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Repeated measurements of motor activity in rats in long-term toxicity studies
Journal of Pharmacological and Toxicological Methods 70 (2014) 241–245
Contents lists available at ScienceDirect
Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox
Original article
Repeated measurements of motor activity in rats in long-term toxicity studies V. Golozoubova ⁎, T.K. Brodersen 1, S. Klastrup, M. Oksama, J. Løgsted, A. Makin CiToxLAB Scantox A/S, Hestehavevej 36A, Ejby, DK-4623 Lille Skensved, Denmark
a r t i c l e
i n f o
Available online 19 July 2014 Keywords: Open field test Behavioural test battery Safety pharmacology endpoints Regulatory toxicology study
a b s t r a c t Introduction: In the light of 3R (replace, reduce, refine) principles in animal experimentation and increased focus on delayed effects of treatment on central nervous system, the incorporation of behavioural tests into standard toxicology studies as a complement or substitution of a stand-alone safety pharmacology study appears very attractive, but poses some challenges. In the present study, we evaluated the results of an open field test (standard part of the behavioural test batte- ries) incorporated into the 3-month regulatory toxicology study. Methods: The study was performed in two rat strains most commonly used in toxicology studies (Wistar and Sprague Dawley (SprD)). Open field test was performed according to the standard protocol for stand-alone behavioural test (modified Irwin test) before the start of treat- ment (Day-7, “naïve” animals), on Day 2, inWeek 6 and inWeek 13 of treatment with saline. Results and Discussion: There was no overall difference between strains, and onlyminor differenceswere detected at the individual time points. With regard to time effect, the average values for most of the parameters were comparable throughout the study but individual variability in the performance in the arena was increased at repeated measurements compared to the start. In conclusion, performance in the open field arena did not differ principally betweenWistar and SprD rats of both genders. However, individual variability in the behaviour in the open field arena increased with time. This has clear implications for deciding the appropriate group size for this type of study and has to be taken into account in the design of a toxicology study with integrated safety pharmacology endpoints. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Functional observation battery (FOB) and the modified Irwin test are two behavioural test batteries commonly utilised to assess the effects of test substances on the central nervous system in accordance with regulatory guidelines for pharmaceuticals (Ewart et al., 2013; S7A, Safety Pharmacology Studies for Human Pharmaceuticals) and chemicals (OECD Guidelines for the Testing of Chemicals, test 408). The importance of including a functional observation battery test as part of the safety pharmacology ‘core battery’ also for the compounds which do not have central nervous system as a target was emphasised earlier (Redfern et al., 2005). In the light of the biomedical industry's focus on the 3R approach to animal experimentation and increased attention to the delayed effects on safety pharmacology parameters, an integrated approach, incorporating neurological evaluation into the standard repeat-
⁎ Corresponding author. Tel.: +45 56861500. E-mail address: [email protected] (V. Golozoubova). 1 Present address: Novo Nordisk A/S, Denmark.
http://dx.doi.org/10.1016/j.vascn.2014.06.007 1056-8719/© 2014 Elsevier Inc. All rights reserved.
dose toxicology study, appears attractive. However, this approach may present some logistical and interpretational challenges. The behavioural test battery typically includes three types of observations: cage-side observations, hand-held observations and open field observations. While the settings of cage-side observations and handheld observations can be standardized throughout the study, open field observations may be more of a challenge, as they include an additional factor, namely, the novelty factor. In the open field test, which, per definition, “… consists of the measurement of behaviors elicited by placing the subject in a novel open space from which escape is prevented by a surrounding wall” (Walsh & Cummins, 1976), the validity of the results of repeated measurements of motor activity performed in the course of a long-term toxicity study may be questioned on the basis of limited “novelty” of the test arena when repeated measurements are performed in the same animals. Therefore, our efforts were concentrated on this part of the test battery. Generally, the repeated open field test is regarded as an acceptable option as long as due care is taken to assure that both control and treatment group(s) undergo exactly the same procedures performed in the same manner. However, this is correct only under the assumption that variation in the data would be the same after repeated measurements
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as it is at a single recording. This, however is not necessarily the case in long-term studies, as the absolute values of the parameters may be different (e.g. due to the age of the animals (Himmel, 2008)). The mean values and the standard deviations of the parameters at the later phases of the study have a direct influence on the study design (group size in particular) for the integrated study, and therefore have to be evaluated. Further, the majority of the single-dose stand-alone behavioural tests are performed in male rats. The incorporation of the test in toxicology studies would typically mean that the test has to be performed both in male and in female animals, and therefore knowledge of the possible gender difference is important. In the present study, we compared the results of repeated open field test performed in both genders of Wistar and Sprague Dawley (SprD) rats integrated into a 3 month-regulatory toxicology study design, and, based on the data obtained, attempted to evaluate whether group size known to be sufficient at acute measurements would give reliable data at repeated dosing with a multiple open field measurement paradigm. 2. Materials and methods 2.1. Animals and housing The study was performed in 40 animals: 10 male and 10 female SPF Wistar rats of the strain HanTac:WH (GALAS), and 10 male and 10 female SPF Sprague Dawley rats of the Ntac:SD strain, all delivered from Taconic Europe A/S, Ejby, Denmark. The rats were approximately 5 weeks old upon arrival to the facility. At arrival, the animals were health checked and a pupil reflex-test was performed to confirm that they were not blind. The rats were kept in polycarbonate cages (floor area: 1500 cm2 — height 21 cm) with two animals/cage. For environmental enrichment, each cage was supplied with bedding (softwood sawdust), wood wool (Aspen Wood Wool (Tapvei Estonia OÜ, Estonia)), autoclaved wood bricks (Tapvei Estonia OÜ, Estonia) and a rat house (Tecniplast Gazzada S.a.r.l., 21020 Buguggiate-Va, Italy). As stereotypic behaviour was observed in three animals towards the end of the study, additional environmental enrichment was offered to these animals (and their cage mates) from Day 71 and until the termination of the study. The study took place in an animal room provided with filtered air at a temperature of 21 °C ± 3 °C and relative humidity of 55% ± 15%. The room was illuminated to give a 12:12 light:darkness cycle (light on from 06:00 h to 18:00 h). Animals had an ad libitum access to complete pelleted rodent diet (Altromin, Gesellschaft für Tierernährung mbH, D-32791 Lage, Germany) and water. Animals were weighed once weekly. The type of study was approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark.
2.2. Treatment In order to reconstruct the experimental settings of the typical toxicology study, animals were treated daily for the whole period of the study. The daily dose of sterile physiological saline (Fresenius Kabi, Denmark) was given subcutaneously (5 ml/kg body weight). 2.3. Open field test All animals were examined in an open field test (5 min recording in ActiMot Motility Measuring System (TSE, Germany), arena size 96 × 96 cm) before the start of treatment (Day-7), after the second dosing (Day 2), in Week 6 (Day 37) and in Week 13 (Day 86) of daily treatment with saline. The animals were tested at each testing occasion by the same operators, at least 1 h after dosing, at approximately the same time, in the same sequence. At the start of the session, the animal was placed in the centre of arena.
On each occasion, the test was performed in parallel in two arenas first in all males, then in all females (same sequence of individual animals on all occasions), at 10.00–12.30 (±15 min). The level of illumination was kept similar to that in the home cage. Parameters evaluated were the same as in a stand-alone behavioural test — either automatically recorded by ActiMot system (time moving, total distance, numbers of rearings (vertical activity), time in centre/ periphery, total corner visits, total activity (moves/counts)), or visually evaluated (abnormal behaviour (e.g. aggression, stereotypic behaviour, cataleptic behaviour) and number of faecal pellets at the end of each session). 2.4. Statistical analysis Overall strain differences (separate for males and females) were assessed by an analysis of covariance (ANCOVA) with pre-dose measurement as a covariate (where applicable). In addition, Student's ttest (unpaired) was used for the direct comparison of strains on the individual days. Time effect (using Day-7 values as a reference) was evaluated by paired t-test. For all tests, the level of significance was defined as p b 0.05. The statistical analyses were made with SAS® procedures (version 8.2) described in “SAS/STAT® User's Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA”. 3. Results 3.1. General As expected, there was a substantial weight gain in the course of the study (gain of approx. 240 g for males and approx. 100 g for females). Body weights were significantly different between the strains from the study start and until Day 29 for males, and throughout the study for the females (SprD heavier than Wistar). Open field measurements were performed successfully in all animals. No abnormal behaviour was observed during the test in any of the animals at any of the time points. No overall difference between the strains was identified either for males or for females. 3.2. First exposure to the arena Male Wistar rats appeared to have a more pronounced reaction to the novelty of the environment at the first exposure to the test arena compared to SprD males: their total activity count and number of rearings were higher, they spent more time moving, spent more time in the centre of arena (Table 1), and had more faecal pellets. For the females, the difference between strains was minimal, the only parameter which differed statistically significantly between strains was total distance travelled (longer in Wistar females). 3.3. Repeated exposure to the arena In both strains time moving, time in the centre and total activity tended to be higher at the first measurement (before, Day-7) compared to the following measurements (Day 2, Week 6 and Week 13), the difference being more pronounced in males than in females (Table 1). The pattern of reaction to the exposure to arena was very similar on all occasions, the peak of activity was, as expected, observed within the first minute in the arena, and was gradually reduced thereafter. Due to an increased individual variation, this trend was more clear after the measurements in the beginning of the study (before and Day 2) compared to Week 6 and Week 13. At all measurement occasions, individual variation was least pronounced during the first minute of exposure to the arena and was increased during the following period (2–5 min) (data not shown).
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Table 1 Overview of parameters measured in the open field in Wistar and Sprague Dawley rats at different time points. Parameter
Time point
Wistar
Sprague Dawley
Males Mean Time moving
Distance travelled
Number of rearings
Time centre
Total corner visits
Total activity
Before Day 2 Week 6 Week 13 Pre Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13
245.5 220.7 201.2# 215.2# 99.4 85.3 75.5 89.2 40.8 30.2 33.5 36.5 32.7 27.7 27.8 23.3 77.3 60.3 55.3# 69.0 7011.7 6113.4 5475.0# 6040.6#
Females S.D. 17.2 47.5 52.9 27.1 16.5 25.6 32.3 23.4 7.7 15.1 15.0 10.4 9.1 14.2 13.3 13.1 15.0 20.9 28.1 18.0 765.5 1489.9 1622.2 1026.2
CV 7.0 21.5 26.3 12.6 16.6 30.0 42.8 26.2 18.8 50.0 44.7 28.4 27.9 51.2 47.9 56.3 19.4 34.7 50.8 26.1 10.9 24.4 29.6 17.0
Mean 228.5 222.3 231.4 195.6 89.9 88.8 97.5 77.5 32.0 34.2 36.3 29.9 23.5 21.2 29.4 9.8# 71.5 66.7 69.4 63.1 6448.4 6185.6 6632.0 5550.6
Males S.D. 17.4 41.2 12.7 44.3 9.7 25.9 18.6 26.3 10.8 16.5 13.8 13.7 13.0 16.2 15.3 7.5 14.4 25.0 18.0 30.0 553.9 1349.2 715.2 1589.2
CV 7.6 18.5 5.5 22.6 10.8 29.2 19.1 34.0 33.7 48.1 38.0 45.9 55.0 76.5 52.1 76.9 20.1 37.4 26.0 47.6 8.6 21.8 10.8 28.6
Mean 229.3⁎ 199.1# 224.4 217.2 88.4 79.0 96.9 91.4 32.1⁎ 32.4 32.7 28.5 21.3⁎ 16.9 27.1 12.1⁎# 73.6 58.9# 75.8 65.5 6371.3⁎ 5564.8# 6467.9 6060.6
Females S.D.
CV
Mean
S.D.
CV
12.2 29.5 22.5 37.2 11.7 13.3 29.2 34.9 10.6 12.3 14.5 10.7 5.2 10.2 14.6 7.5 15.2 13.6 24.2 25.2 490.4 901.1 1172.3 1429.9
5.3 14.8 10.0 17.1 13.2 16.9 30.2 38.1 33.1 38.1 44.3 37.6 24.2 60.3 54.0 62.5 20.6 23.0 31.9 38.5 7.7 16.2 18.1 23.6
220.4 195.0 215.8 227.2 78.7⁎ 71.3 86.1 93.5 29.9 25.1 31.3 35.3 21.8 14.2# 26.1 16.1 64.6 59.2 69.7 78.0# 5943.7 5324.9 6025.2 6293.0
19.1 35.3 43.9 21.6 13.2 17.6 27.8 18.2 8.0 10.7 13.1 8.4 10.1 10.5 11.6 6.4 9.8 18.2 28.4 13.2 733.2 1119.1 1325.0 794.2
8.6 18.1 20.4 9.5 16.7 24.7 32.3 19.4 26.6 42.7 41.8 23.8 46.1 73.9 44.3 39.7 15.2 30.8 40.8 16.9 12.3 21.0 22.0 12.6
8 animals per sex per strain; data collected over a period of 5 min at each recording. S.D. — standard deviation; CV — coefficient of variation. ⁎ Difference between strains (by gender), p b 0.05, tested with Student's unpaired t-test for males and females, separately. # Difference from the pre-values (Before, Day-7) to values obtained on Day 2, Week 6 or Week 13, p b 0.05, tested with Student's paired t-test.
The difference between strains on individual days of repeated measurements was minor (see Table 1) and not statistically significant for most of the parameters. In Week 13, male Wistar rats showed a slightly
more marked reaction to the arena than SprD males as they spent significantly more time in the centre of the cage and had a higher incidence of defecation.
Fig. 1. Pattern of activity of individual animals after placement to the open-field arena on different days. Total activity counts; A — Sprague Dawley males (filled circles);B — Sprague Dawley females (empty circles); C — Wistar males (filled squares); D — Wistar females (empty squares).
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3.4. Individual variation While activity levels in naïve animals were rather homogenous within the groups, individual variation between animals of the same group was more pronounced at repeated measurements (Days 2, Week 6 and Week 13). An increase in standard deviation of the mean as well as in coefficient of variation at repeated measurements (Day 2, Week 6 and Week 13) compared to the pre-treatment (Day-7) was observed for many of the parameters measured (Table 1). The reason for increased variation could not easily be categorised by separation into broad categories such as more and less active individuals; as animals showing low level of activity at one of the measurements showed normal or even relatively high activity at the other time point (Fig. 1). Increased individual variation (lower signal/noise ratio) means, in statistical terms, need for a larger group size in order to reliably determine intergroup differences. 3.5. Stereotypic behaviour Three Wistar rats (1 male and 2 females) showed stereotypic behaviour (walking in a particular circular pattern) in the home cage at the end of the second month of the study. Stereotypic behaviour is a very rare finding, observed predominantly in Wistar rats, occurring in a long term studies, and not as early as at the end of the second month of the study. These three animals did not show any stereotypic behaviour in the open field arena. When compared to the other animals, the male showing stereotypy in the home cage had lower than average number of rearings. The females did not distinguish themselves from the average in any of the parameters. 4. Discussion The results of the present study suggest no principal difference in the outcome of the repeated open-field test between the two rat strains (Wistar and SprD) most commonly used in toxicology studies. The choice of the rat strain for the integrated study would therefore be based on the same considerations as for the “stand-alone” toxicology study. Further, the pattern of behaviour in the open field arena did not differ between males and females of both strains. In spite of difference in state of sexual maturation and substantial weight gain, no major difference in average values was observed either in the pattern or in the degree of activity of animals throughout the study. On the other hand, our study indicates increased variability in the behaviour of the individual animals in the open field. This increased individual variation in many of the parameters measured was the most unexpected finding in the present study. This difference was not directly attributable to specific animals; animals which showed high (or low) activity on one occasion, would show average activity on the other occasion. A possible explanation for this observation could be insufficient “novelty stress” to which animals are presented in the open field arena at repeated measurements. The behaviour of the animal in the open field is the result of interaction with a variety of test factors such as (a) stimulation as a result of removal from a familiar home environment; (b) stimulation involved in transferring the animal to the open field; (c) exposure to the test environment, consisting of both the open field itself and its surroundings; and (d) all prior experience of the test situation (Walsh & Cummins, 1976). Therefore, “… the open field test probably begins as a test of anxiety or response to novelty, and changes over time with the contribution of fear diminishing and baseline activity level coming to the fore” (Blizard, Takahashi, Galsworthy, Martin, & Koide, 2007). Indeed, at the first measurement, the animals had a relatively short time (about a week) to adapt to the new environment and had relatively little “handling experience”. With daily handling (weighing, dosing, bedding/cage
changing, clinical observations) which animals experience during the study, removal from the home cage becomes a routine, which does not induce as much stress as initially, and as such would be less stimulating. It may also be suggested that stimulation by exposure to the arena would be reduced at repeated exposure by learning and memory, in the present study, particularly on Day 2 (8 days after the first test). Although minimally different from the other time points, reaction to the open-field arena was least pronounced on Day 2 of the study. Therefore, while pre-study measurement may be advantageous, care should be taken not to place this measurement too close to the study start (at least a week or, preferably, more) to avoid reduced reaction to the arena at the start of the study. Further, animals are generally less stressed by living in an enriched environment (wood wool, house, sticks) and the presence of a companion in the home cage. Rearing conditions were shown previously to change anxiety-like behaviour in balb/c (but not in Bl/6) mice in spontaneous activity recorder, elevated plus-maze and a free exploration test (Chapillon, Manneché, Belzung, & Caston, 1999). The overall effect of all these factors would be that animals would require less time to adjust to the new situation, become familiar with the environment in the arena and exhibit more “spontaneous” type of behaviour. The direct implication of increased individual variability in the response in the open field for the design of the integrated toxicology/ safety pharmacology study is a potential increase in group size within the study. The recommendation on the group size would be different dependent on which parameter the calculation would be based upon, and, for some parameters may give totally unrealistic values. Therefore, the specific variables relevant for the test compound under investigation should be considered. For example, time in the centre, total locomotion and rearing would be of particular interest for the compounds with potential anxiolytic or anxiogenic effects (Prut and Belzung, 2003), and therefore variation in these parameters should be taken into consideration in the first place for such compounds. Based on the results of the present study, in order to detect 20% effect on total activity and 50% effect on rearing and on time in the centre (power 0.8, p b 0.05), calculated group size for the last measurement (Day 86) was substantially higher (at least double) than for the first (Day-7) for both genders of Wistar rats, and SprD males. No difference or even slightly smaller value for the calculated required group size was seen in SprD females. This calculation indicates that while group size as low as 6 animals per group may be justified for the single open field test, and 10 animals per group would give a good margin, group size of 12 (or, preferably, more) animals would be the minimal requirement for the group size in the integrated study. Our data speak therefore against the notion of performing the open field observations only in a subgroup of the animals (e.g. half of the animals of each group) included in the study. On the other hand, the main study group of the typical toxicology group would consist of at least 12 animals of each sex, and may include satellite groups as well. Therefore, there is no immediate need for including additional animals to the study, which speaks in favour of integrated studies from the 3R point of view. In practical terms, the integration of the behavioural test would often mean a staggered start (maybe over several days), and would be a logistical challenge particularly at the first dosing, when multiple blood sampling for toxicokinetics is performed. The possible solution of evaluating acute effects in a separate study (with a smaller group size), and using the toxicology study only for the evaluation of the long-term effects does not result in the reduction of number of animals but still has an effect on one of the Rs — refinement, but requires additional practical and logistical adjustments. 5. Conclusion The results of the present study suggest no principal difference in the outcome of the repeated open-field test between the two rat strains (Wistar and SprD, either males or females) most commonly used in
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toxicology studies. While the general pattern of behaviour and activity observed in the repeated measurements of motor activity in the open field test in rats was similar to the observations after a single measurement, individual variation in the pattern of behaviour was increased at repeated measurements. Due to this increase in the individual variation in reaction to the open field, an increase in group size has to be considered in order to obtain reliable data in long term integrated safety pharmacology–toxicology studies in rats. Conflict of interest None of the authors have any conflict of interest. References Blizard, D. A., Takahashi, A., Galsworthy, M. J., Martin, B., & Koide, T. (2007). Test standardization in behavioural neuroscience: A response to Stanford. Journal of Psychopharmacology, 21(2), 136–139.
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Chapillon, P., Manneché, C., Belzung, C., & Caston, J. (1999). Rearing environmental enrichment in two inbred strains of mice: 1. Effects on emotional reactivity. Behavior Genetics, 29(1), 41–46. Ewart, L., Milne, A., Adkins, D., Benjamin, A., Bialecki, R., Chen, Y., et al. (2013). A multi-site comparison of in vivo safety pharmacology studies conducted to support ICH S7A & B regulatory submissions. Pharmacological and Toxicological Methods, 68(1), 30–43. Himmel, H. M. (2008). Safety pharmacology assessment of central nervous system function in juvenile and adult rats: Effects of pharmacological reference compounds. Journal of Pharmacological and Toxicological Methods, 58(2), 129–146. ICH harmonised tripartite guideline: Safety pharmacology studies for human pharmaceuticals S7A. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/ Safety/S7A/Step4/S7A_Guideline.pdf OECD guidelines for the testing of chemicals, Section 4: Test no. 408: Repeated dose 90day oral toxicity study in rodents. http://www.oecd-ilibrary.org/environment/testno-408-repeated-dose-90-day-oral-toxicity-study-in-rodents_9789264070707-en Prut, L., & Belzung, C. (2003). The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: A review. European Journal of Pharmacology, 463(1–3), 3–33. Redfern, W. S., Strang, I., Storey, S., Heys, C., Barnard, C., Lawton, K., et al. (2005). Spectrum of effects detected in the rat functional observational battery following oral administration of non-CNS targeted compounds. Journal of Pharmacological and Toxicological Methods, 52(1), 77–82. Walsh, R. N., & Cummins, R. A. (1976). The open-field test: A critical review. Psychological Bulletin, 83(3), 482–504.
Contents lists available at ScienceDirect
Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox
Original article
Repeated measurements of motor activity in rats in long-term toxicity studies V. Golozoubova ⁎, T.K. Brodersen 1, S. Klastrup, M. Oksama, J. Løgsted, A. Makin CiToxLAB Scantox A/S, Hestehavevej 36A, Ejby, DK-4623 Lille Skensved, Denmark
a r t i c l e
i n f o
Available online 19 July 2014 Keywords: Open field test Behavioural test battery Safety pharmacology endpoints Regulatory toxicology study
a b s t r a c t Introduction: In the light of 3R (replace, reduce, refine) principles in animal experimentation and increased focus on delayed effects of treatment on central nervous system, the incorporation of behavioural tests into standard toxicology studies as a complement or substitution of a stand-alone safety pharmacology study appears very attractive, but poses some challenges. In the present study, we evaluated the results of an open field test (standard part of the behavioural test batte- ries) incorporated into the 3-month regulatory toxicology study. Methods: The study was performed in two rat strains most commonly used in toxicology studies (Wistar and Sprague Dawley (SprD)). Open field test was performed according to the standard protocol for stand-alone behavioural test (modified Irwin test) before the start of treat- ment (Day-7, “naïve” animals), on Day 2, inWeek 6 and inWeek 13 of treatment with saline. Results and Discussion: There was no overall difference between strains, and onlyminor differenceswere detected at the individual time points. With regard to time effect, the average values for most of the parameters were comparable throughout the study but individual variability in the performance in the arena was increased at repeated measurements compared to the start. In conclusion, performance in the open field arena did not differ principally betweenWistar and SprD rats of both genders. However, individual variability in the behaviour in the open field arena increased with time. This has clear implications for deciding the appropriate group size for this type of study and has to be taken into account in the design of a toxicology study with integrated safety pharmacology endpoints. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Functional observation battery (FOB) and the modified Irwin test are two behavioural test batteries commonly utilised to assess the effects of test substances on the central nervous system in accordance with regulatory guidelines for pharmaceuticals (Ewart et al., 2013; S7A, Safety Pharmacology Studies for Human Pharmaceuticals) and chemicals (OECD Guidelines for the Testing of Chemicals, test 408). The importance of including a functional observation battery test as part of the safety pharmacology ‘core battery’ also for the compounds which do not have central nervous system as a target was emphasised earlier (Redfern et al., 2005). In the light of the biomedical industry's focus on the 3R approach to animal experimentation and increased attention to the delayed effects on safety pharmacology parameters, an integrated approach, incorporating neurological evaluation into the standard repeat-
⁎ Corresponding author. Tel.: +45 56861500. E-mail address: [email protected] (V. Golozoubova). 1 Present address: Novo Nordisk A/S, Denmark.
http://dx.doi.org/10.1016/j.vascn.2014.06.007 1056-8719/© 2014 Elsevier Inc. All rights reserved.
dose toxicology study, appears attractive. However, this approach may present some logistical and interpretational challenges. The behavioural test battery typically includes three types of observations: cage-side observations, hand-held observations and open field observations. While the settings of cage-side observations and handheld observations can be standardized throughout the study, open field observations may be more of a challenge, as they include an additional factor, namely, the novelty factor. In the open field test, which, per definition, “… consists of the measurement of behaviors elicited by placing the subject in a novel open space from which escape is prevented by a surrounding wall” (Walsh & Cummins, 1976), the validity of the results of repeated measurements of motor activity performed in the course of a long-term toxicity study may be questioned on the basis of limited “novelty” of the test arena when repeated measurements are performed in the same animals. Therefore, our efforts were concentrated on this part of the test battery. Generally, the repeated open field test is regarded as an acceptable option as long as due care is taken to assure that both control and treatment group(s) undergo exactly the same procedures performed in the same manner. However, this is correct only under the assumption that variation in the data would be the same after repeated measurements
242
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as it is at a single recording. This, however is not necessarily the case in long-term studies, as the absolute values of the parameters may be different (e.g. due to the age of the animals (Himmel, 2008)). The mean values and the standard deviations of the parameters at the later phases of the study have a direct influence on the study design (group size in particular) for the integrated study, and therefore have to be evaluated. Further, the majority of the single-dose stand-alone behavioural tests are performed in male rats. The incorporation of the test in toxicology studies would typically mean that the test has to be performed both in male and in female animals, and therefore knowledge of the possible gender difference is important. In the present study, we compared the results of repeated open field test performed in both genders of Wistar and Sprague Dawley (SprD) rats integrated into a 3 month-regulatory toxicology study design, and, based on the data obtained, attempted to evaluate whether group size known to be sufficient at acute measurements would give reliable data at repeated dosing with a multiple open field measurement paradigm. 2. Materials and methods 2.1. Animals and housing The study was performed in 40 animals: 10 male and 10 female SPF Wistar rats of the strain HanTac:WH (GALAS), and 10 male and 10 female SPF Sprague Dawley rats of the Ntac:SD strain, all delivered from Taconic Europe A/S, Ejby, Denmark. The rats were approximately 5 weeks old upon arrival to the facility. At arrival, the animals were health checked and a pupil reflex-test was performed to confirm that they were not blind. The rats were kept in polycarbonate cages (floor area: 1500 cm2 — height 21 cm) with two animals/cage. For environmental enrichment, each cage was supplied with bedding (softwood sawdust), wood wool (Aspen Wood Wool (Tapvei Estonia OÜ, Estonia)), autoclaved wood bricks (Tapvei Estonia OÜ, Estonia) and a rat house (Tecniplast Gazzada S.a.r.l., 21020 Buguggiate-Va, Italy). As stereotypic behaviour was observed in three animals towards the end of the study, additional environmental enrichment was offered to these animals (and their cage mates) from Day 71 and until the termination of the study. The study took place in an animal room provided with filtered air at a temperature of 21 °C ± 3 °C and relative humidity of 55% ± 15%. The room was illuminated to give a 12:12 light:darkness cycle (light on from 06:00 h to 18:00 h). Animals had an ad libitum access to complete pelleted rodent diet (Altromin, Gesellschaft für Tierernährung mbH, D-32791 Lage, Germany) and water. Animals were weighed once weekly. The type of study was approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark.
2.2. Treatment In order to reconstruct the experimental settings of the typical toxicology study, animals were treated daily for the whole period of the study. The daily dose of sterile physiological saline (Fresenius Kabi, Denmark) was given subcutaneously (5 ml/kg body weight). 2.3. Open field test All animals were examined in an open field test (5 min recording in ActiMot Motility Measuring System (TSE, Germany), arena size 96 × 96 cm) before the start of treatment (Day-7), after the second dosing (Day 2), in Week 6 (Day 37) and in Week 13 (Day 86) of daily treatment with saline. The animals were tested at each testing occasion by the same operators, at least 1 h after dosing, at approximately the same time, in the same sequence. At the start of the session, the animal was placed in the centre of arena.
On each occasion, the test was performed in parallel in two arenas first in all males, then in all females (same sequence of individual animals on all occasions), at 10.00–12.30 (±15 min). The level of illumination was kept similar to that in the home cage. Parameters evaluated were the same as in a stand-alone behavioural test — either automatically recorded by ActiMot system (time moving, total distance, numbers of rearings (vertical activity), time in centre/ periphery, total corner visits, total activity (moves/counts)), or visually evaluated (abnormal behaviour (e.g. aggression, stereotypic behaviour, cataleptic behaviour) and number of faecal pellets at the end of each session). 2.4. Statistical analysis Overall strain differences (separate for males and females) were assessed by an analysis of covariance (ANCOVA) with pre-dose measurement as a covariate (where applicable). In addition, Student's ttest (unpaired) was used for the direct comparison of strains on the individual days. Time effect (using Day-7 values as a reference) was evaluated by paired t-test. For all tests, the level of significance was defined as p b 0.05. The statistical analyses were made with SAS® procedures (version 8.2) described in “SAS/STAT® User's Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA”. 3. Results 3.1. General As expected, there was a substantial weight gain in the course of the study (gain of approx. 240 g for males and approx. 100 g for females). Body weights were significantly different between the strains from the study start and until Day 29 for males, and throughout the study for the females (SprD heavier than Wistar). Open field measurements were performed successfully in all animals. No abnormal behaviour was observed during the test in any of the animals at any of the time points. No overall difference between the strains was identified either for males or for females. 3.2. First exposure to the arena Male Wistar rats appeared to have a more pronounced reaction to the novelty of the environment at the first exposure to the test arena compared to SprD males: their total activity count and number of rearings were higher, they spent more time moving, spent more time in the centre of arena (Table 1), and had more faecal pellets. For the females, the difference between strains was minimal, the only parameter which differed statistically significantly between strains was total distance travelled (longer in Wistar females). 3.3. Repeated exposure to the arena In both strains time moving, time in the centre and total activity tended to be higher at the first measurement (before, Day-7) compared to the following measurements (Day 2, Week 6 and Week 13), the difference being more pronounced in males than in females (Table 1). The pattern of reaction to the exposure to arena was very similar on all occasions, the peak of activity was, as expected, observed within the first minute in the arena, and was gradually reduced thereafter. Due to an increased individual variation, this trend was more clear after the measurements in the beginning of the study (before and Day 2) compared to Week 6 and Week 13. At all measurement occasions, individual variation was least pronounced during the first minute of exposure to the arena and was increased during the following period (2–5 min) (data not shown).
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Table 1 Overview of parameters measured in the open field in Wistar and Sprague Dawley rats at different time points. Parameter
Time point
Wistar
Sprague Dawley
Males Mean Time moving
Distance travelled
Number of rearings
Time centre
Total corner visits
Total activity
Before Day 2 Week 6 Week 13 Pre Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13 Before Day 2 Week 6 Week 13
245.5 220.7 201.2# 215.2# 99.4 85.3 75.5 89.2 40.8 30.2 33.5 36.5 32.7 27.7 27.8 23.3 77.3 60.3 55.3# 69.0 7011.7 6113.4 5475.0# 6040.6#
Females S.D. 17.2 47.5 52.9 27.1 16.5 25.6 32.3 23.4 7.7 15.1 15.0 10.4 9.1 14.2 13.3 13.1 15.0 20.9 28.1 18.0 765.5 1489.9 1622.2 1026.2
CV 7.0 21.5 26.3 12.6 16.6 30.0 42.8 26.2 18.8 50.0 44.7 28.4 27.9 51.2 47.9 56.3 19.4 34.7 50.8 26.1 10.9 24.4 29.6 17.0
Mean 228.5 222.3 231.4 195.6 89.9 88.8 97.5 77.5 32.0 34.2 36.3 29.9 23.5 21.2 29.4 9.8# 71.5 66.7 69.4 63.1 6448.4 6185.6 6632.0 5550.6
Males S.D. 17.4 41.2 12.7 44.3 9.7 25.9 18.6 26.3 10.8 16.5 13.8 13.7 13.0 16.2 15.3 7.5 14.4 25.0 18.0 30.0 553.9 1349.2 715.2 1589.2
CV 7.6 18.5 5.5 22.6 10.8 29.2 19.1 34.0 33.7 48.1 38.0 45.9 55.0 76.5 52.1 76.9 20.1 37.4 26.0 47.6 8.6 21.8 10.8 28.6
Mean 229.3⁎ 199.1# 224.4 217.2 88.4 79.0 96.9 91.4 32.1⁎ 32.4 32.7 28.5 21.3⁎ 16.9 27.1 12.1⁎# 73.6 58.9# 75.8 65.5 6371.3⁎ 5564.8# 6467.9 6060.6
Females S.D.
CV
Mean
S.D.
CV
12.2 29.5 22.5 37.2 11.7 13.3 29.2 34.9 10.6 12.3 14.5 10.7 5.2 10.2 14.6 7.5 15.2 13.6 24.2 25.2 490.4 901.1 1172.3 1429.9
5.3 14.8 10.0 17.1 13.2 16.9 30.2 38.1 33.1 38.1 44.3 37.6 24.2 60.3 54.0 62.5 20.6 23.0 31.9 38.5 7.7 16.2 18.1 23.6
220.4 195.0 215.8 227.2 78.7⁎ 71.3 86.1 93.5 29.9 25.1 31.3 35.3 21.8 14.2# 26.1 16.1 64.6 59.2 69.7 78.0# 5943.7 5324.9 6025.2 6293.0
19.1 35.3 43.9 21.6 13.2 17.6 27.8 18.2 8.0 10.7 13.1 8.4 10.1 10.5 11.6 6.4 9.8 18.2 28.4 13.2 733.2 1119.1 1325.0 794.2
8.6 18.1 20.4 9.5 16.7 24.7 32.3 19.4 26.6 42.7 41.8 23.8 46.1 73.9 44.3 39.7 15.2 30.8 40.8 16.9 12.3 21.0 22.0 12.6
8 animals per sex per strain; data collected over a period of 5 min at each recording. S.D. — standard deviation; CV — coefficient of variation. ⁎ Difference between strains (by gender), p b 0.05, tested with Student's unpaired t-test for males and females, separately. # Difference from the pre-values (Before, Day-7) to values obtained on Day 2, Week 6 or Week 13, p b 0.05, tested with Student's paired t-test.
The difference between strains on individual days of repeated measurements was minor (see Table 1) and not statistically significant for most of the parameters. In Week 13, male Wistar rats showed a slightly
more marked reaction to the arena than SprD males as they spent significantly more time in the centre of the cage and had a higher incidence of defecation.
Fig. 1. Pattern of activity of individual animals after placement to the open-field arena on different days. Total activity counts; A — Sprague Dawley males (filled circles);B — Sprague Dawley females (empty circles); C — Wistar males (filled squares); D — Wistar females (empty squares).
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3.4. Individual variation While activity levels in naïve animals were rather homogenous within the groups, individual variation between animals of the same group was more pronounced at repeated measurements (Days 2, Week 6 and Week 13). An increase in standard deviation of the mean as well as in coefficient of variation at repeated measurements (Day 2, Week 6 and Week 13) compared to the pre-treatment (Day-7) was observed for many of the parameters measured (Table 1). The reason for increased variation could not easily be categorised by separation into broad categories such as more and less active individuals; as animals showing low level of activity at one of the measurements showed normal or even relatively high activity at the other time point (Fig. 1). Increased individual variation (lower signal/noise ratio) means, in statistical terms, need for a larger group size in order to reliably determine intergroup differences. 3.5. Stereotypic behaviour Three Wistar rats (1 male and 2 females) showed stereotypic behaviour (walking in a particular circular pattern) in the home cage at the end of the second month of the study. Stereotypic behaviour is a very rare finding, observed predominantly in Wistar rats, occurring in a long term studies, and not as early as at the end of the second month of the study. These three animals did not show any stereotypic behaviour in the open field arena. When compared to the other animals, the male showing stereotypy in the home cage had lower than average number of rearings. The females did not distinguish themselves from the average in any of the parameters. 4. Discussion The results of the present study suggest no principal difference in the outcome of the repeated open-field test between the two rat strains (Wistar and SprD) most commonly used in toxicology studies. The choice of the rat strain for the integrated study would therefore be based on the same considerations as for the “stand-alone” toxicology study. Further, the pattern of behaviour in the open field arena did not differ between males and females of both strains. In spite of difference in state of sexual maturation and substantial weight gain, no major difference in average values was observed either in the pattern or in the degree of activity of animals throughout the study. On the other hand, our study indicates increased variability in the behaviour of the individual animals in the open field. This increased individual variation in many of the parameters measured was the most unexpected finding in the present study. This difference was not directly attributable to specific animals; animals which showed high (or low) activity on one occasion, would show average activity on the other occasion. A possible explanation for this observation could be insufficient “novelty stress” to which animals are presented in the open field arena at repeated measurements. The behaviour of the animal in the open field is the result of interaction with a variety of test factors such as (a) stimulation as a result of removal from a familiar home environment; (b) stimulation involved in transferring the animal to the open field; (c) exposure to the test environment, consisting of both the open field itself and its surroundings; and (d) all prior experience of the test situation (Walsh & Cummins, 1976). Therefore, “… the open field test probably begins as a test of anxiety or response to novelty, and changes over time with the contribution of fear diminishing and baseline activity level coming to the fore” (Blizard, Takahashi, Galsworthy, Martin, & Koide, 2007). Indeed, at the first measurement, the animals had a relatively short time (about a week) to adapt to the new environment and had relatively little “handling experience”. With daily handling (weighing, dosing, bedding/cage
changing, clinical observations) which animals experience during the study, removal from the home cage becomes a routine, which does not induce as much stress as initially, and as such would be less stimulating. It may also be suggested that stimulation by exposure to the arena would be reduced at repeated exposure by learning and memory, in the present study, particularly on Day 2 (8 days after the first test). Although minimally different from the other time points, reaction to the open-field arena was least pronounced on Day 2 of the study. Therefore, while pre-study measurement may be advantageous, care should be taken not to place this measurement too close to the study start (at least a week or, preferably, more) to avoid reduced reaction to the arena at the start of the study. Further, animals are generally less stressed by living in an enriched environment (wood wool, house, sticks) and the presence of a companion in the home cage. Rearing conditions were shown previously to change anxiety-like behaviour in balb/c (but not in Bl/6) mice in spontaneous activity recorder, elevated plus-maze and a free exploration test (Chapillon, Manneché, Belzung, & Caston, 1999). The overall effect of all these factors would be that animals would require less time to adjust to the new situation, become familiar with the environment in the arena and exhibit more “spontaneous” type of behaviour. The direct implication of increased individual variability in the response in the open field for the design of the integrated toxicology/ safety pharmacology study is a potential increase in group size within the study. The recommendation on the group size would be different dependent on which parameter the calculation would be based upon, and, for some parameters may give totally unrealistic values. Therefore, the specific variables relevant for the test compound under investigation should be considered. For example, time in the centre, total locomotion and rearing would be of particular interest for the compounds with potential anxiolytic or anxiogenic effects (Prut and Belzung, 2003), and therefore variation in these parameters should be taken into consideration in the first place for such compounds. Based on the results of the present study, in order to detect 20% effect on total activity and 50% effect on rearing and on time in the centre (power 0.8, p b 0.05), calculated group size for the last measurement (Day 86) was substantially higher (at least double) than for the first (Day-7) for both genders of Wistar rats, and SprD males. No difference or even slightly smaller value for the calculated required group size was seen in SprD females. This calculation indicates that while group size as low as 6 animals per group may be justified for the single open field test, and 10 animals per group would give a good margin, group size of 12 (or, preferably, more) animals would be the minimal requirement for the group size in the integrated study. Our data speak therefore against the notion of performing the open field observations only in a subgroup of the animals (e.g. half of the animals of each group) included in the study. On the other hand, the main study group of the typical toxicology group would consist of at least 12 animals of each sex, and may include satellite groups as well. Therefore, there is no immediate need for including additional animals to the study, which speaks in favour of integrated studies from the 3R point of view. In practical terms, the integration of the behavioural test would often mean a staggered start (maybe over several days), and would be a logistical challenge particularly at the first dosing, when multiple blood sampling for toxicokinetics is performed. The possible solution of evaluating acute effects in a separate study (with a smaller group size), and using the toxicology study only for the evaluation of the long-term effects does not result in the reduction of number of animals but still has an effect on one of the Rs — refinement, but requires additional practical and logistical adjustments. 5. Conclusion The results of the present study suggest no principal difference in the outcome of the repeated open-field test between the two rat strains (Wistar and SprD, either males or females) most commonly used in
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toxicology studies. While the general pattern of behaviour and activity observed in the repeated measurements of motor activity in the open field test in rats was similar to the observations after a single measurement, individual variation in the pattern of behaviour was increased at repeated measurements. Due to this increase in the individual variation in reaction to the open field, an increase in group size has to be considered in order to obtain reliable data in long term integrated safety pharmacology–toxicology studies in rats. Conflict of interest None of the authors have any conflict of interest. References Blizard, D. A., Takahashi, A., Galsworthy, M. J., Martin, B., & Koide, T. (2007). Test standardization in behavioural neuroscience: A response to Stanford. Journal of Psychopharmacology, 21(2), 136–139.
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