Statement of problem: Novel methods for manufacturing intraoral appliances, such as 3-dimensional (3D) printing, offer numerous benefits, but the issue of leaching substances from 3D printed dental polymers requires investigation. Purpose: This study aimed to evaluate the chemical stability of intraoral appliances via monitoring the eluates, weight changes, and in vitro cellular responses. Material and methods: Three materials were compared: Dental LT Clear (for 3D printing), Duran+Durasplint LC (for thermoforming and light-activated polymerization), and Villacryl H Plus (for conventional heat-activated polymerization). After post-processing (no treatment, 24 hours immersion in water, chemical, or ultraviolet C disinfection), specimens were extracted in artificial saliva or methanol for 1, 3, 7, and 30 days at 37 °C. The leachates were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the percentage weight increase was calculated. The cytotoxic effects induced in human gingival fibroblasts (HGFs) were assessed using a 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay and microscopic observation. Two-way and 1-way analyses of variance (ANOVAs) with the post hoc Tukey or Dunnett test was conducted to statistically evaluate the results (α=.05). Results: The release of 7 chemicals (methyl methacrylate (MMA), ethyl methacrylate (EMA), diurethane dimethacrylate (UDMA), 1,4-butanediol dimethacrylate (BDMA), benzoyl peroxide (BPO), diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), and 4-methoxyphenol (MEHQ)) was detected at a level that enabled quantification. The concentration of substances released from Villacryl H Plus was the highest (BPO:12.6 mg/mL). Duran+Durasplint LC was the most stable material. All materials released more substances into methanol than artificial saliva. Dental LT Clear was most susceptible to weight increase upon immersion in artificial saliva (weight change: 2.62%; P<.001), while Villacryl H Plus – in methanol (weight change: 25.15%; P<.001). All the leachates detected exhibited a cytotoxic effect on HGFs. For the lower concentration (0.005 mg/mL), the strongest reduction of the viability of cells was induced by trimethylolpropane triacrylate (TMPTA) (decrease to 47%) and BPO (to 52%). Conclusions: Duran+Durasplint LC revealed favorable chemical stability. For each novel material, the potential biological risks associated with the leakage of compounds must be carefully assessed to ensure the safety of long-term use in intraoral conditions.