SynopsisObjectiveChallenge test (CT) is essential to assure the efficiency of the preservative system in products. A previous study realized by our staff in 2012, carried out to evaluate the influence of three parameters (ethanol, pH and water) on the microbiological cosmetics products conservation. Following this work, a correlation between aw (based on the glycerine concentration) and the selected parameter has been demonstrated. In the present study, smaller limits of ethanol, pH and glycerine were applied to determinate CT necessity.MethodsSixteen stables O/W cosmetics creams with different concentration of ethanol (1–19%), glycerine (3–16%) and different pH (6–11) were formulated. To evaluate the efficiency of the different formulations, CTs were performed according to the International Standard ISO 11930:2012. To determine the influence of the parameters, a D‐optimal plan generated by Design Expert® was applied. Design of Experiments software offers to plan, estimate and control the statistics and models for factorial and no‐factorial designs.ResultsChallenge tests results show that 10 formula passed criteria A, two passed criteria B and four are not conform. Mostly, an ethanol concentration higher than 16% exempts products of CT. It has been shown that an ethanol concentration between 10.5% and 16%, and an glycerine concentration >10%; or if the ethanol concentration is between 5% and 10.5%, glycerine is >6% and pH is ≥10, the CT is not required. Ethanol has a significant impact on conservation and especially when it is correlated with glycerine and pH. Finally, a glycerine concentration higher than 16% exempts products of CT.ConclusionFollowing the analysis of the different concentration, a correlation between glycerine and ethanol that directly influence microbiological protection of cosmetics products has been established. Indeed, by controlling ethanol, pH and glycerine, many products may be exempted from the CT.