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2994. A new sensitivity test
Methods for assessing toxicity
160
METHODS 2993. Mutagenicity
FOR ASSJZSSING
testing in the mouse
Generoso,W. M., Russell,W. L., Huff, Sandra W., Stout, SandraK. & Gosslee,D. G. (1974).Effects of doseon the induction of dominant-lethalmutations and heritable translocationswith ethyl methanesulfonatein malemice.Genetics,N.Y. 77, 741.
TOXICITY
2994. A new sensitivity test
Maurer. Th., Thomann, P., Weirich. E. G. & Hess. R. (1975).The optimization test in the guinea-pig.A method for the predictive evaluation of the contact allergenicityof chemicals.Agents & Actions 5, 174.
As the incidenceof allergicdermatitishasincreased Matter, B. E. & Generoso,W. M. (1974).Effects of appreciablyover the last few years,the authorscited doseon the induction of dominant-lethalmutations abovesawthe needfor an improvedmethodof studywith triethylenemelamine in malemice.Generics,N.Y. ing sensitizationin animals, sinceother commontest 77, 753. methods(the Draize epicutaneoustest in the guineapig, the Magnussonand Kligman maximization test, The first papercited aboveis a study of dominant- epidermalpatch testsand in t&o methods)either fail lethal mutationsinducedin malemiceby ip injection to accountproperly for the sensitivity of humanskin of ethyl methanesulphonate (EMS) in single doses or are insufficientlystandardized.In developingtheir rangingfrom 50 to 300 mg/kg. Effectson spermato- new ‘optimization test’, therefore,comparisonswere zoa and spermatidswere evaluated by mating the made with resultsobtained with the maximization treated mice 6.59.5 days after the injection. test and the Draize intradermal test conducted in a Dominant lethalswere detectedafter a doseof 150 standardmanner. mg EMS/kg or more, with a saturation point, at The three testswere carried oui on guinea-pigs, which determinationwasno longer accurate,of 250 using 1-chloro-24-dinitrobenzene(DNCB), ethyl mg/kg. Betweenthesedoselevelsthe increasein in- Caminobenzoate (benzocaine), penicilloylpolylysin duceddominant-lethalmutationswassharp.A sign& (PPL), penicillin G and 35% formaldehydesolution cant increasein the frequencyof translocationswas as test substances. Groups of 20 maleand 20 female detectablein the male progeny of the treated mice guinea-pigswere usedfor eachexperiment,20 acting evenafter the 50 mg/kg dose,and again rosesharply as test and 20 as control animals. as the dose increased.This is an indication that The optimization test usesFreund’s adjuvant to dominant lethals, although not directly detectable, stimulatethe immuneresponse.In wk 1. dosesof 0.1 were induced at dosesof 50 and 100 mg EMS/kg. ml of the test solution without adjuvant were It is evident that screeningthe male progeny of administeredintracutaneouslyin the flank and back treated malesfor translocationheterozygosityis the on Monday and in the back on Wednesdayand Frimoresensitivemethodof studying the ability of low day, and 21 hr after the treatment the animalswere dosesof EMS to causechromosomaldamage.Since depilated chemically. Reactionswere assessed 3 hr low-dosetesting approximatesmore nearly to the later. The two largestdiametersof the erythematous usualconditionsof exposureof human populations, reaction and the skin-fold thicknesswere measured this is an important observation,particularly since (in mm).The ‘reactionvolume’(in ~1)wascalculated translocationsrepresenttransmissible geneticdamage. from this for each animal and each reaction. In wk The secondpaper cited describesthe effects of 2 and 3, 0.1 ml of the test substancein adjuvant was various dosesof triethylenemelamine (TEM) injected injected into the nuchal skin three times/wk. No into male mice.In one experiment,0.2 mg TEM/kg measurements were taken. The first challengewas was injected into maleswhich were mated 05-7.5, given, at a fresh (flank) site. 14 days after the last 8.521.5 and 22.5-30.5days after the dose.and in induction dose in the samevolume and dosesas in another the effect of dose on the induction of wk 1 of the induction period(without adjuvant).After dominant-lethalmutationswasstudiedby matingthe 24 hr the reactionswere measuredand the reaction males4.7-7’5,11.515.5and 225-32.5daysafter injec- volumes were again calculated. The reaction was tion of a doseof TEM in the 0.035-4.0mg/kg range. assessed by calculatingfor each animal the average The effects on spermatocytes(matings on days reactionvolumefor the first four induction dosesand 225-32.5) indicated true cytotoxicity, and the data the standarddeviation. The meanand the standard for midspermatidand early spermatozoa1 stages(days deviation were then added to give a thresholdvalue 11+15.5 and 45-7.5, respectively) were therefore for each animal and, if the challenge reaction analysed.Increaseof the dominant-lethaleffect with exceeded this threshold value, the animal was doseoccurredlesssharplywith TEM than with EMS. regardedas having been sensitized.The number of The ratio of geneticallyactive doseto lethal dosewas positive animalsin each test and control group was 1 :100for TEM comparedwith 1: 3.5 for EMS, indi- countedand the differencewasassessed by the Fisher cating that TEM hasmutageniceffectsat greatly sub- test.An epidermalchallengeusingthe maximumsubtoxic doses.The most sensitivestagein spermato- irritant dosewasgiven 14 days after the intradermal genesis for the detectionof the effectwasthe midsper- challenge.A 2 x 2 cm patch of the test substance matid phase,followed by the spermatozoa1 and late was applied under occlusionto a shorn untreated spermatidphasesin descendingorder of sensitivity. area of skin for 24 hr, and 21 hr after its removal No inductionof dominant-lethalmutationsappeared the hair was removed chemically.The reaction was to occur during the meiotic stage(matings225-30.5 read 3 hr later by measuringany erythemaand the daysafter the injection). skinfold thickness.Clearly discerniblereddeningof
Methods for assessing toxicity the site was regarded as indicative of an allergic reaction. The results of the three types of test showed the Draize test to be sensitive to potent but not to weak allergens. The optimization test gave more responsive results, the known allergens giving positive results while non-allergenic materials were negative. Weak allergens such as penicillin G, ethyl aminobenzoate and formalin gave positive results, but it is noteworthy that with penicillin G positive results were obtained only with the epidermal patch and not with the intradermal challenge. In general, the results of
161
this test correlated well with those of the maximization test. The authors conclude that direct stimulation of the immune system by adjuvant produces a better result in sensitization studies than the use of croton oil or sodium lauryl sulphate pretreatment or occlusive dressings to improve penetration. ‘The optimization method described has the advantage of smoothing out variations between individual animals and providing. an objective means of assessment of any reaction provoked.
160
METHODS 2993. Mutagenicity
FOR ASSJZSSING
testing in the mouse
Generoso,W. M., Russell,W. L., Huff, Sandra W., Stout, SandraK. & Gosslee,D. G. (1974).Effects of doseon the induction of dominant-lethalmutations and heritable translocationswith ethyl methanesulfonatein malemice.Genetics,N.Y. 77, 741.
TOXICITY
2994. A new sensitivity test
Maurer. Th., Thomann, P., Weirich. E. G. & Hess. R. (1975).The optimization test in the guinea-pig.A method for the predictive evaluation of the contact allergenicityof chemicals.Agents & Actions 5, 174.
As the incidenceof allergicdermatitishasincreased Matter, B. E. & Generoso,W. M. (1974).Effects of appreciablyover the last few years,the authorscited doseon the induction of dominant-lethalmutations abovesawthe needfor an improvedmethodof studywith triethylenemelamine in malemice.Generics,N.Y. ing sensitizationin animals, sinceother commontest 77, 753. methods(the Draize epicutaneoustest in the guineapig, the Magnussonand Kligman maximization test, The first papercited aboveis a study of dominant- epidermalpatch testsand in t&o methods)either fail lethal mutationsinducedin malemiceby ip injection to accountproperly for the sensitivity of humanskin of ethyl methanesulphonate (EMS) in single doses or are insufficientlystandardized.In developingtheir rangingfrom 50 to 300 mg/kg. Effectson spermato- new ‘optimization test’, therefore,comparisonswere zoa and spermatidswere evaluated by mating the made with resultsobtained with the maximization treated mice 6.59.5 days after the injection. test and the Draize intradermal test conducted in a Dominant lethalswere detectedafter a doseof 150 standardmanner. mg EMS/kg or more, with a saturation point, at The three testswere carried oui on guinea-pigs, which determinationwasno longer accurate,of 250 using 1-chloro-24-dinitrobenzene(DNCB), ethyl mg/kg. Betweenthesedoselevelsthe increasein in- Caminobenzoate (benzocaine), penicilloylpolylysin duceddominant-lethalmutationswassharp.A sign& (PPL), penicillin G and 35% formaldehydesolution cant increasein the frequencyof translocationswas as test substances. Groups of 20 maleand 20 female detectablein the male progeny of the treated mice guinea-pigswere usedfor eachexperiment,20 acting evenafter the 50 mg/kg dose,and again rosesharply as test and 20 as control animals. as the dose increased.This is an indication that The optimization test usesFreund’s adjuvant to dominant lethals, although not directly detectable, stimulatethe immuneresponse.In wk 1. dosesof 0.1 were induced at dosesof 50 and 100 mg EMS/kg. ml of the test solution without adjuvant were It is evident that screeningthe male progeny of administeredintracutaneouslyin the flank and back treated malesfor translocationheterozygosityis the on Monday and in the back on Wednesdayand Frimoresensitivemethodof studying the ability of low day, and 21 hr after the treatment the animalswere dosesof EMS to causechromosomaldamage.Since depilated chemically. Reactionswere assessed 3 hr low-dosetesting approximatesmore nearly to the later. The two largestdiametersof the erythematous usualconditionsof exposureof human populations, reaction and the skin-fold thicknesswere measured this is an important observation,particularly since (in mm).The ‘reactionvolume’(in ~1)wascalculated translocationsrepresenttransmissible geneticdamage. from this for each animal and each reaction. In wk The secondpaper cited describesthe effects of 2 and 3, 0.1 ml of the test substancein adjuvant was various dosesof triethylenemelamine (TEM) injected injected into the nuchal skin three times/wk. No into male mice.In one experiment,0.2 mg TEM/kg measurements were taken. The first challengewas was injected into maleswhich were mated 05-7.5, given, at a fresh (flank) site. 14 days after the last 8.521.5 and 22.5-30.5days after the dose.and in induction dose in the samevolume and dosesas in another the effect of dose on the induction of wk 1 of the induction period(without adjuvant).After dominant-lethalmutationswasstudiedby matingthe 24 hr the reactionswere measuredand the reaction males4.7-7’5,11.515.5and 225-32.5daysafter injec- volumes were again calculated. The reaction was tion of a doseof TEM in the 0.035-4.0mg/kg range. assessed by calculatingfor each animal the average The effects on spermatocytes(matings on days reactionvolumefor the first four induction dosesand 225-32.5) indicated true cytotoxicity, and the data the standarddeviation. The meanand the standard for midspermatidand early spermatozoa1 stages(days deviation were then added to give a thresholdvalue 11+15.5 and 45-7.5, respectively) were therefore for each animal and, if the challenge reaction analysed.Increaseof the dominant-lethaleffect with exceeded this threshold value, the animal was doseoccurredlesssharplywith TEM than with EMS. regardedas having been sensitized.The number of The ratio of geneticallyactive doseto lethal dosewas positive animalsin each test and control group was 1 :100for TEM comparedwith 1: 3.5 for EMS, indi- countedand the differencewasassessed by the Fisher cating that TEM hasmutageniceffectsat greatly sub- test.An epidermalchallengeusingthe maximumsubtoxic doses.The most sensitivestagein spermato- irritant dosewasgiven 14 days after the intradermal genesis for the detectionof the effectwasthe midsper- challenge.A 2 x 2 cm patch of the test substance matid phase,followed by the spermatozoa1 and late was applied under occlusionto a shorn untreated spermatidphasesin descendingorder of sensitivity. area of skin for 24 hr, and 21 hr after its removal No inductionof dominant-lethalmutationsappeared the hair was removed chemically.The reaction was to occur during the meiotic stage(matings225-30.5 read 3 hr later by measuringany erythemaand the daysafter the injection). skinfold thickness.Clearly discerniblereddeningof
Methods for assessing toxicity the site was regarded as indicative of an allergic reaction. The results of the three types of test showed the Draize test to be sensitive to potent but not to weak allergens. The optimization test gave more responsive results, the known allergens giving positive results while non-allergenic materials were negative. Weak allergens such as penicillin G, ethyl aminobenzoate and formalin gave positive results, but it is noteworthy that with penicillin G positive results were obtained only with the epidermal patch and not with the intradermal challenge. In general, the results of
161
this test correlated well with those of the maximization test. The authors conclude that direct stimulation of the immune system by adjuvant produces a better result in sensitization studies than the use of croton oil or sodium lauryl sulphate pretreatment or occlusive dressings to improve penetration. ‘The optimization method described has the advantage of smoothing out variations between individual animals and providing. an objective means of assessment of any reaction provoked.