2-isopropyl-N,2,3-trimethylbutyramide (WS-23) is a well-known artificial synthesis cooling agent widely used in foods, medicines, and tobaccos. As a commonly cooling agent in e-cigarette liquids, WS-23 has led to concerns about the inhalation toxicity with the prosperous of e-cigarettes in recent years. Thus, the aim of this study is to assess the acute and subacute inhalation toxicity of WS-23 in Sprague–Dawley (SD) rats according to the Organization for Economic Cooperation and Development (OECD) guidelines. In the acute toxicity study, there was no mortality and behavioral signs of toxicity at the limit test dose level (340.0 mg/m³) in the exposure period and the following 14-day observation period. In the subacute inhalation toxicity study, there was no significant difference observed in the body weights, feed consumption, and relative organ weights. Haematological, serum biochemical, urine, and bronchoalveolar lavage fluid (BALF) analysis revealed the non-adverse effects after 28-day repeated WS-23 inhalation (342.85 mg/m³), accompanied by slight changes in few parameters which returned to normal during the 28-day recovery period. The histopathologic examination also did not show any differences in vital organs. In conclusion, the maximum tolerated dose for WS-23 acute inhalation is not less than 340.0 mg/m³, and the No Observed Adverse Effect Level (NOAEL) of WS-23 subacute inhalation was determined to be over 342.85 mg/m³.

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The authors declare that there are no conflicts of interest.

Supporting Information

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Table S1. The weight values of test SD rats in acute inhalation study (Mean ± SEM).

Table S2. The hematology parameters of female SD rats on 29th day in subacute study (Mean ± SEM, n = 6).

Table S3. The hematology parameters of female SD rats on 57th day in subacute study (Mean ± SEM, n = 4).

Table S4. The hematology parameters of male SD rats on 29th day in subacute study (Mean ± SEM, n = 6).

Table S5. The hematology parameters of male SD rats on 57th day in subacute study (Mean ± SEM, n = 4).

Table S6. The results of salient serum biochemistry parameters on 29th day from female SD rats (Mean ± SEM, n = 6).

Table S7. The results of salient serum biochemistry parameters on 57th day from female SD rats (Mean ± SEM, n = 4).

Table S8. The results of salient serum biochemistry parameters on 28th day from male SD rats (Mean ± SEM, n = 6).

Table S9. The results of salient serum biochemistry parameters on 57th day from male SD rats (Mean ± SEM, n = 4).

Table S10. The results of urine biochemical parameters on 28th day from SD rats (Mean ± SEM).

Table S11. The results of urine biochemical parameters on 56th day from SD rats (Mean ± SEM).

Table S12. The results of absolute organ weights (g) and organ body-weight ratios of SD rats on 29th day (Mean ± SEM).

Table S13. The results of absolute organ weights (g) and organ body-weight ratios of SD rats on 57th day (Mean ± SEM).

Figure S1. Effect of repeated nose-only inhalation of WS-23 for 28-days on histological sections after exposure period (29th day) (Hematoxylin and Eosin staining, 40X) of heart (A–C), liver (D–F), spleen (G–I) lung (J–L), kidney (M–O), brain (P–R), turbinate (S–U), trachea (V–X), and throat (Y–Z₁) of rats. Control group (Fresh air); Solvent group (PG:VG = 1:1); High-dose group (WS-23 = 29 mg/kg/day).

Figure S2. Effect of repeated nose-only inhalation of WS-23 for 28-days on histological sections after recovery period (57th day) (Hematoxylin and Eosin staining, 40X) of heart (A–C), liver (D–F), spleen (G–I) lung (J–L), kidney (M–O), brain (P–R), turbinate (S–U), trachea (V–X), and throat (Y–Z₁) of rats. Control group (Fresh air); Solvent group (PG:VG = 1:1); High-dose group (WS-23 = 29 mg/kg/day).

Figure S3 the graphs of the particle diameter distribution of PG, VG, and WS-23 in different groups in subacute inhalation study.

Figure S3–1 the particle diameter distribution of PG in the solvent group

Figure S3–2 the particle diameter distribution of VG in the solvent group

Figure S3–3 the particle diameter distribution of PG in the low-dose group (5% WS-23)

Figure S3–4 the particle diameter distribution of VG in the low-dose group (5% WS-23)

Figure S3–5 the particle diameter distribution of WS-23 in the low-dose group (5% WS-23)

Figure S3–6 the particle diameter distribution of PG in the high-dose group (10% WS-23)

Figure S3–7 the particle diameter distribution of VG in the high-dose group (10% WS-23)

Figure S3–8 the particle diameter distribution of WS-23 in the high-dose group (10% WS-23)

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Acute and subacute inhalation toxicity assessment of WS-23 in Sprague–Dawley rats

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Acute and subacute inhalation toxicity assessment of WS-23 in Sprague–Dawley rats

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