Reaction mass of 1,1'-(2,2,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione and 1H-Pyrrole-2,5-dione, 1,1'-(2,4,4-trimethyl-1,6-hexanediyl)bis-

Endpoint summary

Description of key information

Skin sensitisation (in vivo, OECD 406, GPMT): 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

Study period:

1989-11-21 to 1989-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

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

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

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.

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.

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

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	/

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A guinea pig maximisation test (GPMT) was conducted according to OECD guideline 406 (version adopted in 1981) and compliant with GLP, to assess the skin sensitising properties of the test item (90-0536-DGT). Ten male and 10 female Himalayan guinea pigs were induced intradermally with 1% test substance formulated in acetone on Day 1 and epicutaneous with 25% test substance formulated in acetone on Day 7, one week after intradermal induction. After 48 h exposure under semi-occlusive conditions, the patch was removed. As the 25% dilution of the test substance did not cause skin irritating properties, the test site was pre-treated with 10% sodium lauryl sulfate (SLS) in petrolatum oil 24 h prior to epicutaneous induction. A concurrent negative control group of 5 animals/sex was included, which was treated in an identical way as the test group but with vehicle (acetone) instead of the test item. Two weeks after epicutaenous induction, challenge was performed. All guinea pigs were treated epicutaneously with 25% test item formulation in acetone for 24 h under semi-occlusive conditions. A similar constituted group of challenge control animals was induced but remained untreated during the challenge. A positive control group was treated with formaldehyde in regular reliability checks performed by the testing laboratory. Skin reactions were evaluated (erythema and edema formation) 24 and 48 h after patch removal.

At the first reading (24 h after patch removal) 10/10 males and 10/10 females showed weak to mild erythema and 4/10 males and 4/10 females showed weak edema. At the second reading (48 h after patch removal) weak erythema was noted in 9/10 males and in 6/10 females. Weak to moderate erythema was also noted in 9/10 females and in 4/10 females after treatment with the positive control formaldehyde (results included as part of a different study) 24 and 48 h after patch removal, thus demonstrating the sensitivity of the test. Challenge with the vehicle (acetone) did not cause skin responses in any animal.

The irritating properties of the test substance at several concentrations were assessed in a range-finding test. An intradermal concentration of 1% caused mild irritation. Although no dermal irritation was noted up to the highest tested concentration of 25%, this dilution was selected as the topical concentration. As the substance caused skin sensitisation, the study is considered to be valid.

Under the conditions of the test and based on the experimental findings, the test item is considered to be sensitising to the skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation meet the criteria for classification as Skin sens. 1A (H317) according to Regulation (EC) No. 1272/2008 (CLP) and according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Respiratory sensitisation: data lacking