N-hydroxymethylglycinate with EDTA is an efficient eye drop preservative with very low toxicity: an in vitro comparative study

Abstract

Purpose: Preservatives are used in multi-dose ophthalmic topical medications in order to prevent contamination by bacteria and fungi. However, prolonged use of preserved eye drops, as it may happen in dry eye or glaucoma, may damage cells of the ocular surface. Therefore, an important goal is to find preservatives with low toxicity which are mild to host cells, still able to prevent drug contamination so to maintain their sterility and efficacy. Hence, aim of this study has been to compare the relative toxicity on a rabbit corneal cell line of a new preservative, made by the association of N-hydroxy-methyl-glycinate (NIG) with disodium-ethylene diamine tetra-acetate (EDTA), with other known and widely used eye-drops preservatives.

Materials and methods: Rabbit corneal cells (SIRC) were tested either in 96-well plates or in suspension culture. Treatments with preservatives (used at known bacteriostatic concentrations) included: benzalkonium chloride (BAK), polyquaternium-1 (PQ-1), sodium perborate (SP: NaBO_{3 *} H₂O), and NIG ± EDTA at different concentrations (0.001% and 0.002%), and different treatment times (from 30 minutes to 120 hours). At the end of treatment, cell survival was evaluated by a specific spectrophotometric method through the metabolic conversion of MTT [3-(4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide] into formazan crystals.

Results: Almost no cell toxicity was evident for NIG and SP at either concentration (0.001% or 0.002%), while a low toxicity was observed for PQ-1 (62% at the highest dose at 120 hours). BAK, as expected, showed the highest toxicity (60-80% at 30 minutes, and over 90% from eight hours onward). EDTA 0.1% alone or in combination with NIG 0.002%, showed no toxicity at 24 hours, and even resulted in cell growth promotion (46% and 38%, respectively), after 48 hours of treatment.

Conclusions: These data show that the new preservative NIG/EDTA, at doses known to have effective antimicrobial properties, has a very low toxicity on corneal cells, and so it can be safely used in multi-dose eye drops.

Keywords: EDTA; MTT; NIG; SIRC; eye drops; preservatives.