Study of the Embryonic Toxicity of TiO2 and ZrO2 Nanoparticles

Currently, the widespread use of TiO2 and ZrO2 nanoparticles (NPs) in various industries poses a risk in terms of their potential toxicity. A number of experimental studies provide evidence of the toxic effect of TiO2 and ZrO2 NPs on biological objects. In order to supplement the level of knowledge and assess the risks of toxicity and danger of TiO2 and ZrO2 NPs, we decided to conduct a comprehensive experiment to study the embryonic toxicity of TiO2 and ZrO2 NPs in pregnant rats. For the experiment, mongrel white rats during pregnancy received aqueous dispersions of powders of TiO2 and ZrO2 NPs at a dose of 100 mg/kg/day. To characterize the effect of TiO2 and ZrO2 NPs on females and the postnatal ontogenesis of offspring, a complex of physiological and biochemical research methods was used. The results of the experiment showed that TiO2 NPs as ZrO2 NPs (100 mg/kg per os) cause few shifts of similar orientation in the maternal body. Neither TiO2 NPs nor ZrO2 NPs have an embryonic and teratogenic effect on the offspring in utero, but both modify its postnatal development.


Introduction
The rapid development of nanotechnology has put the scientific community in front of the need to control the safety of products obtained with their use, the production volumes of which are progressively increasing. Timely assessment of the toxicity and danger of nanomaterials can become a reliable barrier to the unhindered penetration into the life of harmful to human health and environmentally hazardous products and/or contribute to the development of a system of measures to protect against their adverse effects [1][2][3][4][5]. It should also be taken into account that any information shedding light on the peculiarities of the biological action of nanomaterials is of scientific interest per se [6][7][8].
Nanomaterials' environmental exposure represents a real public health issue that will become highly relevant in the near future when people's consciousness of this issue pregnant mice [49]. The authors supposed that detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side. The same conclusion was made by Wang et al. (2021), who discovered that ZrO 2 NPs are able to cross multiple biological barriers and were accumulated in the maternal placenta and fetal brains [50]. Similarly, Zhou et al. (2019) suggested that at maternal exposure, TiO 2 NPs crossed the blood-fetal barrier and blood-brain barrier and deposited in the brain of offspring, which retarded axonal and dendritic outgrowth, including the absence of axonal outgrowth, and decreased dendritic filament length, dendritic branching number, and dendritic spine density [51]. It is important to note that maternal exposure of mice to TiO 2 NPs may affect the expression of genes related to the development and function of the central nervous system, as was reported by Shimizu et al. (2009) [52]. Lee et al. (2019) studied the effect of TiO 2 NPs on pregnant rats on gestation days (GD) 6-19 at dosage levels of 0, 100, 300, and 1000 mg/kg/day [53]. The authors did not find acute toxic effects on pregnant rats in the experiment, but titanium contents were increased in the maternal liver, maternal brain, and placenta, which can affect the embryo.
Thus, the current level of knowledge about the effect of TiO 2 and ZrO 2 NPs on pregnant women and embryo development has shown that these nanoparticles do not cause a direct negative impact on the gestation but are accumulated in the maternal and fetus organs and can have a significant impact on the further development of the fetus. Nevertheless, the described works were aimed at studying a narrow range of indicators of experimental animals, which do not fully explain the mechanism of the effect of TiO 2 and ZrO 2 NPs. Also, as far as we know, no one has conducted a comparative analysis of the toxic effects of TiO 2 and ZrO 2 NPs during pregnancy. Therefore, in order to supplement the level of knowledge and assess the risks of toxicity and danger of TiO 2 and ZrO 2 NPs, in our opinion, it is necessary to conduct a comprehensive experiment to study the embryonic toxicity of TiO 2 and ZrO 2 NPs. The aim of this work was to study the embryotoxic effect of TiO 2 and ZrO 2 NPs on pregnant laboratory rats. The novelty of this work is associated with a comprehensive parallel assessment of the effect of TiO 2 and ZrO 2 NPs on pregnant rats and fetuses in the same experiment under the same conditions.

Synthesis of TiO 2 NPs
In the first stage, 190 cm 3 of distilled water (H 2 O) and 10 cm 3 of a 25% solution of ammonia NH 4 OH (Lenreactiv, Saints Petersburg, Russia) were measured with a measuring cylinder. The resulting solution was poured into a beaker and mixed on a Topolino magnetic stirrer (IKA, Moscow, Russia) for 2 min.
In the second stage, 10 cm 3 of a concentrated aqueous solution of titanium tetrachloride TiCl 4 (Lenreactiv, Saints Petersburg, Russia) was measured with a measuring cylinder and, with intensive stirring, it was poured into an aqueous solution of ammonia, resulting in a white precipitate of TiO 2 · · · nH 2 O. Then a 25% solution of ammonia NH 4 OH (Lenreactiv, Saints Petersburg, Russia) was added drop by drop to a certain pH value. The process was completed at 6 ≤ pH ≤ 8 since it is a gel-formation point. The resulting gel was left in a dark place at room temperature for a day.
In the third stage, the resulting gel was washed 4 times with distilled water and centrifuged using a TSLN-16 centrifuge (Polycom, Moscow, Russia) at 2000 rpm for 6 min. This procedure was carried out to remove impurity ions (Cl − , NH4 + ).
In the fourth stage, the washed TiO 2 gel was dried at a temperature of 125 • C.

Synthesis of ZrO 2 NPs
In the first stage, 26.7 g of dihydrous zirconium nitrate ZrO(NO 3 ) 2 · · · 2H 2 O (Lenreactiv, Saints Petersburg, Russia) was weighed on analytical scales by the method of precise weighting. Then the suspension of zirconium nitrate was quantitatively transferred to a beaker, and 50 cm 3 of distilled water was added. The resulting mixture was stirred on a Topolino magnetic stirrer (IKA, Moscow, Russia) for 30 min and then carefully filtered to remove the insoluble components of zirconium.
In the second stage, a 12% vol. aqueous solution of ammonia NH 4 OH (Lenreactiv, Saints Petersburg, Russia) was added to a filtered aqueous solution of zirconium nitrate at a concentration of 1 mol/L with intensive stirring by a mechanical stirrer drop by drop until pH = 4, at which the formation of a stable gel was observed.
In the third stage, the gel was centrifuged using a TSLN-16 centrifuge (Polycom, Moscow, Russia) at 2000 rpm for 6 min and washed three times with distilled water to remove the impurity ions remaining after the reaction.
In the last stage, the resulting gels were dried and calcined at various temperatures from 125-850 • C in order to remove the remaining water and to decompose zirconium hydroxide to zirconium dioxide.

Research of TiO 2 and ZrO 2 NPs
Identification of TiO 2 and ZrO 2 NPs was carried out by X-ray phase analysis with PANalytical Empyrean installation (Malvern Panalytical, Malvern, UK). The analysis and preparation of drugs were carried out according to works [25,26]. The identification of diffractograms was carried out according to another work [24]. Measurements of NPs sizes were carried out using a Photocor Complex installation (Antek-97, Moscow, Russia). Computer processing of spectroscopy results was carried out using DynaLS software (Antek-97, Moscow, Russia) [54].

Determination of TiO 2 and ZrO 2 NPs Embryotoxicity
The scheme of the experiment with pregnant rats is presented in Figure 1.

Synthesis of ZrO2 NPs
In the first stage, 26.7 g of dihydrous zirconium nitrate ZrO(NO3)2⋯2H2O (Lenreactiv, Saints Petersburg, Russia) was weighed on analytical scales by the method of precise weighting. Then the suspension of zirconium nitrate was quantitatively transferred to a beaker, and 50 cm 3 of distilled water was added. The resulting mixture was stirred on a Topolino magnetic stirrer (IKA, Moscow, Russia) for 30 min and then carefully filtered to remove the insoluble components of zirconium.
In the second stage, a 12% vol. aqueous solution of ammonia NH4OH (Lenreactiv, Saints Petersburg, Russia) was added to a filtered aqueous solution of zirconium nitrate at a concentration of 1 mol/L with intensive stirring by a mechanical stirrer drop by drop until pH = 4, at which the formation of a stable gel was observed.
In the third stage, the gel was centrifuged using a TSLN-16 centrifuge (Polycom, Moscow, Russia) at 2000 rpm for 6 min and washed three times with distilled water to remove the impurity ions remaining after the reaction.
In the last stage, the resulting gels were dried and calcined at various temperatures from 125-850 °C in order to remove the remaining water and to decompose zirconium hydroxide to zirconium dioxide.

Research of TiO2 and ZrO2 NPs
Identification of TiO2 and ZrO2 NPs was carried out by X-ray phase analysis with PANalytical Empyrean installation (Malvern Panalytical, Malvern, UK). The analysis and preparation of drugs were carried out according to works [25,26]. The identification of diffractograms was carried out according to another work [24]. Measurements of NPs sizes were carried out using a Photocor Complex installation (Antek-97, Moscow, Russia). Computer processing of spectroscopy results was carried out using DynaLS software (Antek-97, Moscow, Russia) [54].

Determination of TiO2 and ZrO2 NPs Embryotoxicity
The scheme of the experiment with pregnant rats is presented in Figure 1. In the experiments, the dispersion of the initial samples of TiO2 and ZrO2 NPs obtained ex tempore by diluting the powder with deionized water was used. Aggregation of NPs was prevented by sonification of the solution using Megeon 76,010 ultrasonic bath (Megeon, Moscow, Russia).
Mongrel white rats with an initial body weight of 200-240 g and a regular estrous cycle were used as a biomodel. Working solutions of TiO2 and ZrO2 NPs at a dose of 100 In the experiments, the dispersion of the initial samples of TiO 2 and ZrO 2 NPs obtained ex tempore by diluting the powder with deionized water was used. Aggregation of NPs was prevented by sonification of the solution using Megeon 76,010 ultrasonic bath (Megeon, Moscow, Russia).
Mongrel white rats with an initial body weight of 200-240 g and a regular estrous cycle were used as a biomodel. Working solutions of TiO 2 and ZrO 2 NPs at a dose of 100 mg/kg/day were administered per os to females using a probe at the rate of 1.0 mL per 100 g of body weight practically throughout the entire gestational period from the 1st to the 20th GD. The exposure dose was determined based on data on the multidirectional toxic effect of TiO 2 or ZrO 2 NPs reported in [47][48][49][50][51][52][53]. Parallel control animals received deionized water in an equivalent volume daily. The 1st GD was considered the date of detection of spermatozoa in vaginal smears after mating with intact males. Experimental groups (two experimental and one control) were formed according to the cumulative principle, gradually bringing their number to 25 individuals in each for 4 days. For ease of identification, the following designations of experimental groups were introduced: Group 1 (TiO 2 NPs), Group 2 (ZrO 2 NPs), and Control. Laboratory animals were placed in individual polypropylene cages KMK-1 (UniZoo, Moscow, Russia) measuring 384 mm × 250 mm × 163 mm. The KMK-1 cages were manufactured in full compliance with all the established requirements and norms [55,56], providing convenient and proper animal maintenance ( Figure 2). mg/kg/day were administered peг оs to females using a probe at the rate of 1.0 mL per 100 g of body weight practically throughout the entire gestational period from the 1st to the 20th GD. The exposure dose was determined based on data on the multidirectional toxic effect of TiO2 or ZrO2 NPs reported in [47][48][49][50][51][52][53]. Parallel control animals received deionized water in an equivalent volume daily. The 1st GD was considered the date of detection of spermatozoa in vaginal smears after mating with intact males. Experimental groups (two experimental and one control) were formed according to the cumulative principle, gradually bringing their number to 25 individuals in each for 4 days. For ease of identification, the following designations of experimental groups were introduced: Group 1 (TiO2 NPs), Group 2 (ZrO2 NPs), and Control. Laboratory animals were placed in individual polypropylene cages KMK-1 (UniZoo, Moscow, Russia) measuring 384 mm × 250 mm × 163 mm. The KMK-1 cages were manufactured in full compliance with all the established requirements and norms [55,56], providing convenient and proper animal maintenance ( Figure 2). When compiling the working algorithm of the experiment, we took into account the uniqueness of the relationships that develop in mammals during pregnancy between the organisms of the mother and fetus and proceeded from the need to apply a systematic approach as an adequate method of their analysis [57]. The last stage was implemented by conducting a simultaneous assessment of the state of the main links of the functional system "mother-fetus" at the end of the 3-week exposure cycle by the tested compounds.
Violations of homeostasis in pregnant females were judged by the results of their one-time (on the 21st GD) comprehensive examination, which was completed by euthanasia and necropsy in half of each group. At the autopsy, a targeted revision of the reproductive organs was performed in females. Fetuses were assessed for general physical development according to the somatometric parameters measured in them. In parallel, indicators characterizing the functional activity of the fetal life support system (weight and diameter of the placenta disc) were recorded. Fetal material seized at autopsy was subjected to special fixation for further microanatomic study.
Taking into account the possibility of manifestation in postnatal ontogenesis of disorders, the foundations of which were laid during the period of development in utero, the second half of the numerical composition of each group of pregnant rats was brought to natural delivery, and the postnatal development of their offspring was monitored. When compiling the working algorithm of the experiment, we took into account the uniqueness of the relationships that develop in mammals during pregnancy between the organisms of the mother and fetus and proceeded from the need to apply a systematic approach as an adequate method of their analysis [57]. The last stage was implemented by conducting a simultaneous assessment of the state of the main links of the functional system "mother-fetus" at the end of the 3-week exposure cycle by the tested compounds.
Violations of homeostasis in pregnant females were judged by the results of their onetime (on the 21st GD) comprehensive examination, which was completed by euthanasia and necropsy in half of each group. At the autopsy, a targeted revision of the reproductive organs was performed in females. Fetuses were assessed for general physical development according to the somatometric parameters measured in them. In parallel, indicators characterizing the functional activity of the fetal life support system (weight and diameter of the placenta disc) were recorded. Fetal material seized at autopsy was subjected to special fixation for further microanatomic study.
Taking into account the possibility of manifestation in postnatal ontogenesis of disorders, the foundations of which were laid during the period of development in utero, the second half of the numerical composition of each group of pregnant rats was brought to natural delivery, and the postnatal development of their offspring was monitored.
To identify shifts induced by TiO 2 and ZrO 2 NPs, a set of methods were used, the choice of which was determined by the tasks of a particular stage of the experiment and the features of the anatomical and physiological organization of the object of study.
The functional state of the organism in sexually mature individuals was judged by integral tests: body weight, summation-threshold indicator (STI), heart rate (HR), muscle strength, the morphological composition of blood, and behavioral reactions in the automated system of registration and identification of behavioral acts Metris LABORAS [58][59][60]. The effect of TiO 2 and ZrO 2 NPs on rat metabolism was evaluated based on the analysis of changes in the main biochemical parameters of blood serum, including total protein content, albumin, glucose, triglycerides, cholesterol, urea, creatinine, lactic and pyruvic acids, the activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and gamma-glutamyltransferase (GGT) according to the methods described in [61]. The activity of the mechanisms of antiperoxide protection of the body was judged by the content of reduced glutathione in whole blood and the final product of lipid peroxidation (malondialdehyde, MDA) in serum. The studies were performed using the MNCHIP Celercare V5 biochemical analyzer (MNCHIP, Moscow, Russia) and unified spectrophotometric methods [62]. In infant rats and juvenile animals, speciestypical indicators of physical development and the rate of maturation of sensory-motor reflexes were monitored [63]. The fetuses were examined for the presence of developmental anomalies by conventional microanatomic methods.
The obtained experimental material was subjected to statistical processing. The indicators are taken into account in an alternative form (conception indices, frequency of anomalies, values of embryonic and postnatal death) and were compared by the Fisher method. Statistical processing of graded features with continuous distribution was carried out using the Student's criterion using STATISTICA 12 (Statsoft, Tulsa, OK, USA). The differences were considered significant at p < 0.05.

Characterization of TiO 2 and ZrO 2 NPs
The structure and phase composition of TiO 2 and ZrO 2 NPs samples were determined by X-ray phase analysis. The diffractogram of TiO 2 and ZrO 2 NPs samples is shown in Figure 3.
As can be seen from the diffraction pattern (Figure 3), the samples of TiO 2 and ZrO 2 NPs are amorphous. There are no characteristic peaks that do not allow for determining the structure and phase composition of the samples. Therefore, we used the method of correlation spectroscopy of dynamic light scattering (photon-correlation spectroscopy) to determine the size of the phases of the prepared samples. Figure 4 shows a histogram of the size distribution of TiO 2 and ZrO 2 nanoparticles. To identify shifts induced by TiO2 and ZrO2 NPs, a set of methods were used, the choice of which was determined by the tasks of a particular stage of the experiment and the features of the anatomical and physiological organization of the object of study.
The functional state of the organism in sexually mature individuals was judged by integral tests: body weight, summation-threshold indicator (STI), heart rate (HR), muscle strength, the morphological composition of blood, and behavioral reactions in the automated system of registration and identification of behavioral acts Metris LABORAS [58][59][60]. The effect of TiO2 and ZrO2 NPs on rat metabolism was evaluated based on the analysis of changes in the main biochemical parameters of blood serum, including total protein content, albumin, glucose, triglycerides, cholesterol, urea, creatinine, lactic and pyruvic acids, the activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and gamma-glutamyltransferase (GGT) according to the methods described in [61]. The activity of the mechanisms of antiperoxide protection of the body was judged by the content of reduced glutathione in whole blood and the final product of lipid peroxidation (malondialdehyde, MDA) in serum. The studies were performed using the MNCHIP Celercare V5 biochemical analyzer (MNCHIP, Moscow, Russia) and unified spectrophotometric methods [62]. In infant rats and juvenile animals, species-typical indicators of physical development and the rate of maturation of sensory-motor reflexes were monitored [63]. The fetuses were examined for the presence of developmental anomalies by conventional microanatomic methods.
The obtained experimental material was subjected to statistical processing. The indicators are taken into account in an alternative form (conception indices, frequency of anomalies, values of embryonic and postnatal death) and were compared by the Fisher method. Statistical processing of graded features with continuous distribution was carried out using the Student's criterion using STATISTICA 12 (Statsoft, Tulsa, OK, USA). The differences were considered significant at p < 0.05.

Characterization of TiO2 and ZrO2 NPs
The structure and phase composition of TiO2 and ZrO2 NPs samples were determined by X-ray phase analysis. The diffractogram of TiO2 and ZrO2 NPs samples is shown in Figure 3. As can be seen from the diffraction pattern (Figure 3), the samples of TiO2 and ZrO2 NPs are amorphous. There are no characteristic peaks that do not allow for determining the structure and phase composition of the samples. Therefore, we used the method of correlation spectroscopy of dynamic light scattering (photon-correlation spectroscopy) to determine the size of the phases of the prepared samples. Figure 4 shows a histogram of the size distribution of TiO2 and ZrO2 nanoparticles.  According to the data presented in Figure 4, the average hydrodynamic radius of the obtained TiO 2 NPs was 87 ± 36 nm, and the size of the ZrO 2 NPs was 138 ± 24 nm. The results obtained correspond to other works [25,26]. Thus, the nanoscale nature of the synthesized samples was proved by photon correlation spectroscopy, which made it possible to proceed to the next stage of the experiment. As can be seen from the diffraction pattern (Figure 3), the samples of TiO2 and ZrO2 NPs are amorphous. There are no characteristic peaks that do not allow for determining the structure and phase composition of the samples. Therefore, we used the method of correlation spectroscopy of dynamic light scattering (photon-correlation spectroscopy) to determine the size of the phases of the prepared samples. Figure 4 shows a histogram of the size distribution of TiO2 and ZrO2 nanoparticles.   According to the data presented in Figure 4, the average hydrodynamic radius of the obtained TiO2 NPs was 87 ± 36 nm, and the size of the ZrO2 NPs was 138 ± 24 nm. The results obtained correspond to other works [25,26]. Thus, the nanoscale nature of the synthesized samples was proved by photon correlation spectroscopy, which made it possible to proceed to the next stage of the experiment.

Assessment of the Effect of TiO2 and ZrO2 NPs on Pregnant Rats and Antenatal Development of Offspring
The experiment showed that the 3-week intake of TiO2 and ZrO2 NPs into the body during pregnancy causes few, although significant shifts in behavior, peripheral blood composition, and metabolic processes in rats (Table 1), which corresponds to the results of Younes et al. (2015) [39]. In female rats receiving TiO2 NPs, only eight significant deviations were revealed: an almost threefold reduction of immobility time, leukocytosis, and cholesterol reduction. A 15% decrease in the blood content of the final product of lipid peroxidation (MDA). TiO2 NPs also caused an increase in the duration of grooming, the number of counterclockwise circular rotations, and the concentration of pyruvic acid in the blood. In female rats receiving ZrO2 NPs, there were 16 significant changes related to the control group. Most of the detected changes reflected a similar direction of action of TiO2 nanoparticles. A certain motor stimulation in female rats of this group was indicated by a 25% increase in the maximum speed of movement. Activation of the antiperoxis protection system was indicated by a pronounced decrease in MDA. ZrO2 NPs also caused a reduction of creatinine and leukocyte content and an increase in gamma-glutamyl transferase number (Table 1). Similar effects were reported in relative works [50,53]. Detected changes can be related to TiO2 and ZrO2 NPs' effect on the antioxidant enzyme activity of rats [64] and defective mechanisms on metabolism [65]. Special behavioral parameters indicated in pregnant rats from experimental groups are probably associated with the neurotoxicological and neurodegenerative effect of TiO2 and ZrO2 NPs on the brain and CNS [39,66,67].

Assessment of the Effect of TiO 2 and ZrO 2 NPs on Pregnant Rats and Antenatal Development of Offspring
The experiment showed that the 3-week intake of TiO 2 and ZrO 2 NPs into the body during pregnancy causes few, although significant shifts in behavior, peripheral blood composition, and metabolic processes in rats (Table 1), which corresponds to the results of Younes et al. (2015) [39]. In female rats receiving TiO 2 NPs, only eight significant deviations were revealed: an almost threefold reduction of immobility time, leukocytosis, and cholesterol reduction. A 15% decrease in the blood content of the final product of lipid peroxidation (MDA). TiO 2 NPs also caused an increase in the duration of grooming, the number of counterclockwise circular rotations, and the concentration of pyruvic acid in the blood. In female rats receiving ZrO 2 NPs, there were 16 significant changes related to the control group. Most of the detected changes reflected a similar direction of action of TiO 2 nanoparticles. A certain motor stimulation in female rats of this group was indicated by a 25% increase in the maximum speed of movement. Activation of the antiperoxis protection system was indicated by a pronounced decrease in MDA. ZrO 2 NPs also caused a reduction of creatinine and leukocyte content and an increase in gamma-glutamyl transferase number (Table 1). Similar effects were reported in relative works [50,53]. Detected changes can be related to TiO 2 and ZrO 2 NPs' effect on the antioxidant enzyme activity of rats [64] and defective mechanisms on metabolism [65]. Special behavioral parameters indicated in pregnant rats from experimental groups are probably associated with the neurotoxicological and neurodegenerative effect of TiO 2 and ZrO 2 NPs on the brain and CNS [39,66,67].
The signs of distress detected in females when TiO 2 and ZrO 2 NPs entered the body did not, however, affect their ability to reproduce offspring. The conception indices, which amounted to 88% and 96% in the first and the second experimental groups, respectively, did not significantly differ from the control indicator, equal to 92%. A sufficient number of pregnant individuals in all groups allowed them to be very evenly divided into subgroups in accordance with the stages of the study (Table 2).
At the abdominal autopsy of the first half of the female rats, it was found that the tested preparations of TiO 2 and ZrO 2 NPs did not have a negative effect on the intrauterine development of the offspring, which corresponds well to the result of Lee et al. (2019) [53,68]. In both experimental groups, the fetus carried out in appearance, and the main indicators of viability (craniocaudal size, body weight) corresponded to their physiological age and did not differ from the offspring of control individuals. The placental fetal life support system, judging by the unchanged metric characteristics, had an adequate degree of functional maturity. The same result was reported in other works [49,69]. There were also no significant differences in the values of antenatal death between experience and control (Table 3). Table 1. Results of examination of pregnant female rats exposed to oral exposure to TiO 2 and ZrO 2 NPs during the gestational period, p < 0.05.

Group 2 (ZrO 2 NPs) Control
Integral physiological indicators  A lifetime examination of the fetuses did not reveal the presence of gross malformations in them. In the subsequent microanatomic study of the recorded fetal material, teratogenic effects were also not observed. Thus, the enlightenment of soft tissues and subsequent analysis of the fetal bone system (32 in the Control, 33 in Group 1, and 27 in Group 2) did not reveal any deviations in the structure of their skeleton in the experiment, as well as in the rates of ossification of cartilaginous blastemas of various bone formations.
During the study of transversal sections of the body, it was found that the internal organs of the fetuses retained normal topography and their characteristic structure. Rare disorders of embryogenesis without characteristic specificity were recorded in all experimental groups, each of which did not exceed the level of spontaneous anomalies of a similar type in the Control in frequency. Comparative analysis of all experimental groups showed a tendency to increase embryogenesis disorders. However, due to the lack of statistical confirmation of the significance of the revealed differences, all embryogenesis disorders in the experiment were qualified as having no causal connection with the effects of the tested preparations of TiO 2 and ZrO 2 nanoparticles (Table 4).
Thus, under the described experimental conditions, both TiO 2 and ZrO 2 NPs caused a few pathological shifts in the functional mother-fetus system, concentrated within the maternal organism. The absence of differences between the experiment and the control in the frequency and nature of malformations testified against the presence of teratogenic effects in them. Similar results are declared by Jia et al. (2017) [42] and El Ghareeb et al. (2015) [48]. Table 4. Data of microanatomic analysis of transversal body sections of fetuses from female rats exposed to TiO 2 and ZrO 2 NPs during pregnancy, p < 0.05.

Study of the Effect of TiO 2 and ZrO 2 NPs on Postnatal Development of Offspring
The condition of the offspring obtained from the second half of the female rats was monitored for 2 months after birth. Dynamic monitoring of its development included accounting for mortality, registration of morphological maturation parameters, and the study of the rate of formation of basic sensory-motor reflexes during the lactation period. At the age of 2 months, the animals were subjected to a comprehensive examination. With the exception of body weight, which was determined in all rats, the remaining indicators were recorded selectively. To ensure the representativeness of samples (at least 24 individuals) from each individual female, two rats (female and male) were randomly selected for every two to three tests, which made it possible in this case to achieve almost total inclusion of the entire grown population in the experiment.
The observation showed that the development of offspring in experimental groups occurs with some differences. First of all, this concerns such indicators as postnatal mortality and body weight gain, which are fundamental for judging the nature and severity of the action of xenobiotics [53]. It was found that TiO 2 NPs have some stimulating effect on the increase in body weight by rats during lactation. This indicator in Group 1 was significantly increased relative to the Control on the 14th and 21st days of life. Postnatal mortality had dynamics similar to those observed in the Control (Table 5).
On the contrary, after exposure to ZrO 2 NPs, the body weight of the rats on the 14th day significantly decreased, and at the same time, there was a tendency to increase postnatal death. Starting from the 21st day, the rise in mortality above the control level by~2.4 times has acquired a steadily reliable character (Table 5).
Total testing of infant rats testified that the main signs of progressive morphological maturation, such as detachment of the auricle, eruption of incisors, covering with wool, and opening of the eyes, were formed simultaneously in the experimental groups and control. However, the acquisition of gender-specific reproductive traits occurred in different ways, even within one Group, depending on sex. In males, TiO 2 and ZrO 2 NPs accelerated the rate of testicular descent, and by the 25th day of life, this process was completed in 83.7% (TiO 2 NPs) and 78.7% (ZrO 2 NPs) of males, despite the fact that the same indicator in the Control was only 57.4%. In contrast, the puberty of females exposed to prenatal exposure to ZrO 2 NPs was delayed. Thus, on the 55th day of life, the opening of the vagina occurred only in 78.3% of females of Group 2, while in the Control, it happened in 98.4% (Table 6). The assessment of the timeliness and dynamics of the formation of sensory-motor reflexes of the lactation period in experimental animals indicated the desynchronization of the processes of formation of a number of sensory functions. Thus, in the early neonatal period, the experimental offspring demonstrated a better ability to coordinate movements compared to the control, as indicated by higher indicators obtained in both experimental groups in the "flipping on a plane" test ( Table 7).
The more developed sense of smell in the descendants of the Group 2 could be judged by a certain advance of control in the timing of the formation of positive responses in the "homing" test. However, along with such changes, there were also negative shifts. In particular, if the descendants of the second experimental group had a percentage of individuals who took the correct pose in the "negative geotaxis" test, the magnitude was comparable to the control indicators. In Group 1, it was significantly reduced on the fifth day of life. At 2 months of age, chemically induced modifications of the postnatal development of the experimental offspring persisted. Their manifestations were relatively few but biologically significant. In the first experimental group, four main indicators can be highlighted. The first one characterized changes in the sphere of circular motions and was expressed in an increase in the average diameter of the circle of rotations. The second was leukopenia, and the third and fourth were a decrease in the level of total cholesterol and urea in the blood serum (Table 8). The descendants of Group 2, reaching the same maximum speed as the control animals, could not maintain it for as long, and the time of movement with the maximum speed was reduced in comparison with the Control by~1.6 times. The pyruvic acid level was also lower in Group 2 than in the Control group. Generally, the data obtained correlates with the data presented in Table 1. Table 8. Results of examination of two-month-old offspring (both sexes) of female rats exposed to TiO 2 and ZrO 2 NPs during pregnancy (average of 24-40 definitions).  Thus, the results of the observation indicate that during the life span from the period of newborn to the age of reproductive maturity, the development of the offspring of Group 1 and Group 2 occurred with a number of deviations from the physiological etalon presented by the parallel Control group. The revealed modifications give grounds to conclude that under the described experimental conditions (100 mg/kg/day), TiO 2 and ZrO 2 NPs have an embryotoxic effect which was not reported as a result obtained but was stated as a potential risk in related works [47,48,50,53].

Conclusions
In this work, for the first time, a comparative analysis of the effect of TiO 2 and ZrO 2 NPs on the vital signs of pregnant rats, embryos, and offspring in postnatal development was carried out. It was found that 3-week contact with TiO 2 and ZrO 2 NPs (100 mg/kg per os) leads to very few similar homeostatic shifts in pregnant female rats and does not affect their ability to conceive and gestation offspring. The intrauterine development of offspring proceeded without obvious pathological abnormalities: neither an increase in antenatal death nor an increase in structural defects in the fetuses of the experimental groups was recorded. Nevertheless, follow-up of live-born offspring provided experimental evidence that the real consequences of prenatal exposure to TiO 2 and ZrO 2 NPs turned out to be more serious.
During the discussion of the results obtained, it was found that despite the absence of a direct acute toxic effect, TiO 2 and ZrO 2 NPs caused significant deviations in biochemical indexes and behavior parameters of rats. Apparently, the observed changes are related to changes in the antioxidant enzyme activity of rats, metabolic disorders, and neurotoxicological and neurodegenerative effects of TiO 2 and ZrO 2 NPs. The multidirectional shifts relative to the control of the marker timing of physical and functional maturation probably indicated a chemically induced desynchronization of complex processes of heterogeneous systemogenesis in experimental rats, and the consequences of prenatal exposure to TiO 2 and ZrO 2 NPs differed, which allows us to declare different activity of TiO 2 and ZrO 2 NPs. Even after the descendants reached the age of relative maturity, modification changes retained intergroup differences. Thus, in Group 1 (TiO 2 NPs), certain disorders in the sphere of circular rotations, leukopenia and hypocholesterolemia were noted, and in the Group 2 (ZrO 2 NPs), changes in locomotion in the absence of hematological shifts and signs of impaired hepatic metabolism were detected.
The results obtained will become an important basis for further investigation of the mechanisms of toxic action of TiO 2 , ZrO 2, and other industrial nanoparticles and decisionmaking to ensure safety and reduce their impact on animals and humans. Further research will be related to a detailed study of the mechanisms of the effect of TiO 2 al ZrO 2 NPs on the vital signs of rats and the determination of the tolerance level. The collected data will allow us to draw parallels with the human organism and understand how important to introduce regulations on the contact of pregnant women with materials and micromachines based on TiO 2 and ZrO 2 NPs.