Papers. Safety of a non-adjuvanted therapeutic vaccine for the treatment of feline dermatophytosis

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1 Papers Safety of a non-adjuvanted therapeutic vaccine for the treatment of feline dermatophytosis D. Westhoff, F. X. Orveillon, D. Farnow, M-C. Klös, K. Elbers The safety of a non-adjuvanted inactivated fungal vaccine for the treatment of dermatophytosis in cats was investigated in two studies: a controlled laboratory study, and a placebo-controlled double-blind field study with a cross-over design in Europe. In the laboratory study, two groups of 10 cats each were administered an intramuscular twofold overdose, followed by five single 1 ml doses, of either vaccine or control product at 14-day intervals. In the field study, cats were treated with three intramuscular injections of 1 ml vaccine administered at 14-day intervals, as recommended by the manufacturer. A total of 89 cats were enrolled in the field study and divided into two groups to receive either vaccine or placebo for the first three treatments, followed by the opposite product for the final three treatments. The cats enrolled in the two studies were 12 weeks of age or older, as recommended by the manufacturer. All the cats were monitored closely for possible injection site reactions, systemic reactions (including changes in rectal body temperature) and adverse events. The results from both studies showed no significant differences between the vaccinated cats and the control or placebo-treated cats with regard to local or systemic reactions. A few mild to moderate local reactions were noted, but these were evenly distributed between the vaccinated and placebo-treated cats and resolved within a few days. No severe or serious adverse events related to the vaccinations were observed. FUNGAL infections in cats are caused by dermatophytes of the genera Microsporum and Trichophyton. In most cases, affected cats are infected by the fungus Microsporum canis (Lewis and others 1991, Moriello 2004, Chermette and others 2008). Dermatophytosis is highly contagious and has a great potential for transmission to other animal species and human beings (Mancianti and others 2003, Chermette and others 2008). A combination of systemic and topical treatment is the currently recommended therapy, supported by partial or complete clipping of the cat s coat (Moriello 2004, Chermette and others 2008). Prophylactic and/or therapeutic vaccines against dermatophytosis for cats are controversial and have often been shown to be ineffective in experimental challenge studies (DeBoer and Moriello 1995, Pier and others 1995, Lund and DeBoer 2008). A vaccine for therapeutic and prophylactic treatment has been licensed for use in horses, cats and dogs in several European countries since 1999 (Insol Dermatophyton; Boehringer Ingelheim). A field study has shown that a cat-specific vaccine (Insol Microsporum; Boehringer Veterinary Record (2010) 167, D. Westhoff, Dr rer nat, F. X. Orveillon, DVM, D. Farnow, DVM, M-C. Klös, K. Elbers, Dr rer nat, Boehringer Ingelheim Vetmedica, Binger Strasse 173, Ingelheim, Germany doi: /vr.c4140 Dr Westhoff s present address is RRD International, 7361 Calhoun Place, Suite 510, Rockville, MD 20855, USA for correspondence: dwesthoff@rrdintl.com Provenance: not commissioned; externally peer reviewed Ingelheim), similar to Insol Dermatophyton, is effective in shortening the time to healing of clinical signs of dermatophytosis caused by M canis, Microsporum gypseum or Trichophyton mentagrophytes (Westhoff and others 2010). This paper describes two studies to investigate the safety of the feline vaccine Insol Microsporum. A controlled laboratory study was conducted to observe possible local and systemic reactions after administration of a twofold overdose, and six repeated administrations of single doses, to 12-week-old cats. Histology was performed to investigate the muscle tissue at the injection sites in some of these cats. A second trial was performed as a placebo- controlled, multi- centre field study in Europe, to investigate the safety of the vaccine when administered at the recommended dose of three 1 ml injections given at 14-day intervals to cats kept under typical European husbandry conditions. Material and methods Laboratory study Design A randomised controlled safety study was conducted under the principles of Good Laboratory Practice (GLP) in specific pathogen-free (SPF) cats at a GLP facility (Harlan BioService for Science) in Germany. Twelve cats (six male and six female), approximately 11 to 12 weeks of age on the day of inclusion, and eight female adult cats (approximately five years old) were included in the study. The adult cats were already housed at the facility. The acclimatisation period for the young cats was 10 days. Ten of the young cats were randomly assigned to group II (vaccine) and the remaining two young cats as well as the eight adult cats were assigned to group I (control product: saline solution). Six treatments were given at intervals of 14 days (on days 0, 14, 28, 42, 56 and 70). On day 0, a twofold dose (ie, an overdose) was administered as two intramuscular injections of 1 ml of vaccine, one into the left thigh and one into the right thigh. All the following treatments were given intramuscularly as single doses of 1 ml volume, alternating between the left and right hindlimbs, starting with the left limb.

2 Vaccine The pentavalent, inactivated, non-adjuvanted vaccine (Insol Microsporum) was formulated as an aqueous solution containing 10 x 10 6 to 12 x 10 6 microconidia of each of the following five fungal strains: T mentagrophytes (strain 1032), M canis (strain 1393), M canis var distortum (strain 120), M canis var obesum (strain 1311) and M gypseum (strain 59), and 0.04 mg thiomersal per 1 ml. Saline solution was used as the control product. Clinical observations The cats underwent a general clinical examination on day 1, before and approximately four hours after each treatment, on day 0, days 1 to 4 and day 7 after each treatment, and 14 days after the last treatment. In addition, they were weighed before each treatment. During the clinical examination, the cats were observed for any changes from normal (for example, in their vital signs or behaviour) and the rectal body temperature was measured. The injection sites were observed for redness, local temperature, swelling and signs of pain. The appearance of redness was classed as none (no reddening), slight (reddening of the injection site and immediate surrounding area), moderate (reddening of the injection site and the wider surrounding area) or severe (dark red or black coloration of the injection site and the surrounding area). The temperature at the injection site was assessed as normal (similar to skin temperature), warm (slightly warmer than skin temperature) or hot (markedly warmer than skin temperature). Swellings at the injection site were measured (mm) for the proximodistal and craniocaudal length. Pain was assessed by palpation and classed as absent or present. Postmortem examination and histopathology On day 84, five cats (three randomly selected cats from group II and the two juvenile cats from group I) were euthanased and examined postmortem. An adult cat (group I) that was found dead on day 25 was also examined postmortem. Each cat was examined macroscopically, and pathological changes were recorded. Two samples were collected from each injection site and preserved in 10 per cent formaldehyde solution. Histopathological examination of sections stained with haem atoxylin and eosin (Precision Histology International) was carried out. Histological findings in the skeletal muscle cells, such as purulent, lymphatic, macrophage and plasmacytic inflammations, were scored as 1 Minimal, 2 Mild, 3 Moderate or 4 Marked. Statistical evaluation Statistical analysis was performed using SAS v8.2 software, with the individual animal as the experimental unit. The rectal body temperature was assessed for individual animals and as mean values per group. A two-sided test for comparison between the groups was performed for the changes from baseline. The time point before each of the six treatments served as the baseline for the respective observation period (for example, day 0 Baseline for all time points until the second treatment; day 14 Baseline for all time points until the third treatment, etc). Field study Design The field safety study was conducted as a double-blind, randomised, placebo-controlled multi-centre study with a crossover design under the principles of GCP. A total of 89 cats were enrolled at 15 European sites in Denmark, France and Germany between June 2004 and July Cats were randomised to treatment groups 1 and 2 by site in blocks of two. The randomisation scheme was prepared by the statistician. Group 1 received three treatments with the vaccine first, and then three treatments with the placebo. Group 2 received three treatments with the placebo first, and then three treatments with the vaccine. The treatments were administered at 14-day intervals on days 0, 14, 28, 42, 56 and 70. Cats Cats included in the field study were client-owned and were required to be healthy, except for clinical signs of dermatophytosis, and older than 12 weeks of age. Dermatophytosis was confirmed by a fungal culture test from hair/skin samples taken at day 0 performed at a central laboratory (Serumwerk Memsen WDT, Germany). Concurrent Veterinary Record December 4, 2010 TABLE 1: Mean (sd) rectal body temperature ( C) and the changes from baseline values for cats in a laboratory study of a therapeutic feline dermatophytosis vaccine. The temperature before each treatment served as the baseline for the respective observation period (eg, day 0 Baseline for all time points until next treatment) Rectal temperature ( C) Change from baseline ( C) Day Group* mean (sd) mean (sd) P 14 I (0.517) 0.17 (0.464) II (0.327) 0.30 (0.383) 29 I (0.349) 0.21 (0.793) II (0.246) 0.53 (0.521) 42, 4h I (0.399) 0.70 (0.474) II (0.269) 0.22 (0.286) 43 I (0.583) 0.70 (0.557) II (0.191) 0.10 (0.249) 45 I (0.569) 0.57 (0.512) II (0.321) 0.08 (0.290) 46 I (0.404) 0.77 (0.436) II (0.522) 0.09 (0.530) 56 I (0.468) 0.56 (0.505) II (0.291) 0.04 (0.334) * Group I control (saline-treated) cats, Group II vaccinated cats Only timepoints with significant differences are displayed Day 42, 4 hours after treatment treatment with immunosuppressive drugs, such as corticosteroids or ciclosporin, was contraindicated. Cats known to be serologically positive for feline leukaemia virus or feline immunodeficiency virus were excluded from the study. Vaccine and placebo The same lot of the pentavalent, inactivated, non-adjuvanted vaccine was used in the field study. A placebo not containing the active ingredient, which mimicked the turbidity (optical density 1.8) of the test vaccine, was used (aqueous solution with 0.04 mg thiomersal and Intralipid 10 [Baxter Medication Germany]). Injections were administered as deep intramuscular injections of 1 ml volume into the thighs of the cats, alternating between the right and left hindlimbs. Clinical observations The cats were weighed and examined clinically for general appearance and vital signs before each treatment and at the end of the study (day 84). Rectal temperature was measured before and on the day after each treatment, and on day 84. The injection sites were observed before and on the day after each treatment for visible and palpable local reactions. Redness, hardness and swelling were scored as 0 Normal, 1 Minor (diameter 1 cm), 2 Moderate (diameter >1 cm, <3 cm) and 3 Major (diameter 3 cm). If the cat showed pain at the injection sites, the reaction was differentiated as pain on palpation, pain without palpation or lameness. Owners observations The cats owners received diaries for them to record follow-up observations approximately three hours after each treatment and for the four days following each treatment. The owners were instructed to observe the cats for changes in appetite, general behaviour and grooming behaviour, and to monitor the injection sites for local reactions. Statistical evaluation Statistical analysis was performed using SAS v8.2 software, with the individual animal as the experimental unit. The primary safety parameter was the frequency of local reactions at the injection site. As secondary parameters, the cats rectal temperature, general appearance and vital signs, the frequency of animals with adverse events, and the type and size of local reactions at the injection site were evaluated. Changes in bodyweight over the course of the study were also evaluated. The null hypothesis was that the treatments were equal for the primary variable. Differences in local reactions between the two treatment groups were tested using the Wilcoxon Mann-Whitney U test, and differences between the groups for rectal body temperature and weight development were evaluated using the two-sided test. The

3 TABLE 2: Histological findings* in skeletal muscle tissue samples from the hindlimbs of three vaccinated cats in a laboratory study of a therapeutic feline dermatophytosis vaccine. Two samples (S1, S2) from the right and the left hindlimb were investigated Cat 1 Cat 2 Cat 3 Microscopic findings in Left Right Left Right Left Right the skeletal muscle cells S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 Lymphocytic inflammation Macrophage inflammation Plasmacytic inflammation Purulent inflammation Abscess, pyrogranulomatous wall 3 * No findings, 1 Minimal, 2 Mild, 3 Moderate, 4 Marked Focal parameters heart rate, respiration, status of nutrition and appearance of lymph nodes, as well as data from the owners diaries, were evaluated using Fisher s exact test. Ethics Ethical approval was granted by the national regulatory agencies (Paul- Ehrlich Institute, Germany; Danish Medicines Agency, Denmark; Agence Nationale de Sécurité Sanitaire, France) and the respective local authorities where required. An informed consent form was signed by each owner for all cats participating in the field study. Results Laboratory study One swelling was found at the left injection site in one of the cats in group II on day 15, one day after the second administration of the vaccine. The swelling was 10 x 9 mm in size on day 16, but decreased until day 25 to 3 x 3 mm. On day 29, the swelling was not perceptible. No other local reaction of any kind was observed at the injection sites by clinical examination or palpation in any of the cats at any time point during the 14-day observation period after each administration of vaccine or control product. No significant systemic reaction to the administration of the vaccine or the control product was observed during the course of the study. Because the bodyweight of the cats in group II (which were juvenile and therefore growing) increased continuously, no effect of the vaccine on changes in bodyweight could be identified. No clinically relevant differences with regard to the rectal body temperature were observed. Rectal body temperature was generally higher in group II. The highest temperature post-treatment found in group I (the control group) was 38.9 C (day 7), and in group II (the vaccinated group) it was 39.4 C (day 29). The highest mean (sd) values of rectal temperature were 38.7 (0.36) C on day 2 in the vaccinated cats, and 38.5 (0.56) C on day 42, before treatment, for the controls. As group II consisted of only young cats and group I of mainly adult cats, a comparison between the groups was performed for the changes from baseline temperatures (Table 1). Statistically significant differences were observed at seven time points, but with no distinguishable pattern or clinical relevance. The results more often showed a decrease of the values in comparison to the baseline temperature (Table 1). No specific findings were observed for the cats general health. One cat in group I was found dead on day 25. Gross pathology did not reveal any significant findings. The cause of death of this cat was not investigated further. This cat, three cats randomly selected from group II and the two young cats from group I were examined postmortem. Only the cat that had shown transient swelling at the injection site in the left hindlimb had a whitish, pea-sized structure in the muscle tissue. The histo pathology report described the finding as a single moderate abscess with a pyrogranul omatous, not a primarily fibrous, wall. No skeletal muscle lesions were found in the two control cats or the control cat that was found dead on day 25, having received only two treatments. Four of the 12 injection site samples from the vaccinated cats showed no histological changes. The other eight samples showed minimal, mild or moderate foci of inflammatory cells without a distinguishable pattern (Table 2). The plasmacyte response was unremarkable, and necrosis was not a significant part of the response. The histopathologist concluded that the treatment resulted in mild inflammatory cell changes that were of minor clinical importance. Field study Study cats A total of 89 client-owned cats (48 male and 41 female), aged between 12 weeks and 15.8 years, and of various breeds, were included in the field study. For day 0, data relating to 86 cats, and for day 85, data for 55 cats, were complete and could be evaluated. The other cats (34) did not receive all treatments, were withdrawn or were not available for all study assessments. General health No increase in mean body temperature was observed. The mean temperature was significantly higher on the day after the first treatment in the placebo-treated cats compared with the vaccinated cats (P=0.044) and on the day after the last treatment in the vaccinated cats (P=0.024). Nevertheless, the highest mean value was 38.6 C for both vaccinated and placebo-treated cats. One cat had a high temperature (39.8 C) after the second vaccine treatment but also showed clinical signs of bronchitis, an increased respiratory rate and lymphadenopathy. The evaluation of changes in the bodyweight of the cats was similar for both groups. No significant differences between the groups were observed for heart rate, respiration, status of nutrition and appearance of the lymph nodes during the entire study. An abnormal heart rate was observed in a vaccinated cat (12 years old) on four study days, three times before treatment and once on day 1 after the first vaccination. Abnormal respiration was observed in one cat after the second placebo treatment and in another cat after the second vaccine treatment. Local reactions Vaccine was administered 199 times, and placebo 201 times, during the course of the study. There were no significant differences between the two groups for any of the evaluated parameters (Tables 3, 4). At one investigational site, 11 cats were not treated according to the protocol. These cats received the test vaccine or placebo subcutaneously instead of intramuscularly. Exclusion of these cats from the analysis did not alter the findings (data not shown). Observations by the owners Data recorded by the cats owners were evaluated to support the safety assessment, although these were considered carefully because cat owners may not be objective evaluators. The owners observations of local reactions at the injection site showed no significant differences between cats treated with the vaccine or the placebo. Pain at the injection site was reported by the owners on a few occasions, but a significant difference (P=0.037) was observed only at the time point four hours after treatment, when the owners of four cats recorded pain after the second placebo treatment. Lameness after treatment was noted in three instances, two times for a placebo cat, at four hours and one day after treatment, and once for a vaccinated cat one day after the second treatment. Adverse events One cat vomited six hours after the second placebo treatment, and three cats (two vaccinated and one placebo-treated) showed diarrhoea four hours after the second treatment. None of the additional clinical observations made by the investigators was judged as an adverse event. One cat died four days after the first placebo treatment, due to a cardiovascular failure resulting from hypertrophic cardiomyopathy. Discussion Feline dermatophytosis is a fungal infection that affects cats of all breeds and has high zoonotic potential. A treatment that achieves a rapid cure is desirable to protect other animals and people in the household from fungal spores shed by the infected cat (DeBoer and others 1995, Mancianti and others 2003, Chermette and others 2008). Dermatophytosis is often treated with combinations of systemic and

4 TABLE 3: Number and percentage of local reactions* for cats in a field study of a therapeutic feline dermatophytosis vaccine Number (%) of cats with score Reaction Study day Group P Redness NS (2.4) NS (3.8) Swelling (2.3) NS (4.8) (2.7) NS (3.0) NS (6.9) (3.0) NS (7.1) (3.8) (3.3) NS 2 26 Hardness (4.5) NS (5.4) NS (3.0) NS (10.3) NS (3.6) (3.8) (3.3) NS 2 26 * Local reactions scored as 0 Normal, 1 Minor or 2 Moderate Group 1 received vaccine on day 0, 14 and 28 and placebo on day 42, 56 and 70. Group 2 received placebo on day 0, 14 and 28 and vaccine on day 42, 56 and 70. Study days where no local reaction was recorded are not listed NS Not significant (P>0.05) TABLE 4: Number and percentage of cats in a field study of a therapeutic feline dermatophytosis vaccine that showed pain at the injection site Number (%) of cats with Study day Group* No pain Pain without palpation Pain on palpation P NS (2.3) (2.3) 1 (2.3) NS (4.8) (2.7) NS (2.9) NS (6.9) NS (3.6) (3.8) *Group 1 received vaccine on day 0, 14 and 28 and placebo on day 42, 56 and 70. Group 2 received placebo on day 0, 14 and 28 and vaccine on day 42, 56 and 70. Study days where no local reaction was recorded are not listed NS Not significant (P>0.05) topical treatments. However, the available treatment options are frequently not sufficient to effect a cure. The need for an effective prophylactic or therapeutic vaccine for cats has been discussed for many years (DeBoer and Moriello 1995, Pier and others 1995, DeBoer and others 2002, Lund and DeBoer 2008). The therapeutic vaccine for cats described in this study has shown efficacy in a placebo-controlled study evaluating the differences in the reduction of clinical signs of dermatophytosis. Statistically significant differences were shown in young cats with serious infections and in cats with a first fungal infection when vaccinated cats were compared with cats given a placebo (Westhoff and others 2010). The safety profile of this pentavalent, inactivated, non-adjuvanted vaccine was carefully investigated with regard to local, systemic and allergic Veterinary Record December 4, 2010 reactions as well as the occurrence of adverse events. The results of the laboratory study demonstrated the local and systemic tolerance of the vaccine under investigation. The safety of the vaccine was shown for juvenile cats approximately 12 weeks of age on the day of first treatment, receiving two injections of the vaccine (1 ml into each hindlimb) containing a total of 10 x 10 7 to 12 x 10 7 inactivated microconidia: a twofold overdose. Vaccine safety is generally tested with a 10-fold excess for live vaccines and twofold excess for inactivated vaccines. In the present study, the twofold dose was chosen to avoid any changes to the physical parameters of the vaccine (for example, viscosity), and, as this study was conducted in 11- to 12-week-old cats, the intramuscular application of more than 1 ml per injection was not feasible. The repeated administration of the vaccine (six treatments) demonstrates the safety with regard to allergic or anaphylactic reactions. These reactions can be expected to occur with an increasing number of administrations. No allergic or anaphylactic reactions occurred in the present study. The safety data from the field and laboratory studies were similar. The few local injection site reactions reported in the field study were mild to moderate, and disappeared within a few days. Reports of injection site reactions were evenly distributed between vaccinated and placebo-treated cats. In the laboratory study, only one cat showed a small, transient, local reaction after the second administration of the vaccine a reaction that is not unusual after intramuscular injection of foreign material. Histological investigation of this lesion indicated it was not primarily a fibrous lesion but was a moderate pyrogranulomatous abscess. The histological investigation of other vaccinated cats in the laboratory study showed subacute inflammatory responses of minor importance in the muscle tissue, which were considered to be expected after injection of foreign material. In the laboratory study, higher body temperatures were observed in group II (the vaccinated group), including the measurements made on day 0 before vaccination. This is considered to be related to the fact that group II consisted of only young cats, whereas group I (the control group) consisted predominantly of adult cats (eight of 10). A comparison between the two groups of the changes from the baseline temperatures did not reveal any clinically relevant findings. Systemic reactions in the field study were very rare and not considered to be serious, and were also evenly distributed between the vaccine and placebo groups. Adverse events were observed only on a few occasions and were not considered to be related to the administration of the vaccine. According to the literature, mild local and systemic reactions to vaccines are to be expected as a natural consequence of stimulating the immune system (Roth 1999). Mostly mild vaccine-associated adverse events were found in 1.2 per cent of cats in a feline vaccine safety study including 2288 routinely vaccinated cats (Moore and others 2007). The rate increased in cats older than one year of age and in cats administered multiple vaccines concurrently (Starr 1993, Meyer 2001, Moore and others 2007). Systemic reactions in cats are mostly transient, postvaccinal, non-specific illnesses and can be characterised as changes in the general health status, such as pyrexia, lethargy, anorexia or vomiting (Roth 1999, Day 2006). These reactions have also been attributed to the pyrogenic effects of the antigen or adjuvant contained in the vaccine (Aucouturier and others 2001, Day 2006). The few instances of mild systemic reactions seen in the studies reported here, such as vomiting or diarrhoea, are thus not atypical, but may also be related simply to the excitement of the cats travelling to and being examined and treated at a veterinary practice, as evidenced by the similar frequency of these events in the placebo-treated cats. The few local injection site reactions in the cats that received vaccine or placebo may be considered as non-specific effects associated with the injection of a product and the mechanical irritation during application (for example, trauma to soft tissues caused by the needle and by the administration of a volume of fluid). Subcutaneous vaccine site reactions in cats show cytological characteristics described as inflammatory infiltrates composed primarily of lymphocytes. They are consistent with a simple, first-exposure, immune, non-suppurative, inflammatory reaction, and may be attributable to vaccine adjuvants enhancing the normal immune response (Schultze and others 1997, Roth 1999, Moore and others 2007). The vaccine used in the present study does not contain an

5 adjuvant, which may explain the similar distribution of injection site reactions between the vaccinated and placebo-treated cats. The initiation and development of vaccine site-associated fibro sarcoma in cats is controversial but of particular concern. Fibrosarcomas have been reported since Initially, the fibrosarcomas were thought to be possibly related to a new killed, adjuvanted rabies vaccine (Hendrick and Goldschmidt 1991, Hendrick and others 1992, McEntee and Page 2001). The possible association of injection site reactions with fibrosarcomas in cats was first described by Hendrick and Goldschmidt (1991). Injection site reactions followed by inflammatory processes and a tissue response are reported more often after the application of adjuvanted vaccines than after nonadjuvanted vaccines in cats (Aucouturier and others 2001, Gaskell and others 2002, Day 2006). An increase in volume at the injection site after treatment with aluminium hydroxide adjuvant-containing vaccines, sometimes palpable for up to 10 days, can develop into simple granulomas and, more rarely, into fibrosarcomas (Cohrs and Schulte 1952). The true incidence of the development of fibrosarcomas in cats is unknown, but has been investigated during the past 15 years (Lester and others 1996, McEntee and Page 2001, Morrison and Starr 2001). The incidence of injection site sarcomas attributed to feline vaccines is currently believed to range between one case per 10,000 vaccinations and one case per 30,000 vaccinations (Gaskell and others 2002, Gobar and Kass 2002, Séguin 2002). The vaccine investigated in the present study does not contain any adjuvants, which seem to be a factor in the induction of sarcomas. In conclusion, the results from these two studies a controlled laboratory study and a field study show that the investigated therapeutic vaccine for the treatment of dermatophytosis is safe and well tolerated when applied three times at 14-day intervals to cats of different breeds older than 12 weeks of age. No differences in the number or the severity of local or systemic adverse events were seen when the vaccinated and control groups were compared. Although the duration of the study and the number of cats enrolled does not allow for a full assessment of possible adverse effects, adjuvant-free vaccines, such as the investigated inactivated, non-adjuvanted vaccine, are less likely to cause such events. Acknowledgements The authors thank H. Schmidt (Harlan Bioservice for Science) for the conduct of the GLP study, J. P. Finn (Precision Histology International) for the histopathological investigations, and the investigators involved in the field study: C. Anders, L. Ansay, R. Bauer, P. 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