A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 19, 1990 to March 28, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted following good laboratory practices and was a well conducted standard test with adequate documentation. However the exact guidelines were not specifically cited numerically and the purity and batch number of the test substance was not provided.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: U.S. EPA Federal Insecticide, Fungicide, and Rodenticide Act (40 CFR), the Toxic Substances Control Act (40 CFR), and the OECD Guidelines

Deviations:

no

Remarks:

Study report states there were no deviations.

GLP compliance:

yes

Type of study:

Buehler test

Test material

Test material information

Constituent 1

Reference substance name:

87787-81-3

EC Number:

618-070-1

Cas Number:

87787-81-3

IUPAC Name:

87787-81-3

Constituent 2

Reference substance name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

EC Number:

413-800-3

EC Name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Cas Number:

87787-81-3

Molecular formula:

C44H79NO3

IUPAC Name:

2-(dioctadecylcarbamoyl)benzoic acid; 2-(ditetradecylcarbamoyl)benzoic acid; 2-[(propan-2-yloxy)carbonyl]benzoic acid; 2-[octadecyl(tetradecyl)carbamoyl]benzoic acid; 2-octadecyl-2,3-dihydro-1H-isoindole-1,3-dione; 2-tetradecyl-2,3-dihydro-1H-isoindole-1,3-dione; benzene-1,2-dicarboxylic acid; dioctadecylamine; ditetradecylamine; octadecyl(tetradecyl)amine

Constituent 3

Reference substance name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

IUPAC Name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

Constituent 4

Reference substance name:

Stepan TAB-2

IUPAC Name:

Stepan TAB-2

Details on test material:

No data on test substance purity or composition. It is assumed to be typical commercial material. Test material designation is as Stepan Agent X1401 -91. Test substance was stored at room temperature in a clear colorless glass jar with a black plastic lid..

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Young adult, male and female Hartley albino guinea pigs of a size to fit the restraining device from a U.S.D.A.approved supplier were used. Twenty test animals, ten naive control, four animals per pilot study were used. A total of 38 animals were used. When possible, equal numbers of males and females were included in both test and control groups. Cage cards incorporating an animal group code- arrival date - color code marking system, was used to identify each guinea pig. The animals were randomly caged according to standard operating procedures. Prior to use, all animals were quarantined for at least four days. Animals were housed individually in wire mesh suspension cages. Purina Guinea Pig Chow (or other comparable diet) was fed ad libitum. The animals were maintained on a 12-hour light/12-hour dark cycle. The weights of the test animals fell into a range of 354 to 520 grams.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Induction 2

Concentration / amount:

Concentrations of test material in acetone vehicle are provided in tables below in results section. The concentrations are 75%, 50%, 25% and 10% (w/v).

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Challenge 2

Concentration / amount:

Concentrations of test material in acetone vehicle are provided in tables below in results section. The concentrations are 75%, 50%, 25% and 10% (w/v).

No. of animals per dose:

20 animals were used for the test - 10 for naive control and 4 animals per pilot study were used. There were two pilot studies. In all, 38 animals were used. See Challenge Phase: The test animals which have had three previous exposures to the test substance at appropriate intervals are again exposed in the Challenge Phase. In addition, ten animals (naïve control) which have never been exposed to the test material are treated with the same concentration.
The animals previously exposed during the induction period as well as the previously untreated control animals are challenged two weeks after the last induction exposure. The same patching procedure is used as for the “induction phase,” but the patches are applied to a skin site that has not been previously exposed. The site for challenge may be varied as necessary to achieve the objectives of the experiment (e.g., using multiple samples at the challenge will require using several sites as illustrated in the study appendix).

Details on study design:

The procedure used was based on that of Buehler (Buehler, 1965; Ritz and Buehler, 1980) and is summarized below.
References:
Buehler, E. V. (1965). Arch. Dermatol. 91, 171-175
Ritz, H.L. and Buehler, E.V. (1980). Current Concepts in Cutaneous Toxicity (V.A. Drill and T. Lazar, eds.). pp. 25-40.

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 substance to be used in the induction and/challenge phase. If the test substance 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 substance per animal, formulated in the solvent appropriate for a given study phase, may be evaluated. Generally the concentration selected by the study director may be one which causes mild to moderate irritation. For challenge, the concentration selected should be no more than slightly irritating.

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. These positions on the test animal’s body are illustrated in the study report Appendix A. The irritation evaluations can be done before or during the study phases as appropriate.

In preparation, the hair is removed from the animals’ backs using a small animal clipper. This is done the day before applying the test material. Closed patches are applied to the animals in the following manner: An appropriate volume of each test material is applied into the selected patch system. The animal is placed in the restrainer and the patches are applied to the clipped surface as quickly as possible. The patch is occluded with a rubber dental dam pulled taut and fastened to the bottom of the restrainer with clips. The restrainer is adjusted to minimize movement of the animal during exposure. Approximately six hours later the dental dam and patches are removed and the animal is taken from the restrainer and placed in its cage. Excess material may be removed by an appropriate solvent. The day following the irritation exposure the animals are depilated and scored as described under “observations.”.

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. The level of test material 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.

The left shoulder of each animal is clipped with a small animal clipper the day before exposure. Closed patches are applied to the clipped area of the guinea pigs. The animals are exposed and restrained as described previously under “irritation screen.” The procedure is repeated at the same site once a week for the next two 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 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, the animals are left untreated for approximately two weeks (12-16 days) before primary challenge.

Challenge Phase: The test animals which have had three previous exposures to the test substance at appropriate intervals are again exposed in the Challenge Phase. In addition, ten animals (naïve control) which have never been exposed to the test material are treated with the same concentration.

The animals previously exposed during the induction period as well as the previously untreated control animals are challenged two weeks after the last induction exposure. The same patching procedure is used as for the “induction phase,” but the patches are applied to a skin site that has not been previously exposed. The site for challenge may be varied as necessary to achieve the objectives of the experiment (e.g., using multiple samples at the challenge will require using several sites as illustrated in the study appendix).

Observations: The day following the primary challenge exposure, all animals are depilated with a commercial depilatory. The depilatory is placed on the test sites and surrounding areas and left for no more than fifteen minutes. The depilatory is thoroughly washed off with warm running water, the animals dried with a towel and returned to their cages.

A minimum of two hours after depilation the test sites are graded on a scale of 0-3 (0 = no reaction, ± = slightly patchy erythema, 1 – slight but confluent or moderate patchy erythema, 2 = moderate erythema, 3 = severe erythema with or without edema)(reported as 24 hour scores. The grading is repeated the following day. For reporting purposes the first and second gradings are 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 reactions 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:

Naive controls were used as described above in the details on study design, and in the "Any Other Information on Materials and Methods Including Tables Section."

Positive control substance(s):

yes

Remarks:

1-Chloro-2m4-dinitrobenzene (DNCB)(0.1% in acetone) historical positive control

Results and discussion

Positive control results:

See Tables in the "Any other Information on results incl. tables section.

In vivo (non-LLNA)

Results

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% formulation in acetone

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

mild irritation but no sensitization. A positive + reaction is defined as :slight but confluent or moderate patchy erythema. is

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% formulation in acetone. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: mild irritation but no sensitization. A positive + reaction is defined as :slight but confluent or moderate patchy erythema. is .

Any other information on results incl. tables

Incidence and Severity of Responses

Group	Test Material	Concentration^b (test material formulated in acetone)	Incidence of Responses		Mean Severity       Scores
			24-Hour	48- Hour
			0 ±   1    2      3	0 ±   1    2      3	24-Hour   48-Hour
Primary Challenge
Test	Stepan Agent X1401-91	5%	11 9  0   0    0	17 3  0   0    0	0.2           0.1
Naïve Control	Stepan Agent X1401-91	5%	2  8  0   0    0	7  3   0   0    0	0.4          0.2

 

Table 1 Primary Irritation: Skin Grades in Guinea Pigs 24 and 48 Hours Following a Six Hour Patch Application of Stepan Agent X1401—91 at Various Concentrations in Acetone

Animal No.	Sex	Numerical Score (Sight Location^a)
		5% w/v	2.5% w/v	1% w/v	0.5% w/v
		24-hour 48-hour	24-hour 48-hour	24-hour 48-hour	24-hour 48-hour
		Pilot No. 1
P-31 PN215	M	0             0 (1)	0             0 (2)	0             0 (3)	0             0 (4)
P-32 PN215	M	0             0 (2)	0             0 (3)	0             0 (4)	0             0 (1)
P-33 PN215	F	0             0 (3)	0             0 (4)	0             0 (1)	0             0 (2)
P-34 PN215	F	0             0 (4)	0             0 (1)	0             0 (2)	0             0 (3)

^aPatch site locations 1-4 given in parentheses were used in accordance with those specified in Appendix A of the protocol.

Table 2 Primary Irritation: Skin Grades in Guinea Pigs 24 and 48 Hours Following a Six Hour Patch Application of Stepan Agent X1401—91 at Various Concentrations in Acetone

Animal No.	Sex	Numerical Score (Sight Location^a)
		75% w/v	50% w/v	25% w/v	10% w/v
		24-hour48-hour	24-hour 48-hour	24-hour 48-hour	24-hour 48-hour
		Pilot No. 2
P-35 PN215	M	±             ± (1)	±              ± (2)	±              ± (3)	±              0 (4)
P-36 PN215	M	±             ± (2)	±              ± (3)	±              0 (4)	±              0 (1)
P-37 PN215	F	±              0(3)	0              0 (4)	±              0 (1)	±              0 (2)
P-38 PN215	F	±             ± (4)	±              ± (1)	0              0 (2)	±              ± (3)

^aPatch site locations 1-4 given in parentheses were used in accordance with those specified in Appendix A of the protocol.

Table 3 In report (not shown in this summary) tabulates skin grades following primary challenge.

Table 4 in report (not shown in this summary) tabulates individual body weight data of test animals.

References: 

Buehler, E. V. (1965). Arch. Dermatol. 91, 171-175

Ritz, H.L. and Buehler, E.V. (1980). Current Concepts in Cutaneous Toxicity (V.A. Drill and T. Lazar, eds.). pp. 25-40.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test substance was determined not to be positive for skin sensitization under the conditions of the study.

Executive summary:

The potential of Stepan Agent X1401-91, as a 25% formulation in acetone to produce delayed contact hypersensivity in guinea pigs was evaluated using an adaption 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 (10 of 20) was compared to that of the naive control group (8 of 10). The incidence of these responses in the test group was less than that produced by the naive group, indicating that sensitization had not been induced.