| Background:The current toxicological test methods in China including: acute toxicity study,chronic toxicity study, genetic toxicity study, teratogenicity study and carcinogenicity study, etc. However, these methods could not identify the potential endocrine disruptors. Therefore, establishment the Endocrine Disruptor Screening System has a significant importance for the protection of human health and ecological security.Objective:To estabilish mammalian in vivo endocrine disruptor screening system, to study to potential endocrine disrupting effects of bifenthrin(BIF), to provide science evidence for establishing guidelines of endeocrine disruptor screening system inChina.Methods:Tier 1 Screening1. Uterotrophic assay: 90 female SD rats were randomly divided into 9 groups(10 rats per group): rats in the negative control group received corn oil by gavage; rats in BIF-treated groups were given different doses of BIF(1.47, 4.41 and 13.23 mg/kg bw/d) by gavage; rats in the ethinyl estradiol(EE) oral positive group were administered with 1.0μg/kg bw/d of EE by gavage; rats in EE subcutaneously injection positive group were given 0.6μg/kg bw/d of EE by subcutaneously injection while received corn oil by gavage; rats in EE+BIF groups received different doses of BIF respectively,while given 0.6μg/kg bw/d of EE by subcutaneously injection. 3 days later, all the animals were sacrificed and the wet and blotted uterine weights were weighed, relative uterin weights were calculated, the histology of uterus was observed.2. Hershberger assay: 90 castrated male SD rats were randomly divided into 9 groups (10 rats per group): rats in the negative control group received corn oil by gavage; rats in BIF-treated groups were given different doses of BIF(1.4, 4.2, and 12.6mg/kg bw/d) by gavage; rats in TP+BIF groups received different doses of BIF respectively, while receiving 0.2mg/kg bw/d of testosterone propionate (TP) by subcutaneously injection; rats in flutamide(FLU) positive group received 3.0 mg/kg bw/d of FLU by gavage,while receiving 0.2mg/kg bw/d of TP by subcutaneously injection. 10 days later, all the animals were sacrificed, ventral prostate (VP), seminal vesicle plus fluids and coagulating glands (SVCG), levator ani-bulbocavernosus muscle (LABC), glans penis (GP), paired Cowper’s glands (COW), liver, kidneys and adrenals were weighed. Relative organ weights were calculated. Serum triiodothyronine (TT3) and thyroxine (TT4) were determined.3. Pubertal Development and Thyroid Function in Intact Juvenile/ Peripubertal Female Rats: 70 female SD rats were randomly divided into 7 groups (10 rats per group): rats in the negative control group were received corn oil by gavage; rats in BIF-treated groups were given different doses of BIF(1.1, 3.3 and 9.9 mg/kg bw/d) by gavage; rats in EE positive group, TAM positive group and PTU positive group were received 0.005 mg/kg bw/d of EE, 0.2 mg/kg bw/d of TAM and 5.0 mg/kg bw/d of PTU by gavage, respectively. The administration was given from PND22 to PND42.The age at vaginal opening (VO) and body weight were recorded. On PND42, all animals were sacrificed, uterus (wet and blotted), ovaries, liver, kidneys, adrenals,thyroid(fixed) and pituitary were weighed. Relative organ weights were calculated.Serum TT3, TT4, free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) were determined.4. Pubertal Development and Thyroid Function in Intact Juvenile/ Peripubertal Male Rats: 70 male SD rats were randomly divided into 7 groups (10 rats per group):rats in the negative control group were received corn oil by gavage; rats in BIF-treated groups were given different doses of BIF(1.4,4.2,and 12.6mg/kg bw/d) by gavage;rats in methyl testosterone(MT) positive group, FLU positive group and PTU positive group received 80 mg/kg bw/d of MT, 50 mg/kg bw/d of FLU and 5.0 mg/kg bw/d of PTU by gavage, respectively. The administration was from PND23 to PND53. On PND 53, all animals were sacrificed, VP, DP,SVCG, LABC, testis, epididymis, liver,kidneys, adrenals, thyroid (fixed) and pituitary were weighed. Relative organ weights were calculated. Serum TT3, TT4, FT3,FT4 and TSH were determined。Tier 2 TestingExtended One-Generation Reproductive Toxicity Study100 female and 100 male SD rats were randomly divided into 4 groups (10 rats per group for each sex): rats in the negative control group were received normal diet;rats in BIF-treated groups were given different doses of BIF (1, 3, 9mg/kg bw/d)through diet. The anministration of F0 rats was from pre-mating to the pups weaned on PND21. F1 rats were divided into two studies: F1a group,BIF was administered for 13 weeks (90 days) in the diet at dose levels of 0, 1, 3, 9mg/kg bw/d; F1b group,rats were administered the diet containing BIF at 0, 1, 3, 9mg/kg bw/d for 13 weeks and then fed a recovery (control) diet for an additional 30 days.1. F0 rats: reproductive data; F0 males and females were sacrificed after the pups were weaned on PND21, the serum TT3, TT4, FT3 and FT4 were determined.Brain, pituitary, thyroid (fixed), liver, kidneys, adrenals, spleen, thymus, heart, ovaries,uterus, SVCG, prostate, testis and epididymis were weighed. Relative organ weights were calculated. Pathology examination was conducted on all above organs and mammary gland, vagina. In addition, the ultrastructure of arcuate nucleus, pituitary,testis and ovary of rats in BIF high dose group was investigated.2. F1 rats: reproductive data; age and body weight at vaginal opening (VO) and preputial separation (PPS).(1) Fla group: animals were sacrificed at week 13th (PND90), serum TT3, TT4,FT3 and FT4 were determined, Brain, pituitary, thyroid (fixed), liver, kidneys,adrenals, spleen,thymus, heart,ovaries,uterus, SVCG,prostate, testis and epididymis were weighed. Relative organ weights were calculated. Pathology examination was conducted on all above organs and mammary gland, vagina. In addition, the ultrastructure of arcuate nucleus, pituitary, testis and ovary of rats in BIF high dose group was investigated.(3) F1b group: animals were sacrificed at week 17th (PND 120), the parameters were the same as F1a groupResults:Tier 1 Screening1. Uterotrophic assay: In EE oral positive group and EE subcutaneous group,significant increase in the absolute and relative weights of uterus was observed when compared with the negative control group, and significant increase in the mean relative weight of the wet and blotted uterus was observed in female rats receiving BIF at 13.23 mg/kg for 3 days. Pathology changes observed include thickness and keratinization of endometrium, increase of the height of epithelial cells in EE oral positive group and EE subcutaneous group, hyperplasia and thickness of endometrium,increase of the height of epithelial cells in BIF groups with doses of 4.41 and 13.23 mg/kg.2. Hershberger assay: in TP positive group, the absolute and relative weight of 5 androgen-dependent organs were significantly increased, while in FLU positive group were significantly reduced. The level of TT3 in BIF high dose group was reduced when compared with the negative control group. Significant decrease in the mean absolute and relative weight of VP and LABC, the levels of serum TT3 and TT4 was observed in TP+BIF high dose group when compared with TP group.3. Pubertal Development and Thyroid Function in Intact Juvenile/ Peripubertal Female Rats: in EE positive group and TAM positive group, the age of VO of rats were both advanced and the body weight at VO both reduced; in EE positive group,the weight of uterus was increased and the weight of ovary was reduced. In TAM positive group, the weights of uterus and ovary were reduced. Compared with the negative control group, the age of VO of rats in the mid-dose and high-dose BIF group advanced, the body weight of rats in the high dose BIF group was significantly reduced. In PTU group, the thyroid weight was significantly increased, the levels of serum TT3, TT4, FT3 and FT4 decreased significantly, while serum TSH elevated.Compared with the negative control group, serum TT4 in the mid-dose and high-dose BIF group reduced while serum TSH rised, the serum TT3 in the high-dose BIF group reduced.4. Pubertal Development and Thyroid Function in Intact Juvenile/ Peripubertal Male Rats: in MT positive group, the age of PPS and the body weight of male rats were reduced, organ weight of SVCG, VP, DP, LABC was increased, weight of left testis and left epididymis was decreased. In FLU positive group, the age of PPS delayed and the body weight of male rats were increased; organ weight of SVCG, VP,DP, LABC and left epididymis was decreased, weight of left testis was increased.When compared with the negative control group, the age of PPS of male rats in BIF high dose group was delayed. The thyroid weight of male rats in PTU positive group was significantly increased, serum TT3, TT4, FT3 and FT4 was significantly decreased while serum TSH was increased. The thyroid weight was significantly increased, and serum TT3 and TT4 in BIF high dose group was decreased while serum TSH was increased; the level of TT4 in BIF low dose group was decreased; the level of TSH in BIF mid-dose group was increasedTier 2 TestingExtended One-Generation Reproductive Toxicity Study1. FO rats: No significant difference between groups of the reproductive data.When compared with negative control group, the relative weight of liver and kidneys of female rats in BIF high dose group was increased; when compared with negative control group, the absolute weight of SVCG, VP was decreased, while the relative weight of liver, kidney and brain of male rats in BIF high dose group was increased.Pathology changes observed in FO rats include: lesions of mammary gland of female rats in BIF groups (low, medium and high dose), and thyroid follicular hyperplasia of female rats in BIF groups (medium and high dose); prostate lesions and thyroid follicular hyperplasia of male rats in BIF groups (low, medium and high dose). The level of TT4 of female rats in BIF medium dose group was decreased, the levels of serum TT3, TT4 and FT3 of female rats in BIF high dose group were reduced; the levels of serum TT3 and TT4 of male rats in BIF medium and high dose group were reduced. Changes were observed in the ultrastructure of arcuate nucleus, pituitary and ovary of rats in BIF high dose group.2. F1 rats: No significant difference between groups of the reproductive data.The age of VO in female rats in BIF groups (low, medium and high dose) was advanced, the body weight of rats in BIF high dose group was decreased; the age of PPS in male rats in BIF medium and high dose groups was delayed, the body weight of rats in BIF high dose group was increased.(1) Fla group: compared with the negative control group, absolute ovarian weight of female rats in BIF high dose group was reduced significantly; relative weight of brain, liver and kidneys in females was increased significantly; compared with the negative group, the organ weight of VP in male animals of BIF medium dose group was decreased.The terminal body weight of male rats in BIF high dose group was significantly decreased, absolute weight of brain and liver was increased, the relative weight of brain, pituitary, thyroid, liver, kidneys and adrenals in BIF high dose group were significantly increased while the absolute and relative weight of VP was reduced. Pathology changes observed include: thyroid follicular hyperplasia in females in BIF medium and high dose group; prostate lesions, thyroid follicular hyperplasia and crastration cells in pituitary in male rats in BIF groups (low, medium and high dose). Compared with the negative control group, the levels of serum TT3,TT4 and FT3 of female rats and male rats in BIF groups (medium and high dose)was reduced, the level of FT4 of female rats in high dose group was decreased, while the level of FT3 of male rats in low dose group was reduced. Changes were observed in the ultrastructure of arcuate nucleus, pituitary and ovary of rats in BIF high dose group.(2) F1b group: compared with the negative control group, absolute ovarian weight of female rats was significantly reduced in BIF high dose group, the relative weights of brain, liver and kidneys were significantly increased; compared with the negative control group, absolute weight of VP of male rats in BIF high dose group was significantly reduced, relative weight of brain, liver and kidneys was significantly increased. Pathology changes observed include: thyroid follicular hyperplasia in females in BIF high dose group; prostate lesions, thyroid follicular hyperplasia in male rats in all BIF groups (low, medium and high dose), and crastration cells in pituitary in male rats in BIF medium and high dose group. The level of serum FT3 of female rats in BIF medium dose group was reduced, the levels of serum TT4, FT3 and FT4 in BIF high dose group were significantly reduced; the levels of serum TT3 and FT3 of male rats in BIF medium and high dose group were significantly reduced; the levels of serum TT4 and FT4 in BIF high dose group were also significantly reduced.Changes were observed in the ultrastructure of arcuate nucleus, pituitary and ovary of rats in BIF high dose group.Conclusion:Tier 1 screening1. Uterotrophic assay: to detect estrogenic/anti-estrogenic chemicals. EE, as the positive control, has estrogenic activity; BIF may have estrogenic-like activity.2. Herhsberger assay: to detect androgenic/anti-androgenic chemicals. Two positive controls: TP has androgenic effects, while FLU has anti-androgenic effects;BIF may have anti-androgenic and anti-thyroid effects.3. Pubertal Development and Thyroid Function in Intact Juvenile/ Peripubertal Female Rats (Female pubertal assay): to detect estrogenic/anti-estrogenic also anti-thyroid chemicals. Three positive controls: EE has estrogenic effects, TAM has anti-estrogenic and partial estrogenic effects, PTU has anti-thyroid hormone effects;BIF may have estrogenic-like and anti-thyroid effects.4. Pubertal Development and Thyroid Function in Intact Juvenile/ Peripubertal Male Rats (Male pubertal assay): To detect androgenic/anti-androgenic also anti-thyroid chemicals. Three positive controls: TP has androgenic effects,while FLU has anti-androgenic effects, PTU has anti-thyroid hormone effects; BIF may have anti-androgenic and anti-thyroid hormone effects.Conclusion of Tier 1 screening: BIF may have estrogenic, anti-androgenic and anti-thyroid hormone effects.Tier 2 TestingExtended One-Generation Reproductive Toxicity Study: to more definitively identify and characterize the potential hazard on the endocrine system.The results showed the estrogen-like, anti-androgen and anti-thyroid hormone interference effects of BIF on the endocrine system in rats. In the following reversibility test, the effect of tremors was reversible; however, the reversibility of endocrine disruption is not obvious.In this study, the lowest observed adverse effect level (LOAEL) of BIF was 1.0 mg/kg bw/d.Base on our study, the Mammalian in Vivo of Endocrine Disruptor Screening System is as follows:Note:1. If Tier 1 Screening has positive result(s), then move onto Tier 2 Testing.2. In Hershberger assay, the thyroid hormone endpoints also can be detected, for screening anti-thyroid chemicals. |
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