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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 January - 4 March 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
This study was conducted before the adoption of LLNA guideline

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: white solid
Details on test material:
The test substance, identified as RH-24,573 (2-methyl-3(2H) isothiazolone), Lot No. ASU 2379, was used. RH-24,573 is a white solid containing 99.8% active ingredient.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
One hundred fifty-two outbred Hartley guinea pigs [Crl:(HA)BR] were obtained from Hazleton Research Animals, Denver, PA, on January 06, 1988. Upon arrival animals were placed into an animal room, examined for physical abnormalities, assigned unique animal numbers (identified by cage cards), and acclimated to the study room for approximately three weeks. Fifty of the 152 animals that were originally placed in one study room were used in the present study. Animal care quarantine procedures were in effect during the first week of acclimation. The animals were individually housed in stainless steel cages (16" x 9-1/4" x 7") with wire mesh bottoms and fronts. Cages were suspended above absorbent paper liners which were changed daily. The animals were housed in an environmentally controlled room with controls set to maintain a temperature of 75°F with a relative humidity of
40-60%. Temperature and relative humidity were monitored 24 hrs a day and the light cycle was automatically controlled 12 hrs on and 12 hrs off. All guinea pigs had free access to filtered tap water (via automatic watering) and Certified Purina Guinea Pig Chow #5026, throughout the acclimation and test period except during the 6-hr periods of restraint during dosing. One day prior to the first induction dose, the guinea pigs were randomized into groups using computer generated random numbers. Each animal was then identified by a cage card, indicating the unique animal number, sex, test material, group number and the protocol number. At the initiation of the induction phase, the animals were approximately 5 weeks old and their body weights ranged from 365 to 534 grams. The guinea pigs fit comfortably in the restrainers throughout the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5000, 7500, 15000 and 30000 ppm a.i.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5000, 7500, 15000 and 30000 ppm a.i.
No. of animals per dose:
4 guinea pigs
Details on study design:
Dosinq Procedure
The procedures for dosing are those that are described in the method of Buehler (1). This procedure was followed for the animals of the naive control and induced groups during the induction and challenge phases. The day before application of the test substance, the hair was removed from the back of each guinea pig with electric clippers equipped with a surgical clipping head. Prior to each dosing, the guinea pig was placed in a restrainer, and 0.4 ml of the test substance was pipetted onto a patch*. The patch was then placed on the shaved skin of the guinea pig and the application site was occluded with rubber dental dam (0.01 inches thick). At the end of the 6-hr exposure period, each animal was removed from its restrainer and the patch and dam discarded. The exposure sites were washed with paper towels soaked in lukewarm tap water, the skin dried with paper towels, and the animals returned to their cages. Animals in the naive control group received a "SHAM" treatment during the induction phase. The exposure site was shaved, the animals were restrained, a blank patch was placed on the application site which was occluded for 6 hrs, and the shaved sites were washed in the same manner as the induced group.

Assessment of Primary Skin Irritation
Range-finding skin irritation tests were conducted on four naive guinea pigs to select doses to be used for the induction and challenge phases. Four concentrations of RH-24,573 (i.e., 5000, 7500, 15000 and 30000 ppm a.i.) were dissolved in distilled water and applied to four guinea pigs. The application sites were changed among the guinea pigs for each of the above concentrations to minimize any site-to-site variation in irritation response. The test substance was applied as described below. All application sites were depilated prior to scoring. Primary skin irritation was scored at 24 and 48 hrs after patch removal.

Assessment of Delayed Contact Hypersensitivit(DCH)
The closed patch method of Buehler (1) which has been validated in our laboratory with dinitrochlorobenzene (3) was used. Ten induction doses were applied to the shaved backs of guinea pigs over a 3.5 week period (3 doses/week; Mondays, Wednesdays and Fridays). Each dose (6-hr exposure period) consisted of 0.4 ml of the test material at 1000, 5000, 15000 or 30000 ppm a.i. in distilled water. Guinea pigs in the naive control group received a blank patch. Two weeks after the last induction dose, all guinea pigs in the naive control and RH-24,573 induced groups were challenged with three concentrations of RH-24,573 in distilled water (i.e., 1000, 5000 and 15,000 ppm a.i.). Nineteen to 22 hrs after the challenge application, the backs of the guinea pigs were depilated with Neet lotion hair remover. The depilatory was liberally applied to the clipped application sites and allowed to remain on the animals for approximately 20 minutes. The animals were rinsed with lukewarm running tap water, blotted dry, and returned to their cages. Two to 5 hrs after depilation (24 hrs after removal of the challenge patch) the erythema reactions at the application sites were scored according to the method of Ritz and Buehler (2)

Score Erythema Reaction
0................No reaction
+/-...........Slight patchy erythema
1................Slight confluent or moderate patchy erythema
2................Moderate erythema
3................Severe erythema
The application sites were re-evaluated 24 hrs later (i.e., 48 hrs after the challenge application).

Evaluation of Results
The results were evaluated by comparing the incidence of erythema reactions (at either 24 or 48 hrs) in the control groups to the incidence in the induced group. Grades of + were considered to be representative of insignificant erythema responses. Only those scores of grade 1 or greater were considered positive responses.

References:
1. Buehler, E.V. (1965), Delayed Contact Hypersensitivity in the Guinea Pig, Arch. Dermatol., 91, 171-175.
2. Ritz, H.L., and Buehler, E.V. (1980) Current Concepts in Cutaneous Toxicity, pp. 25-40. Eds. V.A. Drill and P. Lazar. Academic Press, New York.

*The "Hill-top" patches used were composed of a Parke-Davis Readi-Bandage modified by the addition of a centered Webril pad (20 x 20 mrn).
Challenge controls:
Guinea pigs in the naive control group received a blank patch. Two weeks after the last induction dose, all guinea pigs in the naive control groups were challenged with three concentrations of RH-24,573 in distilled water (i.e., 1000, 5000 and 15,000 ppm a.i.)
Positive control substance(s):
yes
Remarks:
Dinitrochlorobenzene has been used in the lab to validate the method.

Results and discussion

Positive control results:
Dinitrochlorobenzene has been used in the lab to validate the method.

In vivo (non-LLNA)

Results
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
15000ppm
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Erythema
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Assessment of Primary Skin Irritation

Minimal to no erythema was observed at 24 or 48 hrs in guinea pigs treated with the test material at the following concentrations; 5000, 7500, 15,000 and 30,000 ppm a.i.. Concentrations of RH-24,573 up to and including 30,000 ppm a.i. could be used for both induction and challenge.

Assessment of Delayed Contact Hypersensitivity (DCH)

No erythema reactions were observed in the noninduced control animals following challenge with 1000, 5000 and 15000 ppm a.i. RH-24,573.

In groups of guinea pigs induced with RH-24,573 at 1000, 5000, 15000 and 30000 ppm a.i., the incidence of erythema responses following challenge with 1000 ppm a.i. RH-24,573 was 0/10, 0/10, 1/10 and 0/10, respectively. In these same induction groups, the incidence of erythema following challenge at 5000 ppm a.i. RH-24,573 was 0/10, 2/10, 1/10 and 2/10, respectively, and at 15000 ppm a.i. RH-24,573 was 1/10, 6/10, 3/10 and 5/10, respectively.

The concentrations of RH-24,573 required to induce and elicit a response in 50% of the animals (EC50) are estimated to be 15,000 ppm a.i. for induction at a challenge concentration of 15,000 ppm a.i. and 15,000 ppm a.i. for elicitation at an induction concentration of 30,000 ppm a.i.. These induction and elicitation concentrations are approximately 20 to 40 fold greater than those required to induce and elicit a 50% incidence of sensitization with Kathon 886 biocide.

Table 1 A Summary of Incidences of Erythema of Grade 1 or Greater in Guinea Pigs During the Challenge Phase*

 Induction        Erythema Responses after Challenge with:
 Treatment

 RH-24,573 at 1000 ppm

in distilled water

 RH-24,573 at 5000 ppm

in distilled water

 RH-24,573 at 15,000 ppm

in distilled water

 None  0/10  0/10  0/10
 RH-24,573 at 1000 ppm a.i. in distilled water  0/10  0/10  1/10
  RH-24,573 at 5000 ppm a.i. in distilled water  0/10  2/10  6/10
  RH-24,573 at 15,000 ppm a.i. in distilled water  1/10  1/10  3/10
  RH-24,573 at 30,000 ppm a.i. in distilled water  0/10  2/10  5/10

*The maximum number of animals exhibiting erythema of grade 1 or greater at either 24 and/or 48 hrs, over the total number of animals in that group.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Under the conditions of this study, RH-24,573 produced delayed contact hypersensitivity in guinea pigs. The EC50's for induction and elicitation are estimated to be >5000 ppm a.i. (at a challenge concentration of 15,000 ppm a.i.) and >15,000 ppm a.i. (at an induction concentration of 30,000 ppm a.i.) RH-24,573, respectively.
Executive summary:

The delayed contact hypersensitivity (DCH) potential of RH-24,573 (Lot No. ASU 2379, TD No. 88-001, 99.8% a.i.) was tested in young adult outbred Hartley guinea pigs using the closed patch method of Buehler. Four groups of guinea pigs (5/sex/group) received ten 6-hr induction doses (3 doses/week, for 3.5 weeks) of 0.4 ml of the test material at the following concentrations: 1000, 5000, 15,000 or 30,000 ppm a.i. in distilled water. These animals and a group of 10 naive control guinea pigs (i.e., receiving no induction treatments) were challenged 2 weeks after the last induction dose. All guinea pigs were challenged with RH-24,573 at 1000, 5000 and 15,000 ppm a.i. in distilled water. Erythema reactions were scored at 24 and 48 hrs after the challenge exposure.

No erythema reactions were observed in the noninduced control animals following challenge with 1000, 5000 and 15,000 ppm a.i. RH-24,573.

In groups of guinea pigs induced with RH-24,573 at 1000, 5000, 15,000 and 30,000 ppm a.i., the incidence of erythema responses following challenge with 1000 ppm a.i. RH-24,573 was 0/10, 0/10, 1/10 and 0/10, respectively. In these same induction groups, the incidence of erythema following challenge at 5000 ppm a.i. RH-24,573 was 0/10, 2/10, 1/10 and 2/10, respectively, and at 15,000 ppm a.i. RH-24,573 was 1/10, 6/10, 3/10 and 5/10, respectively.

The concentrations of RH-24,573 required to induce and elicit a response in 50% of the animals (EC50) are estimated to be ≥5000 ppm a.i. for induction at a challenge concentration of 15,000 ppm a.i. and ≥15,000 ppm a.i. for elicitation at an induction concentration of 30,000 ppm a.i.. These induction and elicitation concentrations are approximately 20 to 40 fold greater than those required to induce and elicit a 50% incidence of sensitization with Kathon® 886 biocide.

CONCLUSION

Under the conditions of this study, RH-24,573 produced delayed contact hypersensitivity in guinea pigs. The EC50’s for induction and elicitation are estimated to be >5000 ppm a.i. (at a challenge concentration of 15,000 ppm a.i.) and >15,000 ppm a.i. (at an induction concentration of 30,000 ppm a.i.) RH-24,573, respectively