2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

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 8-4-1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted similar to OECD 406 guideline study. The study was performed in accordance with GLP.

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)

Principles of method if other than guideline:

The procedure used was based on that of Buehler (Buehler, 1965; Ritz and Buehler, 1980). The study was conducted according to the general conditions of the protocol and specifically as designated in the Project Instruction Sheet and Supplemental Instructions, as applicable. As such, it satisfies the criteria established by the Federal Insecticide, Fungicide, and Rodenticide Act (40 CFR), the Toxic Substance Control Act (40 CFR), and the OECD Guidelines.

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Study performed prior to the establishement of the LLNA protocol as an OECD guideline

Test material

Test material information

Constituent 1

Reference substance name:

2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

EC Number:

296-120-8

EC Name:

2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

Cas Number:

92257-31-3

Molecular formula:

Not applicable

IUPAC Name:

2-​Naphthalenol, 1-​[2-​[4-​(2-​phenyldiazenyl)​phenyl]​diazenyl]​-​, ar-​heptyl ar',​ar''-​Me derivatives *

Test material form:

liquid

Remarks:

Dark Red

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 period. 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

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

6 hours per day, once per week for 3 weeks

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

6 hours on one day

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Ten males and ten females were used in the induction primary challenge. Five males and five females were used in the primary challenge phase as naive controls.

Details on study design:

Irritation Screen:
Exposure of the guinea pigs can be divided into three phases: Irritation, Inductino, 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 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 guickly 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): 100, 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 atotal 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 pahse, 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 (e.G., using multiple samples at challenge will require using several sites).

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 was hoff 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 sesignated as 24 and 48 hour reading 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.

Challenge controls:

Primary Challenge:
Five male and five female naive animals were exposed to the undiluted test material.

Positive control substance(s):

no

Results and discussion

Positive control results:

none

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

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: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No data.

Results 2

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

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: 100%. No with. + reactions: 0.0. 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:

100%

No. with + reactions:

0

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: 100%. No with. + reactions: 0.0. 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:

100%

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: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No data.

Any other information on results incl. tables

The test material at 100% undiluted 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 in Guinea Pigs:

Test Material (100% undiluted) in Induced Animals^aWR

		Numerical Score
Animal Number	Sex	24-Hour	48-Hour
T-1 BK622	M	0	0
T-2 BK622	M	0	0
T-3 BK622	M	0	0
T-4 BK622	M	0	0
T-5 BK622	M	0	0
T-6 BK622	M	0	0
T-7 BK622	M	0	0
T-8 BK622	M	0	0
T-9 BK622	M	0	0
T-10 BK622	M	0	0
T-11 BK622	F	0	0
T-12 BK622	F	0	0
T-13 BK622	F	0	0
T-14 BK622	F	0	0
T-15 BK622	F	0	0
T-16 BK622	F	0	0
T-17 BK622	F	0	0
T-18 BK622	F	0	0
T-19 BK622	F	0	0
T-20 BK622	F	0	0
Mean		0.0	0.0

^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.

R = Red sample stain interfered with scoring.

Test Material (Undiluted) in Naive Animals^aPR

			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	0
C-28 RD/BK622	F	BA	0	0
C-29 RD/BK622	F	AB	0	0
C-30 RD/BK622	F	BA	0	0
Mean			0.0	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 as shown in Appendix A of the protocol.

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

R = Red samples stain interfered with scoring.

Individual Body Weight Data in Guinea Pigs

		Body Weight (grams)
Animal Number	Sex	Initial	Terminal
T-1 BK622	M	519	757
T-2 BK622	M	491	838
T-3 BK622	M	450	688
T-4 BK622	M	485	755
T-5 BK622	M	454	668
T-6 BK 622	M	493	788
T-7 BK 622	M	467	785
T-8 BK 622	M	459	706
T-9 BK 622	M	491	760
T-10 BK 622	M	503	776
T-11 BK 622	F	431	610
T-12 BK 622	F	404	555
T-13 BK 622	F	410	586
T-14 BK 622	F	428	620
T-15 BK 622	F	366	560
T-16 BK 622	F	420	640
T-17 BK 622	F	440	648
T-18 BK 622	F	426	601
T-19 BK 622	F	441	568
T-20 BK 622	F	442	665
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
P-41 RD622	M	482*	ND
P-42 RD622	M	435*	ND
P-43 RD622	F	416*	ND
P-44 RD622	F	457*	ND
P-45 RD622	M	436*	ND
P-46 RD622	M	452*	ND
P-47 RD622	F	458*	ND
P-48 RD622	F	456*	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*.
Following primary challenge, there were no erythema responses produced in any of the test or control animals. These data indicate that the test material did not induce a sensitising response.

Executive summary:

The potential of the test material to produce delayed contact hypersensitivity in male and female guinea pigs was evaluated using an adaptation of the method of Ritz and Buehler*. Study methodology followed was equivalent ot similar to OECD 406 in accordance with the Principles of Good Laboratory practice (GLP).

Following primary challenge, there were no erythema responses produced in any of the test or control animals. These data indicate that the test material was not a sensitiser