Registration Dossier

Administrative data

Description of key information

Skin sensitisation (non-guideline study): sensitising

Respiratory sensitisation (no guideline available): sensitising



Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male guinea pigs were induced with the test item by a series of 3 intradermal injections. After a 2 week rest period the animals received the epicutaneous challenge exposure, and following 2 further weeks of rest the animals were rechallenged. After additional 2 weeks, the animals received a challenge exposure with the respective polymer of the test item, which was spiked with the monomer (the test ietm itself). Naive control animals were included into the test.
GLP compliance:
no
Type of study:
other: intradermal induction without adjuvant and epicutaneous challenge
Justification for non-LLNA method:
Test was done before LLNA as first-choice method for in-vivo testing was set into force.
Species:
guinea pig
Strain:
other: albino
Sex:
male
Details on test animals and environmental conditions:
No details are given in the study report.
Route:
intradermal
Vehicle:
other: dimethyl phthalate (intradermal injections) and acetone (topical applications)
Concentration / amount:
1%
Route:
epicutaneous, open
Vehicle:
other: dimethyl phthalate (intradermal injections) and acetone (topical applications)
Concentration / amount:
- challenge: 0.01, 0.1, and 1% solution- rechallenge: 0.05, 0.1, 0.5, and 1% solution- challenge with polymer: 50% solution, spiked with 0.01, 0.1, and 1% monomer
No. of animals per dose:
9 males
Details on study design:
RANGE FINDING TESTS: A preliminary primary irritation test was conducted by applying, and lightly rubbing in, a drop (=0.05 mL) each of a 0.25 and 2.5% (v/v) solution of the test item (monomer) in acetone on the shaved intact skin of 10 male albino guinea pigs. Skin reactions were investigated 24 and 48 h after exposure. MAIN STUDYA. INDUCTION EXPOSURE- No. of exposures: 3 - Exposure period: 3 single intradermal injections- Test groups: 0.1 mL of the test item (monomer) in dimethyl phthalate- Control group: Naive control animals were included into the study. - Site: sacrum- Frequency of applications: one each week over a two week period- Duration: days 0-14- Concentrations: 1%B. CHALLENGE EXPOSURE- No. of exposures: 3 (challenge and rechallenge with monomer (test item), and a second rechallenge with polymer spiked with the monomer)- Day(s) of challenge: 28 (challenge); 42 (rechallenge); 66 (second rechallenge)- Exposure period: no data- Test groups: 1 drop (=0.05 mL) of the test item in acetone- Control group: naive control animals received 1 drop (=0.05 mL) of the test item in acetone- Site: shoulder- Concentrations: 0.01, 0.1, and 1% (challenge); 0.05, 0.1, 0.5, and 1% (rechallenge); 50% polymer spiked with 0.01, 0.1, and 1% monomer (second challenge)- Evaluation (hr after challenge): 24 and 48 h
Challenge controls:
Only naive control animals were included into the study.
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.01 and 0.1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1%
No. with + reactions:
8
Total no. in group:
9
Clinical observations:
5 animals showed moderate erythema, 3 animals showed mild erythema, and 1 animal did not show any skin reactions.
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.01, 0.1, and 1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.01 and 0.1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1%
No. with + reactions:
7
Total no. in group:
9
Clinical observations:
5 animals showed moderate erythema, 2 animals showed mild erythema, and 2 animals did not show any skin reactions.
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.01, 0.1, and 1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
0.05 and 0.1 %
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
0.5%
No. with + reactions:
4
Total no. in group:
9
Clinical observations:
2 animals showed moderate erythema, 2 animals showed mild erythema, and 5 animals did not show any skin reactions.
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
1%
No. with + reactions:
9
Total no. in group:
9
Clinical observations:
3 animals showed moderate erythema and 6 animals showed mild erythema.
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0.05, 0.1, 0.5, and 1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0.05 and 0.1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0.5%
No. with + reactions:
3
Total no. in group:
9
Clinical observations:
3 animals showed mild erythema and 6 animals did not show any skin reactions.
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
1%
No. with + reactions:
7
Total no. in group:
9
Clinical observations:
1 animal showed moderate erythema and 8 animals showed mild erythema.
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0.05, 0.1, 0.5, and 1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
none stated in the study report

Results of test (ctd.)

Reading

Hours after challenge

Group

Dose level

No. with + reactions

Total no. in group

Clinical observations

second rechallenge

24

test group

50% polymer with 0.01% monomer (test item)

2

9

2 animals showed mild erythema and 7 animals did not show any skin reactions.

second rechallenge

24

test group

50% polymer with 0.1% monomer (test item)

6

9

1 animal showed moderate erythema, 5 animals showed mild erythema, and 2 animals did not show any skin reactions.

second rechallenge

24

test group

50% polymer with 1% monomer (test item)

8

9

1 animal showed erythema plus oedema, 5 animals showed moderate erythema, 2 animals showed mild erythema, and 1 animal did not show any skin reactions.

second rechallenge

24

negative control

50% polymer with 0.01% monomer (test item)

2

9

2 animals showed mild erythema and 7 animals did not show any skin reactions.

second rechallenge

24

negative control

50% polymer with 0. 1% monomer (test item)

6

9

6 animals showed mild erythema and 3 animals did not show any skin reactions.

second rechallenge

24

negative control

50% polymer with 1% monomer (test item)

8

9

8 animals showed mild erythema and 1 animal did not show any skin reactions.

second rechallenge

48

test group

50% polymer with 0.01% monomer (test item)

3

9

3 animals showed mild erythema and 6 animals did not show any skin reactions.

second rechallenge

48

test group

50% polymer with 0. 1% monomer (test item)

6

 9

1 animal showed moderate erythema, 5 animals showed mild erythema, and 3 animals did not show any skin reactions.

second rechallenge

48

test group

50% polymer with 1% monomer (test item)

7

9

2 animals showed moderate erythema, 5 animals showed mild erythema, and 2 animals did not show any skin reactions.

second rechallenge

48

negative control

50% polymer with 0.01% monomer (test item)

1

9

1 animal showed mild erythema and 8 animals did not show any skin reactions.

second rechallenge

48

negative control

50% polymer with 0. 1% monomer (test item)

6

9

6 animals showed mild erythema and 3 animals did not show any skin reactions.

second rechallenge

48

negative control

50% polymer with 1% monomer (test item)

7

9

1 animal showed moderate erythema, 6 animals showed mild erythema, and 2 animals did not show any skin reactions.

Interpretation of results:
other: CLP/EU GHS Category 1A (H317) according to Regulation (EC) No 1272/2008
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In the available key study (DuPont, 1976), which predates the appropriate OECD test guideline and GLP, the test item was investigated for skin sensitising properties. Male guinea pigs were induced with the test item by a series of 3 intradermal injections of a 1% test material solution in dimethyl phthalate, but no additional treatment with adjuvant was included into the study. After a 2 week rest period the animals received the epicutaneous challenge exposure with 0.01, 0.1, and 1% solutions in acetone, and following 2 further weeks of rest the animals were rechallenged with 0.05, 0.1, 0.5, and 1% solutions in acetone. After another 2 weeks, the animals received a challenge exposure with the respective polymer of the test item (50% solution in acteone), which was spiked with the monomer (the test item itself) at concentrations of 0.01, 0.1, and 1%. Naive animals were included as controls into the test. 24 and 48 h after challenge exposure with 1% test material solution, 8/9 and 7/9 animals, respectively, were observed with skin reactions, whereas 5 animals showed moderate erythema after both 24 and 48 h, and 3 animals and 2 animals showed mild erythema after 24 and 48 h, respectively. Animals treated with lower concentrations did not show any skin reactions. Rechallenge with 0.5 and 1% test item solutions revealed 4/9 and 9/9 animals with skin reactions 24 h post treatment, whereas the first exposure revealed 2 animals with moderate erythema and 2 animals with mild erythema, and the latter exposure resulted in 3 animals with moderate erythema and 6 animals with mild erythema. After 48 h 3/9 and 7/9 animals treated with 0.5 and 1% concentrations, respectively, still showed skin reactions, with 3 animals with mild erythema in the 0.5% dose group and 1 animal with moderate erythema and 8 animals with mild erythema in the 1% dose group. Treatment with 0.05 and 0.1% test substance solution again did not result in skin reactions. Exposure to the polymer spiked with the monomer also exhibited skin reactions. However, also the naive control animals showed comparable skin reactions when treated with the spiked polymer.

Based on the outcome of the study, the test material meets the criteria to be classified as a skin sensitiser (Xi, R43) according to 67/584/EEC and (Cat 1A, H317) according to EC/1272/2008.


Respiratory sensitisation

Link to relevant study records
Reference
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.
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
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Breeding Laboratories, Wilmington, Mass., USA- Weight at arrival: 350-400 g- Acclimation period: 10 daysNo further details are given in the publication.
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 STUDYA. 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-IEMB. 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:
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.
Interpretation of results:
other: CLP/EU GHS Category 1 (H334) according to Regulation (EC) No 1272/2008
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In the available key study (Mullin et al., 1983) the test item was investigated for the risk of respiratory sensitisation after exposure via inhalation. No information is available, whether the study was performed in compliance with GLP, and no guideline is available, which adresses this endpoint. Several further test materials were prepared in addition to the submission substance (IEM, monomer) prior to the study: IEM was conjugated to bovine serum albumin (BSA-IEM) or to guinea pig serum albumin (GSA-IEM), and additionally a polymer (styrene/IEM/dodecyl mercaptan polymer (44/46/10) as a 20% solution in dimethyl phthalate), which contained less than 0.0004% total monomer, was prepared. Once daily for 5 days/week male albino guinea pigs were induced only with the BSA-IEM conjugate via inhalation at concentrations of 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. Exposures were carried out until respiratory responses were observed. Challenge was carried out with GSA-IEM conjugate, the IEM monomer, or the polymer, respectively. Control animals were treated with BSA alone.

The study revealed that respiratory 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, if animals were rechallenged after a 1-week rest period. When animals were responsive to the BSA-IEM, 80% also 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.

Based on the outcome of the study, the test material meets the criteria to be classified as a respiratory sensitiser (Xn, R42) according to 67/584/EEC and (Cat 1, H334) according to EC/1272/2008.


Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on this data, the registered substance meets the criteria to be classified for skin sensitisation (Xi, R43) and respiratory sensitisation (Xn, R42) according to 67/584/EEC and skin sensitisation (Cat 1A, H317) and respiratory sensitisation (Cat 1, H334) according to EC/1272/2008.