TOXICOLOGY AND APPLIED PHARMACOLOGY i', 559-565 (1965)



### Comparative Acute Effects of Some Chemicals
on the Skin of Rabbits and Guinea Pigs’

ROBERT L. ROUDABUSH, C.J. TERHAAR, DAVID W. FASSETT, AND STANLEY P. DZIUBA Laboratory of Industrial Medicine, Eastman Kodak Company, Rochester, Sew York

Received April lb, 1964 The potential hazard of human skin contact with achemical is frequently determined from dataobtained from theapplication of thechemical totheskin ofexperimental animals. Exposures of this type permit one to evaluate both the type and degree of dermal reaction aswell as the possibility of percutaneous absorption. The literature records examples of the use of a number of mammalian species for such studies. However, since the publication of Draize etal. (1944)) and the subsequent designation of the rabbit as thesole animal species recognized for such tests in the regulations under the Federal Hazardous Substances Labeling Act (21 CFR 191; hereinafter referred to as the “Regulations”), industrial toxicologists in the United States have restricted themselves largely tothis single species for the routine screeningofskin effects caused by chemicals or chemical formulations. While the rabbit hasbeenuseful in predicting human response toskin irritants or penetrants, there has apparently beenlittle inclination tocarry out systematic comparisons with other species. For the past fifteen years, this laboratory has used the “albino” guinea pig for studies of skin irritation, percutaneous absorption, and skin sensitization. Someof the advantages of the guinea pig are obvious; namely, lower animal cost and food consumption, lesser space requirement, easeof handling, and, especially, the possibility of testing chemicals of which only limited amounts are available. Over theyears the dermal effects ofalarge number oforganic compounds ofextremely diverse types, including most commonly used industrial chemicals, have beentested inthis species. Many of these data can be found in the recent revision of Patty’s book (1962). Sinceour experience with skin tests onthe intact abdomen of guinea pigs has shown this species to bevery useful for such studies, it was felt that itwould beof interest tocompare the reactions of theguinea pigwith those of the rabbit to materials of various types of toxic action and potency. Substances in
common use in industry were included in this study aswell assome household substances. MATERIALS AND METHODS All ofthe guinea pigs used inthis study were males, Hartley derived,” ranging in weight from 400to900g.Thewhite rabbits were of both sexes, were purchased from
alocal supplier, andvaried from 1to4kg inweight.

1 Presented at the Third Annual Meeting of the Society of Toxicology, Williamsburg, Virginia, March 9-11, 1964.

zzTumblebrook Farms, Brant Lake, New York.

559560 ROBERT L. ROUDABUSH ET AL.

The hair was removed from the test area of all animals by clipping. The guinea pigs hadthe hair stubble removed with abarium sulfide depilatory which was allowed to remain on the skin for only afew minutes. After this, the skin was washed in running water and the animals were allowed to dry for several hours before application of the test materials. For the abraded skin tests, abrasions were made longitudinally every 2-3 cm by drawin g a harpoon-shaped needle over the skin with sufficient pressure topenetrate only thestratum corneum. Acute dermal toxicity. The procedure for testing acute dermal toxicity on rabbit skin was that described in the Regulations (21 CFR 191.10). The only exceptions tothis procedure were that theanimals were not immobilized for 24hours but were confined tocages, andthe skin ofall rabbits was abraded. A minimum of 3dosages
was employed, usually with 4animals per dosage, and the LDoo was calculated by the method of Finney (1952). Rubber dental dam was used as the sleeve material except with those materials that react with rubber, inwhich case S-mil fluorocarbon plastic sheeting was used. The test compounds were applied undiluted to guinea pigs on a l-inch square cellulose pad3 held onto the clipped and depilated intact abdomens or the clipped and depilated abraded backs under acuff of rubber dental dam or plastic sheeting
asnecessary. The edgesof the cuff were sealed by attaching them to the skin with black rayon tape,” one side of which was coated with adhesive. The number of dosages, animals perdosage, andthemethod ofcalculation oftheLDSowere thesame aswith rabbits. Primary irritation. The procedure for testing andevaluating primary irritation was identical to that described in the Regulations (21CFR 191.1I), except that the rabbits were not immobilized and the l-inch square cellulose pads were used instead of surgical gauze. Each compound was applied to 6rabbits with intact, and 6with abraded, skin. The test procedure in the guinea pig included application of the materials, or aqueous solutions of them, oncellulose pads held incontact with the skin under an appropriate sleeve. Six guinea pigs with clipped, depilated, intact abdomens and 6 with clipped, depilated, abraded backs were used for each material. Calculations of the primary irritation score were made according to the Regulations (21 CFR 191.11). However, when intact or abraded skin was evaluated alone, the average values at24and 72hours were divided by 2instead of 4. The identity, source, and quantity of the test substances applied to the animals
are listed inTable 1.

Acute Dermal Toxicity RESULTS

Table 2lists the LD6,,‘s obtained asaresult ofthe acute dermal toxicity studies. Benzene, carbon tetrachloride, andacetone,allcompounds which are“practically nontoxic” by skin contact according tothesystem ofclassification of Hodge and Sterner (1949), gave similar responses regardless of whether intact guinea pig abdomen,

3 Multilith pads for Multilith masters, Addressograph-Multigraph Corporation, Cleveland, Ohio. 4 Eastman Kodak Company, Rochester, New York.SKIN OF RABBITS AND GUINEA PIGS 561

TABLE 1 DOSE AND SOURCE OF SUBSTANCES USED IN PRIMARY IRRITATION TESTS Compound Dose and concentration Source
Acetic acid 0.5 ml, 4% inwater (v/v) Ammonia. household 0.5 ml, undiluted Borax 10ml, 5% in water (w/v) Boric acid 5ml, 10% in water (w/v) 2.Butanone 0.5 ml, undiluted Carbon tetrachloride 0.5 ml, undiluted Gasoline 0.5 ml, undiluted Kerosene 0.5 ml, undiluted Phenylhydrazine 50 mg, undiluted Sodium carbonate 2ml, 257,in water (W/V) Sodium sulfite 2ml, 25% in water (w/v) Sucrose 1ml, 50% in water (w/v) Trisodium phosphate 2ml, 25% inwater (w/v) Water. distilled 0.5 ml Eastman Organic Chemical no. P763 Grocery store Kodak Photographic Chemical Kodak Photographic Chemical Eastman Organic Chemical no. 383 Eastman Organic Chemical no. P444 Service station Service station Eastman Organic Chemical no. 329 Kodak Photographic Chemical Kodak Photographic Chemical Grocery store Kodak Photographic Chemical Laboratory

Compounds TABLE 2 ACUTE DEFLMAL LD,, (ML/KG) OF TEST COMPOUNDS Rabbit, Guinea pig, Source” abraded skin intact skin Guinea pig, abraded skin
Acetone Benzene Carbon tetrachloride Butylamine Aniline Rutoxyethanol Acrylonitrile Parathion P297 P777 P444 1261 25 P2270 P.5161

- >9.4

>9.4 >9.4 >1.5 0.82 0.68 0.28 0.07 >9.4 >9.4

>9.4 >9.4 >9.4 >9.4 0.58 >l.S 1.29 2.15 0.23 0.30 0.46 0.84 0.60 ,030

aNumbers refer to Eastman Organic Chemicals sold by Distillation Products Industries, Rochester, New York. The parathion was a sample of 99.6% purity purchased from American Cyanamid Company, Wayne, New Jersey.
abraded guineapig back, orabraded rabbit skin was used. Butylamine, which quickly
causes necrosis incontact with skin, appears much more toxic, based onthe LD;,o’s,
onintact guineapigabdomen than oneither abraded guinea pigorrabbit back. This circumstance is probably due to the fact that extensive and severe necrosis of the skin of theabdomen is more likely to leadto complications anddeath than necrosis
over alarger area involving primarily the back. Aniline appears to be more toxic to rabbits than to guinea pigs regardless of the location of theapplication andthe condition of the skin. However, aniline was more toxic when applied to the intact guinea pig abdomen than it was when applied to the abraded back. Butoxyethanol was less toxic tothe rabbit than itwas totheguinea pigby either type of contact, and, according to Hodge and Sterner’s (1949) classification, would beclassed as“slightly toxic” to rabbits but “moderately toxic” toguineapigs. Acrylonitrile, conversely, was more toxic to rabbits than it was by thesame type ofapplication toguinea pigs, while the LDao for guinea pigs with intact abdominal skin was between these two values.562 ROBERT L. ROUDABUSH ET AL.

The intact guinea pig abdomen was more sensitive than the abraded back, giving lower LD50 values for all compounds for which adifference was demonstrated. This
was not unexpected since previous experience had shown the abdominal skin to bea more responsive area. According to Deichmann et al. (1952), the dermal LDao of parathion to rabbits is870mg/kg, which is similar to our results with guinea pigs. Lehman (1948), on theother hand, reported theacute dermal LDjo for rabbits to be40-50 mg/kg. Our results onrabbits agree more closely with those of Lehman than with those of Deichmann etal. Primary Irritation Table 3summarizes thedata collected from the primary irritation studies. Table 4 lists thematerials indescending order of irritancy according totype oftest, together with somecorrelation coefficients suggested by Snedecor (1946). The average primary irritation scores listed in Table 3 reveal that the abraded rabbit skin was more sensitive to the irritant effects of allfourteen compounds than
was the intact rabbit skin, while ,theintact guinea pig showed equal or greater irritation than theabraded guinea pigskin with twelve of these. Seven ofthe materials produced more irritation on intact guinea pig skin than on abraded rabbit skin. Thirteen compounds produced equal or greater irritation in the intact guinea pig skin test than inthe intact rabbit skin test, and nine gaveequal or greater scores in the intact guinea pig skin test than inthe combination ofabraded and intact rabbit skin. The ranking of compounds according to irritancy in both guinea pig tests is extremely well correlated with the rabbit test. DISCUSSION As pointed out above, of the eight compounds tested for acute dermal toxicity, three gave similar results in both the rabbit and intact guinea pig skin test. According to the classification system of Hodge and Sterner (1949), three compounds, butylamine, aniline, and acrylonitrile, would fall into the same class regardless of thetest animal, thefirst two being “slightly toxic” and thethird “moderately toxic.” Butoxyethanol and parathion were in different classes depending onthe species, the former being “slightly toxic” to rabbits and“moderately toxic” to guinea pigs with intact skin, while thelatter was just the reverse. According to the Regulations [21 CFR 191.1 (f) (3)], acetone, benzene, and carbon tetrachloride would not beclassed asLLtoxic” by dermal application in either species. All theother fivecompounds would beclassed as“toxic” bythe intact guinea pig skin test, and four of the five “toxic” by the rabbit test. Parathion would be “highly toxic” by the rabbit test [21CFR 191.1 (e) (3)]

. The differences inthe LDsO’s calculated from the rabbit and the intact guinea pig skin tests are nogreater than thedifferences frequently recorded for achemical tested bytwo laboratories inasinglespecies. Thedermal LDBO’s for parathion asdetermined by Deichmann etal. (1952) andLehman (1948) havebeencited aboveasanexample of such differences found inthesamespecies. Sincethe Regulations [21CFR 191.1 (g) (2)] define aprimary skin irritant as amaterial having anempirical score of5ormore when tested on rabbits, noneoftheSKIN OF RABBITS AND GUINEA PIGS 563

TABLE 3 SUMMARY OF PRIMARY IRRITATION SCORES

Compound
Acetic acid, 4% aqueous Ammonia, household Borax

Boric acid

2-Butanone

Carbon tetrachloride Gasoline

Kerosene

Phenylhydrazine

Sodium carbonate Sodium sulfite Sucrose

Trisodium phosphate Water, distilled Animal Intact skin Abraded

skin Intact plus

abraded skina
Rabbit 1.5 3.6 2.6 Guinea pig 1.9 1.9 1.9 Rabbit 3.2 5.0 4.1 Guinea pig 5.6 5.O 5.3 Rabbit 1.6 2.3 2.0 Guinea pig 1.5 1.4 1.4 Rabbit 0.8 2.5 1.7 Guinea pig 2.7 1.4 2.1 Rabbit 0.8 2.7 1.8 Guinea pig 1.6 2.4 2.0 Rabbit 2.8 4.0 3.4 Guinea pig 4.1 2.2 3.1 Rabbit 2.0 2.9 2.4 Guinea pig 3.9 2.8 3.3 Rabbit 1.9 3.2 2.5 Guinea pig 3.6 2.6 3.1 Rabbit 1.8 3.o 2.4 Guinea pig 3.6 2.6 3.1 Rabbit 2.1 5.0 3.5 Guinea pig 2.1 2.6 2.3 Rabbit 1.6 3.1 2.4 Guinea pig 2.4 1.7 2.0 Rabbit 0.4 2.3 1.4 Guinea pig I.5 0.9 1.2 Rabbit 3.4 5.1 4.2 Guinea pig 6.3 5.1 5.7 Rabbit 1.1 1.8 1.4 Guinea pig 1.3 1.0 1.1 Range Rabbit 0.4-3.4 1.8-5.1 1.4-t.2 Guinea pig 1.3-6.3 0.9-5.1 1.1-5.7 Average Rabbit 1.79 3.34 2.56 Guinea pig 2.87 2.40 2.69 Correlation coefficient to Rabbit 0.943 0.962 - rabbit, intact plus abraded Guinea pig 0.803 0.861 0.847 0 The calculation of this primary irritation score is described in section 191.11 of the Regulations. In the other columns for intact or abraded skin the sum of the values at 24 and 72 hours
was divided by 2rather than 4.
materials tested could beclassed as irritants according to the results of our tests. However, household ammonia andtrisodium phosphate would fall intheirritant class if the results of the guinea pig tests were calculated in the same manner as in the rabbit test. It isinteresting also that these same results would have beenobtained if only the intact guinea pig skin had beenused inthetest. The results ofthecomparisons made inthis study indicate that theguinea pigskin test provides essentially the same information as the rabbit skin test. Thus, thereScore Guinea pig, intact skin TABLE 4 RANKING OF SUBSTANCFS ACCORDING TO IRRITANCY IN EACH TESTY

: Rabbit, Guinea pig, Rabbit, Guinea pig, Rabbit, intact skin abraded skin abraded skin intact plus abraded intact plus abraded
>4.09

4.09-2.09 Trisodium phosphate Ammonia Carbon tetrachloride Gasoline Kerosene Phenylhydrazine Boric acid Sodium sulfite Sodium carbonate -

Trisodium phosphate Ammonia Carbon tetrachloride Sodium carbonate

<2.09

Spearman’s rank correlation coefficient to rabbit, intact plus abraded Acetic acid

2-Butanone Borax Sucrose Water

0.743 0.896 0.793 0.963 0.752 Trisodium phosphate Ammonia

Gasoline Kerosene Phenylhydrazine Sodium carbonate 2-Butanone Carbon tetrachloride

Gasoline Kerosene Phenylhydrazine Borax Sodium sulfite Acetic acid Water Boric acid 2-Butanone Sucrose Acetic acid Sodium sulfite Boric acid Borax Water Sucrose Trisodium phosphate Ammonia Sodium carbonate Carbon tetrachloride Acetic acid Kerosene Sodium sulfite Phenylhydrazine Gasoline 2-Butanone Boric acid Sucrose Borax Trisodium Trisodium phosphate phosphate Ammonia Ammonia

Gasoline Kerosene Carbon tetrachloride Phenylhydrazine Sodium carbonate Boric acid

Water 2-Butanone Sodium sulfite Acetic acid Borax Sucrose Water Sodium carbonate Carbon tetrachloride Acetic acid TI Kerosene E Gasoline z Sodium sulfite .F Phenylhydrazine tz c $

z Borax Liz 2-Butanone 2 Boric acid ‘Water aF Sucrose

a Braces group substances with identical primary irritation scores.SKIN OF RABBITS AND GUINEA PIGS 565

isasound basis for the useoftheguinea pigasanalternate species tothe rabbit for skin irritation and percutaneous absorption tests specified in governmental regulations, aswell asfor general use inindustrial toxicology laboratories.

SUMMARY
A comparison was made between guinea pigs and rabbits in their response to various compounds in regard to acute dermal toxicity and primary irritation. Of the eight compounds tested for acute dermal toxicity, only one (parathion) would have been placed in a higher toxicity classification by the rabbit test than by the intact guinea pig skin test. The information obtained in the primary irritation tests showed that the intact guinea pig skin test is as sensitive as, or more sensitive than, the rabbit skin test in eliciting dermal reactions. It is concluded that the intact guinea pig skin test serves as a useful alternate for the rabbit test and that there is no scientific basis for preferring the rabbit as a test animal

REFERENCES DEICHMANN, W. B., PUGLIESE, W., and Cnssm~, J. (1952). Effects of dimethyl and diethyl paranitrophenyl thiophosphate on experimental animals. .4.M.A. Arch. Ind. Hyg. Occupational Med. 5, 44-51. DRAIZE, J. H., WOODARD, G., and CALVERY, H. 0. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exptl. Therap. 82, 377-390. FINNEY, D. J. (1952). Probit Analysis, 2nd ed., Chapter 3. Cambridge Univ. Press, London and New York. HODGE, H. C., and STERNER, J. H. (1949). Tabulation of toxicity classes. Am. Znd. Hyg. Assoc. Quart. 10, 93-96. LEHMAN, A. J. (1948). The toxicology of the newer agricultural chemicals. Quevt. Bull. Assoc. Food Drug Ofic. U.S. XII, 82-89. PATTY, F. A. (1962). Industrial Hygiene and Toxicology (David W. Fassett and Don D. Irish, eds.), 2nd ed., Vol. II. Wiley (Interscience), New York. SNEDECOR, G. W. (1946). Statistical Methods, 4th ed., pp. 164-167. Iowa State Univ. Press, Ames, Iowa.