (low IgE responders) gave the highest dpm response to TDI. The other four strains demonstrated high proliferative responses to all three chemicals irrespective of any possible Th1:Th2 predominance.

Regardless of the lower SI values, SJL mice still identified all three chemicals as sensitizers according to 3 -fold SI values, statistical significance and dose responsive proliferation. This initial set of studies highlights the importance of mouse strain when developing assay models. While CBA mice were verified as a good selection for the LLNA, the data from these studies suggest DBA:2, B6C3F1 and BALB:c mice are essentially equal to CBA mice when evaluating the sensitising potential of these three chemicals. While C57BL:6 mice gave similar results as CBA mice following treatment with DNFB and HCA, the LLNA response to TDI was considerably lower than those of DBA:2, B6C3F1, BALB:c and CBA mice. Based on this single series of studies, DBA:2, B6C3F1 and BALB:c mice appear to be reasonable alternative strains for evaluation for use in the LLNA.

Van de Briel and co-workers investigated whether the local lymph node assay (LLNA) was sufficient to not only identify allergens but also mark them as either a contact or a respiratory allergen (van de Briel 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 sensitisation 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.

Prediction using the OECD QSAR Toolbox (v 2.2)

The chemical 2,4,6-triisopropyl-m-phenylene diisocyanate (CAS 2162 -73 -4) was evaluated by the QSAR OECD Toolbox software for its skin sensitisation potential. The prediction was performed by read-across and was based on the measured values of chemicals assigned into the category. The target chemical was classified as "Diisocyanates" by "US EPA New Chemical Categories". Therefore primarily analogues chemicals were searched by this profiling method. 21 chemicals were assigned into the category. The target chemical was predicted "positive" based on the experimental data of the nearest neighbours. Thereafter, the chemicals in the category were scanned for the differences relevant for the investigated endpoint. Although the chemicals in the category possessed different organic functional groups they all exhibited the same mechanism of binding to proteins by "Iso(thyo)cyanateprotein acyltransfer" mechanism. Some of category members showed a quite high level of structural similarity. Taking only maximal values of skin sensitization potential from available data points the chemical 1,1-methylenebis (4-isocyanatocyclohexane) (CAS 5124-30-1) appeared different by test conditions. This chemical was negative in the Guinea Pig Maximization Test while in the Buehler test it was positive. It was the only difference of this chemical to the others and by the reason that the guinea pig maximization test was the single test among 11 available tests on 5 category members the chemical was removed as outlier.

The target chemical falls out of applicability domain because of logPow. TRIDI is the most lipophilic chemical among category members (the highest LogPow). However, the similarity of category members regarding protein binding mechanism because of isocyanate functional group is crucial for exerting their sensitization activity and considered to be meaningful to accept prediction.

The chemical 2,4,6-triisopropyl-m-phenylene diisocyanate (CAS 2162 -73 -4) was predicted to be sensitizing also by the existing SAR model "Danish EPA DB" included in the Toolbox.

Prediction using TOXTREE

According to the results of TOXTREE modelling tool, 2,4,6-triisopropyl-m-phenylene diisocyanate is estimated to be positive for skin sensitization potential. The test substance contains the structural alerts - carbon and nitrogen double bond - are identified for the possibility of nucleophilic substitution, Shiff base formation or addition-elimination reactions. There are no functional groups which can act as Michael acceptors.

 


Migrated from Short description of key information:
1) Read across TDI, Karol et al, 1981, Guinea pig maximisation test, pure test substance, sensitizing to skin and respiratory system
2) Read across TDI, Woolhiser et al., 2000, mice (C57BL:6, SJL:J, BALB:c, B6C3F1, DBA:2 and CBA), LLNA, sensitising
3) Read across TDI, Vandebriel et al., 2000, mouse, LLNA, sensitising
4) Toxtree prediction for TRIDI, Chemservice S.A., 2011
5) Toolbox export file - structural analogues (diisocyanates), test item is predicted to be sensitising
6) Toolbox export file - Danish OSAR model - sensitising
7) Prediction using Toxtree - functional groups which can trigger allergic reaction (Shiff base formation), no functional groups which can act as Michael acceptors

Justification for selection of skin sensitisation endpoint:
The prediction by the OSAR OECD Toolbox is considered to be reliable. Moreover, the prediction is in line the sensitisation result of the nearest analogue TDI (also sensitising).

Respiratory sensitisation

Link to relevant study records
Reference
Endpoint:
respiratory sensitisation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented publication, which meets basic scientific principles
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
An animal exposure experiment, simulating a workplace exposure situation was made to compare toluene diisocyanate (TDI) concentrations which resulted in antibody production with those which elicited pulmonary responses. Groups of guinea pigs were exposed to inhaled TDI from 0.02 to 1.0 ppm (µg/g) for 3 h/day on 5 consecutive days. Three weeks later the animals were challenged with 0.02 ppm of free TDI for 15 min. TDI specific antibodies and pulmonary responses were evaluated. Using an experimental model in which animals were sensitized and challenged by inhalation of free TDI, the present study was performed in order to compare the TDI concentration which resulted in antibody production with that which elicited a pulmonary response, and secondly, to provide an experimental basis for setting up OELs of chemical sensitizers.
GLP compliance:
not specified
Test material information:
Composition 1
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS:
- Source:
- Age at study initiation:
- Weight at study initiation: 300-350 g
- Housing: group housed two per cage
- Diet (e.g. ad libitum): food ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 1 week
Route of induction exposure:
inhalation
Route of challenge exposure:
inhalation
Remarks:
induction: whole body, challenge head-nose-only
Vehicle:
other: clean dry air
Concentration:
Induction: 0 ppm, 0.02 ppm, 0.2 ppm, 0.6 ppm, 1 ppm
Challenge: 0.02 ppm
No. of animals per dose:
6 females
Details on study design:
RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 5
- Exposure period: 5 days
- Test groups: 5
- Control group: 1
- Site: not applicable inhalation
- Frequency of applications: once daily
- Duration: 3 h
- Concentrations: 0 ppm, 0.02 ppm, 0.2 ppm, 0.6 ppm and 1 ppm

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: 15 min
- Test groups: Groups 2 - 6 induced with 0.02 ppm, 0.2 ppm, 0.6 ppm and 1 ppm
- Control group: Group 1 - no induction = 0 ppm TDI
- Site: not applicable inhalation
- Concentrations: 0.02 ppm were used for challenge
- Evaluation (hr after challenge): during challenge and up to 60 min after challenge
- Other: Twenty-six days after the first induction exposure, the animals were placed in individual body plethysmographs with the head of each animal extending through a latex dam into an inhalation chamber, and were challenged by exposure to 0.02 ppm TDI for 15 min. The concentration of TDI at challenge was chosen because a short exposure to that level was previously shown not to be an irritant to guinea pigs.

OTHER:
The actual TDI concentrations (2-+- SD) were 1.128 +/- 0.125, 0.64 +/- 0.078, 0.218 +/- 0.046 and 0.022 +/- 0.003 ppm at induction, and 0.019 +/- 0.002 ppm at challenge.

TDI aerosol was generated by passing air through a midget impinger containing TDI solution and was led into the inhalation chamber after dilution with clean, dry air to achieve the appropriate concentration. TDI concentrations were determined by the method of Marcali (1957).
Challenge controls:
Six animals of an additional group were sham-exposed.
Positive control substance(s):
toluene diisocyanate (TDI)
Negative control substance(s):
other: clean dry air
Table2. Pulmonary responses following ; inhalation challenge to free toluene diisocyanate (TDI)
Group Induction conc,(ppm) Challengeaconc, (ppm) Pulmonary responsea Responding animals/total
I 1.0 0.02 145±12 5/6
II 0.6 0.02 161±40 4/6
III 0.2 0.02 168±32 6/6
IV 0.02 0.02 120±3 0/6
V 0 0.02 114±7 0/6
ax ±SD of % pre-challenge respiratory rate demonstrated in individual animals. Percent pre-challenge rate used here was derived from the most severe response exhibited in individual animals during an observation period.
Production of Specific Antibodies

The time course of IgG antibody production was investigated by ELISA. Serum which gave an absorbance of 0.054 or greater was defined as having a positive IgG response to TDI.

IgG antibodies were detected 6 days after the first exposure in several animals of the 1.0, 0.6 and 0.2 ppm groups (4/6, 2/6, and 2/6, respectively). At 13 days, the antibodies were detected in all animals of these groups. The peak mean absorbance in these groups was reached 20 days following the first exposure. However, IgG antibodies were not detected from animals exposed to 0.02 ppm and control animals. PCA titres showed a linear correlation to TDI concentration at induction, as did IgG antibody production (p < 0.01). The findings were consistent with the results of our previous study using TDI-GSA conjugates at challenge. IgE antibodies were not detected from any animal.

Pulmonary Response

Animals were challenged by inhalation for 15 min of 0.02 ppm free TDI 26 days after the first induction exposure. Pulmonary responses are shown in Table 2. None of the six animals exposed to 0.02 ppm TDI at induction showed significant pulmonary responses (Group IV). By contrast, most of the animals exposed to TDI above 0.2 ppm (1.0, 0.6 and 0.2 ppm groups) displayed significant pulmonary responses. There was no linear correlation between TDI concentration at induction and the intensity of pulmonary response upon challenge to free TDI (p > 0.05). Further, no significant difference in the proportion of animals showing a pulmonary response was found among these three groups (p > 0.05, Fisher's test).

Comparison of Antibody Production and Pulmonary Response

A comparison was made between antibody production and the experience of pulmonary response. As presented in Table 3, the proportion of animals showing a pulmonary response was not closely related to the antibody production. Although animals which had no antibody production did not display pulmonary responses, animals with high antibody production did not inevitably exhibit pulmonary responses. In contrast, some of the animals showing low antibody production were found to give pulmonary responses.

Table3. Comparison of antibody production and pulmonary response in guinea pig exposed to toluene diisocyanate (TDI)
Group Induction conc, (ppm)  ELISA absorbance a PCA titerb Pulmonary responsec
I 1.0 0.285 29 -
0.262 29 +
0.237 28 +
0.228 27 +
0.187 27 +
0.175 26 +
II 0.6 0.274 2io +
0.208 29 +
0.222 26
0.195 2s ++
0.093 25 +
0.171 24 -
III 0.2 0.190 26 +
0.156 25 +
0.153 24 +
0.130 24 + +
0.103 23 +
0.068 22 +
IV 0.02 0.030 nd -
0.028 nd -
0.027 nd -
0.015 nd -
0.003 nd -
0.009 nd -
V 0 0.038 nd -
0.041 nd -
0.001 nd -
0.001 nd -
0.015 nd -
0.003 nd -
nd=Not detected
a An absorbance/>0.054 (2 + 2SD of control responses) was defined as IgG antibody positive
 bTitre of IgG 1 antibodies
cResponse (+): an increase in respiratory rate to 127 % (Mean +/- 3SD of control response) or more of the pre-challenge rate within the challenge and 60 min after challenge. (++): 193 % (Mean +/- 10 SD of control response) or more. (-): 126 % or less.
Interpretation of results:
sensitising
Conclusions:
The publication is based on experiments with guinea pigs exposed via inhalation to toluene diisocyanate and is evaluating the potential of causing respiratory sensitisation. It 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:

Aoyama and co-workers conducted an animal exposure experiment, in which animals were sensitized and challenged by inhalation of free TDI, so

simulating a workplace exposure situation to compare toluene diisocyanate (TDI) concentrations which resulted in antibody production with those which elicited pulmonary responses and secondly, to provide an experimental basis for setting up OELs of chemical sensitizers. Therefore groups of guinea pigs were exposed to TDI from 0.02 to 1.0 ppm for 3 h/day on 5 consecutive days. Three weeks later the animals were challenged with 0.02 ppm of free TDI for 15 min. TDI specific antibodies and pulmonary responses were evaluated. Specific antibody production showed a linear correlation to TDI concentration at induction. An induction level of 0.02 ppm TDI failed to stimulate antibody production in animals. Most of the animals exposed to TDI levels above 0.2 ppm displayed significant pulmonary responses, but no correlation was found between TDI concentration at induction and the intensity of pulmonary response upon challenge to free TDI. These results indicated that there was a threshold concentration of 0.02 ppm TDI for antibody production and for the development of pulmonary response. Although it failed to demonstrate sensitizing potency, 0.02 ppm free TDI did elicit a pulmonary response in some of the animals sensitized to TDI at levels ranging from 0.2 to 1.0 ppm. It was also found that exposure to TDI at a level lower than its threshold concentration for sensitisation may elicit a response in previously sensitized individuals. The intensity of pulmonary response was not notably influenced by TDI concentration at induction levels above 0.2 ppm. This result may be related to several factors such as solubility, hapten concentration, or size of hapten. This finding is also of great importance in extrapolating from the results of an animal model to an industrial situation. However, free TDI was able to elicit immediate-onset reactions, within 60 min following the challenge. No close association was seen between antibody production and the experience of pulmonary response. High antibody production did not stick to the development of pulmonary response. On the other hand, several animals with low antibody titres displayed positive pulmonary responses. This discrepancy might be derived from differences in the ability to release histamine due to the different distribution of specific antibodies in the lungs of animals.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Aoyama and co-workers conducted an animal exposure experiment (Ayama et al., 1994), in which animals were sensitized and challenged by inhalation of free TDI, so simulating a workplace exposure situation to compare toluene diisocyanate (TDI) concentrations which resulted in antibody production with those which elicited pulmonary responses and secondly, to provide an experimental basis for setting up OELs of chemical sensitizers. Therefore groups of guinea pigs were exposed to TDI from 0.02 to 1.0 ppm for 3 h/day on 5 consecutive days. Three weeks later the animals were challenged with 0.02 ppm of free TDI for 15 min. TDI specific antibodies and pulmonary responses were evaluated. Specific antibody production showed a linear correlation to TDI concentration at induction. An induction level of 0.02 ppm TDI failed to stimulate antibody production in animals. Most of the animals exposed to TDI levels above 0.2 ppm displayed significant pulmonary responses, but no correlation was found between TDI concentration at induction and the intensity of pulmonary response upon challenge to free TDI. These results indicated that there was a threshold concentration of 0.02 ppm TDI for antibody production and for the development of pulmonary response. Although it failed to demonstrate sensitizing potency, 0.02 ppm free TDI did elicit a pulmonary response in some of the animals sensitized to TDI at levels ranging from 0.2 to 1.0 ppm. It was also found that exposure to TDI at a level lower than its threshold concentration for sensitisation may elicit a response in previously sensitized individuals. The intensity of pulmonary response was not notably influenced by TDI concentration at induction levels above 0.2 ppm. This result may be related to several factors such as solubility, hapten concentration, or size of hapten. This finding is also of great importance in extrapolating from the results of an animal model to an industrial situation. However, free TDI was able to elicit immediate-onset reactions, within 60 min following the challenge. No close association was seen between antibody production and the experience of pulmonary response. High antibody production did not stick to the development of pulmonary response. On the other hand, several animals with low antibody titres displayed positive pulmonary responses. This discrepancy might be derived from differences in the ability to release histamine due to the different distribution of specific antibodies in the lungs of animals.

Karol and co-workers investigated the potential of toluene diisocyanate to induce respiratory sensitisation in guinea pigs (Karol, et al., 1981). The test substance was solved in olive oil and applied to skin epi- and percutaneous for topical induction and epicutaneous open for challenge purposes. Topical induction was conducted in group I with a total dose of 50 µL 1% TDI, in group II with a total dose of 50 µL 10% TDI, in group III with a total dose of 50 µL 25% TDI, in group IV with a total dose of 50 µL 100% TDI and in group V with a total dose of 100 µL 100% TDI (applied as two 25 µL applications on Day 1 and two 25 µL applications to new dorsal sites on Day 3). Intradermal induction was conducted with injections of 50 µL 100% TDI into each of two dorsal sites. Topical challenge was performed with the application of 25 µL 0.1% TDI onto clean depilated dorsal sites. These concentrations of TDI did not cause dermal irritation in control guinea pigs. Skin sensitivity (contact sensitivity) to TDI was apparent by the seventh day following exposure. After fourteen days, animals were additionally evaluated for TDI sensitivity by serologic analysis and by bronchial provocation challenge. Antibodies to TDI were detected using passive cutaneous anaphylaxis, double diffusion in gel, and radiolabeled antigen binding (Farr) assays.

Dermal exposure to 100% TDI resulted in pulmonary sensitivity in a percentage of animals. Although all animals produced cytophilic antibodies to TDI as a result of dermal TDI exposure, lung reactions were observed in only some of the animals. Respiratory hypersensitivity to TDI could be demonstrated by inhalation challenge with 0.005 ppm TDI, or aerosols of either TDI -protein conjugates or conjugates of p-tolyl isocyanate with protein. Bronchial challenge with 0.005 ppm TDI vapour elicited respiratory reactions in 2 of 12 animals. However, inhalation challenge with a TDI-protein conjugate elicited reactions in 4 of 12 animals, and challenge with a TMI-protein conjugate elicited respiratory reactions in 5 of the 12 animals. All the respiratory reactions were "immediate" and occurred either during challenge or within 30 min following challenge. In challenges using isocyanate-protein conjugates, hapten-specificity of the pulmonary responses was demonstrated by the failure of aerosolised proteins to evoke pulmonary reactions in any of the animals. TDI-specific pulmonary reactions were elicited more effectively when the TDI (or TMI) was inhaled as a hapten-protein conjugate rather than as TDI vapour. This occurrence may be related to several factors such as solubility, hapten concentration, or size of hapten.

This report of immunologic sensitivity resulting from dermal exposure of animals has importance for setting an appropriate TLV for TDI. Repeated inhalation of TDI as well as dermal contact with TDI each resulted in pulmonary sensitisation to TDI. In the case of dermal contact, 1 drop (50 µL) 100% TDI applied to the skin in the absence of any adjuvant caused antibody production in 100% of animals and pulmonary sensitisation in approximately 30 -40 %of animals.

In the study reported here, single dermal exposures to TDI produced strong contact sensitivity in guinea pigs. Dermal sensitivity was highly specific for TDI; no cross-reaction was observed upon challenge of animals with HDI. The single dermal exposure to TDI also resulted in prolonged antibody production. Antibodies had previously been found to result from dermal contact with chemicals in guinea pigs (Chase, 1948) and in mice (Thomas et al,1976). The magnitude of the antibody response in the current study was dependent upon the concentration of TDI used for sensitization. Fifty microliters of 10% TDI was sufficient to stimulate antibody production which was still demonstrable 3 months later. The specificity of the humoral antibody response to TDI was also highly specific for TDI. In gel diffusion studies, precipitin lines were observed only with sera and TDI conjugates; no reactions were seen with HDI, MDI, or HMDI conjugate antigens. Similarly, in the more sensitive radiolabeled antigen-binding assay, antibodies appeared highly specific for TDI.

 


Migrated from Short description of key information:
1) Read across TDI, Ayoama et al., 1994, guinea pigs, sensitising
2) Read across TDI, Karol et al, 1981, guinea pig maximisation test, pure test substance, sensitising to skin and respiratory system

Justification for selection of respiratory sensitisation endpoint:
No respriatory sensitisation studies are available for TRIDI. Therefore, a study in guinea pigs conducted with TDI, the nearest analogue, is used for the derivation of a specific DNEL for respiratory sensitization.

Justification for classification or non-classification

Skin sensitization:

The test material does meet the criteria for classification and will require labelling for skin sensitisation (Category 1) in accordance with European regulation (EC) No. 1272/2008.

Respiratory sensitization:

The test material does meet the criteria for classification and will require labelling for respiratory sensitisation (Category 1) in accordance with European regulation (EC) No. 1272/2008.