Oncogenicity Testing of 2-Ethylhexanol in Fischer 344 Rats and B6C3F1 Mice

2-Ethylhexanol (2EH) is a weak nongenotoxic hepatic peroxisomeproliferator in the rat. It is a high-volume chemical intermediatein the preparation of the plasticizers bis-(2-ethylhexyl) adipate(DEHA), bis-(2-ethylhexyl) phthalate (DEHP), and tris-(2-ethylhexyl)phosphate (TEHP), which are weak hepatocellular tumorigens infemale mice. In consequence, the oncogenic potential of 2EHwas evaluated in male (M) and female (F) rats and mice (50 animals/sex/group).Oral gavage doses of 2EH in 0.005% aqueous Cremophor EL (polyoxyl-35castor oil) were given five times a week to rats: 0 (water),0 (vehicle), 50, 150, and 500 mg/kg for 24 months, and to mice:0 (water), 0 (vehicle), 50, 200, and 750 mg/kg for 18 months.Statistical comparisons of data were made between vehicle controlsand treatment groups. There were no differences of biologicalsignificance between data from vehicle and water control groups.In rats, there were no dose-related changes at 50 mg/kg. Therewas reduced body weight gain at 150 mg/kg (M, 16; F, 12%) and500 mg/kg (M, 33; F, 31%) and an increased incidence of lethargyand unkemptness. There were dose-related increases in relativeliver, stomach, brain, kidney, and testis weights at sacrifice.Female rat mortality was markedly increased at 500 mg/kg. Therewas marked aspiration-induced bronchopneumonia in rats at 500mg/kg; hematologic, gross, and microscopic changes, includingtumors, were otherwise comparable among all rat groups. In miceat 50 and 200 mg/kg there were no dose-related changes and essentiallyno time-dependent or time-independent adverse trends in livertumor incidence at the 5% significance level. At 750 mg/kg mousebody weight gain was reduced (M, 26; F, 24%), and mortalityincreased (M and F, 30%) versus vehicle controls. At 750 mg/kgthere was a slight increase in nonneoplastic focal hyperplasiain the forestomach of mice (M 5/50, F 4/50) versus vehicle controls(M 1/50, F 1/50). There were increases in mouse relative liver(F, 21%) and stomach (M, 13%; F, 19%) weights at 750 mg/kg.There was a 12% incidence of hepatic basophilic foci and an18% incidence of hepatocellular carcinomas in male mice at 750mg/kg, not statistically significant compared with either controlby Fisher's exact test. There was a 12% incidence of hepaticbasophilic foci and a 10% incidence of hepatocellular carcinomasin female mice at 750 mg/kg, statistically significant (p <0.05) compared with vehicle but not with water controls by Fisher'sexact test. There were no metastases. Time-dependent and -independentstatistical analyses showed an adverse trend in the incidenceof hepatocellular carcinomas in male and female mice, correlatedwith toxicity (expressed as mortality) at 750 mg/kg. The time-adjustedincidence of hepatocellular carcinomas in male mice (18.8%)was within the historical normal range at the testing facility(0–22%), but that in females (13.1%) lay outside the normalrange (0–2%). Under the conditions of these studies 2EHwas not oncogenic in rats, but there were weak adverse trendsin hepatocellular carcinoma incidence in mice at high dose levelswhich may have been associated with toxicity. The major effectsof chronic dosing were mortality in female rats at 500 mg/kgand in male and female mice at 750 mg/kg, accompanied by reductionsin body weight gain in rats at 150 and 500 mg/kg and in miceat 750 mg/kg. Direct comparison of any tumorogenic effects of2EH given alone to female mice with those due to 2EH formedin vivo from DEHA, DEHP, or TEHP is limited by the high mortalitycaused by 2EH in female mice at equivalent doses of 2EH. While2EH may be a contributing factor in the hepatocellular carcinogenesisin female mice associated with the chronic administration ofDEHA and DEHP, it is unlikely to be the entire proximate carcinogen.     

The Developmental Toxicity of 2-Ethyihexanol Applied Dermally to Pregnant Fischer 344 Rats

Undiluted 2-ethylhexanol (2-EH) was administered by occludeddermal application for 6 hr per day on Gestation Days 6 through15 to pregnant Fischer 344 rats, in range-finding (R) and main(M) studies. The dermal route is considered to be the most relevantfor human exposure. Treatment levels were (R) 0.0, 0.5, 1.0,2.0, and 3.0 ml/kg/day (equivalent to 0, 420, 840, 1680, and2520 mg/kg/day) and (M) 0.0, 0.3, 1.0, and 3.0 ml/kg/day (equivalentto 0, 252, 840, and 2520 mg/kg/day). Controls (0.0 ml/kg/day,sham controls) received deionized water at 3.0 ml/kg/day. Dermal-positivecontrol groups received undiluted 2-methoxyethanol (2-ME) at(R) 0.5 and 1.5 ml/kg/day and (M) 1.0 ml/kg/day as a referencecompound in a similar regimen. An oral reference compound, valproicacid, was administered by gavage in the range-finding studyon Gestation Days 6 through 15 at 400 mg/kg/day. The range-findingstudy employed an untreated (naive) control group. Numbers ofplug-positive females per group were (R) 8 and (M) 25. Maternalweight gain was reduced for 2-EH at 1680 (R) and 2520 (R andM studies) mg/kg/day. Exfoliation and encrustation were seenat the application site in both studies at 840, 1680, and 2520mg/kg. Maternal liver, kidney, thymus, spleen, adrenal, anduterine weights, and gestational and fetal parameters were unaffectedby treatment with 2-EH. There were no treatment-related increasesin the incidence of individual or pooled external, visceral,and skeletal malformations or variations following the applicationof 2-EH. The NOAELs for the maternal toxicity of 2-EH were 252mg/kg/day based on skin irritation and 840 mg/kg/day based onsystemic toxicity. The developmental toxicity NOAEL was at least2520 mg/kg/day, with no teratogenicity. Administration of 2-MEat 840 mg/kg/day resulted in reduced maternal weight gain andfood consumption, increased postimplantation loss, reduced numbersof live fetuses per litter, and reduced fetal body weights perlitter. The incidence of fetal malformations and variationswas increased. Oral administration of VPA produced maternaltoxicity, developmental toxicity, and teratogenicity. The Fischer344 rat is thus susceptible to known rodent teratogens by boththe dermal and oral routes. It is concluded that 2-EH is notdevelopmentally toxic by the dermal route in the Fischer 344rat at and below treatment levels which produce maternal toxicity.     

Prechronic Toxicity Studies on 2-Ethylhexanol in F334 Rats and B6C3F1 Mice

Data on the subchronic toxicity of 2-ethylhexanol (2EH) wererequired to establish the dose vehicle and dose levels for oncogenicitystudies. In preliminary studies 2EH was given subacutely (11days) to male and female Fischer 344 rats and B6C3F1 mice asan aqueous emulsion by oral gavage (0, 100, 330, 1000, and 1500mg/kg/day). Clinical observations were made, body weights, foodconsumption, clinical chemistries, hematologies, and selectedorgan weights were measured, and gross and micropathologieswere performed. Target organs were the central nervous system,liver, forestomach, spleen, thymus, and kidney in rats and thecentral nervous system, liver, and forestomach in mice. 2EHwas then administered by oral gavage to male and female F344rats and B6C3F1 mice as an aqueous emulsion (0, 25, 125, 250,and 500 mg/kg/day) for 13 weeks. At 500 mg/kg/day in the ratthere was reduced body weight gain (6% male, 7% female), increasedrelative liver (29% male, 15% female), kidney (16% male, 6%female), stomach (11% male, 16% female), and testes (6%) weights,and moderate gross and microscopic changes in the liver andforestomach. There were no behavioral effects or effects onthe spleen or thymus. A no-effect level for target organ effectsin the rat was 125 mg of 2EH/kg/day. At 500 mg of 2EH/kg/dayin the mouse the only effects were increased relative stomachweights in males (13%) and a low incidence of gross and microscopicfindings in the forestomach (male and female) and liver (female).A no-effect level for target organ effects in the mouse was125 mg of 2EH/kg/day. 2EH was a peroxisome proliferator in therat but not in the mouse at subchronic dose levels of 500 mg/kg/day.Dose levels in oncogenicity studies were set at 50 mg/kg/dayfor the absence of treatment-related effects in rats and mice,and 500 and 750 mg/kg/day, respectively, in rats and mice ashigh doses producing minimal toxicity without altering the lifespan.