Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

Endpoint summary

Description of key information

Reliable in vivo skin and eye irritation studies using rabbits

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:

5-5-1982 to 5-9-1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guidelines for Testing Chemicals, 1981

Deviations:

not specified

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Animals and Animal Care:
Six adult New Zealand White rabbits, weighing between 2 and 4 kg initially, were purchased from New York State Rabbit Development, Hartwick, N.Y. (a USDA approved supplier) and used in this study. The rabbits were housed individually in suspended cages in an environmentally controlled room. During an acclimation period of no less than 5 days the animals were examined with respect to their general health to assure their suitability as test animals. NIH Animal Feed A (certified) and water were provided ad libitum. The animals were identified by use of ear tags and color coded cage cards.

Housing: Individually housed in wire mesh bottom cages.
Food: NIH Animal Feed A, certified feed, ad libitum, supplied fresh daily. No knowm contaminants are expected to be present in the basal diet that would interfere with the conduct of this study.
Water: Tap water, ad libitum. Water is monitored for contaminants at periodic intervals according to FDRL Standard Operating Procedures.
Environment: All animals will be housed in environment controlled rooms with temperature and relative humidity regulated as per "Guide for the Care and Use of Laboratory Animals". Filtered air will be supplied to provide 12- 15 air changes per hour. A 12 hour light/dark cycle will be maintained.
Quarantine: Minimum of 5 days. During this conditioning period, the rabbits will be observed for any clinical signs of disease. All rabbits with any evidence of disease or physical abnormalities will be discarded.

Type of coverage:

occlusive

Preparation of test site:

other: abraded and intact

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

A volume of 0.5 ml of the test article (undiluted) was applied to each of two sites per rabbit.

Duration of treatment / exposure:

4 hours

Observation period:

Four hours post-dose, the occlusive binders and patches were removed. The exposure sites were gently wiped with clean gauze to remove as much non-absorbed test article as possible. One-half hour after unwrapping, the exposure sites were examined and scored separately for both erythema and edema on a graded scale of 0 to 4 in accordance with the Skin Reaction Code (method of Draize, 1965). The exposure sites were again examined and scored at 48, 72 and 96 hours. Daily observations were not recorded, however, any extraordinary findings, dermal or toxicological, were noted.

Number of animals:

6 animals.

Details on study design:

Preparation:
On the day prior to dosing, the fur was clipped from the dorsal trunk area of each animal with electric clippers. During this procedure, care was taken to avoid accidental abrasion to the skin.
Method:
A volume of 0.5 ml was applied to each of two sites per rabbit. The skin sites were located directly across from each other, separated by the spinal column. Just prior to administration, test sites on the right dorsal side were abraded with the tip of a 22 gauge hypodermic needle. The abrasions were 4 incisions made in cross-hatch which penetrated the stratum corneum but did not disturb the dermis and/or produce bleeding. Following application, each skin site was occluded ,with a one-inch square gauze patch. The trunk of each animal was then wrapped with an occlusive binder which consisted of a layer of plastic wrap, a protective cloth and a stockinette sleeve all secured in place with masking tape.
Observation:
Four hours post-dose, the occlusive binders and patches were removed. The exposure sites were gently wiped with clean gauze to remove as much non-absorbed test article as possible. One-half hour after unwrapping, the exposure sites were examined and scored separately for both erythema and edema on a graded scale of 0 to 4 in accordance with the Skin Reaction Code (Draize) in Appendix II. The exposure sites were again examined and scored at 48, 72 and 96 hours. Daily observations were not recorded, however, any extraordinary findings, dermal or toxicological, were noted.

Irritation parameter:

erythema score

Basis:

other: Mean score for all animals

Time point:

other: overall at 24.5, 48 and 72 h

Score:

0

Max. score:

4

Remarks on result:

other: Intact skin

Irritation parameter:

edema score

Basis:

other: Mean score for all animals

Time point:

other: overall at 24.5, 48 and 72 h

Score:

0

Max. score:

4

Remarks on result:

other: Intact skin

Individual Rabbit Skin Irritation Scores

			Rabbit Number
Parameter scored	Exposure Time (hours)	Skin	1123	1124	1125	1176	1177	1178
Erythema and Eschar Formation	24.5	Intact	0	0	0	0	0	0
	48	Intact	0	0	0	0	0	0
	72	Intact	0	0	0	0	0	0
	96	Intact	0	0	0	0	0	0
	24.5	Abraded	0	0	0	0	0	0
	48	Abraded	0	0	0	0	0	0
	72	Abraded	0	0	0	0	0	0
	96	Abraded	0	0	0	0	0	0
(A)Erythema and Eschar Sub-total			0	0	0	0	0	0
Edema Formation	24.5	Intact	0	0	0	0	0	0
	48	Intact	0	0	0	0	0	0
	72	Intact	0	0	0	0	0	0
	96	Intact	0	0	0	0	0	0
	24.5	Abraded	0	0	0	0	0	0
	48	Abraded	0	0	0	0	0	0
	72	Abraded	0	0	0	0	0	0
	96	Abraded	0	0	0	0	0	0
(B)Edema Sub-total			0	0	0	0	0	0
(C)Overall Total = (A+B)			0	0	0	0	0	0
(D) Individual Animal Scores = (C/8)			0	0	0	0	0	0

Mean Score (D/6) = 0

Initial Body Weights:

Animal number                     Initial Body Weight (kg)

82 -1123                                   2.00

82 -1124                                   2.46

82 -1125                                   2.35

82 -1176                                   2.30

82 -1177                                   2.92

82 -1178                                   2.57

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

There were no irritative effects observed in any of the animals on intact or abraded skin at any time point during the study. All animals appeared normal throughout the course of the study.

Executive summary:

The skin irritation potential of the test material was evaluated in this study with New Zealand White rabbits. One group of six albino rabbits received a volume of 0.5 ml of the test article applied to two sites per rabbit. One site consisted of intact skin and the other was abraded skin. Following the application, each site was occluded. Four hours post-dose, the occlusive binders were removed. The exposure sites were wiped to remove as much non-absorbed test article as possible. One-half hour after unwrapping, the exposure sites were examined and scored separately for both erythema and edema according to the method of Draize. The exposure sites were again examined and scored at 48, 72 and 96 hours. There were no irritative effects observed in any of the animals at any time-point. All animals appeard normal throughout the course of the study. The individual animal mean scores for the 24.5, 48 and 72 hr time-point for the intact skin indicated that this material would not be classified under EU regulations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

5-5-1982 to 5-12-1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, 1981

Deviations:

not specified

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animals and Animal Care:
Six young adult New Zealand White rabbits, weighing approximately 2.0 - 4.0 kg initially, were purchased from New York State Rabbit Development, Hartwick, NY (a USDA approved supplier) for use in this study. The rabbits were housed individually in wire mesh bottom cages in an environmentally monitored room. Animals were identified by use of ear tags and cage cards. NIH Animal Feed A (certified) and water were provided ad libitum. The animals were acclimated a minimum of 5 days. During this acclimation period, the rabbits were examined with respect to their general health to assure their suitability as test animals.

Housing: Individually housed in wire mesh-bottom cages
Food: NIH Animal Feed A, certified feed, ad libitum, supplied fresh daily. No known contaminants are expected to be present in the basal diet that would interfere with the conduct of this study.
Water: Tap water: Ad libitum. Water is monitored for contaminants at periodic intervals according to FDRL Standard Operating Procedures.
Environment: All animals will be housed in environment-controlled rooms with temperature and relative humidity regulated as per "Guide for the Care and Use of Laboratory Animals". Filtered air will be supplied to provide 12-15 air changes per hour. A 12 hour light/dark cycle will be maintained.
Quarantine: Minimum of 5 days. During this conditioning period, the rabbits will be observed for any clinical signs of disease. All rabbits with any evidence of disease or physical abnormalities will be discarded.

Vehicle:

unchanged (no vehicle)

Controls:

other: Untreated eye served as control.

Amount / concentration applied:

A volume of 0.1 ml of the test article (undiluted) was instilled into one eye of each rabbit.

Duration of treatment / exposure:

Single exposure. Eyes are not irrigated after test article installation.

Observation period (in vivo):

The eyes were examined after treatment using fluorescein dye procedures and the grade of ocular reaction for each animal was recorded at 1, 24, 48 and 72 hours and again at 7 days using the Draize Scale for Scoring Ocular Lesions. If injury persisted the eyes were further examined and scored every 3 days for 13 days or until all signs of reversible irritation subsided.

Number of animals or in vitro replicates:

6

Details on study design:

The eyes were examined after treatment using fluorescein dye procedures and the grade of ocular reaction for each animal was recorded at 1, 24, 48 and 72 hours and again at 7 days using the Draize Scale for Scoring Ocular Lesions.

Irritation parameter:

cornea opacity score

Basis:

other: Mean score for all animals

Time point:

other: 24, 48 and 72 h

Score:

0.5

Max. score:

4

Irritation parameter:

iris score

Basis:

other: Mean score for all animals

Time point:

other: 24, 48 and 72 h

Score:

0.33

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

other: Mean score for all animals

Time point:

other: 24, 48 and 72 h

Score:

0.5

Max. score:

3

Irritation parameter:

chemosis score

Basis:

other: Mean score for all animals

Time point:

other: 24, 48 and 72 h

Score:

0.56

Max. score:

4

Irritant / corrosive response data:

See results section.

Individual Rabbit Eye Irritation Scores

			Reading
Rabbit Number	Item	Tissue	1 hr	24 hr	48 hr	72 hr	Day 7
0896	A	Cornea Opacity	0	1	1	1	0
	B	Cornea Area	0	1	1	1	0
	(1)	Cornea Total=(AxB)x5	0	5	5	5	0
	C	Iris	0	1	0	0	0
	(2)	Iris Total=(C)x5	0	5	0	0	0
	D	Conjunctiva Redness	0	2	1	0	0
	E	Conjunctiva Chemosis	0	2	0	0	0
	F	Conjunctiva Discharge	0	2	1	0	0
	(3)	Conjunctiva Total (D+E+F)x2	0	12	4	0	0
		Totals Added= (1+2+3)	0	22	9	5	0
0897	A	Cornea Opacity	0	1	1	1	0
	B	Cornea Area	0	3	3	3	0
	(1)	Cornea Total=(AxB)x5	0	15	15	15	0
	C	Iris	0	1	1	1	0
	(2)	Iris Total=(C)x5	0	5	5	5	0
	D	Conjunctiva Redness	0	2	1	0	0
	E	Conjunctiva Chemosis	0	2	1	0	0
	F	Conjunctiva Discharge	0	1	1	0	0
	(3)	Conjunctiva Total (D+E+F)x2	0	10	6	0	0
		Totals Added= (1+2+3)	0	30	26	20	0
0898	A	Cornea Opacity	0	0	0	0	0
	B	Cornea Area	0	0	0	0	0
	(1)	Cornea Total=(AxB)x5	0	0	0	0	0
	C	Iris	0	0	0	0	0
	(2)	Iris Total=(C)x5	0	0	0	0	0
	D	Conjunctiva Redness	0	0	0	0	0
	E	Conjunctiva Chemosis	0	0	0	0	0
	F	Conjunctiva Discharge	0	0	0	0	0
	(3)	Conjunctiva Total (D+E+F)x2	0	0	0	0	0
		Totals Added= (1+2+3)	0	0	0	0	0
0901	A	Cornea Opacity	0	0	0	0	0
	B	Cornea Area	0	0	0	0	0
	(1)	Cornea Total=(AxB)x5	0	0	0	0	0
	C	Iris	0	0	0	0	0
	(2)	Iris Total=(C)x5	0	0	0	0	0
	D	Conjunctiva Redness	0	0	0	0	0
	E	Conjunctiva Chemosis	0	0	0	0	0
	F	Conjunctiva Discharge	0	0	0	0	0
	(3)	Conjunctiva Total (D+E+F)x2	0	0	0	0	0
		Totals Added= (1+2+3)	0	0	0	0	0
0902	A	Cornea Opacity	0	0	0	0	0
	B	Cornea Area	0	0	0	0	0
	(1)	Cornea Total=(AxB)x5	0	0	0	0	0
	C	Iris	0	0	0	0	0
	(2)	Iris Total=(C)x5	0	0	0	0	0
	D	Conjunctiva Redness	0	0	0	0	0
	E	Conjunctiva Chemosis	0	0	0	0	0
	F	Conjunctiva Discharge	0	0	0	0	0
	(3)	Conjunctiva Total (D+E+F)x2	0	0	0	0	0
		Totals Added= (1+2+3)	0	0	0	0	0
0904	A	Cornea Opacity	0	1	1	1	0
	B	Cornea Area	0	1	1	1	0
	(1)	Cornea Total=(AxB)x5	0	5	5	5	0
	C	Iris	0	1	1	0	0
	(2)	Iris Total=(C)x5	0	5	5	0	0
	D	Conjunctiva Redness	0	2	1	0	0
	E	Conjunctiva Chemosis	0	2	2	1	0
	F	Conjunctiva Discharge	0	2	2	1	0
	(3)	Conjunctiva Total (D+E+F)x2	0	12	10	4	0
		Totals Added= (1+2+3)	0	22	20	9	0

Initial Body Weights:

Animal Number              Initial Body Weight (kg)

82 -0896                            3.01

82 -0897                            3.05

82 -0898                            3.00

82 -0901                            2.97

82 -0902                            3.15

82 -0904                            2.72

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Three of six animals were noted with positive cornea opacity effects (maximum score grade 1), iridal effects (maximum score grade 1), conjunctival redness (maximum score grade 2) and conjunctival chemosis (maximum score grade 2). The other three animals showed no effects throughout the study. All positive effects observed had resolved by day 7. The individual animal mean scores for the 24, 48 and 72 hr time-point indicated that this material would not be classified under EU regulations.

Executive summary:

The primary ocular irritation potential of the test material was evaluated in this study with New Zealand White rabbits. One group of six albino rabbits received a single, unwashed exposure. A 0.1- ml dose of the test article was instilled into the lower conjunctival sac of one eye. The eyelids were held together for approximately one second and released. The other eye served as an untreated control. The eyes were examined for ocular reactions in accordance with the method of Draize at approximately 1, 24, 48 and 72 hours after dosing and on study day 7. Sodium fluorescein dye was used to aid in revealing possible effects.

Three of six animals were noted with positive cornea opacity effects (maximum score grade 1), iridal effects (maximum score grade 1), conjunctival redness (maximum score grade 2) and conjunctival chemosis (maximum score grade 2). The other three animals showed no effects throughout the study. All positive effects observed had resolved by day 7. The individual animal mean scores for the 24, 48 and 72 hr time-point indicated that this material would not be classified under EU regulations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The skin irritation potential of the test material was evaluated in this study with New Zealand White rabbits. One group of six albino rabbits received a volume of 0.5 ml of the test article applied to two sites per rabbit. One site consisted of intact skin and the other was abraded skin. Following the application, each site was occluded. Four hours post-dose, the occlusive binders were removed. The exposure sites were wiped to remove as much non-absorbed test article as possible. One-half hour after unwrapping, the exposure sites were examined and scored separately for both erythema and edema according to the method of Draize. The exposure sites were again examined and scored at 48, 72 and 96 hours. There were no irritative effects observed in any of the animals at any time-point. All animals appeared normal throughout the course of the study. The individual animal mean scores for the 24.5, 48 and 72 hr time-point for the intact skin indicated that this material would not be classified under EU regulations.

Eye irritation:

The primary ocular irritation potential of the test material was evaluated in this study with New Zealand White rabbits. One group of six albino rabbits received a single, unwashed exposure. A 0.1- ml dose of the test article was instilled into the lower conjunctival sac of one eye. The eyelids were held together for approximately one second and released. The other eye served as an untreated control. The eyes were examined for ocular reactions in accordance with the method of Draize at approximately 1, 24, 48 and 72 hours after dosing and on study day 7. Sodium fluorescein dye was used to aid in revealing possible effects.

Three of six animals were noted with positive cornea opacity effects (maximum score grade 1), iridal effects (maximum score grade 1), conjunctival redness (maximum score grade 2) and conjunctival chemosis (maximum score grade 2). The other three animals showed no effects throughout the study. All positive effects observed had resolved by day 7. The individual animal mean scores for the 24, 48 and 72 hr time-point indicated that this material would not be classified under EU regulations.

Disregarded studies:

In addition to the studies described above, there are skin and eye irritation studies performed by Industrial Biotest. Due to the performance of these studies in a laboratory that was shown to have fraudulent testing practices and that certain information on results was missing from the reports, these studies have been disregarded.

Justification for classification or non-classification

Classification criteria not met.