M. L. Zinovieva¹, P. H. Zhminko¹, V. V. Kirsenko², O. M. Strumenska³

¹  L. I. Medved's Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Ukraine (State Enterprise), Kyiv, Ukraine

² State Institution “Institute of Occupational Health named after Yu. I. Kundiiev of the National Academy of Medical Sciences of Ukraine”, Kyiv, Ukraine

³ O.O. Bohomolets National Medical University Kyiv, Ukraine

ABSTRACT. Validation of the methods at the stage of their implementation and with subsequent application is an important part of ensuring the quality of toxicological study data State Enterprise “Scientific Center for Preventive Toxicology, Food and Chemical Safety named after Academician L. I. Medved of the Ministry of Health of Ukraine” has implemented methods of screening of neurotoxic exposure of chemical substances — a set of clinical and functional tests (CFT) and a method for evaluation of motor activity in the “open field” for rats.

Objective. Determination of specificity, sensitivity and reliability of the implemented methods under exposure to reference neurotropic substances with differently directed effects — chlorpromazine and caffeine on the parameters of the neurological conditions of female Wistar Han rats.

Results. It has been shown that the set of CFT and the method for evaluation of motor activity in the “open field” are relevant for the determination of specific exposure and toxicodynamics of reference substances having differently directed effects on the nervous system of rats. The sensitivity of these methods under the lowest active dose of caffeine and chlorpromazine was determined. Data on the reproducibility of test results have been obtained.

Keywords: validation, neurotoxicity, chlorpromazine, caffeine.

Risks to the health of the population as a result of intensive industrial use of chemicals place high demands on the quality of toxicological study data. Implementation of the concept of mutual acceptance of data (Decision of the Council concerning the mutual acceptance of data in the assessment of chemicals, 1981) and the principles of Good Laboratory Practice (Directive 2004/10/EC of the European Parliament and of the Council, 2004) is aimed directly on the increase of the efficacy of toxicological assessment. These documents reflect the need for global unification and standardization of the research methodology. This provides a greater amount of information on the toxic properties of chemicals and reduces the material and time costs for their research. Validation of toxicological methods (tests) is a key stage of their practical implementation both at the level of international standards of studies and in a separate research institution. This procedure is aimed at ensuring the high quality of the data obtained by one or another method and their effective use in the process of assessing the health risks associated with the use of the investigated chemical substance. The value of the toxicological test consists in its ability to quantify a certain endpoint and thus obtain the most objective information on the exposure of the substance to the body. Practical implementation of a new test requires a preliminary demonstration of its reliability, specificity and sensitivity, that is, validation [1, 2].

Neurotoxicological study takes one of the key positions in assessing body response to the toxic effects of chemicals and other factors since the nervous system (NS) regulates and coordinates the functioning of all organs and systems. For example, a set of neurobehavioural tests at the stage of screening of toxic effects is an integral part of the study of the effect on NS as well as an obligatory component of the modern methodology of the study of subacute, subchronic and embryotoxic effects of chemical substances. Currently, the primary assessment of the neurotoxic chemical potential of chemicals is included in more than 20 European Union guidelines (OECD Test Guidelines for the Chemicals) [3]. Endpoints of neurotoxic effects are indicators of changes in biological processes in NS, its reactions and morphological damages under exposure to toxicants. Chemicals which neurotropic effects are well studied and are specific in assessing the effect on one or another endpoint are used for validation studies. A sufficiently complete list of such reference substances, which are used as a positive control in the neurotoxicological study, is provided in OECD documents [1]. As stated above, validation of the methods is carried out at different organizational levels. For example, the methodological recommendations proposed for national or international use are validated through multi-laboratory studies and their main objective is reproducibility of the testing methodology in various laboratories [4]. The main issues that arise during planning and performance of validation studies are detailed in the paper of J. P. Maurissen and B.R. Marable [5].

Within-laboratory validation neurotoxicological studies are aimed at confirming the appropriateness and relevance of the whole complex laboratory practice system. Validation is of particular importance for in vivo tests since the reactions of NS are integral and depend on the influence of many factors — environmental changes, the competence of staff when handling animals, etc. Furthermore, in spite of the efforts to objectivate animal behaviour assessment through the use of automatic registration tools [6, 7], visual, subjective assessment and its dependence on the qualifications of the researcher may also affect the quality of the data received. Therefore, an obligatory condition for the observance of the impartiality of the staff is “blind” examination of animals, without informing about the substances used.

The process of within-laboratory validation of neurotoxicological methods of the first level [8] (a set of clinical observations and functional tests, studies of motor activity of animals) provides determination of sensitivity of these methods by studying the dependence of observed endpoints on the dose of reference substances [9] and the establishment of the minimum dose affecting. The obtained results can be used for between-laboratory comparative studies, monitoring of the quality of neurotoxicological study in the research laboratory, as well as for the creation of an internal base of the historical control. L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Ukraine (State Enterprise) (RC) has in place a set of clinical and functional tests (CFTs) for rats and methods for assessing their motor activity in the “open field”. Considering the regulatory function of the NS, it is important to establish the sensitivity of the method to detect the exposure of different directions — both stimulating and depressing.

Previous studies [10] have shown that an implemented set of CFTs provides a relevant reflection of NS inhibition in rats in response to a single intragastric administration of chlorpromazine (CPZ) at doses of 8 and 16 mg/kg. It was shown that statistically significant changes (р ≤ 0.5) under exposure to this reference substance in the higher studied dose were detected within 1/2–4 hours after administration and were characterized by a decrease in the total activity of animals, their sensomotor reactions, muscle tone, drowsiness, vocalization in response to a touch, stereotyping, mydriasis. The study of behaviour in the “open field” established the appearance of a latent period of the beginning of animal movement, reduced locomotor activity, and a number of stacks. At a dose of 8 mg/kg, CPZ caused less expressed suppression of NS: attenuation of the reaction of animals to approach and touch, decrease in muscular tone, locomotor activity. The maximum manifestation of changes, according to the criterion of their statistical significance, was observed in a shorter period — 1–4 hours after CPZ administration. Therefore, the specificity of a set of CFTS was shown for diagnostics of the inhibitory effect on NS in rats.

Along with clinical and functional tests, quantification of motor activity of animals in the “open field” is one of the effective predictors of neurotoxic action of chemicals at the screening stage. Currently, various modifications of this method are used, and they adapted for both visual and automated registration of rodent activity [11]. The RC has accumulated considerable experience in using this test under conditions of visual fixation of data; conditions for its performance have been standardized.

Objective. Determination of the specificity and sensitivity of a set of clinical and functional tests under exposure to caffeine, which has a stimulating effect on NS, as well as determination of the sensitivity of the method of assessing motor activity of rats under exposure to the reference neurotropic substances with the action of different directions by studying the dose-effect dependence.

Materials and methods. The experiment was conducted in accordance with the principles of Good Laboratory Practice (GLP) and in compliance with international recommendations for neurotoxicological study [12] in female Wistar Han SPF rats at the age of 10 weeks, with a body weight of 170–220 g. The animals were received from the clinic of laboratory animals of the RC and they underwent acclimatization within 5 days. Animals were kept in T-4 cages (40 × 30 × 15 cm), 1 or 2 animals in each, under conditions of vivarium SPF with artificial light, with 12-hour cycle and free access of rats to feed and water. All procedures with animals were performed in accordance with international rules and regulations (European Communities Council Directives of November 24, 1986, 86/609 / EEC).

Reference substances. The study used chlorpromazine (CPZ) hydrochloride, 2.5 % solution in ampoules manufactured by Arterium (Ukraine), caffeine (C) sodium benzoate, 10 % solution in ampoules manufactured by PJSC “Pharmaceutical Firm “Darnitsa” (Ukraine). Substances were given as a single intragastrically administration under fasting conditions. Drinking water purified by dual osmosis was used as a solvent. Concentrations of the administered solutions were adjusted to the volume of 5 mL/kg. To ensure “blind” assessment of the studied parameters, solutions were prepared by the staff who did not participate in the observation of animals, and ready-made solutions were provided with digital codes.

Study design. A set of CFTs was performed as described in [10]. Parameters were assessed according to the scale shown in Table 1.

Table 1

Rating scale for parameters of the set of clinical and functional tests in points

Note: To determine condition “2”, results of examination of the group of intact animals not participating in the study are used.

Study of the exposure to C was conducted in 3 groups, 5 rats in each, receiving the solvent (control) and C at the doses of 12 and 24 mg/kg. In animals, parameters of the set of CFTs were recorded over time — 1/4, 1/2, 1, 2, 4 and 24 hours after C administration. Assessment of behavioural reactions in the “open field” was performed 2 hours after administration of substances and included determination of the latent period of movement; horizontal (locomotor) activity (general and in the central zone of the “open field”), assessment of vertical motor activity by the number of stacks, intensity of defecation by the number of boluses, recording of the average grooming duration.

There was a separate study of the motor activity of rats in the “open field” in 6 groups of animals, 5 animals in each. Three groups of rats received a solvent (control), CPZ at the doses of 4 mg/kg and 8 mg/kg, respectively. Other groups received a solvent (control), C at the doses of 12 mg/kg and 24 mg/kg, respectively. Parameters were recorded 1 hour after administration. Locomotor (horizontal) and vertical activity were determined.

Statistical processing of the study results was performed out using a t‑test for continuous data, and Mann — Whitney test for descriptive, binary, and rank parameters by the threshold level of significance of changes p < 0.05.

Results. The assessment of the status of rats according to the parameters of the set of CFTs showed that they had no sensomotor disturbances, their muscular tone was the same as in intact rats, animals were free of symptoms that would indicate a pathological condition. Ophthalmoscopy did not reveal any violations. Observations in the “open field” showed that the animals began movement without a latent period. Disturbances of movements and coordination of movements were not observed; there was no defecation and grooming. Locomotor activity of animals in the control group was 51.2 ± 1.9 squares, including in the central zone –4.8 ± 0.6 squares, the number of stacks was 8.0 ± 1.1.

Under exposure to the C in the maximum studied dose — a dose of 24 mg/kg in rats, there were changes in endpoints recorded by the set of CFTs, in the period 1/4–2 hours (Table 2). During this period, the intensity of the auditory-motor reaction of animals and the reaction to approach and touch increased. In isolated cases, stereotypical sniffing was observed and nociceptive reactivity increased. Ophthalmoscopy did not reveal differences from the control group. After administration of C at the dose of 12 mg/kg, individual animals at 1/2–1 hour after administration showed mild symptoms (p > 0.05) similar to that observed under exposure to a higher dose of the reference substance. According to the criterion of statistical significance (p < 0.05) changes of the parameters of the set of CFTs over time, the time of maximum manifestations of symptoms after exposure to C — 1/2–2 hours was determined.

Table 2

Rate of changes of the parameters of the set of clinical and functional tests after caffeine administration at the doses of 12 and 24 mg/kg over time (n = 5)

Notes: 1. The table provides the number of animals per group with detected changes compared to control. 2. SM — stereotypic movements 3. * — p < 0.05 (Mann — Whitney test)

Research of behavioural parameters in the “open field” (Table 3) 2 hours after exposure to C at the dose of 24 mg/kg showed activation of defecation in most animals (p < 0.05), there was a tendency for grooming (p > 0.05). Changes in the parameters of motor activity (locomotor activity and number of stacks), the latent period of the animal movement were not observed. Similar changes in animals were induced by C at the dose of 12 mg/kg, with the intensity of defecation less pronounced than at higher dose levels. The obtained data confirmed that the period of maximal manifestation of symptoms for C is 1/2–2 hours. According to the observation of behavioural reactions of female rats in the open field, the sensitivity of the set of CFTs under exposure to C was at the level of the minimum dose studied — 12 mg/kg.

Table 3

Parameters of rat behaviour in the “open field” after 2 hours of caffeine exposure at the doses of 12 and 24 mg/kg (М ± m, n = 5)

Notes:
1. The rate of behavioural act in the group of animals is provided in quotes
2.* — p < 0.05 (t-test)
3.** — p < 0.05 (Mann — Whitney test)

In general, the applied tests appropriately reflect the effect of C on the functional status of the NS of female rats. The set of CFTs showed an increase in sensomotor reactions characteristic of the action of this substance [12]; there was a dose-dependent activation of defecation in rats with the observation in the “open field”, which is also a specific manifestation of the effect of C [13]. The sensitivity of the method according to the criterion of statistical significance of changes has been established at the level of 12 mg/kg.

Assessment of motor activity in the “open field” after 1 hour (Table 4) allowed revealing the effect of C, which was not observed in the research of these parameters in 2 hours. For example, the number of stacks significantly (p < 0.05) increased depending on the dose of C — by 54 % and 38 % under exposure to the doses of 24 mg/kg and 12 mg/kg, respectively. This allowed confirming the sensitivity of the method of assessing motor activity at the level of 12 mg/kg for the stimulating effect of C. The time of maximum manifestation of the symptoms specific for this reference substance, which should be taken into account when planning further validation studies, has also been specified.

Table 4

Parameters of motor activity of the rats in the “open field” after 1 hour of administration of caffeine and chlorpromazine (M ± m, n = 10)

Note: * — р < 0.05

Upon the use of the reference substance with a depressing effect on the NS to assess parameters of the motor activity in the “open field”, the following results were obtained. After 1 hour of CPZ administration at the doses of 4 and 8 mg/kg, a decrease (p < 0.05) of locomotor activity and the number of stacks was almost doubled under exposure to a higher dose (Table 4). Such data match with the results obtained during assessment evaluation of the behaviour of rats in the “open field” after 2 hours of exposure to CPZ (Table 3). These results indicate the reproducibility of the effect, which in turn reflects the reliability of the method. Under exposure to CPZ at the dose of 4 mg/kg no significant changes in these endpoints were found, which allows establishing the sensitivity of the method of assessment of motor activity at the level of 8 mg/kg CPZ.

Discussion. The obtained data in combination with the previously published results of the assessment of the efficacy of the set of CFTs suggest that the methods of neurotoxicological screening of rats, implemented in the RC, allow identifying the influence on NS of different direction effectively. The tests appropriately reflect the specific action of the reference substances used: depressing effect for CPZ and activating for C. The set of CFTs, which were performed over time, allowed characterizing the kinetics of the influence of reference substances in the doses studied. For example, the time of maximal manifestation of neurotropic effects was determined as 1/2–4 hours for CPZ and 1/2–1 hour for C, which corresponds to the literature [12].

Study of the parameters of motor activity in the “open field” 1 hour after the administration confirmed that this time point corresponds to the maximum exposure to both C and CPZ under the conditions of this experiment. At this time, an increase in the number of stacks in rats, characteristic of C exposure, was found, and this indicates its anxiolytic effect [14]. Under exposure to CPZ, there was a characteristic inhibition of both studied parameters of the motor activity [12].

Dependence of the observed effects of C and CPZ from their dose when given orally to rats confirms their specificity and allows establishing the sensitivity of the applied tests. For example, for the set of CFTs in a female rats study, the sensitivity of the test has been established at the level of minimum tested doses: CPZ — 8 mg/kg, C — 12 mg/kg.

The method of study of motor activity in the period of maximum exposure to the reference substances (1 hour) confirmed the effect of CP at a dose of 8 mg/kg, the sensitivity of the test under exposure to C was limited to the minimum tested dose — 12 mg/kg, which revealed a significant increase in stacks. Since the inactive C dose for this method has not been achieved, further planning should take into account the possibility of studying lower doses of this reference substance. 

Therefore, according to the criteria of specificity and sensitivity, methods of the first level of determination of the neurotoxic influence of chemical substances — a set of CFTs and the method of studying the motor activity of rats in the “open field” — were validated. The obtained data suggest their reproducibility. Further regular validation studies have been substantiated. The success of conducting “blind” neurotoxicological observations became the basis for assessing staff qualification.

Conclusion

1. Validation studies of neurotoxic effects screening methods — a set of CFTs and method for assessing motor activity in the “open field” for rats — implemented in the RC have shown the efficacy of these tests to detect the influence of chemicals of a different direction — activating and stimulating — on the NS. It was shown that in both cases, the effect was dependent on the dose of reference substances.
2. In the course of the study, based on the results of each of the validated tests, the specific effect of C — enhancement of the auditory-motor reaction, anxiolytic effect, activation of defecation was established. The exposure to CPZ in these tests was characterized by inhibition of the general activity of animals and their sensomotor reactions, decreased muscular tone, stereotypy and mydriasis, in the “open field” animals showed a latent period of onset of movement, the locomotor activity and the number of stacks decreased.
3. Both tests provide a relevant reflection of the toxicodynamics of the acute effects of reference substances and allow establishing a period of maximum exposure to C (30–60 min) and CPZ (60–240 min.).
4. The reliability of the method of study of motor activity in the “open field” under exposure to CPZ in the course of repeated study has been shown.

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