Genotoxicity evaluation of orally administered styrene monomer in mice using comet, micronucleus, and Pig‐a endpoints

Abstract

Male B6C3F1 mice were administered styrene monomer by oral gavage for 29 consecutive days at dose levels of 0, 75, 150, or 300 mg/kg/day. The highest dose level represented the maximum tolerated dose based on findings in a 28‐day dose range‐finding study, in which the bioavailability of orally administered styrene was also confirmed. The positive control group received ethyl nitrosourea (ENU; 51.7 mg/kg/day) on Study Days 1–3 and ethyl methanesulfonate (EMS; 150 mg/kg/day) on Study Days 27–29 by oral gavage. Approximately 3 h following the final dose, blood was collected to assess erythrocyte Pig‐a mutant and micronucleus frequencies. DNA strand breakage was assessed in glandular stomach, duodenum, kidney, liver, and lung tissues using the alkaline comet assay. The %tail DNA for stomach, liver, lung, and kidney in the comet assay among the styrene‐treated groups was neither significantly different from the respective vehicle controls nor was there any dose‐related increasing trend in any of the tissues; results for duodenum were interpreted to be inconclusive because of technical issues. The Pig‐a and micronucleus frequencies among styrene‐treated groups also did not show significant increases relative to the vehicle controls and there was also no evidence for a dose‐related increasing trend. Thus, orally administered styrene did not induce DNA damage, mutagenesis, or clastogenesis/aneugenesis in these Organization of Economic Co‐operation and Development test guideline‐compliant genotoxicity studies. Data from these studies can contribute to the overall assessment of genotoxic hazard and risk posed to humans potentially exposed to styrene.

Investigations on the genotoxic potential of styrene in Fischer 344 rats using multiple endpoints

“…The general principle and methodology described in Gollapudi et al (2015) and Dertinger et al (2021) were followed. Details of procedures for the quantification of Pig-a mutant frequencies in red blood cells (RBC) and reticulocytes (RETIC) were described in Gollapudi (2023). Freezing and thawing of blood samples followed the recommendations of Litron Laboratories (Rochester, NY) from whom the reagents (Rodent Blood Freezing and Thawing Solutions) were obtained.…”

“…The frequencies of micronucleated reticulocytes (MN-RETIC) in the blood samples were determined by flow cytometry and using Litron's MicroFlowPLUS Kit reagents per the kit's instructional manual with the minimal modifications described in Gollapudi (2023). Approximately 10,000 immature CD71-positive RETIC were analyzed to determine the frequency of micronucleated RETIC (MN-RETIC).…”

“…Overall while styrene itself is not mutagenic or clastogenic either in vitro or in vivo, it did yield some positive genotoxic results in vitro in the presence of metabolic activation presumably due to the formation of the reactive styrene oxide (SO). Recently, Gollapudi (2023) reported that styrene did not induce DNA strand breakage, clastogenicity/aneugenicity, or mutagenicity in mice Acceted by: B. Engelward.…”