Preclinical Toxicology Studies with Acyclovir: Genetic Toxicity Tests

Preclinical Toxicology Studies with Acyclovir: Genetic ToxicityTests. Clive, D., Turner, N.T., Hozier, J., Batson, A.G. andTucker, W.E., Jr. (1983). Fundam. Appl. Toxicol 3: 587–602.Acyclovir (ACV), an antiviral drug active in the treatment oforal and genital Herpes infections, has been evaluated for mutagenicand carcinogenic potential in a battery of in vitro and in vivoshort-termassays. Negative results were obtained in the following in vitrotests: Ames Salmonella, plate incorporation and preincubationmodification assays; E. coli polA⁺/polA^– DNA repair; yeast(S. cerevisiae D4) gene conversion; Chinese hamster ovary cells(HGPRT, APRT loci and ouabain-resistance marker); L5178 Y mouselymphoma cells (HGPRT locus and ouabain-resistance marker);and C3H/10Tmouse fibroblast neo-plastic transformation assay.All except the last assay were performed in the presence andabsence of an exogenous metabolic activation system. ACV waspositive at high concentrations x exposure times in the absenceof exogenous metabolic activation in the following in vitrosystems and at the indicated concentrations: BALB/c-3T3 neoplastictransformation (50 /µg/mL, 72 h exposure); human lymphocytecytogenetics (250–500 µg/mL, 48 h exposure); andL5178Y mouse lymphoma cells (TK locus, 400–2400 µg/mL,4 h exposure; predominantly small colony mutants of chromosomalorigin produced). No effects were seen in vivo (mouse dominantlethal assay; rat and Chinese hamster bone marrow cytogenetics)at up to maximum tolerated doses (MTD). An unusual clastogeniceffect was seen in Chinese hamsters at 5 times the MTD. Overall,positive effects were seen only at either high concentrations(250 µg/mL in vitro or plasma levels) or prolonged exposure(72 hr in the BALB/ c-3T3 neoplastic transformation assay).These studies support the view that ACV is a chromosomal mutagen,i.e., one which causes multi-locus damage but not single geneeffects. The significance of these results for the genetic riskof ACV to man is discussed.     

Nonclinical Toxicology Studies with Zidovudine: Genetic Toxicity Tests and Carcinogenicity Bioassays in Mice and Rats

Zidovudine (ZDV), an antiviral drug active in the treatmentof acquired immunodeficiency syndrome (recommended human dose,100 mg every 4 hr while awake), was evaluated for mutagenicand carcinogenic potential in a battery of short-term in vitroand in vivo assays and in lifetime studies in mice and rats.In L5178Y mouse lymphoma cells (tk^+/– locus), a weak positiveresult was obtained only at the highest concentrations tested(4000 to 5000 µg/ml) in the absence of metabolic activation.In the presence of metabolic activation, the drug was weaklymutagenic at concentrations of 1000 µg/ml and higher.Following 24 hr treatment in the absence of metabolic activation,ZDV was moderately mutagenic at concentrations up to 600 µg/ml;dose-related structural chromosomal alterations were seen atconcentrations of 3 µg/ml and higher in cultured humanlymphocytes. Such effects were not noted at the two lowest concentrationstested, 0.3 and 1 µg/ml, and BALB/c-3T3 cells were transformedat concentrations of 0.5 µg/ml and higher. No effectswere seen in the Ames Salmonella plate incorporation and preincubationmodification assays (possibly due to bacteriocidal activityof ZDV at low concentrations) at concentrations ranging from0.01 to 10 µg/plate or in a single-dose intravenous bonemarrow cytogenetic assay in CD rats. In multidose micronucleusstudies, increases in micronucleated erythrocytes were seenin mice at doses of 100 to 1000 mg/kg/day. Similar results wereseen in rats and mice after 4 or 7 days of dosing at 500 mg/kg/day.In carcinogenicity bioassays, adjusted doses of 20, 30, or 40mg/kg/day and 80, 220, and 300 mg/kg/day were given to CD-1mice and CD rats, respectively, for up to 22 months in miceand 24 months in rats. ZDV caused a macrocytic, normochromicanemia in both species. No evidence of carcinogenicity was seenin male mice or rats. In female mice, five malignant and twobenign vaginal epithelial neoplasms occurred in animals given40 mg/kg/day. A single benign vaginal epithelial tumor was seenin a mouse given 30 mg/kg/day. In rats, two malignant vaginalepithelial neoplasms were seen in animals given 300 mg/kg/day.In a 7-day study in mice, ZDV was shown to be devoid of estrogenicactivity. In an oral pharmacokinetics study, the AUC was 17and 144 µg/ml hr in female mice and rats given 40 or 300mg/kg of ZDV, respectively. In contrast, the average steady-stateconcentration in humans at the recommended daily dose is 0.62µg/ml. Twenty-four hour urine concentrations were 1245and 4417 µg/ml in female mice and rats given 40 or 300mg/kg of ZDV, respectively. These values were approximately26-and 136-fold higher than the human urine concentration atthe recommended daily dose. In a one- to three-day study withintravenously administered sodium fluoroscein in rats and mice,retrograde flow of urine into the vagina was demonstrated. Ina subsequent lifetime carcinogenicity bioassay in mice in whichZDV was given intravaginally at concentrations of 5 or 20 mgZDV/ml in saline, 13 vaginal squamous cell carcinomas were seenat the highest concentration tested. It was concluded that thevaginal tumors seen in the oral carcinogenicity studies werethe result of chronic local exposure of the vaginal epitheliumto high urine concentrations of ZDV.     

Potentiated Toxicity of 2-sec-Butylphenyl Methylcarbamate (BPMC) by O,O-Dimethyl O-(3-Methyl-4-nitrophenyl)phosphorothioate (Fenitrothion) in Mice; Relationship between Acute Toxicity and Metabolism of BPMC

Potentiated Toxicity of 2-sec-Butytpbenyl Methylcarbamate (BPMC)by O,O-Dimethyl O-(3-Methyl-4-nitrophenyl)phosphorothioate (Fenitrothion)in Mice; Relationship between Acute Toxicity and Metabolismof BPMC. TAKAHASHI, H., MIYAOKA, T., TSUDA, S., AND SHIRASU,Y. (1984). Fundam. Appl. Toxicol. 4, 718–723. Fenitrothionof oral subtoxic dose (100 mg/kg; 4 hr pretreatment) decreasedacute oral LD50 of BPMC from 360 to 66 mg/kg in male mice. Thetreatment prolonged the hexobarbital sleeping time and increasedthe plasma BPMC concentrations. The BPMC toxicity and its plasmaconcentrations were significantly reduced by phenobarbital treatment(80 mg/kg/day, 2 days, ip). This treatment diminished the effectsof fenitrothion on BPMC toxicity and plasma BPMC concentrations.BPMC was metabolized by mixed-function oxidases of the liverin vitro. The metabolism of BPMC was competitively inhibitedby the addition of fenitrothion (5 µg/ml). Fenitrothionremained in the liver (7 µg/g liver). These results suggestthat competitive inhibition of BPMC metabolism by fenitrothionmay, at least in part, play a role in inhibition of BPMC detoxication,resulting in potentiation of its toxicity.     

Synthesis and biological activity of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine deoxyuridine diastereomers.

The synthesis of the two diastereomers (9 and 10) of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine are described. Their antiviral and cytotoxic activities were determined, in comparison with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 5-fluoro-2'-deoxyuridine (FDU), respectively. 5-[(1R)-2,2-Difluorocyclopropyl]-2'-deoxyuridine (10) was the most active antiviral agent against HSV-1 (IC50 = 5 micrograms/ml) relative to BVDU (IC50 = 0.082 micrograms/ml), and cytotoxic agent in the CCRF-CEM (IC50 = 230 microM) screen relative to FDU (IC50 = 4.7 x 10(-3) microM). The 5-[(1S)-2,2-difluorocyclopropyl] diastereomer was inactive in both screens. Partition coefficients (P) and affinity for the mouse erythrocyte nucleoside transporter (Ki) were not determinants of antiviral or cytotoxic activities. However, the (1R)-diastereomer (10) was more resistant to glycosidic bond cleavage by thymidine phosphorylase than the (1S)-diastereomer (9).     

Sublethal Acute Toxicity of Carbosulfan [2,3-dihydro-2,2-dimethyl-7-benzofuranyl(di-n-butylaminosulfenyl)(methyl)carbamate] in the Rat after Intravenous and Oral Exposures

Sublethal Acute Toxicity of Carbosulfan [2,3-dihydro-2,2-dimethyl-7-benzofuranyl(di-n-butylaminosulfenyl)(methyl)carbamate]in the Rat after Intravenous and Oral Exposures. RENZI, B. E.,AND KRIEGER, R. I. (l986). Fundwn. Appl. Toxicol. 6, 7–15.Sublethal toxicity of carcarbosulfan, 2,3-dihydro-2,2-dimethyl-benzofuranyl(di-n-butylaminosulfenyl)(methyl)carbamate,was evaluated in female Sprague-Dawley rats. Erythrocyte acetylcholinesterase(AChE) activity was maximally inhibited 1 min after iv administration(38, 23, and 15% of pretreatment activity after 86, 250, and690 µg/kg, respectively) and recovered by 4 hr. MaximumAChE inhibition (63% of pretreatment activity) was measured45 min after oral dosing (690 µg/kg) and activity recoveredafter 5 hr. Signs included urination, defecation, facial musclefasciculations, salivation, and tremors. Carbosulfan was lesstoxic when given orally. Metabolic activation of carbosulfanto carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate)was in vestigated by measuring plasma concentrations 4, 30,and 240 min after iv (80–120 or 620–640 µg/kg)and oral (540–700 or 2030–2190 µ/kg) dosagesof [carbonyl-¹⁴C]carbosulfan. Peak plasma concentrations weremeasured at 4 and 30 min after iv and oral exposure, respectively.Carbosulfan was rapidly activated to carbofuran. Reduction inAChE activity was better correlated (r = 0.97) with plasma concentrationof [carbosulfan + carbofuran] and plasma carbofuran (r = 0.96)than with plasma carbosulfan (r = 0.73). Signs generally occurredwhen AChE activity was less than 65% of pretreatment levels,corresponding to 40 pmol/ml [carbosulfan + carbofuran] in plasma.Based on regression analysis and metabolic studies, both carbosulfanand carbofuran contributed to the observed AChE inhibition;however, carbofuran, a more potent in vitro inhibitor and theusual predominant inhibitor in plasma, was responsible for mostof the erythrocyte AChE inhibition.     

Glyphosate Skin Binding, Absorption, Residual Tissue Distribution, and Skin Decontamination

Glyphosate Skin Binding, Absorption, Residual Tissue Distribution,and Skin Decontamination. Wester, R. C., Melendres, J., Sarason,R., McMaster, J., and Maibach, H. I. (1991). Fundam. Appl. Toxicol.16, 725–732. Glyphosate is a broad-spectrum postemergencetranslocated herbicide. Its interactions with skin and potentialsystemic availability through percutaneous absorption was studiedby skin binding, skin absorption, residual tissue distribution,and skin decontamination. Glyphosate in a final formulation(Roundup) undiluted and diluted with water 1:20 and 1:32, wouldnot partition into powdered human stratum corneum (<1%).In vitro percutaneous absorption through human skin into humanplasma as receptor fluid was no more than 2% over a concentrationrange of 0.5–154 µg/cm² and a topical volume rangeof 0.014–0.14 ml/cm². Disposition of glyphosate followingiv administration of 93 and 9 µg doses to rhesus monkeyswas mainly through urine excretion, 95 ± 8 and 99 ±4% in 7 days, respectively. Percutaneous absorption in vivoin rhesus monkey was 0.8 ± 0.6% for the low dose (25µg/cm²) and 2.2 ± 0.8% for the high dose (270 µg/cm²).No residual ¹⁴C was found in organs of the monkeys euthanized7 days after the topical application. Washing the skin applicationsite with soap and water removed 90 ± 4% of applied dose,and washing with water only removed 84 ± 3% of applieddose. Both soap and water and water only were equal in abilityto remove glyphosate from skin over a 24 hr skin applicationperiod. About 50% of the initially applied dose could be recoveredafter 24 hr. Glyphosate is very soluble in water and insolublein most organics (octanol/water log P = –1.70) and thereforenot compatible with the lipid-laden stratum corneum. This isconsistent with the low skin binding and skin absorption andalso consistent with the efficient removal from skin with soapand water or water-only wash.     

Structure Elucidation and in Vitro Reactivity of the Major Metabolite of 4,4'-Methylenebis(2-chloroaniline) (MBOCA) in Canine Urine

Structure Elucidation and in Vitro Reactivity of the Major Metaboliteof 4,4'-Methylenebis(2-chloroaniline) (MBOCA) in Canine Urine.Manis, M. O., and Braselton, W. E. Jr. (1984). Fundam. Appi.Toxicol. 4, 1000–1008. The structure of the major urinarymetabolite of 4,4'-methylenebis(2-chloroaniline) (MBOCA) indogs was identified and the reactivity of the metabolite wascharacterized in vitro. Arylsulfatase but not ß-glucuronidasehydrolyzed the metabolite in a time- and enzyme concentration-dependentmanner. Electron impact mass spectrometry following derivatizationand transesterification indicated that the major metabolitewas ring hydroxylated and fas atom bombardment mass spectrometryconfirmed the molecular weight as a sulfate ester. Proton nuclearmagnetic resonance studies indicated that the ring substitutionwas ortho to an arnine. These analytical and enzymatic datasupported the proposed structure of the major urinary metaboliteof MBOCA in dogs as 5-hydroxy-3,3'-dichloro-4,4'-diaminodiphenylmethane-5-sulfate.Protein and DNA binding in vitro and mutagenicity were investigated.During hydrolysis with arylsulfatase, time- and enzyme concentration-dependentprotein binding and time-dependent DNA binding were observed.Mutagenicity during enzymatic hydrolysis in the presence ofSalmonella typhimurium TA1538 with up to 20 µg metabolite/platewas negative and at 50 µ/plate the metabolite was cytotoxic.These results indicated that the metabolite was the sulfateconjugate of a reactive molecule. This study demonstrated thatthe major metabolite of MBOCA in canine urine is an ortho-hydroxysulfaleand thus is similar to the major metabolites of bcnzkline, 2-naphthylamine,and 4-aminobiphenyl     

Absence of Mutagenic Activity for Monosodium Cyanurate

Absence of Mutagenic Activity for Monosodium Cyanurate. HAMMOND,B. G., BARBEE, S. J., WHEELER, A. G., AND CASCIERI, T. (1985).Fundam. Appl. Toxicol. 5, 655–664. The mutagenic potentialof monosodium cyanurate was evaluated using in vitro and invivo tests. All in vitro tests were carried out in the presenceand absence of metabolic activation. In each assay, the highestconcentration tested generally exceeded the solubility of monosodiumcyanurate in the incubation medium. In the Salmonella microbialassay, monosodium cyanurate was not mutagenic towards test strainsTA 98, 100, 1535, and 1537 up to a concentration of 10,000 µg/plate.Monosodium cyanurate did not induce forward mutations at theTK. locus of L5I78Y mouse lymphoma cells up to a concentrationof 2000 µg/ml. No significant increases in sister chromatidexchanges were observed when monosodium cyanurate was incubatedwith Chinese hamster ovary cells at concentrations up to 1500µg/ml. In an in vivo test, rats were administered monosodiumcyanurate by gavage at single dosages up to 5000 mg/kg and killed24 and 48 hr after dosing. Bone marrow cells were collectedand examined for chromosomal aberrations. At the time pointsexamined, there was no evidence of monosodium cyanurate-inducedchromosomal aberrations in rat bone marrow cells     

In Vivo and in Vitro Percutaneous Absorption and Skin Decontamination of Arsenic from Water and Soil

The objective was to determine the percutaneous absorption ofarsenic-73 as H₃A_sO₄ from water and soil. Soil (Yolo County65-California-57-8) was passed through 10-, 20-, and 48-meshsieves. Soil retained by 80 mesh was mixed with radioactivearsenic-73 at a low (trace) level of 0.0004 µg/cm² (microgramsarsenic per square centimeter skin surface area) and a higherdose of 0.6 µg/cm². Water solutions of arsenic-73 at alow (trace) level of 0.000024 µg/cm² and a higher doseof 2.1 µg/cm² were prepared for comparative analysis.In vivo in Rhesus monkey a total of 80.1 ± 6.7% (SD)intravenous arsenic-73 dose was recovered in urine over 7 days;the majority of the dose was excreted in the first day. Withtopical administration for 24 hr, absorption of the low dosefrom water was 6.4 ± 3.9% and 2.0 ± 1.2% fromthe high dose. In vitro percutaneous absorption of the low dosefrom water with human skin resulted in 24-hr receptor fluid(phosphate-buffered saline) accumulation of 0.93 ± 1.1%dose and skin concentration (after washing) of 0.98 ±0.96%. Combining receptor fluid accumulation and skin concentrationgave a combined amount of 1.9%, a value less than that in vivo(6.4%) in the Rhesus monkey. From soil, receptor fluid accumulationwas 0.43 ± 0.54% and skin concentration was 0.33 ±0.25%. Combining receptor fluid plus skin concentrations gavean absorption value of 0.8%, an amount less than that with invivo absorption (4.5%) in the Rhesus. These absorption valuesdid not match current EPA default assumptions. Washing withsoap and water readily removed residual skin surface arsenic,both in vitro and in vivo. The partition coefficient of arsenicin water to powdered human stratum corneum was 1.1 x 10⁴andfrom water to soil it was 2.5 x 10⁴. This relative similarityin arsenic binding to powdered human stratum corneum and soilmay indicate why arsenic absorption was similar from water andsoil. This powdered human stratum corneum partition coefficientmodel may provide a facile method for such predictions.     

The Fate of Chronically Consumed Caffeine in the Monkey (Macaca fascicularis)

The Fate of Chronically Consumed Caffeine in the Monkey (Macacafascicularis). GILBERT, S. G., STAVRIC, B., KLASSEN, R. D.,AND RICE, D. C. (1985) Fundam. Appl. Toxicol. 5, 578–587.The metabolic fate of chronically administered caffeine wasexamined in monkeys. Caffeine and equal parts of citric acidwere added to the drinking water of four female monkeys (Macacafascicularis). The concentration was gradually increased overa 10-week period to 0.35 mg/ml for three of the monkeys. A monkeythat was lactating, but had no infant, was exposed to caffeinein the drinking water at a concentration of 0.30 mg/ml. At thesedoses, administered for up to 50 weeks, there were no overtsigns of toxicity as indicated by food and fluid consumption,body weight, or general condition of the monkey. Mean plasmacaffeine concentrations were 3.8, 5.7, and 5.9 µg/ml,while mean plasma theophylline concentrations were 11.8, 13.0,and 20.1 µ/ml. respectively for the monkeys receiving0.35 mg/ml. Mean plasma caffeine and theophylline concentrationsfor the lactating monkey were 10.7 and 21.4 µg/ml, whilemean milk concentrations were 10.5 and 17.6 µ/ml, respectively,indicating that caffeine and its major metabolite theophyllineare readily excreted in milk. The high plasma theophylline levelsindicate that caffeine metabolism in the monkeys differs fromthat in humans. Theophylline was the main urinary metabolite.In addition, large amounts of 1,3-dimethyluric acid were excretedin the urine but only traces of this metabolite were found inthe plasma. After withdrawal of caffeine, plasma caffeine levelsdecreased to almost zero in the first 24 hr with a half-lifeof 5.5 hr, and plasma theophylline levels declined with a half-lifeof 12.7 hr     

Bacterial and Human Cell Mutagenicity and Mouse Lung Tumorigenicity of the Oxygenated Polynuclear Aromatic Hydrocarbon Phenalenone

Phenalenone (perinaphthenone) is a major oxygenated polynucleararomatic hydrocarbon (oxy-PAH) atmospheric pollutant formedfrom the combustion of fossil fuels. Mutagenicity of phenalenonewas measured in quantitative forward mutation assays with Salmonellatyphimurium TM677 and metabolically competent human B-lymphoblastoidcell lines (MCL-5 and hlAlv2 cells), and its tumorigenicitywas also assessed in a newborn mouse assay. Phenalenone wasmutagenic in Salmonella in the presence of rat liver postmitochondrialsupernatant (PMS) at a minimum detectable mutagen concentration(MDMC) of 12 /µg/ml, but was not mutagenic in the absenceof PMS at concentrations up to 100 /µg/ ml. Phenalenonewas not significantly mutagenic in either human cell line after28 hr treatment, although mutant fractions were increased bynearly fivefold in hlAlv2 cells (at the tk locus) exposed at30 µg/ml. However, after 72 hr treatment, phenalenonewas mutagenic at the hprt locus in hlAlv2 cells with an MDMCof 3 µg/ml Phenalenone was also tumorigenic in male BLU:Hamice with a lung tumor incidence of 33% 6 months after injectionwith 4.2 mg phenalenone, the highest dose tested. Lung tumormultiplicity in this treatment group was 0.5 tumor/mouse. Noincrease in lung tumors in female mice was observed. Indicesof lung tumor incidence (ED₅₀) and multiplicity (TM_1.0) formale mice were 29.3 and 34.9 µxmol, respectively. Thesedata suggest that phenalenone does not contribute significantlyto the mutagenicity or carcinogenicity of combustion emissionextracts.     

Allosteric Binding of Nickel(II) to Calmodulin

The binding of Ni(II) to calmodulin (CAM) in the presence andin the absence of Ca(II) was investigated by equilibrium dialysisin order to test the physicochemistry of direct Ni(II)-CAM interactionsthat might be responsible for the effects of this metal on CAMobserved in vivo. Samples containing 5 µm CAM, 5 mM Tris/HClbuffer (pH 7.4), and NaCl to maintain the ionic strength I =3600 µm, with or without 200 µm CaCl₂, were dialyzedat 37?C against 1–300 µm ⁶³NiCl₂. In the presenceof Ca(II), the CAM molecule has two binding sites for Ni(II)(K₁, = 7.25 ? 10⁵m^–1; = 3.79 ? 10³ M^–1) with markedcoopera-tivity (Hill coefficient = 1.20 ? 0.03 SE). In the absenceof Ca(II), a complicated Ni(II)-binding curve is obtained indicatingformation of many mutually interacting complex species. Bindingof Ni(II) to CAM in the presence of Ca(II) is inhibited slightlyby added MnCl₂ (50 µM) and very strongly by CuCl₂ andZnCl₂ (10 µm). To elucidate the mechanism of this inhibition,binding of Zn(II) (0.5–50 µm ⁶⁵ZnCl₂) to CAM inthe presence of Ca(II) (200 µM) was also studied. Themaximum molecular ratio of Zn(II) to CAM in the Zn(II)/Ca(II)/CAMcomplex approached 0.5. Thus, the observed inhibition by Zn(II)of the Ni(II) binding to Ca(II)/CAM does not involve competitionfor the same binding sites but is rather caused by a conformationalarrangement of CAM in its Ca(II)/Zn(II) complex that is differentthan the Ca(II) complex. This fact, as well as the observeddifference in binding of Ni(II) in the presence and absenceof Ca(II), stress the importance of conformation of the CAMmolecule to Ni(II) binding.     

Lack of Genotoxicity of the Cancer Chemopreventive Agent N-(4-Hydroxyphenyl)retinamide

Lack of Genotoxicity of the Cancer Chemopreventive Agent N-(4-Hydroxyphenyl)retinamide.PAULSON, J. D., OLDHAM, J. W., PRESTON, R. F., AND NEWMAN, D.(1985). Fundam. Appl. Toxicol. 5, 144–150. As part ofthe preclinical drug safety evaluation of the cancer chemopreventiveagent N(4-hydroxyphenyl)retinainide (HPR) in vitro and in vivotests were conducted to assess its genotoxic activity. Negativefindings from HPR testing were demonstrated in the Ames Salmonella/microosomalactivation test, the L5178Y mouse lymphoma assay, and a ratbone marrow cytogenetics study. These data imply that HPR lacksthe ability to induce point mutations or chromosomal aberrations,and is therefore not genotoxic. Limited testing of retinyl acetatein the Ames test, the L5178Y mouse lymphoma assay, and the primaryrat hepatocyte/DNA repair assay yielded consistently negativeresults. These findings and previously published results concerningretinoid genotoxicity are discussed.     

Percutaneous Absorption/Metabolism of Phenanthrene in the Hairless Guinea Pig: Comparison of in Vitro and in Vivo Results

Percutaneous Absorption/Metabolism of Phenanthrene in the HairlessGuinea Pig: Comparison of in Vitro and in Vivo Results. NG,K. M. E., CHU, I., BRONAUGH, R. L., FRANKLIN, C. A., AND SOMERS,D. A. (1991). Fundam. Appl. Toxicol. 16, 517–524. Thein vitro and in vivo percutaneous absorption/metabolism of phenanthrenewas investigated in hairless guinea pigs. Flow-through diffusioncells and Hepes-buffered Hanks' balanced salt solution (HHBSS)as receptor fluid were used in the in vitro system. When phenanthrenewas applied to excised guinea pig skin mounted on the cellsat dose levels of 6.6 and 15.2 µg/cm², 89.7 and 79.1%of the administered doses were respectively absorbed into theskin and receptor fluids during a 24-hr perfusion period. Theseresults are consistent with the in vivo data which showed approximately80% absorption over the same period of time. Phenanthrene wasmetabolized in vitro into phenanthrene 9,10-dihydrodiol, 3,4-dihydrodiol,1,2-dihydrodiol, and traces of hydroxy phenanthrenes. Of thematerials absorbed in vitro, 92% was the parent compound and7% the dihydrodiol metabolites. When a nonviable in vitro systemwas used, 68% of the applied 15.2 µg/cm² dose was absorbed.Data from the present study demonstrate that the in vitro systemis a good model for predicting in vivo percutaneous absorptionof phenanthrene, and that penetration of phenanthrene throughthe skin is controlled more by the passive rate of diffusionthan by metabolism.     

Deposition, Clearance, and Shortening of Kevlar Para-aramid Fibrils in Acute, Subchronic, and Chronic Inhalation Studies in Rats

The deposition and clearance of lung-deposited Kevlar paraaramidfibrils (subfibers) have been investigated as part of a subchronicand chronic inhalation toxicity testing program. Fibrils recoveredfrom lung tissue in para-aramid-exposed Sprague-Dawley ratswere microscopically counted and measured after exposures toairborne fibrils which were about 12 µm median length(ML) and <0.3 µm median diameter. In each of threestudies lung-recovered fibrils were progressively shorter withincreasing residence time in the lungs. Twenty-eight days aftera single 6-hr exposure at 400 respirable fibrils per cubic centimeter(f/cm³) the ML of recovered fibrils decreased to about 5 µm.Twenty-four months after a 3-week exposure to 25 or 400 (f/cm³)fibrils reached about 2 µm ML. After 2 years of continuousexposure at 2.5, 25, or 100 f/cm³ or 1 year exposure plus 1year recovery at 400 f/cm fibril ML approached 4 µm. Inthe 2-year study, the lung-fiber accumulation rate/exposureconcentration was similar for the three highest concentrationsand was about 3x greater than that seen at 2.5 f/cm³ indicatingthat concentrations of about 25 f/cm or more may overwhelm clearancemechanisms. Time required for fibrils to be reduced to <5µm in the lung was markedly less at lower exposure concentrationand shorter exposure time. The primary shortening mechanismis proposed to be long fibril cutting by enzymatic attack atfibril defects. However, length-selective fibril depositionand clearance may contribute to shortening in the first fewdays after exposure. The enzymatic cutting hypothesis is supportedby measured increases in numbers of short fibers following cessationof exposures, continued shortening of the fibril length distributionup to 2 years following exposure, and in vitro fibril shorteningafter 3 months in a proteolytic enzyme preparation. The conclusionis that para-aramid fibrils are less durable in the lungs ofrats than expected from the known chemical resistance of commercialyarn. These data suggest that at the low para-aramid fibrilexposures found in the workplace, this fibril-shortening mechanismmay limit the residence time of long fibers in the lungs ofexposed workers. In addition, associated cascade impactor aerodynamicmeasurements indicate that due to their ribbon shape and curlynature, para-aramid fibrils behave aerodynamically larger thanstraight fibers.     

Genotoxicity testing of esterified propoxylated glycerol (EPG)

Four versions of esterified propoxylated glycerols (EPGs) were evaluated for potential genotoxicity using a range of in vitro and in vivo assays. H-EPG-05 HR/SO 9:1, H-EPG-05 soyate, and H-EPG-14 soyate were non-mutagenic in reverse mutation assays (maximum concentration 1000 μg/plate) using Salmonella typhimurium and Escherichia coli. Heated and unheated H-EPG-05 HR/SO 9:1 and EPG-05 HR/ST 45:55 were likewise non-mutagenic in reverse mutation assays in S. typhimurium strains TA98 and TA100 (maximum concentration 5000 μg/plate). H-EPG-05 HR/SO 9:1, H-EPG-05 soyate, and H-EPG-14 soyate, were devoid of mutagenic activity in a mouse lymphoma assay in L5178Y tk +/− cells (maximum concentration 200 μg/plate for H-EPG-05 HR/SO 9:1; 100 μg/plate for H-EPG-05 soyate and H-EPG-14 soyate), and a chromosomal aberration test using human lymphocytes (maximum concentration 50 μg/plate for H-EPG-05 HR/SO 9:1 and H-EPG-05 soyate; 60 μg/plate for H-EPG-14 soyate). All assays were conducted with and without metabolic activation. Additionally, H-EPG-05 HR/SO 9:1, H-EPG-05 soyate, and H-EPG-14 soyate were non-genotoxic in unscheduled DNA synthesis tests in rats (maximum dose 2000 mg/kg). Based on the results of these assays it was concluded that these versions of EPG were not genotoxic under any of the conditions of the assays performed.     

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPase Activity in Rat Brain Synaptosomes

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPaseActivity in Rat Brain Synaptosomes. PRASADA RAO, K. S., TROTTMAN,C. H., MORROW, W., AND DESAIAH, D. (1986) Fundam. Appl. Toxicol.6, 648–653. Effect of toxaphene on Ca⁺²-ATPase activityin rat brain synaptosomes was studied in vitro and in vivo.Ca⁺²-ATPase in calmodulin-depleted synaptosomes was inhibitedin vitro to a maximum of about 50% at 150 µM toxaphenc.Substrate activation kinetics of Ca⁺²-ATPase in synaptosomesrevealed that toxaphene inhibited the enzyme activity noncompetetivelyby decreasing V_max values, without affecting the enzyme-substrateaffinity. Toxaphene inhibited the calmodulin activated Ca⁺²-ATPaseactivity in a concentration-dependent manner with an IC50 of10 µM, a concentration at which no significant effectwas observed on basal enzyme activity. Nuclear and P₂ fraction(synaptosomes) calmodulin levels were reduced significantlyin toxaphene-treated rats. The synaptosomal Ca⁺²-ATPase wasalso reduced to about 45% in toxaphene-treated rats and theactivity was restored to normal levels by the exogenously addedcalmodulin. These results suggest that toxaphene may cause synapticdysfunction by in terfering with calmodulin and its regulationof neuronal calcium.     

Synthesis and antiviral activity of the carbocyclic analogues of (E)-5-(2-halovinyl)-2'-deoxyuridines and (E)-5-(2-halovinyl)-2'-deoxycytidines

The carbocyclic analogues of the potent and selective antiherpes agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) were synthesized by conventional methods with use of carbocyclic 2'-deoxyuridine as starting material. C-BVDU, C-IVDU, and C-BVDC were equally selective, albeit slightly less potent, in their antiherpes action than BVDU, IVDU, and BVDC. Although resistant to degradation by pyrimidine nucleoside phosphorylases, C-BVDU did not prove more effective than BVDU in the systemic (oral, intraperitoneal) or topical treatment of HSV-1 infections in mice.     

4,4'-Methylenebis(2-chloroaniline) (MOCA): The Effect of Multiple Oral Administration, Route, and Phenobarbital Induction on Macromolecular Adduct Formation in the Rat

The effect of multiple oral administration of MOCA, a suspecthuman carcinogen, was studied in the adult male rat. As manyas 28 consecutive daily doses of [¹⁴C]MOCA at 28.1µmol/kgbody wt (5 µC1/day) were administered and rats were euthanizedat weekly intervals for 7 weeks. MOCA adduct formation for globinand serum albumin was evaluated by determination of [¹⁴C]MOCAcovalent binding. The covalent binding associated with globinshowed a linear increase over the 28-day exposure period with342 fmol/mg globin 24 hr after the final dose. More extensivecovalent binding was detected for albumin with 443 fmol/mg albuminafter the final dose, but increases were not linear. After cessationof dosing, the albumin adduct levels decreased rapidly (t _1/2=4.6 days) in relation to globin adduct levels (t _1/2 =16.1days). The MOCA-globin adduct t _1/2 is consistent with thatdetermined after a single 281 µmol/kg oral dose of MOCA.Significant differences related to route of administration weredetected for 24-hr globin covalent binding with ip > po >dermal. Distribution of undifferentiated [¹⁴C]MOCA was highestin the liver at 24 hr with tissue levels for liver > kidney> lung > spleen > testes > urinary bladder. Inductionof cytochrome P450 enzymes by administration of phenobarbital(100 mg/kg/day/3 days) resulted in a significant (p < 0.05)increase in MOCA-globin adduct formation detected with 33.5pmol/ mg globin for induced rats versus 13.6 pmol/mg globinfor control rats. Although MOCA-globin and albumin adducts showdiffering stability, quantification of such MOCA adducts maybe useful for long-term industrial biomonitoring of MOCA.     

Teratology of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in a Complex Environmental Mixture from the Love Canal

The organic phase of a leachate (OPL) from the Love Canal chemicaldump site contains more than 100 organic compounds including2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The teratogenicpotential of OPL was determined in two inbred and one hybridmouse strain which differ in their sensitivity to aromatic hydrocarbon(Ah) receptor-mediated toxicity. OPL was orally administeredin corn oil on Days 6–15 of gestation to C57BL/6J mice(Ah^b/ Ah^b) at doses of 0, 0.1, 0.3, 0.5, and 0.7 g kg^–1day^–1 and to DBA/ZJ (Ah^d/Ah^d) females, which were matedwith either DBA/2J or C57BL/6J males, at 0, 0.5, 1, and 2.0g kg^–1 day^–1. In C57BL/6J mice, which express ahigh-affinity Ah receptor that avidly binds TCDD, the ED50'sof OPL for cleft palate and hydronephrosis were 0.44 and 0.11g OPL kg^–1 day^–1, respectively. Maternal mortalitywas 5% at the highest dose. In DBA/2J fetuses, which expressa low-affinity receptor, neither treatment-related cleft palatenor hydronephrosis was induced by dose levels that caused 36%maternal mortality. In hybrid D2B6F1 fetuses, the incidenceof cleft palate reached only 8% at 2 g OPL kg^–1 day^–1but the ED50 for hydronephrosis was 0.76 g OPL kg^–1 day^–1.TCDD was similarly administered to pregnant C57BL/6J mice at0, 0.5, 1, 2, and 4 µg kg^–1 day^–1 and to DBA/2Jmice at 0, 0.5, 2, 4, and 8 µg kg^–1 day^–1.In C57BL/6J fetuses, the ED50's for cleft palate and hydronephrosiswere 4.6 and 0.73 µg TCDD kg^–1 day^–1, respectively.In DBA/2J fetuses the ED50's for cleft palate and hydronephrosiswere 15.0 and 6.4 µg TCDD kg^–1 day^–1, respectively.Both the OPL and TCDD caused maternal hepatomegaly and thymicatrophy in all strains, but increased only C57BL/6J fetal weights.OPL decreased the number of fetuses per C57BL/6J dam at thetwo highest doses but there were no other reproductive effectsin any of the groups. It was concluded that the OPL is teratogenicand that hydronephrosis is a sensitive measure of TCDD toxicityin a complex organic mixture. Based on the ED50's of OPL- andTCDD-induced cleft palate and hydronephrosis in the C57BL/6Jstrain, the OPL had TCDD equivalence of 6.6 and 10.5 ppm, respectively.These values compare closely with the chemical analysis of 3ppm. The results suggest that the teratologic effects are dueprimarily to the TCDD in the OPL and that these effects aremediated through the Ah receptor, but that the maternal thymicatrophy and hepatomegaly were due primarily to the non-TCDDcomponents of the OPL.