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Environ Anal Health Toxicol > Volume 37:2022 > Article
Baek, Park, Kim, Im, Seo, Park, and Nah: Reproductive and developmental toxicity screening test of new TiO2 GST in Sprague-Dawley rats


TiO2 nanoparticles are widely used in paints, plastics, cosmetics, printing ink, rubber, food products, pharmaceuticals and other products (photocatalyst, etc.). However, there is little toxicological information during reproduction and developmental period. This study was performed to obtain safety data for new TiO2 powder, GST (Green Sludge Titanium) produced through sludge recycling of the sewage treatment plant for Reproduction/developmental toxicity screening test in Sprague-Dawley (SD) rats in according to the OECD test guideline (TG 421). Based on the results of the dose-range finding study (14-day repeated toxicity), GST was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg B.W/day. Males were dosed for 35 days beginning 14 days before mating, and females for a maximum of 53 days beginning 14 days before mating to day 13 of lactation, including throughout the mating, gestation and lactation periods. In the reproductive and developmental examinations, there were no marked toxicities in terms of general clinical signs, body weight, food consumption, organ weights, macroscopic / microscopic findings, stages of spermatogenesis in the testis, reproductive finding (estrous cycle, copulation-fertility-gestation index), developmental finding (number of corpora lutea and implantations, pups parameters including live birth and viability index). The NOAEL for reproductive/developmental screening toxicity was concluded to be 2000 mg/kg/day under the present study conditions.


The TiO2 (Titanium Dioxide) has been widely used as a photocatalyst in many environmental and energy applications due to its efficient photoactivity, high stability, low cost, and safety to the environment and humans because of its potential effect to use solar energy to solve environmental problems and provide a recyclable and sustainable energy [1]. In addition, TiO2 has been produced for consumer products, paints, pigments, orthodontic composites, food and cosmetic products [25]. It is often used in the environmental and energy fields, including self-cleaning surfaces, air and water purification systems, sterilization, hydrogen evolution, and photo-electrochemical conversion. It has a characterization such as high photocatalytic activity, excellent physical and chemical stability, low cost, non-corrosive, nontoxicity and high availability [69]. For this reason, the toxicological studies of TiO2 have been performed currently with several investigates through various routes of exposure including dermal, inhalation, oral and in vitro studies [1017]. However, the potential biological and safety effects of TiO2 for reproductive toxicology have not been well studied and more investigations on the potential health hazards of the TiO2 nanoparticles are needed. Morgan et al. [18] reported that the treatment-related effects were observed in adult male albino rats for TiO2 P25 (Aeroxide®, anatase form with particle size of 10 nm); reduction / histopathological alterations in relative sex organ weights, sperm mobility, concentration and viability percentage and increase of sperm morphological abnormalities [18]. Also, Gao et al. [19] reported severe testicular pathological changes including decreased serum sex hormone levels and abnormal semen picture in male mice exposed to TiO2 nanomaterials [19] and Fartkhooni et al. [20] showed the results for alteration in sex hormone (LH, testosterone) without histological findings as testis tissue, count of sperm cells in adult male Wistar rats exposed to TiO2 (size: 18 nm) [20]. Recently, Lee et al. [21] reported that there were no marked toxicities in examinations including macroscopic findings, cesarean section parameters, fetal morphological findings in prenatal developmental toxicity study for TiO2 nanoparticles (primary size: 17.8±5.46 nm) [21].
New TiO2 material used in this article, GST (Green Sludge Titanium, 100% anatase) prepared from the precipitated sludge using TiCl4 used as a coagulant to remove total phosphorus in the wastewater was manufactured to have cost-competitive lower than price of commercial material (P-25, Evonik Corp., a flame-made multiphasic TiO2 nanoparticles containing anatase and rutile) with excellent photocatalytic function [22, 23]. In previous research using GST, we have been studied toxicological test as acute oral / dermal toxicity (TG 402, 423) in female rats by Seol et al. [24], eye or skin irritation/corrosion in rabbit (TG 404, 405) by Kim et al. [25] and 90-day oral repeated toxicity in rats by Kim et al. [25]. Through these studies, we confirmed that GST have no treatment-related effect for oral acute / repeated exposure or acute dermal and eye or skin. But the study for toxicological information on reproductive/developmental effect of GST and dermal repeated effect has been not conducted.
Therefore, the objective of this study was to confirm the potential reproduction/developmental toxicological effect of orally exposed TiO2 such as gonadal function, mating behavior, conception, development of the conceptus and parturition exposure in accordance with OECD test guideline (TG 421).

Materials and Methods

Test facility

This study was conducted in compliance with the principles of Good Laboratory Practice (GLP) at KTR (Korea Testing & Research Institute), Hwasun based on the Korea Good Laboratory Practice (KGLP) and OECD “Principle of Good Laboratory Practice, ENV/MC/CHEM (98)17 (as revised in 1997)”. The study protocol was reviewed and approved (IAC2020–2055) by the Institutional Animal Care and Use Committee (IACUC) of KTR Hwasun based on the Animal Protection Act [Enforcement Date: 2021-02-12] [No.16977 (2020-02-11, partial revision)] [26] and the Laboratory Animal Act [Enforcement Date: 2019-03-12][No. 15944 (2018-12-11, partial revision)] [27]. The KTR Hwasun has been fully accredited by the association for assessment and accreditation of laboratory animal care (AAALAC). Also, this study was conducted in accordance with the OECD Guidelines for the Testing of Chemicals, Section 4, TG 421 “Reproduction / developmental toxicity screening test” [28].

Animal husbandry and maintenance

This study (Study No. TGK-2020-000234) employed 52 male (281.9 g – 359.0 g, 9 week-old) and 55 female (169.6 g-198.7 g, 7 week-old) Sprague-dawley rat [(Crl;CD(SD), SPF] obtained from the ORIENT BIO Inc. (8, Hwaaksan-ro 124, Buk-myeon, Gapyeong-gun, Gyeonggi-do, Republic of Korea) to test for the reproductive performance such as gonadal function, mating behavior (pre-mating and mating period), conception, development of the conceptus and parturition (gestation and 13 days lactation period) and these were kept carefully following an acclimation period of 5 days to ensure their suitability for the study. Test animals were kept within a limited-access rodent facility with environmental conditions set to a temperature of 22±3 °C (value: 21.1–22.9), a relative humidity of 30 – 70% (value: 51.8–62.2) and a 12-h light / 12-h dark cycle (08:00-20:00 / 20:00-08:00). For study, the healthy animals were used after examining body weight changes, healthy condition and pre-dosing estrus cycle evaluation (female for 2 weeks) and were randomly divided into four groups listed in Table 1. Animals were kept in stainless steel wire cages with enrichment (Gnawing bricks, TAPVEI, Estonia) and allowed R/O (reverse osmosis) water via a water bottle and irradiation-sterilized pellet diet (Rodent Diet 20 5053, Labdiet, USA), ad libitum. In female rats, dams were kept in bottom of cage with bedding (Laboratory animal bedding aspen, ABEDD, Austria) from gestation period to lactation period (13 days).

Test materials and preparation

The new TiO2 materials, GST (pale yellow powder, crystalline composition of 100% anatase) was provided by Bentec Frontier Co., Ltd (139, Nanosandan-ro, Nam-myeon, Jangseong-gun, Jeollanam-do, Republic of Korea). The characterization of GST was evaluated as follows; zeta potential, particle size image (SEM), TEM (transmission electron microscopy) image, size distribution.
  • Zeta potential & particle size distribution: Particle size & Zeta potential analyzer (Zetasizer Nano ZSP, Malvern Instruments LTD., UK) by Korea TECH

  • particle size image: FE-SEM(Field Emission Scanning Electrong Microscope, Tescan Corp., Czech)] equipped with EDS systems (Thermo scientific, USA) by KRICT

  • TEM image & size distribution: FE-EF-TEM (Field Emission Energy Filtered Transmission Electron Microscopy, JEOL, Japan) by Korea Basic Science Institute and Image J software (https://imagej.nih.gov/ij/download.html)

Test procedure

Properties of GST

The characterization of GST showed in Figure 1, 2 and 3, respectively. The zeta potential is the potential between droplet surface and dispersing liquid medium and can be used to estimate surface charge of the droplets in the dispersion medium. Also, it was known for indicator of the droplet stability, where values more positive than +30 mV and more negative than −30 mV indicate good stability against coalescence [29]. The estimated value for GST showed that GST has a negative value (−35.4 mV) and this value was thought to be good stability and it was thought to be a property to be less agglomeration nature (Figure 1). The particle size value and distribution (95.8 ± 46.3 nm, 46–270 nm) showed that GST was considered to be have materials of various sizes and was difficult to be classified as a nanomaterial considering the definition of nanomaterials (<100 nm) (Figure 2 and 3).

Selection of doses and treatment

Previously, we studied dose-range finding (DRF) study (Study No.: TNK-2020-000259, 2-week repeated oral toxicity; 0, 250, 500, 1000, 2000 mg/kg B.W/day) for the dose selection to reproductive/developmental toxicity screening test. As a result of DRF study, there were no treatment-related effects in mortality, body weight changes, food consumption, gross finding at necropsy, organ weight. In clinical signs, compound-colored stool was observed on day 8 to 14 in 1000 and 2000 mg/kg B.W/day. Based on these results, the high dose was selected as 2000 mg/kg B.W and doses of 1000 and 500 mg/kg B.W were set as the middle and low doses, respectively, using a common ratio of 2 (Table 1). The vehicle control group was treated with sterile distilled water for injection only (Sterile distilled water for injection, DAI HAN PHARM. CO., LTD, Korea). Male rats were administered for 35 days such as 14 days before mating, 4 days during the mating period and 17 days after mating until necropsy. Female rats were treated for a maximum of 53 days from 2 weeks before mating, during the mating period (4 days) and gestation days, and during 14 days of post-natal days.

Clinical signs, body weight and food consumption

Throughout the test period, general clinical observations, signs of toxicity including mortality and prolonged parturition (dams) were made at once a day for acclimation period (including non-treatment period) and twice a day for treatment period (before/after administration) in parent animal. In pups, clinical observations were made until 13 days after delivery. For males, body weights were recorded once weekly from the start of the study until necropsy and food consumption was recorded weekly except for mating period. Females were weighted once weekly during the pre-mating period, on pregnancy (gestation days; GD 0, 7, 14, 20 days), on lactation period (post-natal days; PND 0, 4, 7, 13) and on the day of necropsy (fasting weight).

Reproductive and developmental findings

Estrous cycle was monitored daily by vaginal smear during the pre-mating period and mating period until evidence of mating. The smears were examined under light microscopy and the stage of the estrous cycle was determined by the type of cell present. Estrous cycles of 4–5 days were judged as normal. Also, vaginal smear was examined on the day of necropsy for historical examination. After the 14 days pre-mating period, each male was housed individually with a female (1:1 basis) from same group until successful copulation occurred or the mating period 2 weeks had elapsed. During mating period, the females were examined for the presence of sperm in vagina or vaginal plug (once/day) and pre-coital time (day) was calculated. After successful mating occurred, mating index were calculated as follows (copulation index, fertility index, gestation index).
  • Male copulation index (%) = (No. of males with confirmed mating / Total No. of males cohabited) ×100

  • Male fertility index (%) = (No. of males impregnating a female / Total No. of males cohabited) ×100

  • Female copulation index (%) = (No. of sperm-positive females / Total No. of females cohabited) ×100

  • Female fertility index (%) = (No. of pregnant females / No. of sperm-positive females) ×100

  • Gestation index (%) = (No. of females with live born pups / No. of pregnant females) ×100

Day 0 of gestation (GD 0) was defined as the day on which successful mating evidence was confirmed. Pregnant females (dams) were allowed to deliver spontaneously and nurse their pups, and the day on which the delivery was completed was designated as day 0 of lactation or postnatal day 0 (PND). Each litter were examined after delivery to establish the number and sex of pups, stillbirths, live births, runts. Live pups were counted and sexed and litters weighed within 24 hours of parturition and on day 4 and 13 post-partum. Also, the AGD (anogenital distance; distance between the anus and the genital tubercle) of each pup was measured on PND 4 (on day 4 after birth) and was normalized to compensate for differences in weight among pups by using AGD/cube root of body weight ratio. The size of each litter was adjusted by eliminating extra pups by random selection (culling target: 8 pups per litter) and the surplus pups were used for hormone level (serum T4). On day 14 post-partum (PND 14), number of corpora lutea and implantation sites were examined. The offspring parameters in pups were calculated as follows.
  • Live birth index (%, PND 0) = (No. of live pups on PND 0 / Total no. of pups born) ×100

  • Viability index (%, PND 4) = (No. of live pups on PND 4 / No. of live pups born on PND 0) ×100

Clinical biochemistry

Blood samples were collected for analysis of circulating thyroid hormones [Thyroxine (T4), Triiodothyronine (T3), Thyroid stimulating hormone (TSH)], from all parental animals, pups on PND 4 and PND 13 (pooled sample per litter). Blood samples were put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature and the serum was separated by centrifugation at 3000 rpm (4 °C) for 10 minutes and investigated using hormone analyzer (Immulite xpi, Siemens, Germany). The T4 for samples of adult males and pups on PND 13 were analyzed and significant differences were not observed in treatment group. Further assessment of T4 in blood samples from the dams and pups on PND 4 were not done. Also, other hormones were not measured (T3, TSH).

Necropsy and histopathology

After blood collection under anesthesia with isoflurane (Forane, JW Pharmaceutical, Korea), the animals were sacrificed by exsanguination from the aorta. The external surface, all orifices, cranial cavity, thoracic (abdominal) organs and their contents were macroscopically checked. The internal organs were fixed with 10% neutral buffered formalin individually except testes / epididymides (Bouin’s fixative solution) and eyes (Davidson’s fixative solution). Those organs were processed routinely for embedding in paraffin, and sections were prepared for staining with hematoxylin-eosin.
After gross necropsy, absolute and relative (organ-to-body weight ratio) weights for following list of organs were measured in all animals. For bilateral organs, the weight of the left and right organ was measured respectively and combined weight was calculated. The list of organ weight was as follows; Liver, Spleen, Heart, Kidneys, Lung, Thymus, Testes, Epididymites, Uterus, Ovaries, Adrenal glands, Brain, Pituitary gland, Cowper’s glands, Glans penis, Levator ani plus bulbocavernosus muscle (LABC), Prostate gland plus Seminal vesicle with coagulating glands.
In dams at necropsy, index for ovaries and uterus were calculated as follows (implantation ratio, pre-implantation loss, post-implantation loss).
  • Implantation ratio (%) = (No. of implantation / No. of corpora lutea) ×100

  • Pre-implantation loss (%) = [(No. of corpora lutea - No. of implantation) / No. of corpora lutea] ×100

  • Post-implantation loss (%) = [(No. of implantation – litter size) / No. of implantation] ×100

Histopathological examination was performed on the ovaries, uterus (and cervix), prostate glands (including seminal vesicles with coagulating glands), testes, and epididymis (on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals of the high dose group and the control group after tissue processing. And then residual organs and fixed organs were preserved in 10% neutral buffered formalin.
The PND 4 pups were euthanized by decapitation and PND 13 pups were killed by exsanguination from the aorta under anesthesia with isoflurane. These pups, any pups found dead during lactation, and all pups killed on PND 13, were examined externally for gross abnormalities, with particular attention paid to the external reproductive organs. Where possible, the thyroid from 1 male and 1 female pup per litter were preserved in 10% neutral buffered formalin for additional histopathological examination.

Statistical analysis

Data were presented as means±standard deviation (S.D.) for each group. The body weight, food consumption, organ weight, pre-coital time, number of pups, numbers of corpora lutea/implantations sites for parental animals and pup body weight were analyzed using SPSS (Ver 19.0) program. The Levene’s test was performed for homogeneity of variances and then One way ANOVA analysis was performed to evaluate the significant differences between groups. If there is no significant difference, additional analysis was not performed, but in case of significant difference is confirmed, post-hoc test was performed according to the result of variance homogeneity (homogeneity; Scheffe test, heterogeneity; Dunnett’s T3 test). The live birth index, viability index and pre/post-implantation loss were examined by using the Kruskal-Walis test. The confidence interval was 95% level.

Results and Discussion

Clinical signs for parental

The treatment-related death was not observed at any dose group in both sexes. The common clinical signs, compound-colored feces, was constantly observed from on day 8 in middle and high dose group and on day 15 in low dose group to the end of administration (up to Day 35 in males and PND 13 in dams). In the degree of signs, there were observed from slight to moderate in the high dose group and the color of the feces was dose-dependently observed and lighter in the treatment group as compared with control as Table 2 and Figure 4. Some study using TiO2 showed that no abnormal clinical sings were observed in any of treatment group of repeated dose 90-day in rat (TiO2 P25, AEROXIDE® or synthesized TiO2) [30,31]. Therefore, this clinical sign was interestingly difference from other study and it was considered to be due to difference in absorption by the various particle size.

Body weight and food consumptions

For the experimental period (2-week pre-mating period, 2 week mating period, gestation period, post-natal period), the body weight showed a normal increase and there were no treatment-related effects in food consumptions between the treatment and dosing group Figure 5.

Stages of spermatogenesis in the tests

Testicular tissue that stained by hematoxylin and eosin and examined by light microscope. The Table 3 and Figure 6 showed the number of spermatogenic cells in each stage of spermatogenesis in the testes and these parameters did not significantly differ in treatment group as compared with control group. In the other studies, it showed that there were TiO2-related effect in exposed male animals as decreased serum sex hormone levels, abnormal semen picture in male mice and alteration in sex hormone (LH, testosterone), but histological studies does not show any important changes in testis tissue, count of spermatogonia, spermatocytes and spermatid, etc. [19, 20]. Also, the other reported that the study using SD rats for oral gavage of pigment-grade TiO2 at 1000 mg/kg body weight according to OECD TG 421 did not reveal any reproductive or developmental toxicity [5]. Based on these results of this study, the reason that GST treatment-related effect was not observed in hormones, etc. was considered to be related to the difference in animal species and absorption of the test substances. Therefore, the observation of compound-colored feces in clinical signs is shown to have been excreted with poor absorption in the body. Less certain is the extent of intestinal absorption, but an elegant vanadium (V) radiotracer study established that the vast majority of ingested TiO2 nanoparticle is directly excreted in the feces [32].

Reproductive and developmental findings

All dams survived through the end of the study and there was no treatment-related change in caesarean section parameters including corpora lutea, implantation (ratio, loss), copulation index (male and female), fertility index (male and female) and gestation index in Table 4. The mating was confirmed within 4 days (pre-coital time, control: 2.3 days, low dose: 3.1 days, middle dose: 2.1 days, high dose: 2.8 days) and almost dams were pregnant except 4 animals (2 females in control group, 1 female in low dose group and 1 animal in high dose group; non-pregnancy confirmed by using ammonium sulphide staining at necropsy). Also, the parameters for pregnancy (dams, litter) and pups (dead, live, sex ratio, live birth index, viability index, body weight) are presented in Table 5 and there were no treatment-related effects in all treatment groups. All parameters for reproduction and developmental showed as summary tubular table in Table 8.

Hormone (serum T4)

No significant difference in mean total serum T4 (Thyroxine, thyroid hormone) concentrations were observed for parental males and PND 13 pups. The samples of parental males and pups on PND 13 were analyzed and significant differences were not observed in treatment group.

Necropsy and Histopathological examination

At the end of treatment, the treatment-related gross finding was not observed in any dosing group for both sexes and no gross findings were observed in recovery group. In the absolute / relative reproductive organ weight, there were no treatment-related significant effects in both sexes in any of treatment group (Table 7).
Also, the histopathological examination was performed to identify the treatment-related effects for reproductive organ in high dose group (Figure 7). Regarding the microscopic examination (ovaries, uterus, prostate glands including seminal vesicles with coagulating glands), there were no treatment-related effect in all reproductive organs.


The present study was conducted to evaluate the biological effects concerning the effects of GST (TiO2, Titanium dioxide) on male and female reproductive performance such as gonadal function, mating behavior (pre-mating and mating period), conception, development of the conceptus and parturition (gestation days and 13 days lactation) by oral administration in Sprague-Dawley rat at 0, 500, 1000 and 2000 mg/kg · B.W./day. There was no treatment-related for parental toxicity, reproductive finding and developmental findings. As a result of the study, the NOAEL (No Observed Adverse Effect Level) value for reproductive and developmental toxicity was considered to be 2000 mg/kg B.W/day under the present study conditions.


This work was supported by a grant (19SCIP-B145906-02) from the Korea Agency for Infrastructure Technology Advancement (KAIA) by Ministry of Land, Infrastructure and Transport of Korea government (MOLIT), Republic of Korea.

Conflict of interest

The authors declare that they have no conflict of interest.


CRediT author statement
HSB: Conceptualization, Methodology, Writing-Original draft preparation; MKP: Supervision, Writing-Reviewing and Editing; HMK: Methodology, Data curation; JMI: Visualization; HSS: Visualization; HJP: Resources; SSN: Project administration, Writing-Reviewing and Editing


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crossref pmid

Figure 1
Characterization of TiO2 particles (GST) analyzed by Korea TECH: (a) negative zeta potential (−35.4 mV, 30 mg/mL); (b) size distribution by intensity (mean: 336.8 nm).
Figure 2
Characterization of TiO2 particles (GST): SEM (scanning electron microscope) image analyzed by KRICT.
Figure 3
Characterization of TiO2 particles (GST); (a) A particles dispersed in 99.9% EtOH was deposited on a copper grid and analyzed using TEM (Transmission electron microscope) image by Korea Basic Science Institute, (b) Size distribution (95.8±46.3 nm, 46–270 nm,) of the imaged GST(image J software).
Figure 4
Compound-colored stool at treatment group (①: control group, ②: low dose, ③: middle dose, ④: high dose).
Figure 5
Changes for body weight and food consumptions for treatment period except mating period (PM; post-mating, M; mating, GD; gestation days, PND; postnatal day): (a) body weight (male); (b) Body weight (female); (c) Food consumptions (male); (d) food consumptions (female).
Figure 6
Histopathological examination for stages of spermatogenesis of testis: (a) Control group; (b) High dose group.
Figure 7
Histopathological examination of reproductive organ (Left: control group, Right: high dose group): (a) Testis; (b) Epididymis; (c) Ovary; (d) Uterus.
Table 1
Groups for reproductive and developmental toxicity screening test
Group Dose (mg/kg B.W/day) Fluid volume (mL/kg) Animal number

(Male) (Female)
G1 0 10 12 (1101–1112) 12 (2101–2112)
G2 500 10 12 (1201–1212) 12 (2201–2212)
G3 1000 10 12 (1301–1312) 12 (2301–2312)
G4 2000 10 12 (1401–1412) 12 (2401–2412)
Table 2
Summary of clinical signs (compound-colored feces) for treatment period.

G1 G2 G3 G4
Males Not observed Day 15–35a (Slight) Day 8–35a (Slight) Day 8–10 (Slight)
Day 11–35a (Moderate)
Females Not observed Day 15-PND 13 a (Slight) Day 8-PND 13a (Slight) Day 8–10 (Slight)
Day 11-PND 13a (Moderate)

a Last day of the treatment (male: Day 35, female: PND 13)

Table 3
Stage of spermatogenesis of GST exposed males
Spermatogenic cells in seminiferous tubules Group

G1 G4
Stage II
 Spermatogonia 21.7±2.5 a 21.3±2.3
 Pachytene spermatocytes 50.8±3.5 48.5±4.8
 Roundspermatids 158.3±6.5 157.4±6.5
 Elongated spermatids 152.8±4.4 152.8±3.5
 Sertoli cells 17.5±1.3 18.5±1.6
Stage V
 Spermatogonia 37.8±1.5 36.2±2.0
 Pachytene spermatocytes 51.5±4.3 52.8±3.4
 Round spermatids 152.0±3.5 155.4±5.2
 Elongated spermatids 167.5±8.6 170.2±4.9
 Sertoli cells 18.8±2.6 19.4±2.0
Stage VII
 Spermatogonia 2.2±0.7 2.2±0.6
 Preleptotene spermatocytes 38.8±2.1 38.9±1.8
 Pachytene spermatocytes 56.3±3.9 55.3±4.0
 Round spermatids 155.3±6.8 154.1±4.6
 Elongated spermatids 154.3±5.6 155.5±5.1
 Sertoli cells 17.3±1.7 17.6±1.0
Stage XII
 Spermatogonia 3.8±0.9 3.9±0.8
 Zygotene spermatocytes 46.3±3.1 48.1±1.4
 Pachytene spermatocytes 61.6±3.4 61.9±3.3
 Elongated spermatids 171.9±6.9 172.3±9.9
 Sertoli cells 19.2±5.0 20.8±2.5

a values are presented as mean±S.D.

Table 4
Caesarean section results of GST exposed pregnant females during the pregnancy.

G1 G2 G3 G4
Pregnant females (N) 10 11 12 11
Estrous cycles (day) 4.0±0.1 a 4.0±0.1 4.1±0.3 4.0±0.0
Irregular cycle (N) 0 0 0 0
Dams with live born pups (N) 10 11 12 11
Pre-coital time (day) 2.3±1.2 a 3.1±1.1 2.1±0.8 2.8±0.6
No. of Corpora lutea (N) b 16.0±1.3 16.5±1.4 16.8±1.7 16.5±0.8
Implantation ratio (%)c 94.5±5.8 91.3±6.2 86.0±12.9 91.3±5.5
No. of implantation (N)b 15.1±1.4 15.0±1.5 14.6±2.9 15.1±0.9
Copulation index
 Male (%) 100.0 100.0 100.0 100.0
 Female (%) 100.0 100.0 100.0 100.0
Fertility index
 Male (%) 83.3 91.7 100.0 91.7
 Female (%) 83.3 91.7 100.0 91.7
Gestation index (%) 100.0 100.0 100.0 100.0

a values are presented as mean±S.D.,

b number

c (Implantation ratio, %) = (No. of implantation / No. of corpora lutea) ×100

Table 5
Pregnancy and litter data of rats.

G1 G2 G3 G4
Pregnant females (N) 10 11 12 11
No. of dams with live born pups (N) 10 11 12 11
No. of live pup on PND 0 (N) 13.1±2.6 13.8±1.5 13.5±3.0 14.5±0.8
No. of dead pup on PND 0 (N) 0.4±1.3 0.1±0.3 0.0±0.0 0.0±0.0
Live birth index on PND 0 (%) b 97.6±7.4 99.4±1.9 100.0±0.0 100.0±0.0
Sex ratio of live pups on PND 0 (N) c 0.459±0.131 0.504±0.202 0.478±0.089 0.501±0.121
No. of live pup on PND 4 (N) 13.0±2.7 13.7±1.6 13.3±2.9 14.3±0.8
No. of dead pup on PND 4 (N) 0.1±0.3 0.1±0.3 0.2±0.4 0.1±0.3
Viability index on PND 4 (%) d 99.2±2.4 99.2±2.5 98.9±2.6 98.8±2.7
Body weight of live pup on PND 0 (g)
 Male 7.14±0.94 6.96±0.54 6.94±0.64 6.83±0.45
 Female 6.80±0.81 6.56±0.48 6.59±0.61 6.39±0.65
Body weight of live pup on PND 4 (g)
 Male 11.94±2.24 11.23±1.00 11.51±1.64 11.24±0.93
 Female 11.38±2.08 10.61±0.83 10.95±1.66 10.61±1.07
Body weight of live pup on PND 13 (g)
 Male 36.21±2.41 34.02±3.38 35.37±2.55 36.73±2.73
 Female 35.00±3.04 33.09±2.63 34.14±2.32 35.44±2.27

a values are presented as mean±S.D.

b (No. of live pups on PND 0 / Total no. of pups born) ×100

c (No. of live male pup / No. of live pup on PND 0)

d (No. of live pups on PND 4 / No. of live pups on PND 0) ×100

Table 6
Serum T4 level in male and pup.

G1 G2 G3 G4
Males (N) 12 12 12 12
 Serum T4 (ng/mL) 55.56±9.40 a 57.68±10.43 59.63±10.75 56.77±8.51
Live pup on PND 13 10 b 11 12 11
 Serum T4 (ng/mL) 61.81±9.61 a 57.83±11.66 58.08±9.57 55.75±11.65

a values are presented as mean ± S.D.

b Dams with live born pups

Table 7
Absolute and relative weight of reproductive organ.
GST (mg/kg)

0 500 1000 2000
Males (N) 12 12 12 12
 Left Testis (g) 1.76±0.21 a 1.72±0.16 1.77±0.15 1.70±0.17
  Organ to terminal body weight ratio (%) 0.37±0.06 0.37±0.04 0.38±0.03 0.36±0.04
 Right Testis (g) 1.77±0.20 1.72±0.16 1.79±0.14 1.73±0.14
  Organ to terminal body weight ratio (%) 0.37±0.05 0.37±0.04 0.38±0.03 0.37±0.04
 Total Testis (g) 3.53±0.41 3.44±0.32 3.56±0.28 3.43±0.31
  Organ to terminal body weight ratio (%) 0.73±0.11 0.74±0.08 0.76±0.07 0.74±0.08
 Left epididymis (g) 0.76±0.07 0.76±0.08 0.74±0.05 0.73±0.06
  Organ to terminal body weight ratio (%) 0.16±0.02 0.16±0.02 0.16±0.01 0.16±0.01
 Right epididymis (g) 0.80±0.05 0.77±0.08 0.76±0.07 0.77±0.06
  Organ to terminal body weight ratio (%) 0.17±0.02 0.16±0.02 0.16±0.02 0.17±0.01
 Total epididymis (g) 1.56±0.10 1.53±0.15 1.50±0.12 1.50±0.11
  Organ to terminal body weight ratio (%) 0.32±0.03 0.33±0.03 0.32±0.03 0.32±0.02
Pregnant Females (N) 10 11 12 11
 Left Ovary (g) 0.046±0.009 0.050±0.006 0.044±0.008 0.045±0.006
  Organ to terminal body weight ratio (%) 0.015±0.003 0.016±0.003 0.014±0.002 0.014±0.002
 Right Ovary (g) 0.050±0.004 0.049±0.005 0.050±0.006 0.047±0.005
  Organ to terminal body weight ratio (%) 0.016±0.001 0.016±0.002 0.016±0.002 0.015±0.001
 Total Ovary (g) 0.096±0.011 0.099±0.006 0.094±0.010 0.092±0.007
  Organ to terminal body weight ratio (%) 0.031±0.003 0.033±0.003 0.030±0.003 0.030±0.002
 Uterus Ovary (g) 0.71±0.24 0.63±0.13 0.60±0.11 0.64±0.14
  Organ to terminal body weight ratio (%) 0.23±0.07 0.21±0.05 0.19±0.04 0.20±0.04

a values are presented as mean±S.D.

Table 8
Summary report of effects on reproduction/ development*1
Dosage (mg/kg)

0 500 1000 2000

Pairs started (N) 12 12 12 12 12 12 12 12
Oestrus cycle (at least mean length and frequency of irregular cycles) 4.0 4.0 4.1 4.0
Females showing evidence of copulation (N)*2 - 12 - 12 - 12 - 12
Females achieving pregnancy (N) - 22.1 - 21.8 - 21.7 - 21.6
Conceiving days 1–5 (N) - 12 - 12 - 12 - 12
Conceiving days 6–14*3 (N) - 0 - 0 - 0 - 0
Pregnancy ≤21 days (N) - 1 - 2 - 4 - 4
Pregnancy =22 days (N) - 7 - 9 - 8 - 7
Pregnancy ≥23 days (N) - 2 - 0 - 0 - 0
Dams with live young born (N) - 10 - 11 - 12 - 11
Dams with live young at day 4 pp (N) - 10 - 11 - 12 - 11
Implants/dam (mean) - 15.1 - 15 - 14.6 - 15.1
Live pups/dam at birth (mean) 13.1 13.8 13.5 14.5
Live pups/dam at day 4 (mean) 13.0 13.7 13.3 14.3
Sex ratio (m/f) at birth (mean) 6.1 7.4 7.0 6.9 6.5 7.0 7.3 7.2
Sex ratio (m/f) at day 4 (mean) 5.9 7.1 6.9 6.9 6.4 6.9 7.4 6.9
Litter weight at birth (mean) 91.8 93.3 90.2 95.7
Litter weight at day 4 (mean) 146.8 146.3 145.9 156.9
Pup weight at birth (mean) 7.1 6.8 7.0 6.6 6.9 6.6 6.8 6.4
Pup weight at the time of AGD measurement (mean, PND 4) 11.9 11.4 11.2 10.6 11.5 11.0 11.2 10.6
Pup AGD*4 on the same postnatal day, birthday 4 (mean, PND 4) 2.7 1.5 2.6 1.5 2.6 1.5 2.4 1.4
Pup weight at day 4 (mean) 11.9 11.4 11.2 10.6 11.5 11.0 11.2 10.6
Male pup nipple retention at day 13 (mean) 0.0 0.0 0.0 0.0
Pup weight at day 13 (mean) 36.2 35.0 34.0 33.1 35.4 34.1 36.7 35.4
Abnormal Pups
 Dams with 0 10 11 12 10
 Dams with 1 0 1*5 0 0
 Dams with ≥2 0 0 0 1*5
Loss of offspring
Pre-implantation (corpora lutea minus implantations)
 Females with 0 4 2 1 2
 Females with 1 4 4 4 3
 Females with 2 1 3 3 5
 Females with ≥3 1 2 4 1
Pre-natal/post-implantaions (implantaions minus live births)
 Females with 0 4 2 3 7
 Females with 1 3 6 6 1
 Females with 2 1 2 2 3
 Females with ≥ 3 2 1 1 0
Post-natal (live births minus alive at post-natal day 4)*6
 Females with 0 9 10 10 9
 Females with 1 1 1 2 2
 Females with 2 0 0 0 0
 Females with ≥3 0 0 0 0

*1 OECD test guideline 421 (summary form).

*2 Vaginal plug and sperm-positive evidence.

*3 Last day of the mating period.

*4 The ratio of AGD to the cube root body weight (%).

*5 runt.

*6 adjustment on PND 4 (culling target; 8/litter).

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