Sub-chronic and developmental toxicity of transdermal delivery of Renzhu ointment in young SD rats

Abstract

Purpose: Renzhu ointment (Renzhuqigao, RZQG) is a patented herbal drug derived from Chinese traditional medicine formula and modern clinical experience for the transdermal treatment of non-infectious infantile diarrhoea. The safety of RZQG in preclinical studies has not been reported.

Materials and methods: In this study, the pups of parent rats were examined for sub-chronic toxicity and developmental toxicity. After 21 days of birth, they were exposed to RZQG through their abdominal skin at doses of 0.1, 0.3, and 0.9 g/kg/day for 4 weeks and then were observed for another four weeks during their recovery period.

Results: During the administration period, RZQG had no significant toxicological effect on body weight, food consumption, external eye examination, urinalysis, bone marrow examination, histopathology, central nervous system, reproductive system, or skeletal development. However, in the 0.9 g/kg/day group, the skin of some rats became dry and cracked, red and swollen, forming a white scab, while the white blood cells (WBC) count in female rats was lower and cholesterol (CHOL), triglycerides (TG), and glutamyl-transferase (GGT) were higher (p < 0.05).

Conclusions: Rats receiving 0.9 g/kg/day exhibited skin irritation and were suspected to have a mild liver injury. There was no evidence of delayed toxicity four weeks after withdrawal. Therefore, the no-observed adverse effect level of RZQG was 0.3 g/kg/day (30 times the clinical dose planned and 4.92 times the human equivalent dose).

Keywords:

• Renzhu ointment
• transdermal delivery
• sub-chronic toxicity
• developmental toxicity

Disclosure statement

The authors declare that we have no declaration of interest.

Supplementary material

Gas chromatography (GC) analysis of RZQG was performed. Reference standards, bornyl acetate (99.6%), atractylodin (99.7%), cinnamaldehyde (98.9%), and eugenol (99.0%) were purchased from China National Institute for Food and Drug Control (Beijing, China). Reserve solutions of 7.4 mg/ml cinnamaldehyde, 4.2 mg/ml bornyl acetate, 9.8 mg/ml eugenol, and 0.6 mg/ml atractylodes of these reference standards were prepared using anhydrous ethanol, and then diluted by 5 ml of each to 25 ml to form a mixed standard reserve solution and stored at 4 °C. RZQG (0.5 g) in 25 ml of ethanol was weighed precisely and ultrasonicated (280 W, 40 kHz) for 30 min. After standing for 30 min at 4 °C, the extract was left at room temperature and the loss of weight was replenished with ethanol. Then the sample was filtrated. The ointment lacking FA, RA, CC, FC (Guangzhou Zhixin Pharmaceutical Co., LTD, Guangzhou, China) were taken respectively, and the negative solution was prepared according to the preparation method of RZQG reserve solution. Shimadzu GC-2010 Plus gas chromatograph (Shimadzu Company, Kyoto, Japan) was used to analyse the four marker compounds in RZQG. A Phenomenex ZB-1ms capillary column (30 m × 0.25 mm, 0.25 μm) was used, the pressure was controlled at 100.0 kPa, the injector temperature was 240 °C, and the detector was flame ionisation detector (FID). The temperature rise procedure of the column is as follows: the initial temperature was 130 °C, and the temperature was kept for 10 min; The temperature was heated at 10 °C/min to 150 °C for 5 min; The temperature was heated at 10 °C/min to 180 °C for 5 min. The temperature was heated at 20 °C/min to 230 °C for 12.5 min. The split ratio and injection volume were 50:1 and 1 μL, respectively. Figure A.1, Table A.1 and Table A.2 record the qualitative and quantitative analysis of the main components of RZQG by Gas chromatography.

Figure B.1. Dosage design of 200 g rat. Table B.1. Effect of RZQG on the urinalysis of SD rats. Table C.1. Effect of RZQG on the blood coagulation of SD rats. Table D.1. Effect of RZQG on the bone marrow of SD male rats at rD29. Table E.1. Effects of RZQG on FOB in SD rats. Table E.2. Effects of RZQG on rotating test in SD rats. Table E.3. Effects of RZQG on inner open field test in SD rats. Table F.1. Effects of RZQG on the reproductive system in SD rats. Table G.1. Effects of RZQG on skeletal development in SD rats.

Additional information

Funding

The experimental scheme protocol was approved by the Guangzhou University of Chinese Medicine Ethics Committee [approval number 20210311001] and conformed to the Regulations of Guangzhou University of Chinese Medicine on Ethical Review of Animal Experiments. This research was funded by the Science and Technology Planning Project of Guangdong Province [2016B020239005], the Guangdong Provincial Ministry Industry University Research Combination Project [2013B090500119], and the National Nature Science Foundation of China [No. 81973830].