Nov. 1983 HLA AND ANTI-l-ILA ANTIBODY STUDIES IN HERPES GESTATIONIS 409

combination is too rare to provide an adequate study group. We assume t hat t here is no peculiar antigen distribution in t he husbands of these women. We also made no attempt to type the children of t hese couples, primarily because of their age. Such informat ion would obviously be of great interest.

Antibodies·against lymphocytes or monocytes occur in most women with HG . Most of the women in our study were 1- 5 years beyond their last pregnancy at the time antibody reactiv­ity was measured. Since anti-HLA antibodies decrease with time, it is likely that an even higher percentage of our patients would have had anti -HLA antibodies if studied at the time of parturition.

HLA-A, B, and C antigens are present on essentially all nucleated cells, including skin cells (keratinocytes). Only about 4% of epidermal cells express HLA-DR antigens (Langerhans ce lls) . There is no evidence that any of these antigens are represented within the BMZ. Further, extensive absorptions of Reunala's case with HLA-B8 positive lymphocytes failed to alter BMZ staining by t he patient's serum. Thus, anti -HLA antibodies are unlikely to play a direct role in the fixation of C at the dermal-epidermal junction in patients with HG.

It is possible that anti-HLA antibodies exert their effect on the development of HG indirectly by influencing some aspect of immune regulation. It is also possible that the production of anti-HLA antibodies, although more common in patients with HG , has no relationship to the pathogenesis of this disease. The production of these antibodies may be stimulated by the same factors that induce anti-BMZ antibody production. Fur­t her research into the factors that influence product ion of anti­BMZ and anti-HLA antibodies in this group of patients may provide insight into the fundamenta l abnormality of HG.

The authors wish to thank Gabriel Nunez, M.D. for his help and expertise.

0022-202X/83/8105-0409$02.00/0 THE JOUilNAL o~- INVESTIGATIVE DEHMATOLOGY, 81:409-4 11, 1983 Copyright © 1983 by The Williams & Wilkins Co.

REFERENCES 1. Jordon RE, Heine KG, T appeiner G, Bushkell LK, Provost TT:

The immunopathology of herpes gestationis. J Clin Invest 57:1426- 1433, 1976

2. Katz Sl , Hertz YC, Yaoita H: Herpes gestationis: immunopathol­ogy and cha racte rization of t he HG factor. J Clin Invest 57:1434-1441, 1976

3. Dupont C: Herpes gestationis with hydatidiform mole. Trans St Johns Hosp Dermatol Soc 60: 103, 1974

4. Tillman WG: Herpes gestationis with hydatidiform mole and cho­rion epithelioma. Br Med J 1:1471, 1950

5. Foidart JM, Yaar M, Hall R, Gaspard U, Katz SI: Immunopath­ological and clinical studies in herpes gestationis. Br J Obstet Gynecol 88:153-159, 1981

6. Rocklin RE, Ki tzmiller JL, Kaye MD: Immunobiology of t he materna l-fetal relationship. Annu Rev Med 30:375-404 , 1979

7. Reunala T , Karvonen J, Tii lika inen A, Salo OP: Herpes gestation is: a high t iter of anti-HLA B8 ant ibody in t he mother and pem­phigoid-like immunohistological findings in t he mother and child. Br J Dermatol 96:563- 568, 1977

8. Shornick JK, Stastny P, Gi lliam JN: High frequency of histocom­patibili ty antigens HLA-DR3 and DR4 in herpes gestationis. J Glin Invest 68:553-555, 1981

9. Ray J G, Hare DB, Pederson PD, Mullelly Dl: Manual of Tissue Type Techniques. DHEW publication (National Institutes of Health) , 78:545. 1976, pp 1-212.

10. Danilovs J , Ayoub G, Terasaki PI: B lymphocyte isolation by thrombin -nylon wool, Histocompatibili ty Testing 1980. Edited by PI Terasaki. Los Angeles, UCLA Tissue Typing Laboratory, 1980, pp 287- 288

11. Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 2l(suppl 97):77-89, 1968

12. Colbaugh P, Stastny P: Antigens in monocytes. III. Use of mono­cytes in typing for the HLA-D related (DR) antigens. Transplant Proc 10:871- 874, 1978

13. Bodmer JG, Bodmer WF, P iazza A: Inclusion analysis of Fifth Histocompatibili ty Testing Workshop sera in 25 populations. Tissue Antigens 5:315-366, 1975

14. Lawler SD, Klodna PT, Bayshawe KD: Immunogenicity of mola r pregnancies in the HLA system. Am J Obstet Gynecol 120:857-861, 1974

Vol. 81, No.5 Printed in U.S.A.

Kathon Biocide: Manifestation of Delayed Contact Dermatitis in Guinea Pigs Is Dependent on the Concentration for Induction and Challenge

PING KWONG C HAN, PH.D., ROBERT C . BALDWIN, PH.D., RICHARD D. PARSONS, B.S., JACK N. Moss, B.S.,

RoBERT STIRATELLI, Sc.D. , JERRY M. SMITH , PH.D., AND A. WALLACE HAYES, PH.D.

Toxicology Department, Rohm and Haas Company, Spring Hou.~e. Pennsylvania, U.S.A.

The potential of Kathon biocide, an aqueous solution containing, as active ingredients (a.i.), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (14.4% a.i.), to produce delayed con­tact dermatitis, a sensitization response, was evaluated in outbred Hartley guinea pigs by a modified Buehler's

Manuscript received May 13, 1983; accepted for publication July 11, 1983.

Reprint requests to: Dr. P ing Kwong Chan, Toxicology Department, Rohm and Haas Co., 727 Norristown Road, Spring House, Pennsylva­nia 19477.

Abbrev iations: a.i. : active ingredients EC50: concentration at wh ich delayed contact dermatitis was seen in 50% of the population

occluded epicutaneous patch technique. The relationship of the response as a function of induction/elicitation concentrations was investigated. Groups of guinea pigs received 9 induction doses of the biocide, 3 times a week, at concentrations ranging from 25- 2000 ppm a.i. These guinea pigs were challenged with the biocide at concen­trations ranging from 20-2000 ppm a .i. , and the appli­cation sites were scored for erythema 24 and 48 h after the challenge. The incidence of delayed contact derma­titis in induced guinea pigs was dependent on both the induction and challenge concentrations. The EC5 0 (con­centration at which delayed contact dermatitis was seen in 50% of the population) for induction at a challenge concentration of 2000 ppm a.i., a nonirritating concen­tration, was estimated to be 88 ppm a.i. with a slope of

410 CHAN ET AL

3.47 probitsjunit log concentration. The EC50 for elici­tation at an induction concentration of 1000 ppm a.i. was estimated to be 429 ppm a.i. with a slope of 2.74 probits/unit log concentration. These data demonstrate that for Kathon biocide, there is an induction/elicitation concentration dependency for delayed contact dermati­tis response, and there is a "no response concentration" zone where the biocide can be used without concern for clinically significant delayed contact dermatitis. In com­parison with a previous study, these data also suggest that the number of induction doses may be an important factor in demonstrating the sensitization potential of a chemical.

Kathon biocide,* a widely used, broad-spectrum antimicro­bial agent, contains, as active ingredients (a.i.), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothia­zolin -3-one in an approximate ratio of 3:1, respectively, with MgCl2 (9%) and Mg(N0 3h (16%) present as stabilizers. The biocide is an effective preservative for toi letries, cosmetics, and household cleaning products. It is also used in some heavy industrial applications such as cooling-tower water, metal­working fluid, and latex emulsions.

A variety of industrial chemicals, cosmetic fragrances, and therapeutic agents, as well as several naturally occurring sub­stances such as poison ivy, can cause delayed contact dermatitis in animals and humans [1- 3]. These sensitizers fa ll into diver­sified chemical categories ranging from simple inorganic metals such as nickel to complex organic chemicals. Determination of the potential for a chemical to cause delayed contact dermatitis is often needed for the development of a risk assessment process to protect workers and consumers. Since cutaneous contact with products containing Kathon biocide is a primary route of exposure, the capacity of Kathon biocide to produce delayed contact dermatitis was investigated in guinea pigs.

Beuthet reported that no incidence of delayed contact der­matitis was observed in guinea pigs even at induction and elicitation concentrations as high as 1500 ppm a.i. when the biocide was applied once a week for 3 weeks. Parsons:j: reported that one of the active ingredients of Kathon biocide, 2-methyl-4-isothiazolin-3-one, induced delayed contact dermatitis at 16,000 ppm a.i ., but no response was noted when these induced guinea pigs were challenged at 1600 ppm a.i. A concentration­dependent delayed contact dermatitis response to the biocide was also reported in humans [4]. Therefore, another objective of this study was to characterize the induction/elicitation con­centration response relationship for Kathon biocide in guinea pigs.

MATERIALS AND METHODS Test Substance and Animals

An aqueous solution of Kathon biocide (commerc ia l sample) con­ta ining 14.4% a.i. was used for prepa ring va rious aqueous dilutions. All fin al concentrations were confirmed by high -pressure liquid chroma­tography analysis.

Outbred Hartley guinea pigs weighing 200-300 g (Charles River Breeding Laboratories, Kingston , New York) were individually housed in cages with wire bottoms in an airconditioned room with controls set to mainta in 20- 22 · c , 45-55 % relative humidity, and a 12-h light cycle. Standard laboratory guinea pig chow (Ralston Purina Co., Richmond,

* Kathon is a Rohm and Haas Company registered tradema rk . The active ingredients descr ibed in this report are commercially available from Rohm and Haas Co. under the names Kathon 886 MW, Kathon WT, Kathan LX, and Kathan CG.

t Beuthe HF (1982). Expert opi nion concerning the experimental sensitivity test of Kathan CG for contact allergy in guinea pigs. Univ­ersitiit Hamburg, Un iversitiits-Krankenhaus (University Hospita l). Private communication.

:j: Parson RD (1979) . Private communication .

Vol. 81, No.5

Indiana) and water were ava ilable ad libitum except during exposure (induction or cha llenge dosing). The a nimals were qua rant ined for at least 7 days prior to the study.

Primary Irritation

A range-findin g irri tation test was conducted with 4 naive gu inea pigs to determine the highest nonirri tating concentration of Kathan biocide. Four conce ntrations were applied in a patch to the closely clipped backs of t he guinea pigs. The method of app lication and t he erythema scoring system were t he same as described in the assessment of delayed contact dermatitis.

Assessment of Delayed Contact Dermatitis

A modified technique described by Ritz and Bueh ler [5] was em­ployed to assess delayed contact dermatitis. Nine induction doses, each consisting of 0.4 ml of the appropriate aqueous dilution of Katha n biocide, were applied under cover to the clipped bac ks of guinea pigs for 3 6-h periods per week for 3 consecutive weeks. The patch was occluded with a rubber "dental da m," and anima ls were placed in a restrainer dur ing eac h of t he exposures. The application s ite was washed wi t h wate r a fter the exposure. The treated guinea pigs were challenged with 0.4 ml of Kathon biocide by means of a n occluded patch, 12- 15 days afte r t he last induction dose. The cha llenge concen­t rations ra nged from 20- 2000 ppm a.i . Noninduced naive guinea pigs were ·aJso challenged with Kathon biocide in the same ma nner a nd at t he same concentrations for comparison to erythema due to primary irri tation.

Approximately 24 h after t he challenge exposure, t he backs of the guinea pigs were depi lated with Neet lotion hair remover. Two to five hours after depilation, t he guinea pigs were scored for erythema reac­t ions according to the system listed in T able I. Erythema reactions of grade 1 or greater were considered positive responses, indicative of sensitization in anima ls that received the induction dose.

EC50 (concentration at which delayed contact dermatit is was seen in 50% of t he population) values for induction or elicitation of delayed contact dermatitis were estimated by probit analys is as described by F inney [6].

RESULTS

The highest nonirritating concentration for Kathon biocide was 2000 ppm a.i. All concentrations used in the induction or challenge phase of the study were equal to or less than 2000 ppm a.i.

The incidences of delayed contact dermatitis are listed in Table II. No erythema was observed in the noninduced naive control guinea pigs. The incidence of delayed contact dermatitis was related to the induction concentration; 20/20, 10/ 10, 9/15, 2/15, and 1/20 guinea pigs induced with 2000, 1000, 500, 100, 50, and 25 ppm a. i. , respectively, responded to a challenge concentration of 2000 ppm a.i. Kathon biocide. Th_e incidence of delayed contact dermatitis was also dependent on the elici­tation concentration. At an induction concentration of 1000 ppm a.i., 4/5, 3/5, 3/15, and 0/20 guinea pigs responded when challenged with 1000, 500, 200, and 50 ppm a.i., respectively. A "no response concentration" . zone which is encompassed by induction and elicitation concentrations of 2000 and 20 ppm, 1000 and 50 ppm, 500 and 100 ppm, 50 and 100 ppm, and 25 arid 200 ppm is suggested by the data in Table II. These concentrations are above the recommended final use concen­tration range of Kathon biocide. The induction/elicitation con­centration dependency of delayed contact dermatitis response is illustrated in a 3-dimensional graph (Fig 1) resulting from fitting the sensitization response vs induction or elicitation

TABLE l. Erythema. scoring system

Erythema reactions

No reaction Slight patchy erythema Sl ight confluent or moderate patchy ery­

thema Moderate erythema Severe erythema (with or without edema)

Assigned score

0 0.5 1

2 3

Nov. 1983 KATHON BIOCIDE 411

TABLE II. Incidence of delayed contact dermatitis in gw:nea pigs induced and challenged by various concentrations of Kathan biocides

Induction" con-Induction treatment centration (ppm

a.i.) 2000 1000

Noninduced cotitrol 0 0/20 Kathon biocide 2000 20/20 2/2

1000 4/5 500 10/ 10 100 9/15 50 2/15 25 1/20

500

0/10 1/2 3/5 3/10

Incidences of delayed contact dermatitis• Elicitation Concentration (ppm, a.i.)"

250

1/2

200

0/ 10 2/10 3/15

1/15 0/20

100

0/10 1/ 15 0/15 0/20

50

0/30

0/20

0/ 15

25

0/ 10

0/ 20

20

0/ 10

"Dosage volume = 0.4 ml/patch. b The number of animals that responded at either 24 or 48 h after the elicitation exposure over the total number of animals challenged in that

group.

PROB

I. 00

0. 67

O.H

2. 2

LOG I BASE 101 Of IN

(lnductl on Concentration) I. I

0. 000. 00

3 . <0

LOG !BASE I OJ Of Ell C (E licitati on Concentnltion)

I. 13

FtG 1. A 3-dimensional illustration of the concentration depend­ency of the delayed contact dermatitis manifested in guinea pigs induced and challenged with Kathon biocides.

concentrations to a logistic model: Log ( p/1-p) = A + Bt (induction concentration) + B2 (elicitation concentration) + B:J (induction concentration) (elicitation concentration), where P = probability of response, and. A = -4.069618, B, = 0.000128, B2 = 0.000919, and B:J = 0.000007.

The calculated EC50 for induction in guinea pigs challenged with 2000 ppm a.i. of Kathan biocide was 88 ppm a.i. with 95% confidence limits of 66-145 ppm a.i. and a slope of 3.4 7 probits/ unit log concentration. The calculated ECr,o for elicitation in guinea pigs induced with 1000 ppm a.i. of Kathan biocide was 429 ppm a.i. with 95 % confidence limits of 272-995 ppm a.i . and a slope of 2. 75 probits/unit log concentration.

DISCUSSION

The dose-response relationship for a particular toxic re­sponse is useful information in assessing the risk of a chemical. Therefore, it is important to conduct sensitization studies with several concentrations of the test substance to evaluate this relationship. Sensitization reactions have been considered by some as lacking a dose-response relationship [7]; however,

Marzulli and Maibach [8] have shown a dose-dependent re­sponse for the induction of delayed contact dermatitis in hu­mans for several sensitizers including mafenide, benzocaine, bronopol, p-phenylenediamine, formalin, and glutaraldehyde. They also demonstrated that the delayed contact dermatitis response with o-chlorobenzylidine malononitrile and 2-chlo­roacetophenone was related to the challenge concentration. Our data suggest that the potential of Kathan biocide to cause delayed contact dermatitis is dependent on both the induction concentration and the challenge concentration. These data support the conclusion reported by Marzulli and Maibach [8]. Although the mechanism of the dependency of delayed contact dermatitis on the induction concentration may be different from that of the challenge concentration, the clinical signifi­cance is the same, i.e ., the manifestation of a sensitization reaction. Therefore, both concentrations should be taken into consideration in any risk assessment.

Beuthet reported that no incidence of delayed contact der­matitis was observed in Kathon biocide-treated guinea pigs even at induction and challenge concentrations as high as 1500 ppm a.i. when only 3 induction doses (1 dose per week for 3 weeks) were employed. In our study, however, 9 induction doses at lower concentrations (e.g., 1000 ppm a.i. , 3 doses per week for 3 weeks) resulted in sensitization in guinea pigs. These data demonstrate that by topical application in guinea pigs, t he number of induction doses may be an important factor in demonstrating the sensitization characteristic of a chemical.

On the basis of these results, there is an induction/elicitation concentration dependency for delayed contact dermatitis re­sponse for Kathan biocide, and there is a "no response concen­t ration" zone where the biocide can be used without concern for clinically significant delayed contact dermatitis.

The authors thank Ms. M. E. De Crescente for her excellent technical assistance and Ms. Karen Hendler for typing the manuscript.

REFERENCES 1. Fisher AA: Contact Dermatitis, 2d ed. Philadelphia, Lea & Febiger,

1973 2. Andersen KG, Maibach HI: Allergic reaction to drugs used topi­

cally. Clin Toxicol 16:415-465, 1980 3. Rao KS, Betso JE, Olson KJ: A collection of guinea pig sensitiza­

tion test results-grouped by chemical class. Drug Chern Toxicol 4:331-351, 1981

4. Rohm and Haas Co. Bulletin: Kathon CG. Cosmetic and Toiletry Preservative, 1982

5. Ritz HL, Buehler EV: Planning, conduct, and interpretation of guinea pig sensitization patch test, in Current Concepts in Cu­taneous Toxicology. Edited by VA Drill , P Lazar. New York, Academic Press, 1970, pp 25- 40

6. Finney DJ: Probit Analysis, 3d ed. London , Cambridge Univ Press, 1971

7. Klaassen CD, Doull J: Evaluation of safety: toxicologic evaluation , in Casarett and Doull's Toxicology- The Basic Science of Poi­son, 2d ed. Edited by J Doull, CD Klaassen, MO Amdur. New York , Macmillan, 1980, p 15

8. Marzulli FN, Maibach HI: The use of graded concentrations in studying skin sensit izers: experimental contact sensitization in man. Food Cosmet Toxicol 12:219- 227, 1974