2-isocyanatoethyl methacrylate

Respiratory sensitisation

Administrative data

Endpoint:

respiratory sensitisation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No official guidance is available for the investigation of respiratory sensitisation. However, the study presented here meets generally accepted scientific standards and reveals positive responses. Hence, the data were considered to be suitable for classification.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1983

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Male guinea pigs were investigated for the development of respiratory sensitisation after inhalation exposure to protein-conjugated test item (conjugate). Challenge with protein-conjugated test material, the unconjugated test material (monomer), as well as copolymerised test item (polymer) was conducted to determine the nature of respiratory response.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

2-isocyanatoethyl methacrylate

EC Number:

250-284-7

EC Name:

2-isocyanatoethyl methacrylate

Cas Number:

30674-80-7

Molecular formula:

C7H9NO3

IUPAC Name:

2-isocyanatoethyl methacrylate

Test material form:

other: aerosol (BSA-IEM conjugate and polymer), and vapour (IEM monomer)

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass., USA
- Weight at arrival: 350-400 g
- Acclimation period: 10 days

No further details are given in the publication.

Test system

Route of induction exposure:

inhalation

Route of challenge exposure:

inhalation

Vehicle:

other: air

Concentration:

- Monomer: 0.01, 0.1, 0.3, 0.4, 0.5, and 0.6 ppm
- Conjugate: 24, 72, 89, 90, 92, and 98% conjugation with 37±10, 54±14, 31±9, 49±24, 47±7, 42±8 g/L average chamber protein concentration, respectively
- Polymer: 0.5 mg/L

No. of animals per dose:

4 males

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
- Exposure period: 5 min control period; 10 min exposure to aerosolised BSA-IEM; and 5 min recovery period
- Test groups: BSA protein conjugate (BSA-IEM)
- Control group: unconjugated BSA
- Frequency of applications: once daily, 5 days/week
- Duration: until a positive respiratory response occurred
- Concentrations: 24, 72, 89, 90, 92, and 98% conjugated BSA-IEM

B. CHALLENGE EXPOSURE
- No. of exposures: 4
- Day(s) after positive response during induction exposure: 1 (unconjugated BSA and GSA-IEM, respectively, with 1 h rest period inbetween); 10 (unconjugated BSA, only if no positive response with GSA-IEM was obtained); animals induced with 92 and 98% conjugated BSA-IEM were challenged with monomer on days 1 and 12, and additional with BSA-IEM on days 5 and 6
- Exposure period: 5 min control period; 10 min exposure to aerosolised test material; and 5 min recovery period (BSA-IEM) or 6 h (monomer or polymer, respectively, due to human data reporting that delayed response)
- Test groups: unconjugated BSA and GSA-IEM, unconjugated BSA and monomer, or unconjugated BSA and polymer, respectively
- Control group: unconjugated BSA
- Concentrations: 24, 72, 89, 90, 92, and 98% conjugated BSA-IEM
- Other: Animals of the 90% BSA-IEM test group were treated with disodium cromoglycate (DSCG), a bronchial asthma prophylactic agent (60 µg/L for 10 min) 30 min prior to exposure on test days 10 and 16. These animals were killed immediately after exposure during the 3rd exposure week and the lungs were examined histopathologically. One of these animals were killed immediately after a positive response.

Negative control substance(s):

other: unconjugated BSA

Results and discussion

Results:

Results:
Responses to the conjugate generally occurred after several minutes of exposure and subsided during the 5-min recovery period following exposure. The number of animals developing respiratory responses was related to the degree of IEM conjugation on protein. 2/4 animals exposed to the 24% conjugate, 3/4 animals exposed to the 72% conjugate, and all animals exposed to 89% and greater conjugates developed positive responses during the 2nd or 3rd week of exposure. Often the responses were transient and lasted only several days. However, the responses could be evoked again and if animals were rechallenged after a 1-week rest period. When animals responsive to the BSA-IEM, 80% responded to the GSA-IEM conjugate, while none responded to unconjugated BSA. Those challenged with GSA alone also responded negatively. The authors considered this result as a strong evidence that IEM portion of the molecule was critical for eliciting the response.
All animals exposed to the 90% BSA-IEM conjugate developed respiratory responses within 8-15 days after study initiation. They were treated with DSCG, which was administered 30 min prior to exposure on test days 10 and 16. None of the animals had positive respiratory responses on the days when DSCG was preadministered.1 animal responded on the subsequent day after DSCG preadministration again, while others responded several days later when challenged with the BSA-IEM conjugate aerosol. The authors conclude that DSCG diminished the respiratory response to BSA-IEM. No exposure related gross or histopathologic abnormalities were observed in any of these animals.
Two groups of animals exposed to the BSA-IEM conjugate (92 and 98%) were challenged with the IEM monomer. No immediate or delayed response occurred in animals challenged with 0.01 ppm monomer. Exposure to 0.1-0.4 ppm monomer vapour elicited repiratory responses in some of the BSA-IEM responsive animals. The responses were qualitatively identical to those induced by the conjugate, i.e. similar increases in respiratory rate. However, the responses to the monomer were delayed, occurring 1-5 h post exposure and generally lasted longer than those induced by the conjugates. No responses were seen, when control animals were exposed to the monomer. Challenge with 0.5 and 0.6 ppm monomer produced severe upper respiratory tract irritation, as indicated by a 30 and 50% decrease in respiration during exposure, respectively, in both control animals and and those previously induced with the conjugate. No delayed responses were detected at these concentrations. The authors stated that the recovery from irritation was slow and may have masked a positive respiratory response.
None of the animals responsive to BSA-IEM and challenged with the polymer displayed a positive response when monitored during exposure or the 6 h recovery period.

Discussion:
The authors conclude from this study that IEM is likely to act as hapten, inducing an immune response after conjugation to a larger molecule. The positive response of the test animals to challeng wit GSA-IEM conjugate and to the IEM monomer was evidence that the haptenic group rather than the carrier was responsible for eliciting the asthmatic response. The delayed response to challenge with monomer supports the likelihood that a protein-isocyanate conjugation reaction occurred in vivo prior to respiratory reaction.
The authors suggested that the test material induced an immediate (Type I) hypersensitivity, since an increase of the respiratory rate followed in some cases by gasping respiration was observed already within 10-14 days of exposure. Further support consists of inhibition of the response by DSCG, which prevents allergic asthmatic attack by inhibiting the release of mediators of anaphylaxis initiated by the interaction of antigens with reagenic (IgE-type) antibodies. In the present study the respiratory responses lasted for only a few days. The authors suggest that this finding might correspond to a built-up first of reagenic (IgE-type) antibodies followed within days by the production of blocking (IgG) antibodies. However it may be that the loss of response corresponded to a depletion of mediator (reagenic antibodies) due to the frequency of challenge. Therefore, a rest period allowed the mediator to again built up and responses could be reinduced.
The authors also suggest that the respiratory response in sensitised guinea pigs is to some extent dose related. In the BSA-IEM treatment the number of animals which became responsive was related to the degree of conjugation of the carrier protein. However, there was no correlation between degree of conjugation and the time of onset of response (min after exposure), response severity, or duration of response. The response to challenge with the IEM monomer was concentration related at levels below 0.5 ppm in both the number of animals responding and and duration and severity of response. Higher concentrations resulted in sensory irritation and respiratory slowing, which may have obscured any potential asthmatic respinses. The lack of response to the polymer also suggested that a critical level of monomer was needed to trigger a response.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS Category 1 (H334) according to Regulation (EC) No 1272/2008