Toxicological and Genotoxicity Assessment of a Dihydroquercetin-Rich Dahurian Larch Tree ( Larix gmelinii Rupr) Extract (Lavitol)

Test preparation

Lavitol was administered per os in 1% ethanol at 0.5 mL of solution, allowing for complete dissolution. The reagents methyl methanesulfonate, Tris, NaOH, and disodium EDTA were supplied by Sigma-Aldrich (St Louis, Missouri), in addition to, low-melting temperature agarose, normal-melting temperature agarose, NaCl, dimethyl sulfoxide (DMSO), and Triton X-100 (pancreas). The fluorescent SYBR Green I dye was provided by Invitrogen (Carlsbad, California).

Animals and housing

Twenty pathogen-free CBAxC₅₇Bl/6 male mice used in the study were obtained from the Research Laboratory of Pharmacogenetics, State Zakusov Research Institute of Pharmacology of the Russian Academy of Medical Sciences, Moscow. Animals were 8 to 12 weeks old and weighed 18 to 20 g. Upon arrival in the laboratory they were kept, 10 animals/cage, in polycarbonate cages (940 cm²), at 20°C ± 2°C, at a relative humidity (RH) of 60%, and a 12-hour light/12-hour dark cycle. They were fed a standard rodent laboratory chow (MEST, Moscow, Russia) and provided water ad libitum. After acclimatization, animals were randomly divided into 4 groups with each group (n = 5) caged separately. Bedding consisted of steam-sterilized hardwood chips.

Experimental design

Methanesulfonate was administered intraperitoneally for 3 hours at 40 mg/kg and used as a positive control. A 1% dose of ethanol (Synthesis, Moscow, Russia) was administered 0.1 mL/10 g body weight (bw) per os for 5 days as a negative control. A single dose of 15 and 2000 mg/kg bw of Lavitol was orally administered to males, as shown in Table 1, corresponding to the daily therapeutic dose of 15 mg/kg bw and a subchronic dose that exceeds the therapeutic dose by >100 times that dose. Cytogenic preparations of the femoral bone marrow, blood samples, and liver samples were prepared using the methods of Singh et al, ¹⁵ Durnev et al, ¹⁶ and Kharbiev. ¹⁷ Gel electrophoresis of isolated cells was performed in an alkaline medium. Preparations were stained with SYBR Green 1 for 30 minutes in total darkness. Cytogenic analysis was performed using an epifluorescent microscope Micmed-2 12T model (Lomo, St Petersburg, Russia), with a high-resolution digital camera (model VEC-335, EBC, St Petersburg, Russia), set at 200× magnification. The images of DNA comets were analyzed using CASP software v.1.2.2 (CASPlab, Wroclaw, Poland) using an automatic imaging system that scores the extent of DNA damage. ¹⁷

OPEN IN VIEWER

Table 1.

Administration of Tested Compound.

Substance	Sex	# of animals	Dosage	Way of introduction
Methyl methanesulfonate	M	5	40 mg/kg bw	Intraperitoneally, single
Lavitol	M	5	15 mg/kg bw	Orally, single
Lavitol	M	5	2000 mg/kg bw	Orally, single
Negative control	M	5	0.1 mL/10 g bw	Orally, 5 days

Abbreviation: M, male.

Statistical analysis

At least 100 individual cells for each micropreparation were analyzed for the DNA content in the tail of the DNA comet as an index of DNA damage. Statistical analysis was performed using the analysis of variance (ANOVA) post hoc Dunnet test (Statistica v. 6; StatSoft, Moscow, Russia).

Test preparation

Prior to commencing testing, the solubility of the test substance in a vehicle compatible with this test system was assessed. Lavitol was found to be soluble at 304.3 mg/mL in ACS reagent grade DMSO, the vehicle for this study. The highest concentration of Lavitol tested in this study was 304.3 mg/mL in the chosen vehicle, which provided a final concentration of 3043 μg/mL, when dosed at 1% (v/v), in order to test up to 10 mmol/L. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations. The pH and osmolality of Lavitol in medium were tested at a concentration of 3043 μg/mL. No fluctuations in pH of the medium of more than 1 unit were observed compared with the vehicle control. No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.

S9 fraction, prepared from male Sprague-Dawley-derived rats, is dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at about −80°C. The S9 mix consists of S9 fraction (10%, v/v), MgCl₂ (8 mmol/L), KCl (33 mmol/L), sodium phosphate buffer pH 7.4 (100 mmol/L), glucose-6-phosphate (5 mmol/L), and nicotinamide adenine dinucleotide phosphate (4 mmol/L). The cofactors were filter sterilized with a 0.2-μm nonpyrogenic sterile filter prior to use. The dose levels of S9 mix were selected so as to reach an acceptable toxicity profile per protocol. Dose levels between plus and minus S9 may therefore not be the same as test compounds respond different in either the absence or presence of S9.

Human blood was collected aseptically from 2 healthy, nonsmoking, adult donors, pooled (in equal volumes from each donor) and diluted with Roswell Park Memorial Institute 1640 tissue culture medium supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin/20 μg/mL streptomycin, and 2.0 mmol/L l-glutamine. Lymphocytes were stimulated by the addition of phytohemagglutinin (PHA). Cultures were established from the prepared (pooled) sample and dispensed as 5-mL aliquots (in sterile universal containers) so that each contained blood (0.4 mL), culture medium (4.5 mL), and PHA solution (0.1 mL). All cultures were then incubated at 37°C, and the cells resuspended (at least once daily) by gentle inversion. The human lymphocytes in whole blood culture were stimulated to divide by addition of PHA 48 hours prior to treatment, then exposed to Lavitol for 3 hours in both the absence and the presence of exogenous metabolic activation (S9 mix), and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 3043 μg/mL, dosed at 1% (v/v), in order to test up to 10 mmol/L, the maximum concentration as recommended in the guidelines that this assay follows. Vehicle (DMSO) and positive control cultures were included in all test conditions.

Cells were harvested by centrifugation at 500g for 5 minutes. The supernatant was removed and the cell pellet resuspended and treated with a 4-mL hypotonic solution (0.075 mol/L KCl) at 37°C, cultures were then incubated for 3 minutes at 37°C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1, v/v methanol:acetic acid) was added slowly onto the culture surface, and the cultures were slowly inverted to mix. The cultures were centrifuged at 500g for 5 minutes. The supernatant was removed, and the cell pellet resuspended. A further 4 mL of fresh fixative was then added and the cells stored at 4°C until slide preparation. Cytokinesis was blocked following mitosis using Cytochalasin B (Cayman Chemical, Ann Arbor, Michigan), the cells harvested, and slides prepared so that binucleate cells could be examined for micronucleus induction. In order to assess the cytotoxicity of Lavitol to cultured human lymphocytes, the cytokinesis-block proliferative index (CBPI) was calculated for cultures treated with the test substance, the vehicle and positive controls. Five Lavitol concentrations were assessed for determination of induction of micronuclei. The highest concentration selected (650 μg/mL) was that which caused a reduction in CBPI equivalent to 55% ± 5% cytotoxicity. Following 3-hour treatment, reductions in CBPI equivalent to 57.8% and 58.0% cytotoxicity were obtained with Lavitol at 650 μg/mL in the absence and presence of S9 mix, respectively. Concentrations of Lavitol selected for micronucleus analysis were 100, 300, 400, 500, and 650 μg/mL in the absence of S9 mix and 100, 350, 450, 600, and 650 in the presence of S9 mix. In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 53.5% cytotoxicity was obtained with Lavitol at 200 μg/mL. Concentrations of Lavitol selected for micronucleus analysis were 50, 100, 150, 175, and 200 μg/mL.

Slide preparation and examination

The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Precleaned microscope slides were prepared for each culture by aliquoting the resuspended cells onto the slides and allowing the slides to air dry. Two slides were prepared per culture. The remaining cell cultures were stored at approximately 4°C until slide analysis was complete. Slide staining was performed by rinsing in purified water, staining in acridine orange solution (0.0125 mg/mL) for 4 minutes, washed in purified water for 5 minutes, rinsed in cold tap water for 2 minutes, stored at room temperature protected from light, and immediately prior to scoring, slides were wet mounted with glass coverslips using purified water.

For the microscopic examination of the preliminary test, the prepared slides were examined by fluorescence microscopy. The incidence of mononucleate, binucleate, and polynucleate cells per 500 cells was assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells, and debris was also noted. From these results, concentrations were selected for treatment in the main test. The highest concentration was intended to be that which caused a depression in the CBPI equivalent to 55% ± 5% cytotoxicity (approximately) when compared with the concurrent vehicle control.

For the main test, the prepared slides were examined by fluorescence microscopy. The incidence of mononucleate, binucleate, and polynucleate cells per 500 cells was assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells, and debris was also noted. From these results, concentrations were selected for micronucleus analysis. The highest concentration was intended to be that which caused a depression in the CBPI equivalent to 55% ± 5% cytotoxicity (approximately) when compared with the concurrent vehicle control. Prior to micronucleus analysis, all slides were randomly coded. Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture was scored where possible. Positive control micronucleus counts are reported for the following treatments only: cyclophosphamide: 5 μg/mL (3 hours, +S9 mix); mitomycin C: 0.2 μg/mL (3 hours, −S9 mix) and 0.1 μg/mL (20 hours, −S9 mix); and colchicine: 0.06 μg/mL (3 hours, −S9 mix) and 0.02 μg/mL (20 hours, −S9 mix). The analyses for micronucleated cells were based on the methods of Fenech and Morley, ¹⁹ Fenech, ²⁰ Fenech et al, ²¹ and Albertini et al. ²²

Statistical analysis

The analysis assumed that the replicate was the experimental unit. An arcsine transformation was used to transform the data. Lavitol was compared to control using Williams tests. ^23,24 Positive controls were compared to control using t tests. Trend tests were carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups. Statistical significance was declared at the 0.5 level for all tests. Data were analyzed using SAS 9.1.3 (Cary, North Carolina) and QuaSAR Quantitative Statistics v. 1.4 (Seattle, Washington).

Test preparation

Lavitol was administered per os in 1% ethanol at 0.5 mL of solution, which allows for complete dissolution of DHQ (taxifolin). Reagents used in the experiment were supplied by Sigma-Aldrich: methanesulfonate, Tris, NaOH, disodium EDTA, low-melting temperature agarose and normal-melting temperature agarose, NaCl, DMSO, and Triton X-100 (pancreas), and fluorescent dye SYBR Green I (Invitrogen Life Technologies).

Test animals and housing

The Animal Care and Use Committee of the Research Laboratory of Pharmacogenetics, State Zakusov Research Institute, Russian Academy of Medical Sciences, Moscow, approved the study. Pathogen-free CBAxC₅₇Bl/6 mice of both sexes were divided into 2 groups of 25 males and 10 females and were obtained from the Research Laboratory. Animals weighed 18 to 20 g and were kept quarantined in the laboratory’s vivarium under observation for clinical signs of ill health following arrival for 7 days. These animals were used for the DNA-Comet assay and the chromosomal aberration test. Once randomly assigned in groups of 5 animals to either study, 20 for the Comet assay and 15 for the chromosomal aberration test, and given unique identification numbers, the animals were then housed in polycarbonate cages (940 cm²) that included steam-sterilized hardwood chips, maintained at 20°C ± 2°C and a RH of 60%, in a 12-hour light/12-hour dark cycle, while allowed free access to tap water and a standard rodent lab chow (MEST) ad libitum.

Experimental design

Positive controls were administered a single intraperitoneal dose of 20 mg/kg of cyclophosphamide (Sigma-Aldrich). A 1% dose of ethanol (Synthesis) was administered 0.1 mL/10 g bw per os for 5 days as a negative control.

During the first experiment, a single dose of 15 mg/kg bw and 2000 mg/kg bw of Lavitol was orally administered to males, corresponding to the daily therapeutic dose for humans, as shown in Table 1. In the second experiment, Lavitol was administered orally at a dose of 15 mg/kg for 5 consecutive days to both males and females, as shown in Table 2.

OPEN IN VIEWER

Table 2.

Administration of the Tested Compounds.

Substance	Sex	Number of animals	Dosage	Way of introduction
Cyclophosphamide	M	5	20 mg/kg bw	Intraperitoneally, single
Lavitol	M	5	15 mg/kg bw	Orally, single
Lavitol	M	5	2000 mg/kg bw	Orally, single
Lavitol	M	5	15 mg/kg bw	Orally, 5 days
Lavitol	F	5	15 mg/kg bw	Orally, 5 days
Negative Control	M	5	0.1 mL/10 g bw	Orally, 5 days
Negative Control	F	5	0.1 mL/10 g bw	Orally, 5 days

Abbreviation: F, female; M, male.

Cytogenic analysis

The cytogenic preparation of the femoral bone marrow was prepared according to the standard dry-air method. ^9,11 For bone marrow metaphase analysis, each dose and week group, including negative controls, consisted of male mice; 2.5 hours before sacrifice, mice were injected intraperitoneally with colchicine (Sigma-Aldrich), at a dose of 4 mg/kg bw. Euthanasia of animals was carried out by cervical dislocation.

Chromosome specimens were prepared from the femoral bone marrow as follows. The femurs of animals were removed, and the femoral marrow cells were flushed out with medium for chromosomal analysis. Bone marrow cell preparations were made by heating up a hypotonic solution of potassium chloride (0.5 mL) and fixative (glacial acetic acid:ethanol, 1:3) to 37°C. All slides were coded and stained with azure-eosin (5 parts of azure [0.1%], 2 parts of eosin [0.1%], and 10 parts of distilled water). The cytogenetic analysis was carried out on a Standart-20 (Carl Zeiss, Gottingen, Germany) microscope with the oil immersion increased 1000×.

Statistical analysis

Results were determined by comparing the “gaps” and ruptures in cells for chromosomal aberrations for both control and experimental groups. Among the experimental group of 5 mice, 100 metaphases were evaluated for each animal. During microscopic analysis, singular and paired chromosomal fragments were evaluated as well as genes and cells with multiple (more than 5 chromosmal aberrations) per cell. Mean standard deviation was calculated for each group of animals. Differences between the control and the experimental groups were analyzed by means of φ-criteria. Statistical analysis was performed using the ANOVA post hoc Dunnet test of the data with Statistica v. 6 (StatSoft, Tulsa, Oklahoma).

Subacute 7-day oral toxicity study

Upon arrival, animals were housed observed for 14 days in type E, 1032 cm² polycarbonate cages (MEST), 5 to a cage, by sex, with each animal tail marked by tattooing, maintained at a temperature of 21°C ± −1°C, RH of 50% to 55%, and 12-hour light–dark cycle. All females were nulliparous and nonpregnant. Through the observation period and during the study, heat-treated sawdust was provided as bedding material, cages cleaned daily, purified water provided ad libitum, and a basal diet given daily in accordance with Russian State Standard R 50258-92 that meets the nutritional requirements for the species. Food consumption was recorded daily for each group of animals and quantity of food adjusted for weight of animals.

After the observation period, animals were randomly assigned to treatment groups. All administrations were performed by gavage. Fifty animals (25 female [F] and 25 male [M]) were divided into 4 groups. Group 1 of 20 animals (10 F and 10 M) received 10 000 mg/kg bw of Lavitol daily for 7 days. Group 2 of 10 animals (5 F and 5 M) served as controls, group 3 of 20 animals (10 F and 10 M) were administered 15 000 mg/kg of Lavitol for 7 days, and group 4 of 10 animals (5 F and 5 M) served as controls. Individual doses were calculated based on the body weight of the animal, modified after each weighing. On the day of randomization, the mean weight of males weighed 184.4 g (170.3-200.0 g) and females weighed 165.5 g (150.0-185.5 g). The phytochemical composition of Lavitol was DHQ 90.94%, aromadendrin 2.75%, eriodictyol 1.03%, quercetin 0.63%, naringenin 0.20%, and pinocembrin 0.03%.

After administration of the test vehicle for 7 days, twice-daily posttreatment observation period was performed to determine mortality, general state, external appearance, behavior, clinical symptoms, sensory reactivity to auditory, visual and proprioceptive stimuli, and muscle strength by the wire netting method, also known as the invested test. ¹⁰ Muscle strength was measured by inside a 40 × 40 cm² screen. The wire net was tightened and divided into 1 × 1 cm² segments. The screen is raised 50 cm over a soft surface, and the experimental animals place in the center of the screen, which is slowly inverted 180°. The animal should hold onto the screen for 15 seconds before it falls. Results were recorded. Body weight was recorded daily for each animal during the 14-day period and on the day of necropsy. Hematological laboratory tests were carried out on days 0 and 8. Blood samples were obtained from the tail vein following 14 to 15 hours of fasting, and testing performed at the Veterinary Center of the Far East State University (Blagoveschensk, Russia), which included hemoglobin (Hb), erythrocyte (RBC), hematocrit (HCT), white blood cell (WBC), lymphocytes, eosinophils, monocytes, and basophiles. Biochemical testing included total protein (TP), blood urea, cholesterol, and glucose, all by the photoelectrocolorimetric method and albumin by the photometric method. Urine samples were collected on day 0 and at the end of the administration period on day 8 and included the following parameters: color, transparency, pH, density, proteins, and RBCs.

Following necropsy on day 8 by decapitation, organ weights were determined (as absolute and relative values), and gross pathology was performed including appearance of tissues. Any abnormality was recorded. Tissues were preserved in a 10% solution of buffered formalin for histopathological examination. If no toxic lesions occurred, tissue samples would not be preserved for histopathological examination.

Statistical analysis

Statistical analysis of the differences between the control and the experimental groups was performed using the Student t test. In the case of abnormal distributions, groups would be compared using the Mann-Whitney U test. Depending on the results, pooled or separate variance estimates would be carried out using the 2-sample t test. Statistical significance was set at P < 0.05. Statistical analysis was performed using STATISTISA v. 6 software (StatSoft, Tulsa, Oklahoma).

Animals

Ninety-six 2-month-old outbred albino Rattus norvegicus rats (48 M and 48 F) were obtained from the breeding colony of the Far East State University, which maintains historical control data on the strain of animals used in the study. Body weight at the start of the study for males was 208.7 to 239.7 g (mean 224.7 g) and 207.7 to 239.9 g (mean 222.4 g) for females. Females were nulliparous and nonpregnant.

Animals were quarantined for 12 days after arrival, then assigned to either 1 of 3 treatment or control groups. Randomization was by body weight of animals; the individual index of body mass did not deviate from the mean by sex by more than 20%. Each treatment group consisted of 24 animals (M and F). Once assigned, animals were housed in groups of 6 animals per cage, in 1032 cm² T3S polycarbonate cages (MEST) that contained heat-treated sawdust bedding and cleaned daily. Identification of animals was by the tail tattooing method. Temperature was maintained between 20°C and 22°C at a RH of 50% and 55%, measured daily using a Thermohydrometer TKA-PKM (Ntap TKA, St Petersburg, Russia), and a 12-hour dark/12-hour light cycle maintained. Animal identification was verified whenever removed from or placed back in its cage, as well as, verified prior to dosing and observations.

The animal’s diet contained not less than 13% proteins, or more than 8% fats, or less than 20% carbohydrates, and not more than 10% cellulose: 100 g of feed provided 240 kcal. Distilled water was given ad libitum.

Dosing, route of administration, and observations

Animals were divided into 4 groups and administered either the test material or a control solution daily by intubation as a single dose, at the same time each day to avoid diurnal influences. The individual doses were calculated based on the body weight of the animal and modified after each weigh in. Group assignment of 24 animals of both sexes (12 F and 12 M) was made as follows: 3 experimental groups received 50, 150, or 1500 mg/kg bw of Lavitol each day, while a fourth group served as the control, which received a 1% potato starch solution (Extra Calls, Promixa, Novosibirsk, Russia), and the same basal diet, at a volume equal to the volume received by the experimental groups. The test product was prepared daily before introduction. The required amount of Lavitol was dissolved in the 1% starch to create the solution. One gram of the soluble starch was mixed with 20 mL of distilled water, brought to a boil, then brought to 100 mL by adding cold distilled water to the solution. Lavitol was then mixed with 3 mL of 1% starch solution. This method, prepared daily, allows the test substance to completely dissolve without precipitation prior to administration. The phytochemical composition of Lavitol used in this study was DHQ 92.20%, aromadendrin 2.35%, eriodictyol 0.53%, quercetin 0.26%, naringenin 0.17%, and pinocembrin 0.11%.

All animals were observed daily for clinical signs, with animals observed twice daily for mortality during the study period. Time of onset, intensity, and duration of symptoms, if any, were recorded. Weight of each animal was recorded at the start of the study and 3 times a week on the same days of the week and throughout the course of the study and at termination when relative organ weights were calculated. The group mean of the body weight and percentage of body weight gain were calculated as g/100 g of body weight. The quantity of food consumed by groups consisting of 6 animals of each sex was recorded. The average daily feed and water consumption per head were calculated by registrating the ratio of total volume of consumed feed and water in each cage to a total weight of animals, found in the cage, multiplied by the individual weight of each animal. The registration of average daily feed and water consumption was carried out at least 3 times a week throughout the study period.

Functional observations were conducted 3 times a day for clinical signs of pharmacological and toxicological effects of the test substance. The parameters observed included outward appearance, pose, behavior, and the presence of convulsions, trepidation, abnormal motion, and/or diarrhea. Observations when handling the animals included muscle tone, skin and mucous color, breathing abnormalities, fur state, and lacrimation. Other observations included general mobility, activity, convulsions, abnormal motions, and/or abnormal pose. Observations were recorded daily in individual cards. Ophthalmological examination included state of mucous membranes, presence of normal corneal reflexes, pupil size, and width of palpebral fissure, observed and measured on days 0, 30, 60, and 90 of the study. As part of physiological examinations, and as required by the Ministry of Health’s guidelines, electrocardiogram measures were taken of voltage peaks, intervals, and heart rate monitoring and recorded. During open-field tests conducted on days 0, 30, 60, and 90, the duration of the latent period, horizontal activity, vertical activity, grooming, and number of defecation acts were documented. The detoxification function of the liver function was evaluated by the duration of hexanal-induced sleep, as measured on days 0, 30, 60, and 90 of the study. The rate of latency and duration of hexanal-induced sleep depend on the functional state of the liver. Disturbances in liver detoxification bioactivity results in the induction of the hexanal-induced sleep duration. Thus, to determine whether Lavitol has any negative impact on the liver, 2% aqueous solution of hexanal was administered intraperitonealy based on 80 mg/kg bw to a pair of animals selected from both the tested and the control groups.

Laboratory testing

Laboratory tests were carried out by the Veterinary Center, Far East State University, on days 0, 30, 60, and 90. Blood samples were collected from the tail vein following 14 to 15 hours of fasting. Hematological parameters measured included Hb, RBC, platelet (PLT), WBC, HCT, eosinophils, and monocytes. Biochemical parameters measured included TP, alkaline phosphatase, aspartate aminotransferase (AST), alanine transaminase (ALT), total bilirubin, urea, creatinine, glucose, cholesterol, and triglycerides. Urine samples and urine analyses were collected on all animals on day 0 and on days 30, 60, and 90 of the study and included color, transparency, specific gravity, nitrites, pH, proteins, ketone bodies, glucose, urobilinogen, bilirubin, mucous, and salts.

Necrospy and histopathological examinations

All of the animals were sacrificed on day 91 by decapitation. Complete gross necropsy and histopathological examination were conducted on all animals. Complete gross necropsy included examination of external surfaces, orifices, cranial, thoracic and abdominal cavities, carcass, and all organs.

Macroscopic and microscopic evaluations were conducted on the lymphatic glands, thyroid glands, salivary glands, heart, trachea, lungs and bronchi, esophagus, stomach, brain, small and large intestine, liver, spleen kidneys, adrenal glands, urethra, pancreas, and organs of the reproductive system. The weights of the heart, lungs, liver, kidneys, spleens, testicles, adrenal glands, thymus, and brain were recorded for all surviving animals. Organ weights were recorded as absolute values and relative values as percentage of body weight calculated. Tissue samples of organs from animals were preserved in a 10% solution of buffered neutral formalin. The following organs were subjected to histopathological examination: brain, hypophysis, salivary glands, thyroid glands, thymus, lungs and bronchi, heart, adrenal glands, kidneys, liver, pancreas, stomach, spleen, and lymphatic glands.

Statistical analysis

Statistical analysis of the differences between the control and experimental groups was performed using the Student t test. In the case of abnormal distributions, groups would be compared using the Mann-Whitney U test. Depending on the results, pooled or separate variance estimates would be carried out using the 2-sample t test. Statistical significance was set at P < 0.05. Statistical analysis was performed using STATISTISA v. 6 software (StatSoft, Tulsa, Oklahoma).

Animals

Eighty 3-month-old female rats were acclimated in the laboratory upon arrival and observed for 14 days prior to dosing, meeting the maximum age limits on the first day of dosing. Females weighed 199 to 270 g (mean, 231.5 g). After the quarantine period, 1 adult male rat was placed into a cage to cohabitate with 2 females during 2 estrus cycles (2 females:1 male). Mating was carried out over a 13-day period. All female rats were examined for signs of fertilization by taking vaginal smears before quarantine. The fertilization was determined by analyzing vaginal smears. Successful mating was confirmed by the presence of sperm in the vaginal smear. The day of finding spermatozoids in vaginal smear was considered the first day of gestation.

Behavioral and state assessments were performed daily by a veterinary upon arrival and during the acclimation period, including twice daily visual inspection of the animals, to ensure that only healthy animals were enrolled in the study. Identification of animals was achieved by recording each animal’s cage and animal numbers and fur markings. Female animals were housed 2 to a cage, in 720 cm² T3S polycarbonate cages (MEST), provided heat-treated sawdust bedding, and cleaned daily. Temperature was maintained between 20°C and 22°C at a RH of 50% to 55%, using a Thermohydrometer TKA-PKM20 (NTP TKA) and 12-hour dark/12-hour light cycle. Noise levels were kept below 85 dB, measured by a Voltcraft sound level meter (Kirschau, Germany). Animals were provided certified rat chow (Laboratorsnab, Moscow, Russia), meeting Russian State Standard R 50258-92, and provided purified water ad libitum. Animal identification was verified each time an animal was removed from or placed back in its cage, as well as, verified prior to dosing and observations.

Dosing

Animals were divided into 4 groups and administered either the test material Lavitol or the control daily by gavage. Administrations occurred at the same time each day to avoid diurnal influences. Assignment to groups was made by randomization. The mean weights and weight distributions between groups was determined and varied insignificantly (from 227.1 to 234.7 g).

Group assignments by randomization were as follows: group 1 (n = 20), the control group, received 3 mL of a 1% starch solution; group 2 (n = 20) was administered 75 mg/kg bw of Lavitol during the period of organogenesis—from the 6th to 16th day of gestation; group 3 (n = 20) was given 1500 mg/kg bw of Lavitol; and group 4 (n = 20) received 75 mg/kg bw of Lavitol during the period of implantation, organogenesis, and fetogenesis—from the 1st to the 19th days of gestation. The diet of both experimental and control groups was identical in volume. The individual dose concentration was modified according to the animal’s body weight after each weighing. Lavitol was dissolved and administered in a single dose in a 1% solution of potato starch (Extra calls, Proxima). Although all 4 groups received the 1% potato starch solution, only groups 2 through 4 received the potato starch in a suspension that included the required amount of Lavitol. One gram of soluble starch was mixed with 20 mL of distilled water, brought to a boil, and then brought to 100 mL by adding cold distilled water to the solution. Lavitol was then mixed with 3 mL of the 1% starch solution.

Experimental Phase

Animals were observed 3 times a day, and all observations recorded daily, for clinical signs of pharmacological and/or toxicological effects, including general appearance, behavioral changes, and locomotor activity, starting with the first day of dosing (day 1). Maternal body weight was recorded on day 1, day 8, and day 14, as well as during days 17 to 21 of gestation. All animals were divided into 4 groups of 20 pregnant animals per group. In each group, 10 of the 20 animals survived for birth, 5 were randomly sacrificed on day 18 of gestation, and the remaining 5 randomly sacrificed on day 20 of gestation. The group mean of the body weight and the percentage of body weight gain, as well as feed and water consumption, were calculated and recorded on days 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, and 42, of the study. All the offspring were sacrificed after 42 days of observation by decapitation. Male rats were weaned on day 25 (on the 4th week after birth). Male rats of each litter were housed in 720 cm² cages type T/3C (drinking bowls and dish—polycarbonate; grid and divisor—stainless steel). One cage for 1 litter of each female rat (total 40 cages). No further analyses on dams were conducted within this experiment. All dams were removed from cages with female dams on day 25. Complete physical growth and development of offspring came to an end on days 25 to 30 after birth. The study of emotional–motor behavior of offspring was conducted up to day 45.

Laboratory investigations were carried out at the beginning (before placing males with females together in cages and on the day 20 of gestation). For mating, females were placed in cages with males for 2 astral cycles (2 females:1 male). Three to four days before the expected day of birth, the pregnant females were placed into single cages. Blood draws were performed and analyzed before mating, on the 18th day of gestation or 20th day of gestation, for those animals sacrificed. The first day of pregnancy was the day on which sperm was found in the vaginal smear.

Laboratory testing and physical examinations

All laboratory tests were performed in the Veterinary Center of the Far East State University. Hematological parameters included Hb, RBCs, PLTs, WBCs, eosinophils, basophiles, lymphocytes, and monocytes. Clinical chemistry and urinalysis testing was obtained at before females began mating and on the 20th day of gestation left for delivery and before necropsy on either the 18th or 20th day of gestation, and before necropsy of pups, conducted at the Central Research Laboratory of Amur State Medical Academy. Fetuses were given a gross external examination consisting of an evaluation of the shape of the body, head size, limb extension, and internal and external sexing of pups. Inspection of each fetus included digits, skin, umbilical region, anus and genitalia, the nares, pinna, eyes, and oral cavity. Two-thirds of the fetuses from each litter were subject to skeletal examination, performed by the Department of Embryology, Research Institute of Experimental Medicine (St Petersburg, Russia) using a method reported by Dawson. ¹⁷ Skeletons were examined under a stereoscopic microscope and the number of ossification points and skeletal abnormalities were evaluated. One-third of the fetuses from each litter were fixed in Bouin fluid and subject to visceral examination. Visceral examinations were performed using the Wilson method ¹¹ by the same Department of Embryology. Examinations were performed on fetuses in all treatment and control groups and included the facial area of the skull, brain, heart, lungs, bronchi, esophagus, spinal cord, liver, kidneys, intestines, bladder, urethra, and reproductive organs.

Litters examined after delivery (lactation day 0) were evaluated for the number and sex of pups, stillbirths, live birth, and the presence of gross anomalies. Thereafter, the day any appearance of primary fur, ear unfolding, incisor eruption, eye opening, testes descent, and vaginal patency was recorded. Live pups were weighed every 3 days, from day 3 to day 42 of the study. In addition, the crania-caudal size of each animal was measured up to the 42nd day to determine whether somatic neural growth was affected. The following sensory-motor reflex changes during the maturation stages were observed or measured in the following chronological sequence: surface-righting reflex (from day 2), negative geotaxis test (from day 5), cliff avoidance (from day 2), open field (from day 8), olfactory (from day 10), pupil (from day 14), auditory (from day 8), muscle tone (from day 15), and open-field motor activity (from day 40).

Clinical chemistry of offspring was carried out on 120 animals (15 of each sex from each group). Blood samples were collected from the tail vein following 14 to 15 hours of fasting and tested (Veterinary Center, Far East State University) and included the following hematological parameters: Hb, RBC, PLT, WBC, HCT, TP, AST, ALT, total bilirubin, urea, and glucose.

Necropsy and histopathology

All offspring, fasted overnight before necropsy, were sacrificed on day 43 by decapitation by trained technicians, supervised by a veterinary pathologist. Complete gross pathology was carried out including examination of external surfaces, orifices, cranial, thoracic and abdominal cavities, and organs, including the carcass of dead or sacrificed animals. Macro- and microscopic evaluation was conducted on the heart, liver, kidneys, spleens, adrenal gland, and testicles. The pathologist was blinded to treatment. Organ weights were recorded as absolute and relative values (% bw) and calculated.

Statistical analysis

Statistical analysis of the differences between the control and experimental groups was performed using the Wilcoxon-Mann-Whitney and Student t test, with significance set at P < 0.05, using STATISTICA v.6 software (StatSoft, Tulsa, Oklahoma).

Subacute 7-day oral toxicity study

No mortality occurred following single administration of 10 000 or 15 000 mg/kg bw of Lavitol. There were no treatment-related clinical or behavioral effects observed. Quantity of food intake and feed consumption patterns was similar between the control and dose groups. Weight gain during the dosing period of 7 days was comparable for the control and dose groups, as shown in Table 5.

OPEN IN VIEWER

Table 5.

Dynamics of Body Weight, Grams (M ± m).^a

Dose	Background	Third day	Seventh day
Males
Controla	187.9 ± 4.34	200.9 ± 4.06	217.7 ± 3.50
10 g (group I)	182.9 ± 2.93	194.9 ± 3.43	210.0 ± 3.44
15 g (group II)	184.3 ± 2.53	197.9 ± 2.57	216.85 ± 2.58
Females
Controla	167.5 ± 5.09	175.3 ± 5.0	190.8 ± 3.98
10 g (group I)	165.5 ± 4.30	173.4 ± 4.28	184.1 ± 4.22
15 g (group II)	164.4 ± 3.04	172.35 ± 3.07	183.2 ± 3.05

^aMean values of quantitative indices were calculated as M ± m, where M, sample mean and m, error of the mean.

Administration of the test substance had no effect on animals’ behavior, skin, and fur condition, or mucous membranes, as the fur was smooth and clear, skin was elastic and mobile, and membranes pale and clear, with no pathological excretions observed. Hematological parameters shown in Table 6, found no significant differences other than decreased RBC values for females at both tested doses on day 8, and significant differences in RBC volumes between the control group of female rats compared to the experimental group of females, as shown in Table 7, which were within normal biological and laboratory historical limits. The RBC volumes in females in the control group shown in Table 8 were higher than historical laboratory limits, which would account for the significant differences observed. As shown in Table 9, significant changes in the values of albumin in males in the 10 000-mg/kg group were seen, yet were within normal biological and laboratory limits. Urine analysis of control and treated animals revealed no abnormalities. No significant difference in organ weights was calculated in either control or treated animals.

OPEN IN VIEWER

Table 6.

Hematological Blood Analysis, Day 8.

Parameters	Laboratory range, 1012/L	Max and min levels in group 1 (10 g/kg), females	Max and min levels in group 2 (15 g/kg), females
RBC	5-11	From 5.2 to 10.6	From 5.6 to 9.4
Dose, g/kg	RBC, 1012/L	WBC, 109/L	HGB, g/L	HCT, %	Basophiles, %	Eosinophils, %	Monocytes, %	Lymphocytes, %
Males
Control	7.2 ± 0.67	10.8 ± 0.94	144.6 ± 10.67	46.1 ± 1.62	0.3 ± 0.12	0.4 ± 0.12	1.0 ± 0.34	70.5 ± 2.0
10	7.5 ± 0.74	10.5 ± 0.65	141.7 ± 5.32	45.2 ± 1.53	0.2 ± 0.09	0.4 ± 0.13	2.6 ± 0.44	71.5 ± 1.40
15	7.4 ± 0.74	10.0 ± 0.60	147.6 ± 5.13	45.4 ± 1.19	0.3 ± 0.08	0.45 ± 0.09	2.1 ± 0.42	70.0 ± 1.16
Females
Control	9.6 ± 0.53	8.5 ± 0.21	141.3 ± 8.53	47.7 ± 1.50	0.2 ± 0.10	0.2 ± 0.05	1.5 ± 0.72	72.4 ± 1.81
10	6.8 ± 0.54a	9.7 ± 0.5	144.1 ± 6.3	46.7 ± 0.87	0.3 ± 0.06	0.4 ± 0.08	1.2 ± 0.44	69.8 ± 1.15
15	6.7 ± 0.39a	8.8 ± 0.28	146.9 ± 4.91	44.2 ± 0.76	0.3 ± 0.07	0.4 ± 0.12	1.9 ± 0.48	69.2 ± 1.11

Abbreviations: HCT, hematocrit; HGB, hemoglobin; max, maximum; min, minimum; RBC, erythrocyte; WBC, white blood cell.

^a P < 0.05.

OPEN IN VIEWER

Table 7.

Erythrocyte (RBC) Changes in Subacute Toxicity Study.

	Males	Females
Control	7.2 ± 0.67	9.6 ± 0.53
10	7.5 ± 0.74	6.8 ± 0.54a
15	7.4 ± 0.74	6.7 ± 0.39a

^a P < 0.05.

OPEN IN VIEWER

Table 8.

Erythrocyte (RBC) Changes in Subacute Toxicity (Compare to Control).

	Males	Females
Control	7.2 ± 0.67	9.6 ± 0.53
10	7.5 ± 0.74	6.8 ± 0.54a
15	7.4 ± 0.74	6.7 ± 0.39a

^a P < 0.05.

OPEN IN VIEWER

Table 9.

Biochemical Blood Analysis, Day 8.

Parameters	Laboratory range, g/L	Max and min levels in group 1 (10 g/kg), males
Albumin	20.0-47.9	From 27.9 to 45.6
Dose, g/kg	Total protein, g/L	Total cholesterol, mmol/L	Albumin, g/L	Glucose, mmol/L	Urea, mmol/L
Males
Control	51.2 ± 3.16	1.9 ± 0.09	28.4 ± 2.14	4.1 ± 0.1	12.3 ± 0.87
10	47.3 ± 3.22	1.5 ± 0.16	36.5 ± 1.74a	4.9 ± 0.21	10.4 ± 0.60
15	48.9 ± 3.17	1.85 ± 0.17	30.3 ± 1.72	4.6 ± 0.35	10.15 ± 0.66
Females
Control	43.5 ± 1.94	2.0 ± 0.26	30.64 ± 2.13	4.3 ± 0.16	10.6 ± 0.75
10	48.5 ± 3.25	1.9 ± 0.14	30.7 ± 2.58	5.3 ± 0.24	10.0 ± 0.56
15	51.6 ± 2.52	1.8 ± 0.22	33.9 ± 2.88	5.2 ± 0.34	11.7 ± 0.71

Abbreviations: max, maximum; min, minimum.

^a P < 0.05.

Gross pathology revealed no abnormalities. Histopathological examination of the internal organs and tissues of the animals from control and experimental groups revealed single-type morphological changes observed in both males and females from controls groups and different dose groups with similar quality and quantity, considered incidental and physiologically related and not induced by the test substance. No morphological changes induced by the test substance were observed.

Subchronic 90-day toxicology study

Animals from the control and dose groups exhibited comparable weight gain throughout the 90-day dosing period, as shown in Table 10, and changes in RBC is shown in Table 11. The quantity of food and water consumed by animals in each experimental group during the dosing period was not significantly different than that of the control group.

OPEN IN VIEWER

Table 10.

Body Weight of Rats During Administration of Test Substance (in Grams).

Day of the study	Lavitol dose groups, M ± m,a mg/kg
	0	50	150	1500
Males
Day 0	224.4 ± 2.22	222.7 ± 2.17	227.2 ± 2.28	224.6 ± 2.12
Day 30	261.6 ± 2.13	262.7 ± 2.41	269.5 ± 2.53	265.3 ± 2.07
Day 60	284.5 ± 2.39	286.9 ± 2.56	292.4 ± 2.26	289.0 ± 1.98
Day 90	295.9 ± 2.16	294.3 ± 2.26	297.0 ± 2.04	293.9 ± 1.82
Females
Day 0	223.2 ± 1.63	220.7 ± 2.29	220.2 ± 1.21	225.6 ± 2.39
Day 30	264.8 ± 1.87	260.8 ± 2.44	260.5 ± 1.45	266.9 ± 2.47
Day 60	288.0 ± 2.16	285.9 ± 2.62	284.5 ± 1.85	289.9 ± 2.2
Day 90	294.8 ± 2.01	292.4 ± 2.38	291.8 ± 1.62	296.1 ± 2.01

^aMean values of quantitative indices were calculated as M ± m, where M, sample mean and m, error of the mean.

OPEN IN VIEWER

Table 11.

Erythrocyte (RBC) Changes in 90-Day Subchronic Toxicity.

Day	Control	50 mg/kg	150 mg/kg	1500 mg/kg
Males
0	7.47 ± 0.16	7.49 ± 0.17	7.58 ± 0.16	7.52 ± 0.15
30	7.62 ± 0.17	7.20 ± 0.16	7.56 ± 0.17	7.48 ± 0.18
60	7.54 ± 0.18	7.53 ± 0.16	7.23 ± 0.12	7.50 ± 0.17
90	7.61 ± 0.21	7.78 ± 0.17	7.96 ± 0.17	8.11 ± 0.17
Females
0	7.53 ± 0.15	7.24 ± 0.16	7.50 ± 0.13	7.16 ± 0.1
30	7.05 ± 0.13	7.40 ± 0.19	7.18 ± 0.17	7.43 ± 0.21
60	7.31 ± 0.2	7.50 ± 0.9	7.07 ± 0.17	7.26 ± 0.16
90	7.59 ± 0.14	7.09 ± 0.2	7.38 ± 0.18	7.30 ± 0.17

No abnormal changes in the skin and fur appearance were observed in any of the experimental groups or controls, other than animals administered 50 and 150 mg/kg that had significantly thicker and fluffier fur compared to the 1500-mg/kg group or controls. No abnormalities in movement or posture, or presence of convulsions or tremors, were observed in any of the groups. During the first month of the study, males in the 150-mg/kg group were more active than males in either of the other 2 experimental groups or controls, while in the third month of the study, males in the 50- and 150-mg/kg group were significantly more active than males in the 1500-mg/kg or control groups. Stool disturbances were observed in all groups during the study, but only significantly lower in both males and females in the 150-mg/kg groups compared to controls. There were no significant differences measured in rectal temperature between the experimental groups and controls. Mucous membranes were normal in all groups. No edema, hyperemia, or pathological excretions were observed in any of the experimental groups. No deviations were seen in the corneal reflex of any animals tested, even at the maximal dose of 1500 mg/kg on pupil size or width of the palpebral fissure.

Physiological examinations, hematology, and biochemistry

No significant changes in heart function or electrophysiology were observed in the 40 animals evaluated at the beginning and at the end of the study on day 0, day 60, and day 90. The auricular (peak value P, interval size P-Q, and ventricular ECG indices [peak T value]) were within normal levels in all tested animals.

Open-field examination data, revealed effects on behavior in some males and females, as follows: males – defecation in the 50 mg dose group at day-30 (P < 0.05), vertical motor activity in the 1500 mg dose group at day-90 (P < 0.05), and looking into holes, in the control group at day-90 (P < 0.05); females – latent period, vertical motor activity, and defecation in 1500 dose group at day-30 (P < 0.05). A statistically significant increase in the latency (time required to leave the center) was observed in females in the 1500-mg/kg group after 30 days compared to day 0. During the same time period, a statistically significant increase in the number of stands (vertical motor activity) and decrease in number of defecations were observed in the same group. A statistically significant decrease in number of stands was observed in males administered 1500 mg/kg for 90 days. Otherwise, no other significant differences between control and experimental groups were seen in other open-field indices.

No statistically significant differences were seen in the duration of hexanal-induced sleep among 40 animals evaluated given Lavitol at any of the dose groups on day 30, 60, and 90, compared to day 0. No statistically differences were found in hematological indices between any of the 96 experimental and control animals for either sex during the study period, the exception of two female control groups or in RBC changes (Table 11), as all indices were within normal values.

Urine analysis of control and treated animals revealed no abnormalities. No significant differences in organ weight between animals of either sex in the control and experimental groups were observed on day 91. Gross pathological examination did not reveal any abnormality within and between the control and experimental groups.

Histopathological examination of internal organs and tissues of the animals in each of the experimental groups did not reveal any significant morphological differences between that of controls, as shown in Table 12. Moderate plethora of the frontal lobe of the hypophysis, insignificant edema of the interstitial tissue of the thyroid gland, a small area of fresh hemorrhages in the lungs, grained dystrophy of single hepatocytes, and a plethora of blood vessels in the cortical area of kidneys observed in the few male and female animals were of similar quality and quantity and hence not considered clinically relevant. Testicles were whitish, of normal size and density. The body of the uterus was of normal density, size, and form. The uterus horns were thin and showed clear to pale mucous membranes. The ovaries were of dark red color, moderate density, usual form, and size.

OPEN IN VIEWER

Table 12.

Morphological Differences Between Groups.

	Control		Lavitol dose groups
	0		50		150		1500
	M	F	M	F	M	F	M	F
Moderate plethora of the frontal lobe of the hypophysis	1/12	1/12	0/12	1/12	0/12	0/12	1/12	0/12
Insignificant edema of the interstitial tissue of the thyroid gland	0/12	1/12	0/12	0/12	0/12	0/12	1/12	0/12
A small area of fresh hemorrhages in the lungs	1/12	0/12	1/12	0/12	0/12	0/12	0/12	0/12
Grained dystrophy of single hepatocytes	0/12	1/12	0/12	1/12	0/12	0/12	0/12	0/12
A plethora of blood vessels in the cortical area of kidneys	1/12	1/12	0/12	0/12	0/12	1/12	0/12	0/12

Clinical chemistry

Blood sampling occurred prior to gestation and during the period of gestation on days 18 or 20. No statistical differences were seen between control or experimental groups in clinical chemistries, as indices were within normal values, as shown in Table 14. Differences in hematological parameters of females between treatment and control groups were within normal laboratory limits, with nonstatistical differences of values for treatment and control groups, and nonstatistical differences of values for RBC changes, as shown in Table 15. Clinical chemistry of dams is shown in Table 16. As shown in Table 17, a decrease in RBC volume was observed in all 4 groups at the end of the gestation period, where the volume of RBCs in rats of the control group was 1.1 times lower than historical norms, and groups 2, 3, and 4 were 1.3, 1.4, and 1.2 times lower as well, respectively. The observed decrease in volume of RBC could be considered a common phenomenon during gestation.

OPEN IN VIEWER

Table 14.

Clinical Chemistry of Dams.

Indices	0 (6th-16th gestation days)		75 (6th-16th gestation days)		1500 (6th-16th gestation days)		75 (1st-19th gestation days)
	Before	After	Before	After	Before	After	Before	After
Hemoglobin	143.7 ± 2.37	135.95 ± 1.86a	142.45 ± 2.24	134.25 ± 1.99	139.85 ± 2.22	136.55 ± 1.99	137.1 ± 2.52	137.4 ± 1.84
Erythrocytes	7.7 ± 0.28	5.85 ± 0.12a	7.5 ± 0.31	5.96 ± 0.12a	8.03 ± 0.39	5.8 ± 0.12a	7.24 ± 0.34	5.97 ± 0.13a
Color indices	1.11 ± 0.03	1.08 ± 0.03	1.14 ± 0.04	1.08 ± 0.04	1.14 ± 0.04	1.08 ± 0.03	1.09 ± 0.03	1.03 ± 0.03
Platelets	504.27 ± 29.51	537.7 ± 14.07	542.25 ± 10.67	563.5 ± 12.7	534.45 ± 11.26	529.05 ± 13.55	541 ± 13.23	555.15 ± 12.36
Leucocytes	9.93 ± 0.23	9.87 ± 0.31	9.85 ± 0.26	10.08 ± 0.31	9.8 ± 0.24	9.88 ± 0.28	10.16 ± 0.24	9.93 ± 0.38
Neutrophils, p/nucleus	1.75 ± 0.18	1.6 ± 0.18	1.7 ± 0.18	1.8 ± 0.17	1.65 ± 0.17	2.0 ± 0.18	1.75 ± 0.18	1.7 ± 0.16
Neutrophils, s/nucleus	25.85 ± 0.56	26.3 ± 0.43	25.6 ± 0.5	26.45 ± 0.41	26.8 ± 0.49	26.35 ± 0.36	26.2 ± 0.61	25.45 ± 0.44
Eosinophils	0.54 ± 0.05	0.45 ± 0.05	0.58 ± 0.05	0.46 ± 0.06	0.53 ± 0.06	0.51 ± 0.06	0.57 ± 0.05	0.53 ± 0.05
Basophiles	0.55 ± 0.06	0.55 ± 0.05	0.57 ± 0.06	0.51 ± 0.06	0.48 ± 0.06	0.43 ± 0.05	0.53 ± 0.06	0.52 ± 0.05
Lymphocytes	70.25 ± 0.67	67.5 ± 0.3	70.75 ± 0.73	67.55 ± 0.29a	69.05 ± 0.54	67.65 ± 0.27	71.1 ± 0.75	67.05 ± 0.33a
Monocytes	1.8 ± 0.3	1.95 ± 0.18	2.2 ± 0.25	1.7 ± 0.15	2.1 ± 0.26	1.75 ± 0.19	1.60 ± 0.23	1.65 ± 0.2

^a P < 0.05 compared with results within a group.

OPEN IN VIEWER

Table 15.

Hematological Values for Assigned Groups.

Parameters	Laboratory range, g/L	Max and min levels in group 1 (control)
Hb	120-170	From 143.7 ± 2.37 to 135.95 ± 1.86
Parameters	Laboratory range, 1012/L	Max and min levels in group 1 (control)	Max and min levels in group 2	Max and min levels in group 3	Max and min levels in group 4
RBC	5-11	From 7.7 ± 0.28 to 5.85 ± 0.12	From 7.5 ± 0.31 to 5.96 ± 0.12	From 8.03 ± 0.39 to 5.8 ± 0.12	From 7.24 ± 0.34 to 5.97 ± 0.13
Parameters	Laboratory range, %	Max and min levels in group 2 (75 mg/kg [6th-16thgestation days])	Max and min levels in group 4 (75 mg/kg [1st-19th gestation days])
Lymphocytes	65-80	From 70.75% to 67.55%	From 71.1% to 67.05%

Abbreviations: max, maximum; min, minimum.

OPEN IN VIEWER

Table 16.

Clinical Chemistry of Dams in Developmental Study.

Indices	0 (6th-16th gestation days)		75 (6th-16th gestation days)		1500 (6th-16th gestation days)		75 (1st-19th gestation days)
	Before	After	Before	After	Before	After	Before	After
Lymphocytes	70.25 ± 0.67	67.5 ± 0.3a	70.75 ± 0.73	67.55 ± 0.29a	69.05 ± 0.54	67.65 ± 0.27	71.1 ± 0.75	67.05 ± 0.33a

^a P < 0.05.

OPEN IN VIEWER

Table 17.

Erythrocyte (RBC) Changes in Developmental Toxicity (Dams, Prior Gestation, and on 18th or 20th Day of Gestation).

Dose	Prior gestation	On 18th or 20th of gestation
Control (Gr 1)	7.7 ± 0.28	5.85 ± 0.12a
75 mg (Gr 2)	7.5 ± 0.31	5.96 ± 0.12a
1500 (Gr 3)	8.03 ± 0.39	5.8 ± 0.12a
75 mg (Gr 4)	7.24 ± 0.34	5.97 ± 0.13a

^a P < 0.05.

Physiological examinations and fetometrics

There were no cases of spontaneous abortion after administration of Lavitol in either the 75- or 1500-mg/kg groups. Based on the assessments of the animal’s corpora lutea, females in group 4 had a greater ability to be impregnated than any of the other groups; the lowest percentage occurred in the control group, as shown in Table 18.

OPEN IN VIEWER

Table 18.

Fetal Survival After Maternal Exposure to Lavitol.

	Lavitol, mg/kg bw
	0 (6th-16th gestation days)	75 (6th-16th gestation days)	1500 (6th-16th gestation days)	75 (1st-19th gestation days)
No corpora luteaa	77/7.7	89/8.9	83/8.3	96/9.6
No of implantation sitesa	64/6.4	79/7.9	67/6.7	89/8.9
Number of live fetuses	61/6.1	76/7.6	63/6.3	88/8.8
Number with resorption	3/0.3	3/0.3	4/0.4	1/0.1
Preimplantation death
Absolute	13/1.3	10/1	15/1.5	7/0.7
% to number of corpora lutea (A)	16.9/1.7	11.2/1.1	18.1/1.8	7.3/0.7
Postimplantation death
Absolute	2/0.2	3/0.3	4/0.4	1/0.1
% to number of implantation (B)	2.6/0.3	3.4/0.3	4.8/0.5	1.0/0.1

^a P < 0.05

One hundred forty-six fetuses from the dams euthanized on the 18th day of gestation were observed. One hundred forty-one fetuses from the dams euthanized on the 20th day of gestation were observed. The results are shown in Tables 19 and 20. Fetal weight of males on the 18th day of gestation varied from 2.15 ± 0.08 g in group 1 to 2.30 ± 0.07 g in group 3, while fetal weight of females varied from 1.98 ± 0.08 g in group 1 to 2.24 ± 0.08 g in group 3. Placental weight varied from 0.63 ± 0.01 g to 0.65 ± 0.01 g (for males) and from 0.60 ± 0.01 g to 0.63 ± 0.01 g (for females). The craniocaudal size varied from 25.28 ± 0.56 to 27.05 ± 0.55 g (for males) and 22.11 ± 0.82 to 25.83 ± 0.61 (for females). The fetus–placental index varied from 0.28% ± 0.01% to 0.30% ± 0.01% (for males) and from 0.28% ± 0.01% to 0.32% ± 0.01% (for females).

OPEN IN VIEWER

Table 19.

Fetometrics of Fetuses Euthanized on 18th day of Gestation.

	Lavitol exposure, mg/kg bw
	0 (6th-16th gestation days)	75 (6th-16th gestation days)	1500 (6th-16th gestation days)	75 (1st-19th gestation days)
No. of fetuses examined
Males	18	18	18	20
Females	18	16	14	24
Male:female ratio	1:1	1.1:1	1.3:1	1:1.2
Fetal weights, g
Males	2.15 ± 0.08	2.16 ± 0.07	2.30 ± 0.07	2.25 ± 0.03
Females	1.98 ± 0.08	2.08 ± 0.05	2.24 ± 0.08	2.17 ± 0.03
Combined	2.07 ± 0.08	2.12 ± 0.06	2.27 ± 0.08	2.21 ± 0.03
Placental weights, g
Males	0.63 ± 0.01	0.63 ± 0.01	0.65 ± 0.01	0.64 ± 0.004
Females	0.60 ± 0.01	0.62 ± 0.01	0.63 ± 0.01	0.62 ± 0.004
Fetus–placental index, %
Males	0.28 ± 0.01	0.30 ± 0.01	0.29 ± 0.01	0.29 ± 0.004
Females	0.32 ± 0.01	0.30 ± 0.01	0.28 ± 0.01	0.29 ± 0.004
Craniocaudal size, mm
Males	25.67 ± 0.86	25.28 ± 0.56	26.67 ± 0.39	27.05 ± 0.55
Females	22.11 ± 0.82	23.69 ± 0.73	24 ± 0.72	25.83 ± 0.61

OPEN IN VIEWER

Table 20.

Fetometric Indices of Fetuses Euthanized on the 20th Day of Gestation.

	Lavitol exposure, mg/kg bw
	0 (6th-16th gestation days)	75 (6th-16th gestation days)	1500 (6th-16th gestation days)	75 (1st-19th gestation days)
No. of fetuses examined
Males	13	23	15	23
Females	11	19	16	21
Male:female ratio	1.2:1	1.2:1	1:1.1	1.1:1
Fetal weights, g
Males	3.82 ± 0.18	3.24 ± 0.12	3.52 ± 0.13	3.47 ± 0.09
Females	3.15 ± 0.16	2.79 ± 0.06	3.35 ± 0.12	3.19 ± 0.1
Combined	3.49 ± 0.17	3.02 ± 0.09	3.44 ± 0.13	3.33 ± 0.05
Placental weights, g
Males	0.70 ± 0.02	0.65 ± 0.01	0.66 ± 0.01	0.70 ± 0.01
Females	0.61 ± 0.02	0.59 ± 0.01	0.64 ± 0.01	0.66 ± 0.01
Fetus–placental index, %
Males	0.19 ± 0.01	0.21 ± 0.005	0.19 ± 0.01	0.20 ± 0.004
Females	0.19 ± 0.01	0.20 ± 0.006	0.19 ± 0.01	0.21 ± 0.005
Craniocaudal size, mm
Males	42.6 ± 2.74	35.17 ± 1.22	36.47 ± 1.01	36.04 ± 0.9
Females	35.36 ± 1.71	30.05 ± 0.83	34.56 ± 0.78	34.76 ± 0.89

Fetal weight of males euthanized on the 20th day of gestation varied from 3.24 ± 0.12 g in group 2 to 3.82 ± 0.18 g in group 1, while fetal weight of females varied from 2.79 ± 0.06 g in group 2 to 3.35 ± 0.12 g in group 3. Placental weight varied from 0.65 ± 0.01 to 0.70 ± 0.01 g (for males) and from 0.59 ± 0.01 to 0.66 ± 0.01 g (for females). The craniocaudal size varied from 35.17 ± 1.22 to 42.6 ± 2.74 mm (for males) and from 30.05 ± 0.83 to 35.36 ± 1.71 mm (for females).

No abnormalities were found during visual observation of 287 fetuses from all groups. Fetal defects of the viscera were seen more so in the control group, as seen in Table 21, than any of the treatment groups.

OPEN IN VIEWER

Table 21.

Fetal Abnormalities of Viscera per Litter in Developmental Study.

	Lavitol exposure, mg/kg bw
	0 (6th-16th gestation days)	75 (6th-16th gestation days)	1500 (6th-16th gestation days)	75 (1st-19th gestation days)
No. of fetuses examined	23	29	24	32
Fetuses with visceral defects	4	2	3	1
Percent of fetuses with visceral defects	17.4%	6.9%	1.5%	3.1%
Hydronephrosis	1 (3.1%)	0	1 (3.1%)	0
Plethoric auricles	2 (6.9%)	1 (3.1%)	0	1 (3.1%)
Plethoric sagittal sinuses	1 (3.1%)	1 (3.1%)	1 (3.1%)	0
Plethoric lung vessels	0	0	1 (3.1%)	0

No treatment-related skeletal anomalies were seen in any of the groups as shown in Table 22. Degree of ossification of each bone (other than the skull) was unremarkable in any group.

OPEN IN VIEWER

Table 22.

Craniocaudal Size of Offspring in Developmental Study.

Day of the study	Craniocaudal size, mm
	Lavitol, mg/kg bw
	0 (6th-16th gestation days)	75 (6th-16th gestation days)	1500 (6th-16th gestation days)	75 (1st-19th gestation days)
Males
Day of birth	53.03 ± 0.87	57.33 ± 0.85	58.21 ± 1.24	54.47 ± 1.04
Day 3	59.72 ± 0.99	66.02 ± 0.95	65.21 ± 1.24	62.06 ± 1.11
Day 6	66.69 ± 1.06	73.43 ± 0.98	72.62 ± 1.23	68.97 ± 1.17
Day 9	74.75 ± 1.06	80.64 ± 1.12	80.26 ± 1.31	76.56 ± 1.26
Day 12	81.69 ± 1.15	87.90 ± 1.22	87.62 ± 1.38	83.28 ± 1.28
Day 15	88.42 ± 1.26	95.86 ± 1.38	95.32 ± 1.53	90.69 ± 1.33
Day 18	95.50 ± 1.41	103.48 ± 1.59	102.62 ± 1.60	99.00 ± 1.49
Day 21	104.03 ± 1.57	112.05 ± 1.73	110.32 ± 1.63	108.72 ± 1.65
Day 24	111.5 ± 1.71	120.10 ± 1.77	118.29 ± 1.69	117.59 ± 1.71
Day 27	119.78 ± 1.73	128.14 ± 1.83	126.21 ± 1.65	124.91 ± 1.70
Day 30	128.19 ± 1.51	136.81 ± 1.85	133.74 ± 1.65	131.78 ± 1.65
Day 33	135.31 ± 1.33	145.21 ± 1.78	140.94 ± 1.66	139.0 ± 1.76
Day 36	141.64 ± 1.50	152.14 ± 1.81	148.71 ± 1.64	146.5 ± 1.90
Day 39	146.83 ± 1.65	159.81 ± 1.75	156.53 ± 1.71	152.28 ± 1.97
Day 42	154.72 ± 1.87	166.21 ± 1.74	164.03 ± 1.79	158.38 ± 2.12
Females
Day of birth	55.16 ± 0.80	54.18 ± 1.09	55.17 ± 1.03	54.64 ± 0.86
Day 3	62.13 ± 0.80	62.03 ± 1.16	63.24 ± 0.98	61.87 ± 0.86
Day 6	70.56 ± 1.09	69.58 ± 1.29	71.0 ± 0.94	69.15 ± 0.89
Day 9	78.41 ± 1.10	76.58 ± 1.30	78.21 ± 0.98	76.03 ± 0.92
Day 12	85.53 ± 1.38	83.48 ± 1.42	85.76 ± 1.11	83.67 ± 0.91
Day 15	92.09 ± 1.48	90.94 ± 1.63	92.83 ± 1.24	90.18 ± 1.03
Day 18	98.53 ± 1.46	98.48 ± 1.75	99.71 ± 1.36	97.10 ± 1.25
Day 21	106.09 ± 1.74	106.21 ± 1.88	106.83 ± 1.43	105.18 ± 1.49
Day 24	114.63 ± 1.71	114.64 ± 1.89	114.26 ± 1.50	113.92 ± 1.47
Day 27	122.97 ± 1.68	123.06 ± 1.86	121.79 ± 1.51	122.97 ± 1.31
Day 30	130.66 ± 1.54	130.88 ± 1.78	129.26 ± 1.50	130.08 ± 1.27
Day 33	136.97 ± 1.48	138.06 ± 1.83	136.90 ± 1.43	137.72 ± 1.31
Day 36	141.94 ± 1.51	145.21 ± 1.87	145.12 ± 1.37	144.79 ± 1.23
Day 39	147.22 ± 1.67	152.97 ± 1.75	153.64 ± 1.32	151.72 ± 1.34
Day 42	153.88 ± 1.77	159.76 ± 1.72	161.19 ± 1.28	157.56 ± 1.39

Postnatal development

Postnatal developmental observations of 291 live pups on day 0 and day 42 were carried out. Body weights of newborns in all test groups were within normal limits, varying from 4.72 ± 0.16 g (the mean body weight of newborns in group 4) to 4.89 ± 0.22 g (the mean body weight of newborns in group 2). A steady increase in body weight was observed for all pups. By the 42nd day, body weight of pups varied from 70.36 ± 3.00 to 74.67 ± 2.92 g. In group 1 and group 2, the number of male offspring exceeded that of female offspring: 36 males and 40 males over 32 females and 33 females, respectively. In groups 3 and 4, the number of female offspring exceeded that of male offspring: 42 females and 39 females compared to 34 males and 32 males, respectively. Two dead pups were found in the control group and in group 2, caused by birth trauma in the control group, and the mothers eating the pups following birth. Earflap detachment occurred on the second day in pups from all groups. Hair growth was observed on the 5th day; incisor eruption on the 7th day in the control group and on the 8th day in the treatment groups; and eye opening observed on the 14th day. Testes descent was observed on day 25, while opening of the vagina on day 30 (but on day 29 in group 2).

There was no statistically significant difference in the maturation date of sensory-motor reflexes among the offspring. Observation of emotional–locomotor behavior of 40-day-old pups was unremarkable. The time required to leave the center varied from an average of 5.73 ± 0.12 seconds (group 2) to 7.01 ± 0.22 seconds (group 4). The number of visited squares varied from 21.33 ± 1.23 (group 3) to 27.48 ± 1.76 (group 1); the number of racks varied from 0.51 ± 0.12 (group 1) to 0.72 ± 0.19 (group 3); the number of different types of grooming varied from 0.51 ± 0.06 (group 2) to 0.62 ± 0.07 (group 1); and the number of defecations varied from 0.32 ± 0.06 (group 2) to 0.44 ± 0.07 (group 3).

Necropsy and histopathology

Autopsy of animals revealed no significant pathological changes. Serous membranes of all animals were smooth and shiny. Mucous membranes were pale pink. Heart, liver, kidneys, spleen, organs of the gastrointestinal tract, adrenals, thymus, testicles, and uterus were of normal consistency, color, and size. Postmortem evaluation of constitution, external surfaces, orifices, and mucous membranes as well as thoratic and abdominal cavities and the organs located in the cavities was conducted. The histological examination of the internal organs and tissues of offspring from control and experimental groups did not reveal any significant morphological differences between groups.