Reaction mass of 1,4-bis[(2-ethylhexyl)amino]-9,10-dihydroanthracene-9,10-dione and 1-(butylamino)-4-[(2-ethylhexyl)amino]-9,10-dihydroanthracene-9,10-dione and 1-[(2-ethylhexyl)amino]-4-(methylamino)-9,10-dihydroanthracene-9,10-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6-23-1989 to 8-11-1989

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.

Justification for type of information:

The study was performed in 1989, prior to the common use of the LLNA for assessing skin sensitising potential.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

not specified

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The study was performed in 1989, prior to the common use of the LLNA for assessing skin sensitising potential.

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

911-360-1

Molecular formula:

variable structures

IUPAC Name:

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

Test material form:

other: Dark blue liquid.

Details on test material:

- Name of test material (as cited in study report): Automate Blue 8 7121-536
- Physical state: Dark blue liquid.
- Storage condition of test material: The test material was stored at room temperature throughout the study in a small, silver metal can with a metal lid.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST SYSTEM JUSTIFICATION:
The guinea pig has historically been used for this test and allows the data to be compared to that of other materials.

TEST ANIMALS:
Young adult, male and female Hartley guinea pigs of a size to fit the restraining device from a U.S.D.A. approved supplier will be used. The animals will be of a size to fit the restrainers. The supplier and date of arrival will be documented.

NUMBER OF ANIMALS:
Twenty (20) test animals, ten (10) naive control, four (4) animals per pilot study will be used. When possible, equal numbers of males and females will be included in both test and control groups.

IDENTIFICATION OF ANIMALS:
Cage cards incorporating an animal group code - arrival date - color code marking system, will be used to identify each guinea pig.

SELECTION:
The animals will be randomly caged according to Standard Operating Procedures.

HOUSING AND ANIMAL CARE:
Prior to use, all animals will be quarantined for at least four days. Animals will be housed individually in wire mesh suspension cages. PURINA GUINEA PIG CHOW (or other comparable diet) will be fed ad libitum during both quarantine and test periods. Tap water will be provided ad libitum. No analysis is required since there are no known contaminants expected that would interfere with the study. The animals will be kept on a 12-hour light/12-hour dark cycle.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

other: Acetone

Induction 2

Concentration / amount:

A 50% w/v concentration of Automate Blue 8 7121-536 in acetone was chosen for use at primary challenge and rechallenge.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Acetone

Challenge 2

Concentration / amount:

A 50% w/v concentration of Automate Blue 8 7121-536 in acetone was chosen for use at primary challenge and rechallenge.

No. of animals per dose:

Ten males and ten females were used in the induction, primary challenge and rechallenge phase. Five males and five females were used in the primary challenge phase as naive controls. A different set of five males and five females was used as the naive controls in the rechallenge phase.

Four males and four females were also used for a Primary Irritation test.

Details on study design:

Irritation Screen:
Exposure of the guinea pigs can be divided into three phases: Irritation, Induction, and Challenge.
The Irritation phase has the purpose of determining the proper level of test article to be used in the Induction and (or) Challenge Phase. If the test article is known not to be an irritant, the irritation evaluation need not be run. If the irritation potentials are unknown, from one to four levels of test article per animal, formulated in the solvent appropriate for a given study phase, may be evaluated. Generally, the concentration selected by the study director for induction may be one which causes mild to moderate irritation as defined by the study director. For Challenge, the concentration selected by the study director should be no more than slightly irritating as defined by the study director.
If all tested levels cause irritation, additional levels may need to be tested. The position of the different concentrations of test materials on the animals should be varied to adjust for possible site-to-site variations in response. The irritation evaluations can be done before or during the study phases as appropriate.
In preparation, remove the hair from the animals' backs using a small animal clipper. Do this the day before applying the test article. Apply closed patches to the animals in the following manner: Apply an appropriate volume of each test article into the selected patch system. place the animal into the restrainer and apply the patch(es) to the clipped surface as quickly as possible. Occlude the patch with a rubber dental dam pulled taut and fastened to the bottom of the restrainer with clips. Adjust the restrainer to minimize movement of the animal during exposure. Approximately six (6) hours later remove the dental dam and patch(es), take the animal from the restrainer and place it in its cage. Excess material may be removed by an appropriate solvent. The day following the irritation exposure, depilate and score all animals as described under "Observations".

Primary Irritation Concentration Levels:
Pilot Number 1 (2 males and 2 females): 5, 2.5, 1 and 0.5 % w/v in acetone
Pilot Number 2 (2 males and 2 females): Undiluted and 50, 25 and 10% w/v in acetone

Induction Phase:
The purpose of this phase is to dermally expose the animals so that, if the material is a sensitizer, the animal can develop an immunological response. Although it is not necessary, the level of test article used may be irritating, but should not cause an excessive response. A level several times greater than the anticipated human exposure should be used if possible.
Clip the left shoulder of each animal with a small animal clipper the day before exposure. Apply closed patches to the clipped area of the guinea pigs. Expose and restrain the animals as previously described under Irritation Screen. Repeat the procedure at the same site once a week for the next two (2) weeks for a total of three approximate 6-hour exposures (the interval between induction exposures may vary from 5 to 9 days). Should excessive irritation (as defined by the study director) appear at the induction sites during the induction phase, patches may still be placed within the same left shoulder area, taking care to avoid this irritation. After the last induction exposure, leave the animals untreated for approximately two (2) weeks (12-16 days) before primary challenge.

Induction Phase Concentration Level: 100%

Challenge Phase:
The test animals which have had three previous exposures to the test material at appropriate intervals are again exposed in the Challenge Phase. In addition, ten animals (naive control) which have never been exposed to the test article are treated with the same concentration.
Challenge the animals previously exposed during the induction period as well as the previously untreated control animals approximately two (2) weeks after the last induction exposure. Use the same patching procedure as for the "Induction Phase," but apply the patches to a skin site that has not been exposed previously. The site for challenge may be varied as necessary to achieve the objectives of the experiment.

A 50% w/v concentration of Automate Blue 8 7121-536 in acetone was chosen for use at primary challenge and rechallenge.

Observations:
The day following the primary challenge exposure, depilate all animals with a commercial depilatory. Place the depilatory on the test sites and surrounding areas, and leave it on for no more than fifteen (15) minutes. Thoroughly wash off the depilatory with warm, running water, dry the animals with a towel, and return them to their cages.
A minimum of two (2) hours after depilation, grade the test sites on a scale of 0 to 3 (0 = no reaction, + = slightly patchy erythema, 1 = slight but confluent, or moderate patchy erythema, 2 = moderate erythema, 3 = severe erythema with or without edema) (report as 24 hour scores). Repeat the grading the following day. For reporting purposes the first and second gradings will be designated as 24 and 48 hour readings respectively.
Grades of 1 or greater in the test group generally indicate sensitization, provided grades of less than 1 are seen on control animals. If grades of 1 or greater are noted on control animals, then the reactions of test animals that exceed the most severe control reaction are presumed to be due to sensitization. Occasionally, test animals may have a higher incidence of skin reactions that are comparable in intensity (e.g., +) to controls, without a single animal being more reactive. In these Instances, a rechallenge may be necessary to clearly define the mechanism.

Rechallange Phase:
If animals are to be rechallenged, it should be scheduled at least 6 days after primary challenge.

Challenge controls:

Primary Challenge:
Five male and five female naive animals were exposed to the test material at 50% w/v in acetone.

Rechallenge:
Five male and five female naive animals were exposed to the test material at 50% w/v in acetone.

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

12

Total no. in group:

20

Clinical observations:

No data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 12.0. Total no. in groups: 20.0. Clinical observations: No data.

Results 2

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

No data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: No data.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

other: Naive Control

Dose level:

50%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

No data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Naive Control. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: No data.

Results 4

Reading:

1st reading

Hours after challenge:

48

Group:

other: Naive Control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: Naive Control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No data.

Results 5

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

11

Total no. in group:

20

Clinical observations:

No data

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 11.0. Total no. in groups: 20.0. Clinical observations: No data.

Results 6

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

20

Clinical observations:

No data

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: No data.

Results 7

Reading:

rechallenge

Hours after challenge:

24

Group:

other: Naive Control

Dose level:

50%

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

No data

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: other: Naive Control. Dose level: 50%. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: No data.

Results 8

Reading:

rechallenge

Hours after challenge:

48

Group:

other: Naive Control

Dose level:

50%

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

No data

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: other: Naive Control. Dose level: 50%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: No data.

Any other information on results incl. tables

The test material at 50% w/v in acetone was determined to be the appropriate concentration for the Primary Challenge based on the results of the Primary Irritation phase studies (Pilot no.1 and 2).

Skin Grades Following Primary Challenge using Automate Blue 8 7121-536 in Guinea Pigs:

Test Material (50% w/v in acetone) in Induced Animals^aWS

		Numerical Score
Animal Number	Sex	24-Hour	48-Hour
T-1 RD622	M	0	0
T-2 RD622	M	0	0
T-3 RD622	M	0	0
T-4 RD622	M	0	0
T-5 RD622	M	+	0
T-6 RD622	M	+	+
T-7 RD622	M	0	0
T-8 RD622	M	+	+
T-9 RD622	M	+	0
T-10 RD622	M	+	+
T-11 RD622	F	+	0
T-12 RD622	F	+	0
T-13 RD622	F	0	0
T-14 RD622	F	0	0
T-15 RD622	F	0	0
T-16 RD622	F	+	0
T-17 RD622	F	+	0
T-18 RD622	F	+	0
T-19 RD622	F	+	0
T-20 RD622	F	+	0
Mean		0.3	0.1

^apatch site location # 2 was used in accordance with that specified in Appendix A of the protocol.

W = At second and third inductions, and primary challenge the respective sites were wiped with a dry paper towel to remove excess sample.

S = Blue sample stain interfered with scoring.

Test Material (50% w/v in acetone) in Naive Animals^aLP

			Numerical Score
Animal Number	Sex	Site Order^b	24-Hour	48-Hour
C-21 RD/BK622	M	AB	0	0
C-22 RD/BK622	M	BA	0	0
C-23 RD/BK622	M	AB	0	0
C-24 RD/BK622	M	BA	0	0
C-25 RD/BK622	M	AB	0	0
C-26 RD/BK622	F	BA	0	0
C-27 RD/BK622	F	AB	+	0
C-28 RD/BK622	F	BA	0	0
C-29 RD/BK622	F	AB	+	0
C-30 RD/BK622	F	BA	0	0
Mean			0.1	0.0

^aAnimals were common to this study and one other Morton-Thiokol, Inc., study (HTB Project No. 89-3809-21).

^bA = Automate Blue 8 7121-536 (HTB project No. 89-3808-21);

B = Automate Red B 7111-2818 (HTB Project No. 89-3809-21). The letter which appears first was placed at site # 2 and the letter which appears second was placed at site # 5.

L = Blue sample stain interfered with scoring.

P = All sites were wiped with a dry paper towel following removal.

Skin Grades Following Rechallenge using Automate Blue 8 7121-536 in Guinea Pigs:

Test Material (50% w/v in acetone) in Induced Animals^aB

		Numerical Score
Animal Number	Sex	24-Hour	48-Hour
T-1 RD622	M	0	0
T-2 RD622	M	0	0
T-3 RD622	M	+	0
T-4 RD622	M	0	0
T-5 RD622	M	+	+
T-6 RD622	M	+	+
T-7 RD622	M	+	+
T-8 RD622	M	0	+
T-9 RD622	M	0	0
T-10 RD622	M	+	+
T-11 RD622	F	+	+
T-12 RD622	F	+	+
T-13 RD622	F	+	+
T-14 RD622	F	+	+
T-15 RD622	F	0	0
T-16 RD622	F	+	0
T-17 RD622	F	+	0
T-18 RD622	F	0	+
T-19 RD622	F	0	0
T-20 RD622	F	0	0
Mean		0.3	0.3

^aPatch site location # 4 was used in accordance with that specified in Appendix A of the protocol.

B = Blue sample stain interfered with scoring.

Test Material (50% w/v in acetone) in Naive Animals^aB

		Numerical Score
Animal Number	Sex	24-Hour	48-Hour
E-31 RD/BK622	M	+	+
E-32 RD/BK622	M	+	0
E-33 RD/BK622	M	0	+
E-34 RD/BK622	M	+	0
E-35 RD/BK622	M	0	+
E-36 RD/BK622	F	+	0
E-37 RD/BK622	F	+	+
E-38 RD/BK622	F	0	0
E-39 RD/BK622	F	+	+
E-40 RD/BK622	F	0	0
		0.3	0.3

^aPatch site location#4 was used in accordance with that specified in Appendix A of the protocol.

B = Blue sample stain interfered with scoring.

Individual Body Weight Data in Guinea Pigs

		Body Weight (grams)
Animal Number	Sex	Initial	Terminal
T-1 RD622	M	477	736
T-2 RD622	M	461	761
T-3 RD622	M	491	769
T-4 RD622	M	492	771
T-5 RD622	M	481	706
T-6 RD622	M	473	730
T-7 RD622	M	544	833
T-8 RD622	M	467	800
T-9 RD622	M	469	718
T-10 RD622	M	461	720
T-11 RD622	F	475	691
T-12 RD622	F	435	649
T-13 RD622	F	469	628
T-14 RD622	F	467	665
T-15 RD622	F	434	556
T-16 RD622	F	445	639
T-17 RD622	F	422	655
T-18 RD622	F	428	567
T-19 RD622	F	447	689
T-20 RD622	F	462	679
C-21 RD/BK622	M	491	768
C-22 RD/BK622	M	471	720
C-23 RD/BK622	M	476	708
C-24 RD/BK622	M	490	792
C-25 RD/BK622	M	464	610
C-26 RD/BK622	F	467	621
C-27 RD/BK622	F	450	670
C-28 RD/BK622	F	398	600
C-29 RD/BK622	F	425	607
C-30 RD/BK622	F	455	619
E-31 RD/BK622	M	466	753
E-32 RD/BK622	M	487	810
E-33 RD/BK622	M	525	803
E-34 RD/BK622	M	470	746
E-35 RD/BK622	M	475	784
E-36 RD/BK622	F	412	458
E-37 RD/BK622	F	451	720
E-38 RD/BK622	F	415	617
E-39 RD/BK622	F	437	723
E-40 RD/BK622	F	482	756
P-41 RD622	M	437*	ND
P-42 RD622	M	428*	ND
P-43 RD622	F	433*	ND
P-44 RD622	F	429*	ND
P-45 RD622	M	429*	ND
P-46 RD622	M	426*	ND
P-47 RD622	F	451*	ND
P-48 RD622	F	460*	ND

*=Weights taken prior to dosing.

ND=No data.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The potential of the test material to produce delayed contact hypersensitivity in guinea pigs was evaluated using an adaptation of the method of Ritz and Buehler*. Results of a primary challenege indicated a need for a rechallenge. Following a single patch rechallenge, there were again no grades of 1 produced in the test or control animals. The incidence of grade ± responses in the test group (13 of 20) was compared to that of the naive control group (8 of 10). The incidence of these responses in the test group remained essentially unchanged from that of primary challenge and was now less than that produced by the naive group indicating that sensitization had not been induced.

Executive summary:

The potential of the test material to produce delayed contact hypersensitivity in guinea pigs was evaluated using an adaptation of the method of Ritz and Buehler*.

Following primary challenge, there were no grades of 1 produced in the test or control animals. The incidence of grade ± responses in the test group (12 of 20) was compared to that of the naive control group (2 of 10). Although there were no grades of 1 elicited, the incidence and severity of these ± responses in the test group were greater than those produced by the naive group suggesting that sensitization might have been induced. A rechallenge was carried out to more clearly define the mechanism.

Following a single patch rechallenge, there were again no grades of 1 produced in the test or control animals. The incidence of grade ± responses in the test group (13 of 20) was compared to that of the naive control group (8 of 10). The incidence of these responses in the test group remained essentially unchanged from that of primary challenge and was now less than that produced by the naive group indicating that sensitization had not been induced.