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 irritation (in vivo, OECD 404): irritating

Eye irritation (in vivo, OECD 405): irreversible damage

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-10-27 to 1989-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 24 Feb 1987

Deviations:

yes

Remarks:

7 days observation period instead of 14 days

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

Version: March 1984

Deviations:

yes

Remarks:

7 day observation period instead of 14 days

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Biological Research Laboratories Ltd., Fuellinsdorf, Switzerland
- Age at study initiation: 14 weeks (males) and 15 weeks (females)
- Weight at study initiation: 2.4 - 2.6 kg (males) and 2.5 kg (females)
- Housing: individually in stainless steel cages
- Diet: pelleted standard Kliba 341 rabbit maintainance diet (Kliba, Klingentalmuehle AG, Kaiseraugst, Switzerland) ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Oct 1989 To: 07 Nov 1989

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: The test article was applied moistened with polyethylene glycol (PEG 400)

Controls:

no

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

4 h

Observation period:

7 days
Reading time points: 1, 24, 48 and 72 h and 7 days

Number of animals:

3 (2 males, 1 female)

Details on study design:

TEST SITE
- Area of exposure: 6 cm² shaved skin of the dorsal area
- Type of wrap if used: The test material was covered with a 3.0 cm x 3.0 cm patch of surgical gauze and covered with an semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: After removal of the dressing the skin was rinsed with lukewarm water
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS: 1, 24, 48 and 72 h and 7 days

SCORING SYSTEM:
- Method of calculation: Draize scoring system

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

male

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

male

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

female

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

male

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

male

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

female

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Slight (score 1, 1/3 rabbits) or well defined erythema (score 2, 2/3 rabbits) and very slight edema (score 1, 3/3 rabbits) was observed within 1 hour after removal of the patches. At the 24, 48 and 72 h reading time point, all animals had well defined erythema (score 2). Very slight edema (score 1) remained in all rabbits until 48 h after path removal. At study Day 7 there were no local signs of erythema and edema. However, slight scales were observed in 2/3 animals and moderate scales were observed in 1/3 animals on day 7 of observation.

Other effects:

Coloration:
In the area of application, brown staining of the treated skin by pigment or coloring of the test article was observed in all animals at each of the reading time points.

Body weights:
The body weight gain of all rabbits was similar.

Clinical symptoms / Mortality:
No acute clinical symptoms were observed in the animals during the test period, and no mortality occurred.

Necropsy:
No necropsy was performed. All animals were killed and discarded.

Table 1: Skin reactions and scores for the individual animals

Sex	Erythema score			Edema score
	male	male	female	male	male	female
Animal #	63	64	65	63	64	65
Reading time point
1 h	2	1	2	1	1	1
24 h	2	2	2	1	1	1
48 h	2	2	2	1	1	1
72 h	2	2	2	0	0	0
7 days	0	0	0	0	0	0
24/48/72 h mean	2	2	2	0.67	0.67	0.67

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

All animals showed erythema and edema, which were fully reversible within 7 days. However, slight to moderate scales were observed in all three rabbits until the end of the 7 days observation period. Due to the shortened observation period of 7 instead of 14 days the reversibility of scales formation is unclear. Based on these findings and on a worst case approach, the substance is classified as Skin Irrit. 2 (H315) according to Regulation (EC) No. 1272/2008.
CLP: Skin irrit. 2, H315. Classification according to Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-11-2 to 1989-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 24 Feb 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Version: March 1984

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Biological Research Laboratories Ltd., Fuellinsdorf, Switzerland
- Age at start of treatment: 14 weeks (males) and 15 weeks (females)
- Weight at study initiation: 2.6 - 2.7 kg (males) and 2.0 kg (females)
- Housing: individually in stainless steel cages
- Diet: pelleted standard Kliba 341 rabbit maintainance diet (Kliba, Klingentalmuehle AG, Kaiseraugst, Switzerland) ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 Nov 1989 To: 27 Nov 1989

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g / animal

Duration of treatment / exposure:

Single exposure, no washing

Observation period (in vivo):

21 days
Reading time points: 1, 24, 48 and 72 h, 7, 14 and 21 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM: Draize scoring system

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3.33

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritant / corrosive response data:

Corneal opacity of grade 1 was noted in 2/3 rabbits 1 h following administration, which increased to grade 2 in all rabbits within 24 h and to grade 3 at the 48 and 72 h reading time point. Corneal opacity of grade 4 was observed in all animals up to 7 days after test item instillation. In 2/3 animals the effects remained unchanged at grade 4 until study termination, whereas in 1/3 rabbits a corneal opacity score of grade 2 was observed at the 14 and 21 day reading time point.
Iris reactions of grade 1 were observed in 2/3 rabbits from 24 h after test item instillation onwards and in 1/3 rabbits 48 h after test item instillation. 7 days after test item instillation, all 3 rabbits showed iris reactions of grade 2. At the 14 day reading time point the iris reaction score was of grade 1 in all 3 rabbits and fully reversible until study termination on Day 21.
Conjunctiva redness of score 2 was noted in 2/3 rabbits from 1 h after test item instillation until study Day 14 and decreased to score 1 until study termination on Day 21. In the third rabbit, conjunctival redness was of score 2 at the 1 and 24 h reading time point, increased to grade 3 on reading time points 48 h, 72 h and 7 days and decreased to a score of grade 2 on study Day 14 and to a score of grade 1 at study termination on Day 21.
Conjunctival chemosis was observed at grade 3 to 4 in all rabbits until 72 h post-test item instillation. At 7 days after instillation, all rabbits had a chemosis score of grade 2, which remained of grade 2 until study Day 14 in 2/3 rabbits and declined to grade 1 in the third rabbit. At study termination, 2/3 rabbits had a chemosis score of grade 1, whereas the finding of conjunctival chemosis was fully reversible until study Day 21 in the third rabbit.
Partly corrosion of the conjunctivae was observed in all animals from 24 h to 7 days after treatment.

Other effects:

No staining of the cornea and conjunctivae of the treated eyes by pigment or coloring of the test article was observed.
The body weight development of the animals was not affected by treatment.

Table 1: Eye irritation scores for individual animals

Animal Number		1 h	24 h	48 h	72 h	7 days	14 days	21 days	Individual mean 24-48-72 h
Cornea Opacity	75 male	1	2	3	3	4	4	4	2.67
	76 male	0	2	3	3	4	2	2	2.67
	77 female	1	2	3	3	4	4	4	2.67
Iris	75 male	0	1	1	1	2	1	0	1.00
	76 male	0	0	1	1	2	1	0	0.67
	77 female	0	1	1	1	2	1	0	1.00
Conjunctiva Redness	75 male	2	2	2	2	2	2	1	2.00
	76 male	2	2	3	3	3	2	1	2.67
	77 female	2	2	2	2	2	2	1	2.00
Conjunctiva Chemosis	75 male	3	3	3	3	2	2	1	3.00
	76 male	4	4	3	3	2	2	1	3.33
	77 female	3	3	3	3	2	1	0	3.00

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item caused irreversible damage to the eyes of all animals, evident as corneal opacity, iritis, conjunctival redness and conjunctival chemosis which were not fully reversible within 21 days of observation.
CLP: Eye Damage 1, H318 according to Regulation (EC) No. 1272/2008

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The test item was assessed for its skin irritating and corrosive properties in an in vivo study conducted according to OECD guideline 404 (version adopted in 1987) and in compliance with GLP (89-0646-DGT). An amount of 0.5 g was moistened in polyethylene glycol (PEG 400) and applied to the clipped dorsal skin of three New Zealand White rabbits. After 4 h exposure under semi-occlusive conditions, the skin was washed with lukewarm water, and dermal reactions (erythema and edema formation) were evaluated based on Draize scoring criteria 1, 24, 48 and 72 h and 7 days post-administration.

One hour after patch removal, slight (grade 1) or well defined erythema (grade 2) were noted in 3/3 rabbits. In addition, 3/3 animals had very slight edema (grade 1). At the 24, 48 and 72 h reading time point, 3/3 animals showed well defined erythema (grade 2). Very slight edema was observed in all rabbits until 48 h after patch removal. The mean scores over 24, 48 and 72 h for the individual animals were 2.0, 2.0 and 2.0, respectively, for erythema. For edema, the mean scores over 24, 48 and 72 h for the individual animals were 0.67, 0.67 and 0.67, respectively. The erythema and edema in all the animals was fully reversible by Day 7. Slight scales were observed in 2/3 animals and moderate scales in 1/3 animals on Day 7. As this was the last day of the observation period it was not possible to establish if the effect was reversible within 14 days.

Based on the results of the study and as a worst-case approach, the test item is considered to be irritating to the skin.

 

Eye irritation

The eye irritating potential of the test substance was investigated in an acute eye irritation/corrosion study in vivo conducted according to OECD guideline 405 (version adopted in 1987) and in compliance with GLP (90-0534-DGT). Three New Zealand White rabbits received a single test item dose of 0.1 g, which was instilled into the conjunctival sack of the left eye. The eye was not washed following instillation. The right eye remained untreated and served as control. Local irritation effects on the eyes were scored according to Draize criteria 1, 24, 48 and 72 h, and 7, 14 and 21 days post-instillation.

All animals showed severe eye damage, with irritation reactions starting 1 h after test item instillation. Corneal opacity of grade 1 was noted in 2/3 rabbits within 1 h following administration and the severity increased gradually to grade 4 in 3/3 animals on day 7 post- instillation. The severity of the corneal opacity grade decreased in 1/3 rabbits to grade 2 by study termination on Day 21, but remained grade 4 in the two remaining animals.

Iris reactions were observed in 2/3 rabbits from 24 h after test item instillation and in 1/3 rabbits 48 h after test item instillation. At the 7 day reading time point, 3/3 rabbits showed iris reactions of grade 2. At the 14-day reading time point the severity of the iris reaction decreased to a score of grade 1 in all 3 rabbits, and the effect was fully reversible by study termination on Day 21.

Conjunctival redness of score 2 to 3 was observed in 3/3 rabbits from 1 h post-administration up to the 7-day reading time point. On the 14-day reading time point the score for 3/3 animals was 2, and the severity decreased to a score of grade 1 at study termination on Day 21. In addition, all rabbits showed conjunctival chemosis of grade 3 to 4 from 1 h until 72 h post-instillation. The severity of chemosis decreased within one week post-administration to grade 2 and gradually the severity decreased to grade 1 in 2/3 rabbits on Day 21.

The mean scores over 24, 48 and 72 h for the individual animals were 2.67, 2.67 and 2.67 for corneal opacity, while the mean individual scores over 24, 48 and 72 h for iris were 1.0, 0.67 and 1.0, respectively. The mean scores over 24, 48 and 72 h for the individual animals were 2.0, 2.67 and 2.0 for conjunctival redness; and the mean scores over 24, 48 and 72 h for the individual animals were 3.0, 3.3 and 3.0 for chemosis, respectively. The experimental findings clearly show that the test item causes irreversible damage to the eye.

Justification for classification or non-classification

The available data on skin irritation meet the criteria for classification as Skin Irr. 2 (H315) 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.

The available data on eye irritation meet the criteria for classification for causing serious eye damage (Cat. 1, H318) 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 irritation: data lacking