FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
19, 157- 158 ( 1992)
LETTERS TO THE EDITOR To the Editor:
Reply
The purpose of this letter is to question two points appearing on page 803 of the recent publication by Thorne et al. (199 I), which are relevant to our study of the mouse ear swelling test (MEST) (Cornacoff et al., 1988). The authors note that we reported a minimal response to the prototypic contact sensitizer DNFB in our evaluation of the MEST and go on to provide several putative explanations for our “problem.” We would be most appreciative if the authors would clarify points 2 and 5 regarding “other injections administered to these mice” and particularly “determination of ear swelling in dead mice,” respectively. At first glance, these statements appear to have a negative connotation and imply that the mice used in our study had been used or were currently being used for other studies. In addition, we are unable to think of any instance in which an investigator would collect data from dead animals except for the purpose of histomorphologic interpretation. We acknowledge that both of these conditions would adversely affect the outcome of the assay. However, neither of these situations applied to our study, and we take exception to Thorne et al.3 implication to the contrary.
To the Editor: In our recent evaluation of the mouse ear swelling assay, or MESA (Thorne et al., 199 la,b), we employed a noninvasive protocol and refined the method using five fragrance solutions as well as using dinitrofluorobenzene (DNFB) over a 2000-fold dose range. In reviewing the literature regarding the MESA we noted that Cornacoff, House, and Dean had employed an invasive MESA protocol that had not led to the expected degree of responsiveness to DNFB. We quote their contact sensitivity protocol (Cornacoff et al., 1988) in its entirety (the bold typeface is added): Contact sensitivity protocol. An adaptation of the method of Gad et al. (1986) was employed for sensitization. Mice were anesthetized with a mixture containing Rompun (87 mg/kg) and Ketaset ( I3 mg/kg) prior to shaving a 1.5 cm2 patch on the abdomen and tape stripping the epidermal layers of skin. A total of 0.05 ml of Freund’s complete adjuvant (FCA, Difco, Detroit, MI) was injected intradermally (id) at two separate sites. Twenty-five microliters of sensitizer was pipetted onto an A 1-test filter disc (Hollister-Steir, Marietta. GA) previously attached to a 2.5 X lo-cm strip of Elastoplast tape. The tape was wrapped around the abdomen and secured in place with extra fast setting readi-cast material (2.5 X 12 cm). The mice were sensitized for 3 additional consecutive days by pipetting the sensitizer onto the filter disc. One day prior to challenge, the mice were reanesthetized and the bandages were removed. Each animal was injected intraperitoneally (ip) with 1 mg/ kg of FUdR followed 1 hr later with 1 pCi of [‘251]UdR. The mice were challenged the following day by applying 25 ~1 of diluted sensitizer on the left ear. The vehicle was placed on the right ear as a control. Mice were killed 24-48 hr later and the ears were evaluated for both skin thickness with a Model D-1000 Oditest gauge (Dyer Co., Lancaster, PA) and the infiltration of [‘ZSl]UdR-labeled cells. An g-mm Keyes cutaneous punch was used to obtain uniform tissue sections for gamma scintillation counting.
REFERENCES Cornacoff, J. B., House, R. V., and Dean, J. H. (1988). Comparison of a radioisotope incorporation method and the mouse ear swelling test (MET) for contact sensitivity to weak sensitizers. Fundum. Appl. Toxicol. 10,4044. Thorne, P. S., Hawk, C., Kaliszewski, S. D., and Guiney, P. D. (1991). The noninvasive mouse ear swelling assay. I. Refinements for detecting weak contact sensitizers. Fundam. Appl. Toxicol. 17, 790-806. JOEL B. CORNACOFF
Sterling Winthrop Pharmaceuticals Research Division ROBERT
V. HOUSE
UT Research Instituie JACK H. DEAN Sterling Winthrop Pharmaceuticals Research Division
On the basis of the methodology described and careful reading of the rest of the paper, we concluded that the decreased responsiveness reported by Cornacoff et al. “could have arisen from (1) their use of Rompun and Ketaset anesthesia at sensitization and 1 day prior to challenge, (2) other injections administered to these mice, (3) casting of the mice, (4) two site intradermal injections of Freund’s complete adjuvant, or (5) determination of ear swelling in dead mice” (Thorne et al., 1991a). The “other injections” in (2) refers to the FUdR and the [‘251]UdR injections described in their methods. The penultimate sentence in their methods reprinted above indicated to us that the mice were killed and then ears were evaluated. In addition, we find nowhere in the Cornacoff et al. article where it is stated that the mice entered in the [‘251]UdR incorporation study are distinct from 157
0272-0590/92 $5.00 Copyright 0 1992by the Societyof Toxicology. All rights of reproduction in any form reserved.
AND
APPLIED
TOXICOLOGY
19, 157- 158 ( 1992)
LETTERS TO THE EDITOR To the Editor:
Reply
The purpose of this letter is to question two points appearing on page 803 of the recent publication by Thorne et al. (199 I), which are relevant to our study of the mouse ear swelling test (MEST) (Cornacoff et al., 1988). The authors note that we reported a minimal response to the prototypic contact sensitizer DNFB in our evaluation of the MEST and go on to provide several putative explanations for our “problem.” We would be most appreciative if the authors would clarify points 2 and 5 regarding “other injections administered to these mice” and particularly “determination of ear swelling in dead mice,” respectively. At first glance, these statements appear to have a negative connotation and imply that the mice used in our study had been used or were currently being used for other studies. In addition, we are unable to think of any instance in which an investigator would collect data from dead animals except for the purpose of histomorphologic interpretation. We acknowledge that both of these conditions would adversely affect the outcome of the assay. However, neither of these situations applied to our study, and we take exception to Thorne et al.3 implication to the contrary.
To the Editor: In our recent evaluation of the mouse ear swelling assay, or MESA (Thorne et al., 199 la,b), we employed a noninvasive protocol and refined the method using five fragrance solutions as well as using dinitrofluorobenzene (DNFB) over a 2000-fold dose range. In reviewing the literature regarding the MESA we noted that Cornacoff, House, and Dean had employed an invasive MESA protocol that had not led to the expected degree of responsiveness to DNFB. We quote their contact sensitivity protocol (Cornacoff et al., 1988) in its entirety (the bold typeface is added): Contact sensitivity protocol. An adaptation of the method of Gad et al. (1986) was employed for sensitization. Mice were anesthetized with a mixture containing Rompun (87 mg/kg) and Ketaset ( I3 mg/kg) prior to shaving a 1.5 cm2 patch on the abdomen and tape stripping the epidermal layers of skin. A total of 0.05 ml of Freund’s complete adjuvant (FCA, Difco, Detroit, MI) was injected intradermally (id) at two separate sites. Twenty-five microliters of sensitizer was pipetted onto an A 1-test filter disc (Hollister-Steir, Marietta. GA) previously attached to a 2.5 X lo-cm strip of Elastoplast tape. The tape was wrapped around the abdomen and secured in place with extra fast setting readi-cast material (2.5 X 12 cm). The mice were sensitized for 3 additional consecutive days by pipetting the sensitizer onto the filter disc. One day prior to challenge, the mice were reanesthetized and the bandages were removed. Each animal was injected intraperitoneally (ip) with 1 mg/ kg of FUdR followed 1 hr later with 1 pCi of [‘251]UdR. The mice were challenged the following day by applying 25 ~1 of diluted sensitizer on the left ear. The vehicle was placed on the right ear as a control. Mice were killed 24-48 hr later and the ears were evaluated for both skin thickness with a Model D-1000 Oditest gauge (Dyer Co., Lancaster, PA) and the infiltration of [‘ZSl]UdR-labeled cells. An g-mm Keyes cutaneous punch was used to obtain uniform tissue sections for gamma scintillation counting.
REFERENCES Cornacoff, J. B., House, R. V., and Dean, J. H. (1988). Comparison of a radioisotope incorporation method and the mouse ear swelling test (MET) for contact sensitivity to weak sensitizers. Fundum. Appl. Toxicol. 10,4044. Thorne, P. S., Hawk, C., Kaliszewski, S. D., and Guiney, P. D. (1991). The noninvasive mouse ear swelling assay. I. Refinements for detecting weak contact sensitizers. Fundam. Appl. Toxicol. 17, 790-806. JOEL B. CORNACOFF
Sterling Winthrop Pharmaceuticals Research Division ROBERT
V. HOUSE
UT Research Instituie JACK H. DEAN Sterling Winthrop Pharmaceuticals Research Division
On the basis of the methodology described and careful reading of the rest of the paper, we concluded that the decreased responsiveness reported by Cornacoff et al. “could have arisen from (1) their use of Rompun and Ketaset anesthesia at sensitization and 1 day prior to challenge, (2) other injections administered to these mice, (3) casting of the mice, (4) two site intradermal injections of Freund’s complete adjuvant, or (5) determination of ear swelling in dead mice” (Thorne et al., 1991a). The “other injections” in (2) refers to the FUdR and the [‘251]UdR injections described in their methods. The penultimate sentence in their methods reprinted above indicated to us that the mice were killed and then ears were evaluated. In addition, we find nowhere in the Cornacoff et al. article where it is stated that the mice entered in the [‘251]UdR incorporation study are distinct from 157
0272-0590/92 $5.00 Copyright 0 1992by the Societyof Toxicology. All rights of reproduction in any form reserved.
