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EC number: 915-671-3 | CAS number: -
Toxicological information
Skin sensitisation
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Administrative data
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1989-11-21 to 1989-12-22
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 989
- Report date:
- 1990
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- adopted 12 May 1981
- Deviations:
- yes
- Remarks:
- topical induction did not cause irritation, semi-occlusive dressing induction
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.6 (Skin Sensitisation)
- Version / remarks:
- Version: March 1984
- Deviations:
- yes
- Remarks:
- topical induction did not cause irritation, semi-occlusive dressing induction
- GLP compliance:
- yes
- Type of study:
- guinea pig maximisation test
- Justification for non-LLNA method:
- The test was performed in 1989 before the alternative OECD guidelines (LLNA and in vitro test methods) were available.
Test material
- Reference substance name:
- Reaction mass of 1,1'-(2,2,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione and 1,1'-(2,4,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione
- EC Number:
- 915-671-3
- Molecular formula:
- C17H22N2O4
- IUPAC Name:
- Reaction mass of 1,1'-(2,2,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione and 1,1'-(2,4,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione
Constituent 1
In vivo test system
Test animals
- Species:
- guinea pig
- Strain:
- other: Himalayan
- Sex:
- male/female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Biological Research Laboratories Ltd., Fuellinsdorf, Switzerland
- Age at study initiation: 7 weeks (males) and 8 weeks (females)
- Weight at study initiation: 366 - 404 g (males) and 299 - 373 g (females)
- Housing: individually in Macrolon type 3 cages with standard softwood bedding
- Diet: pelleted standard Kliba 342 guinea pig breeding / maintainance diet (Kliba, Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Study design: in vivo (non-LLNA)
Induction
- Route:
- intradermal and epicutaneous
- Vehicle:
- other: acetone
- Concentration / amount:
- intradermal induction on Day 1: 1%
epicutaneous induction on Day 7: 25% - Day(s)/duration:
- epicutaneous induction on Day 7: 48 h
- Adequacy of induction:
- other: induction doses based on dose range-finding test; injected concentrations of 0.3 to 5% caused erythema and edema of grade 1 (0.3 - 15%) and 2 (3 - 5%), topical application at concentrations in the range of 5 to 25% did not induce any skin reactions
Challenge
- No.:
- #1
- Route:
- epicutaneous, semiocclusive
- Vehicle:
- other: acetone
- Concentration / amount:
- epicutaneous challenge on Day 20: 25%
- Day(s)/duration:
- 24 h
- Adequacy of challenge:
- other: the dose for challenge was selected based on the results of the dose range-finding test; concentrations up to 25% did not induce local reactions
- No. of animals per dose:
- 10 male and 10 female (test group), 5 male and 5 female (control group)
- Details on study design:
- RANGE FINDING TESTS:
Two guinea pigs were dermally injected with test item concentrations of 5% (right flank) and 3, 1, 0.5 and 0.3% (left flank) in acetone. Dermal reactions were assessed 24 h later and revealed erythema and edema of grade 1 at concentrations of 0.3 - 1% and erythema and edema of grade 2 at 3 and 5%. Thus, 1% was chosen as concentration for the intradermal injection.
In addition, patches with test item concentrations of 25, 15, 10 and 5% were applied to the clipped and shaved flanks of each of four guinea-pigs for 24 h. Skin reactions were assessed immediately after patch removal and 24 and 48 h after patch removal. No signs of irritation were found for any concentration at any observation time. Thus, the 25% concentration was chosen for topical applications.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: 1:1 (v/v) mixture complete Freund's adjuvant (FCA) and bidistilled water
Injection 2: 1% test substance in acetone
Injection 3: 1% test substance in a 1:1 mixture of complete Freund's adjuvant (FCA) and acetone
Epicutaneous: a 2 x 4 cm patch of filter paper was saturated with the test article (25% in acetone) and applied to the clipped and shaved scapula area one week after the injections
- Control group:
Intradermal (3 pairs of injections):
Injection 1: 1:1 (v/v) mixture complete Freund's adjuvant (FCA) and bidistilled water
Injection 2: 100% acetone
Injection 3: a 1% formulation of acetone in a 1:1 mixture of complete Freund's adjuvant (FCA) and acetone
Epicutaneous: a 2 x 4 cm patch of filter paper was soaked with acetone and applied to the clipped and shaved scapula area one week after the injections
- Site: an area of dorsal skin from the scapula region, the three pairs of injection were made at the border of a 4 x 6 cm area in the clipped region
- Frequency of applications: topical induction was performed 1 week after the intradermal induction
- Duration: Days 0 - 6
- Concentrations: intradermal 1%, epicutaneous 25%
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after epidermal induction (Day 20)
- Exposure period: 24 h
- Test groups: test substance and vehicle
- Control group: test substance and vehicle
- Site: left flank (test item) and right flank (vehicle)
- Concentrations: 25%
- Evaluation (hr after challenge): 48 and 72 h
- Challenge controls:
- The control group is actually a challenge control.
- Positive control substance(s):
- yes
- Remarks:
- formaldehyde-solution (HCHO) tested twice per year, but not within this study.
Results and discussion
- Positive control results:
- Challenge with formaldehyde (15% in bidistilled water) induced erythema of grade 1 immediately after patch removal and erythema of grade 1 in 7/10, of grade 2 in 1/10 and of grade 3 in 1/10 rabbits, thus meeting the reliability criteria for the GPMT test.
In vivo (non-LLNA)
Resultsopen allclose all
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- induction: 1% intradermal, 25% epicutaneous; challenge: 25% epicutaneous
- No. with + reactions:
- 20
- Total no. in group:
- 20
- Clinical observations:
- weak to mild erythema in 10/10 males and 10/10 females and weak edema in 4/10 males and 4/10 females
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- induction: 1% intradermal, 25% epicutaneous; challenge: 25% epicutaneous
- No. with + reactions:
- 15
- Total no. in group:
- 20
- Clinical observations:
- weak erythema in 9/10 males and 6/10 females
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- challenge: 25% epicutaneous
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Clinical observations:
- no effects
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- challenge: 25% epicutaneous
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Clinical observations:
- no effects
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- positive control
- Dose level:
- induction: 20% formaldehyde in bidistilled water; challenge: 15% formaldehyde in bidistilled water
- No. with + reactions:
- 9
- Total no. in group:
- 10
- Clinical observations:
- weak to moderate erythema in 9/10 females
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- positive control
- Dose level:
- induction: 20% formaldehyde in bidistilled water; challenge: 15% formaldehyde in bidistilled water
- No. with + reactions:
- 4
- Total no. in group:
- 10
- Clinical observations:
- weak to moderate erythema in 4/6 females
- Remarks on result:
- positive indication of skin sensitisation
Any other information on results incl. tables
Table 1: Summary on skin sensitisation results after challenge
| Animal no. (sex) | Group | Treatment | Erythema | Edema | ||
| 24 h | 48 h | 24 h | 48 h | |||
| 531-535 males right flank |
negative control |
Induction: FCA, acetone, FCA/acetone Challenge: acetone |
0/5 | 0/5 | 0/5 | 0/5 |
| 531-535 females right flank |
negative control |
Induction: FCA, acetone, FCA/acetone Challenge: acetone |
0/5 | 0/5 | 0/5 | 0/5 |
| 531-535 males left flank |
negative control |
Induction: FCA, acetone, FCA/acetone Challenge: 25% test item in acetone |
0/5 | 0/5 | 0/5 | 0/5 |
| 531-535 females left flank |
negative control |
Induction: FCA, acetone, FCA/acetone Challenge: 25% test item in acetone |
0/5 | 0/5 | 0/5 | 0/5 |
| 536-545 males right flank |
test group | Induction: FCA, 1% test item in acetone, FCA/1% test item in acetone Challenge: acetone |
0/10 | 0/10 | 0/10 | 0/10 |
| 551-560 females right flank |
test group | Induction: FCA, 1% test item in acetone, FCA/1% test item in acetone Challenge: acetone |
0/10 | 0/10 | 0/10 | 0/10 |
| 536-545 males left flank |
test group | Induction: FCA, 1% test item in acetone, FCA/1% test item in acetone Challenge: 25% test item in acetone |
10/10 | 9/10 | 4/10 | 0/10 |
| 551-560 females left flank |
test group | Induction: FCA, 1% test item in acetone, FCA/1% test item in acetone Challenge: 25% test item in acetone |
10/10 | 6/10 | 4/10 | 0/10 |
| 636-645 females |
positive control | Induction: FCA, 20% HCHO in bidistilled water, FCA/ 20% HCHO in bidistilled water Challenge: 15% HCHO in bidistilled water |
9/10 | / | 4/10 | / |
HCHO: formaldehyde, positive control substance
Table 2: Skin sensitisation results of individual test group animals (1% intradermal induction, 25% topical induction, 25% topical challenge)
| Animal No. | Sex | immediately after challenge |
24 h after challenge | 48 h after challenge | |||
| Erythema | Edema | Erythema | Edema | Erythema | Edema | ||
| 536 | male test item after challenge |
0 | 0 | 1 | 0 | 1 | 0 |
| 537 | 1 | 0 | 1 | 0 | 1 | 0 | |
| 538 | 1 | 0 | 2 | 1 | 1 | 0 | |
| 539 | 0 | 0 | 1 | 0 | 1 | 0 | |
| 540 | 1 | 0 | 2 | 1 | 1 | 0 | |
| 541 | 0 | 0 | 1 | 0 | 1 | 0 | |
| 542 | 1 | 0 | 2 | 1 | 1 | 0 | |
| 543 | 0 | 0 | 1 | 0 | 1 | 0 | |
| 544 | 0 | 0 | 1 | 0 | 0 | 0 | |
| 545 | 1 | 0 | 2 | 1 | 1 | 0 | |
| 551 | female test item after challenge |
1 | 0 | 1 | 0 | 1 | 0 |
| 552 | 0 | 0 | 1 | 0 | 1 | 0 | |
| 553 | 1 | 0 | 2 | 1 | 1 | 0 | |
| 554 | 1 | 0 | 1 | 0 | 1 | 0 | |
| 555 | 0 | 0 | 1 | 0 | 0 | 0 | |
| 556 | 1 | 0 | 2 | 1 | 0 | 0 | |
| 557 | 0 | 0 | 1 | 1 | 1 | 0 | |
| 558 | 1 | 0 | 1 | 0 | 0 | 0 | |
| 559 | 0 | 0 | 1 | 1 | 1 | 0 | |
| 560 | 0 | 0 | 1 | 0 | 0 | 0 | |
| 636 | female positive control after challenge |
1 | / | 1 | / | 0 | / |
| 637 | 1 | / | 1 | / | 0 | / | |
| 638 | 1 | / | 0 | / | 0 | / | |
| 639 | 1 | / | 2 | / | 2 | / | |
| 640 | 1 | / | 1 | / | 1 | / | |
| 641 | 1 | / | 1 | / | 1 | / | |
| 642 | 1 | / | 1 | / | 0 | / | |
| 643 | 1 | / | 1 | / | 0 | / | |
| 644 | 1 | / | 3 | / | 3 | / | |
| 645 | 1 | / | 1 | / | 0 | / | |
Applicant's summary and conclusion
- Interpretation of results:
- Category 1A (indication of significant skin sensitising potential) based on GHS criteria
- Conclusions:
- In the guinea pig maximisation test, 1% intradermal and 25% topical induction followed by a topical challenge at 25% induced erythema and/or edema in 10/10 males and 10/10 females after the first reading and in 9/10 males and in 6/10 females after the second reading. Based on the experimental findings, the test item is considered to be sensitising to the skin.
CLP: Skin sens. 1A, H317 according to Regulation (EC) No. 1272/2008
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