Results of the L5178Y mouse lymphoma assay and the Balb/3t3 cell in vitro transformation assay for eight phthalate esters.

Eight phthalate esters, with alcohol chain lengths of 1-11 carbon atoms and with various degrees of branching, were tested in vitro in the L5178Y mouse lymphoma mammalian cell mutation assay and in the Balb/3T3 cell transformation assay. The tests were performed as part of a voluntary testing agreement between the Chemical Manufacturers Association's Phthalate Esters Panel and the United States Environmental Protection Agency (US EPA). The esters tested were: dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), di-¿n-hexyl, n-octyl, n-decyl¿ phthalate (610P), di-isononyl phthalate (DINP), di-¿heptyl, nonyl, undecyl¿ phthalate (711P), di-isodecyl phthalate (DIDP) and di-undecyl phthalate (DUP). Both DMP and DBP were found to produce significant increases in the mutant frequency in the mouse lymphoma assay in the presence but not in the absence of an Aroclor-induced rat liver activation system (S-9). Ester 610P gave equivocal results in the mouse lymphoma assay in the presence and absence of rat liver S-9. There was no indication of mutagenic potential for any of the other test materials in the mouse lymphoma assay, and none of the test materials increased transformation frequency in the Balb/3T3 cell transformation assay. Aldehyde metabolites of the de-esterified alcohols are postulated to play a role in the positive results for DMP and DBP.

Hydroquinone: genotoxicity and prevention of genotoxicity following ingestion.

Plant-derived polyphenolics and other chemicals with antioxidant properties have been reported to inhibit the expression of genotoxic activity by pro-oxidant chemicals (Sai et al., 1992, 1994; Teel and Castonguay, 1992). In vitro and in vivo studies with ionizing radiation suggest that hydroquinone (HQ) may have similar protective effects (Babaev et al., 1994). The present study was conducted to determine whether HQ is capable of inhibiting the induction of micronuclei in the bone marrow of mice following exposure to an oxidant, potassium bromate or KBrO3 (Nakajima et al., 1989; Sai et al., 1992, 1994). To be able to interpret the results of this work, it was also necessary to determine whether HQ is itself genotoxic when fed in the diet. HQ diets (0.8%) fed to mice for 6 days reduced the background incidence of micronuclei compared with the basal diet. KBrO3 dosed ip (12.5-100 mg/kg) produced a dose-dependent increase in micronuclei as reported by others. Mice fed 0.8% HQ diets 6 days, and then dosed intraperitoneally with KBrO3, showed a 36% reduction in micronuclei across the range of KBrO3 dose levels. This effect was associated with a reduction in the background micronucleus response as well as a reduction in response to KBrO3. Statistical significance (P < or = 0.05), observed at a dose of 25 mg/kg KBrO3 in the mice fed the control diet, was abolished in the group fed 0.8% HQ. When mice were given 50 mg HQ/kg by oral gavage and then given 50 mg KBrO3/kg ip 20 min later, the micronucleus response induced by KBrO3, was lower in animals given HQ. The results of this study demonstrate that large doses of HQ may be given orally without induction of micronuclei or bone marrow depression, that HQ reduces the background micronucleus response in animals fed a basal diet, and that the HQ reduces the micronucleus response to KBrO3 as well as background incidence of micronuclei in KBrO3-dosed animals. The protective effect of HQ may be due to enzyme induction or a direct antioxidant effect of HQ against oxidants commonly present in the diet.

The lack of binding of methyl-n-amyl ketone (MAK) to rat liver DNA as demonstrated by direct binding measurements, and 32P-postlabeling techniques.

It has been reported that 14C-labeled methyl-n-amyl ketone (MAK, 2-heptanone) is able to bind spontaneously, in vitro, to isolated rat liver DNA to the extent of 400 pmol/mg DNA; and that 14C-MAK, when given by gavage to female Fischer 344 rats, resulted in HPLC chromatograms of isolated, hydrolyzed liver DNA in which some radiolabel was not associated with the four normal DNA bases dA, dT, dC, and dG. The present studies were undertaken to re-examine the hypothesis that MAK is able to bind to rat liver DNA. In the in vitro study, liver nuclear DNA was incubated with [2-14C]-labeled MAK (25 mCi/mmol) in the absence, or in the presence of rat liver microsomes, precipitated, washed free of unbound MAK, and counted by scintillation spectrometry. No binding to DNA by MAK was detectable. In the in vivo study, groups of five female F344 rats were exposed by inhalation to 0, 80, 400, or 1000 ppm MAK for 6 h/day for 10 days. DNA was purified from the liver nuclei of the 0 and 1000 ppm dosed animals, and 32P-postlabeling techniques were used to assay for adducts. No DNA adducts were detected using these techniques. It was concluded that MAK lacks the ability to bind to rat liver DNA in vitro and in vivo.

Subchronic 90-day oral toxicology of di(2-ethylhexyl) terephthalate in the rat.

Di(2-ethylhexyl) terephthalate (DEHT), the 2-ethylhexyl diester of terephthalic acid (1,4-benzenedicarboxylic acid) was administered in the diet to groups of 20 male and female Sprague-Dawley rats for 90 days at 1.0, 0.5, 0.1 or 0.0% by weight. No major organ or systemic toxicity resulted from consumption of the diets in any group of animals. Changes that were observed included slight effects on some haematology parameters including haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin at the 1.0% dose; and slight increases in relative liver weights (11.2% in the males, 8.9% in the females), also at the 1.0% dose level. Marginal changes of less than 3% in some red blood cell indices were observed at the 0.5% dietary dose level; however no anaemia or changes in relative liver weights were observed at this dose level. Thus, no significant adverse effects attributable to the test material were identified in animals consuming the two lower doses. In a morphometric study of liver sections, DEHT was found not to induce hepatic peroxisomes at the 1.0% dose level. The positive control material (2-ethylhexanol at 1000 mg/kg, 5 days/wk for 3 wk), produced a 30% increase in liver-to-body weight ratio, and increases in peroxisome cell fraction, and in peroxisome density. The no-effect levels of DEHT in rats consuming the material for 90 days in the diet were 277 and 309 mg/kg/day in males and females, respectively. These results are contrasted with those reported for di(2-ethylhexyl) phthalate (DEHP) in similar feeding studies. While DEHP at 1% in the diet is reported to produce significant effects on the liver, testes, kidney, brain, stomach and adrenal weights, DEHT has been shown in this study to have only a minor effect on liver weight in 90 days at 1.0% in the diet.

The percutaneous absorption of hydroquinone (HQ) through rat and human skin in vitro.

Because of the potential for human contact with photographic developer solutions containing hydroquinone (HQ), the rates of percutaneous absorption of HQ through human stratum corneum and full-thickness rat skin have been measured in vitro using 5% aqueous solutions of HQ as the donor solutions. The studies were performed using infinite doses of aqueous solutions containing 14C-labeled HQ in Franz-type diffusion cells. The measured absorption rate (mean +/- S.D.) of HQ through human stratum corneum was 0.52 +/- 0.13 micrograms/cm2/h, while that for full-thickness rat skin was 1.1 +/- 0.65 micrograms/cm2/h. The ratio (rat/human) of the permeability constants (Kp) was 2.4. Using the definitions suggested by Marzulli et al. (1969) Toxicol. Appl. Pharmacol. Suppl. 3, 76-83, HQ would be classified as 'slow' with respect to its absorption through human stratum corneum.

Hydrolysis, absorption and metabolism of di(2-ethylhexyl) terephthalate in the rat.

1. The hydrolysis of di(2-ethylhexyl) terephthalate (DEHT) and di(2-ethylhexyl) phthalate (DEHP) were studied using rat gut homogenate fractions in vitro. Both isomers were hydrolysed by the intestinal fraction; however, DEHP was hydrolysed to 2-ethylhexanol (2-EH) and mono(2-ethylhexyl) phthalate (MEHP) in about equal proportions, whereas DEHT was hydrolysed to 2-EH and terephthalic acid (TPA). The half-lives for disappearance of the diesters were determined to be 12.6 min for DEHP and 53.3 min for DEHT. 2. The absorption and metabolism of DEHT were studied by administering [hexyl-2-14C]DEHT (in corn oil) by oral gavage at a dose level of 100 mg/kg to 10 adult male Sprague-Dawley rats. Urine, faeces and expired air were collected for 144 h and analysed for the presence of radioactivity, and faeces and urine were analysed for unlabelled metabolites. 3. Radioactivity was eliminated in faeces (56.5 +/- 12.1% of dose) primarily as unchanged DEHT, small amounts of MEHT and polar metabolites; excreted in urine (31.9 +/- 10.9% of dose) principally as MEHT and metabolic products of 2-EH; and expired as 14CO2 (3.6 +/- 0.9% of dose). Less than 2% of the administered radioactivity was found in the carcass. Small amounts of 14C were found in the tissues with the highest amounts found in liver and fat. 4. Metabolites identified in urine included terephthalic acid (equivalent to 51% of dose), oxidized metabolites of 2-EH and MEHT, and glucuronic and sulphuric acid conjugates (equivalent to about 10% of dose).(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic toxicology testing of di(2-ethylhexyl) terephthalate.

Di(2-ethylhexyl) terephthalate (DEHT) is a commercially produced chemical (Kodaflex DOTP) that is used as a general purpose, low-volatility plasticizer for polyvinyl chloride and other polymeric materials. Less than 30 million kilograms of DEHT are produced annually. DEHT is isomeric with di(2-ethylhexyl) phthalate (DEHP), a nongenotoxic rodent carcinogen whose mode of action has been suggested to derive from its ability to produce hepatocellular proliferation and/or hepatic peroxisome proliferation. Thus it is important to know the behavior of DEHT in genotoxicity assays in order to compare it with that of DEHP and other phthalate ester plasticizers. It is known from previously published studies that rats fed DEHT in the diet at 2,000 mg/kg produce urine that is negative in the Ames Salmonella bacterial mutagenicity assay in the presence and absence of induced rat liver S-9 and in the presence and absence of beta-glucuronidase/aryl sulfatase. Reported here are the results of direct testing of DEHT in the Ames plate incorporation assay, the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) in vitro mammalian cell mutagenicity assay, and an in vitro chromosome aberrations assay using CHO cells. The results for mono(ethylhexyl) terephthalate (MEHT), a metabolite of DEHT, in the Ames Salmonella bacterial mutagenicity assay are also presented. All test results for both DEHT and MEHT were found to be negative, and it is therefore concluded that DEHT, like its isomeric relative DEHP, is not genotoxic.

A comparative study of the rates of in vitro percutaneous absorption of eight chemicals using rat and human skin.

In vitro percutaneous absorption studies were carried out for eight chemicals using full thickness rat skin and human stratum corneum. The purpose of the studies was to compare the rates of absorption for the two species. For each of the chemicals, the observed rate using full thickness rat skin was greater than that observed for human stratum corneum. The ratios of the rates (rat/human) varied from 1.7 to 5.8 with a mean value of 3.1. The chemicals tested were tritiated water, 2-ethoxyethyl acetate, diethylene glycol monobutyl ether, urea, di(2-ethylhexyl) phthalate, 2-ethylhexanol, ethyl 3-ethoxypropionate, and 2-propoxyethanol. The chemicals were chosen to represent a wide range of physical properties and permeability constant values. It was concluded that rat skin was more permeable than human skin for each of these eight chemicals. This conclusion is supported by similar findings from studies in other laboratories and suggests that results from studies in the rat overestimate skin absorption in man.

Unit orientation for experienced nurses. Process and evaluation.

In order to recruit and retain registered nurses during an era of nursing shortages, staff development educators must provide orientation programs that are highly satisfactory. This article describes a program that effectively links the perspectives of nurse managers, nursing staff, and nurse educators. Orientation evaluation questionnaires completed by orientees and staff nurses who served as their buddies show that new employees believe this program meets their learning needs. Such satisfied nurses can recruit their colleagues to employment.

Peroxisome induction studies on seven phthalate esters.

Seven phthalate esters, representing a variety of chain lengths and degrees of branching in the alcohol moiety, were tested for their ability to produce peroxisome proliferation in the Fischer 344 rat. Di(2-ethylhexyl)adipate (DEHA) was tested using the same protocol and di(2-ethylhexyl)phthalate (DEHP) was run with each study as an internal control. Each ester was administered in the feed for a period of 21 days at levels of 2.5%, 1.2% and either 0.6% or 0.3%. DEHP and DEHA were also fed at levels of 0.1% and 0.01%. The animals were sacrificed and samples of liver were prepared for both light and electron microscopy. Serum samples were assayed for both triglyceride and cholesterol. The remaining portion of the liver was homogenized and assayed for cyanide-insensitive palmitoyl-CoA oxidation, lauric acid 11-hydroxylase and lauric acid 12-hydroxylase. The results show that there is approximately a ten-fold difference between the weakest and strongest esters in terms of their potency to induce changes in relative liver weight and in several of the biochemical parameters. In general, the longer chain esters were more potent than the shorter chain ones, and branched chain esters seemed more potent than straight. Several statistical analyses of the dataset have been performed and all render similar conclusions. The results of one of these evaluations are presented elsewhere in this volume (Lin, 1987).

Methods for measuring mutagenicity in urine of rats dosed with [14C]di(2-ethylhexyl)phthalate.

Di(2-ethylhexyl)phthalate (DEHP) is extensively used as a plasticizer for vinyl plastic articles. It has been found to be positive in an NCI rodent bioassay but has generally given negative results in in vitro genotoxicity tests. We therefore decided to test the urine of rats fed [14C]DEHP for mutagenic activity in the Ames Salmonella test. The recovery of radioactivity from the urine of rats dosed with [14C]DEHP was examined by solvent extraction and XAD-2 resin absorption procedures. Both of these procedures were inadequate for quantitative recovery of urinary metabolites required for subsequent mutagenicity testing using the Ames Salmonella/microsome procedure. Recoveries of less than 5% were observed using standard solvent extraction techniques whereas the XAD-2 adsorption technique gave about 67% at high resin/urine ratios. Treatment of the urine with beta-glucuronidase/aryl sulfatase did not affect these recoveries. The direct urine plating procedure represents a viable alternative to the above concentration procedures for this phthalate ester. The effects of L-histidine and the beta-glucuronidase/aryl sulfatase preparation on the background reversion frequencies of the Ames tester strains is discussed.

Bacterial mutagenicity testing of urine from rats dosed with 2-ethylhexanol derived plasticizers.

Di-(2-ethylhexyl)phthalate (DEHP) produced hepatocellular carcinomas in rodents at high doses in a NTP/NCI bioassay. DEHP has not shown evidence of genotoxic activity in in vitro mutagenicity tests. We extended these studies by examining the mutagenicity of urine from rats dosed with DEHP, 2-ethylhexanol (2-EH), and several other 2-EH derived plasticizers, i.e. di-(2-ethylhexyl)adipate (DEHA), di-(2-ethylhexyl)terephthalate (DEHT) and tri-(2-ethylhexyl)trimellitate (TEHT). A modified Ames Salmonella/microsome assay was used to determine mutagenicity. Urine was pooled from male Sprague--Dawley rats dosed daily for 15 days with 2000 mg/kg of each test substance with the exception of 2-EH which was given at 1000 mg/kg. Direct plating procedures were used to determine the presence of mutagens in urine. Urine from rats dosed with 8-hydroxyquinoline was used as a positive control. There was no evidence that mutagenic substances were excreted in the urine by rats dosed with either DEHP, DEHA, DEHT, TEHT or 2-EH as determined in the presence or absence of rat liver microsomes, and with or without treatment with beta-glucuronidase/aryl sulfatase. Our findings indicate that the above test compounds were not converted to urinary metabolites that were mutagenic. These observations provide no evidence for a genotoxic mechanism for DEHP carcinogenicity in rodents.

The relationship between growth and reversion in the Ames Salmonella plate incorporation assay.

Growth curves of the 5 commonly used Ames Salmonella tester strains have been measured turbidimetrically in semi-solid agar. Lag times, doubling times and maximum cell densities have been calculated for each of the 5 strains. The time dependence of reversion has been studied in the standard plate incorporation assay using 1-h pulsed doses of (a) bromoethane, a volatile chemical mutagen, and (b) 1-h exposures to visible light. Essentially no reversion takes place during the first 4 h after plating. Reversion is detectable between hours 4 and 16. The cumulative or integrated revertants versus time curve has the characteristics of a growth curve. Conversely the derivatives of the growth curves resemble the curves obtained in the pulsed mutagenicity studies. Thus, the reversion rate in any given 1 h interval is proportional to the growth rate during that same interval. These results suggest that mutagenic chemicals must be present during the bacterial growth cycle (about 4-16 h after plating) in order to revert the tester strains. Short-lived chemical mutagens, then, should produce enhanced results if plated 6-8 h after the bacteria. We have confirmed this for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 9-aminoacridine and 2-aminoanthracene (with S9).

A procedure for the quantitative measurement of the mutagenicity of volatile liquids in the Ames Salmonella/microsome assay.

We have designed a closed, inert incubation system for testing the mutagenicity of volatile compounds. The containment properties of this system have been investigated using carbon-14 labelled 1,2-dibromoethane. The recovery of this solvent was about 95% following a 48-h incubation at 37 degrees. Using the Ames Salmonella/microsome mutagenicity assay we have determined the mutagenic potency of 10 common halogenated alkane solvents. Of these 10 compounds, only 1,2-dibromoethane and 1-bromo-2-chloroethane give positive results in the standard test procedure, whereas 7 of the 10 give positive results in the closed system. The specificity observed for reversion of the tester strains and the lack of any significant effect of added rat-liver "S9" fractions suggest that these haloalkanes are direct-acting "base-pair" type mutagens. The mutagenic potencies of the 7 positive compounds range from 0.001 revertants per nanomole for 1,2-dichloroethane to 0.172 revertants per nanomole for 1,2-dibromoethane. A minimum or threshold response level for each material has been calculated.

Quantitative effects of unsaturated fatty acids in microbial mutants. VI. Selective growth responses of yeast and bacteria to cis-octadecenoate isomers.

The full series of positional isomers of cis-octadecenoate were tested for their suitability in meeting the nutritional requirement for unsaturated fatty acids by mutants of Escherichia coli and Saccharomyces cerevisiae that were unable to synthesize unsaturated fatty acids. Quantitative comparisons of the efficiencies of the various isomers showed a range from 0-48 cells per femtomole for the prokaryotic cells and 0-5 for eukaryotic cells. The delta 5 isomer was much more effective than the delta 6 isomer with the bacterial cells whereas the reverse was true with the yeast cells. In general, isomers containing a cis ethylenic bond between carbons 7 and 12 were able to support extensive growth of either type of mutant. Since all of the various isomers were incorporated into cellular lipids by both types of microorganism, the different efficiencies observed in supporting growth were not a simple reflection of the inability of an acid to be esterified. The differences may reflect the suitability of the resultant esterified product to function as a normal membrane lipid. The contents of various fatty acids in the cellular phospholipids when growth ceases may have a linearly cumulative relationship to the degree of expansion of the acyl chains.

Quantitative measurement of the effectiveness of unsaturated fatty acids required for the growth of Saccharomyces cerevisiae.

The growth response of a mutant of Saccharomyces cerevisiae which is unable to synthesize unsaturated fatty acids has been measured in the presence of variable concentrations of exogenous unsaturated fatty acids. Final cell yields, doubling times, and lag times were all found to vary as a function of the initial concentration of the added unsaturated acid. The cell yield was found to be a convenient quantitative measurement to use in comparing the effectiveness of various unsaturated acids. Values for the acids ranged from 1.7 to 11 cells per femtomole with values for oleate and palmitoleate at 2.7 and 4.3 cells per femtomole, respectively. In general, the effectiveness of unsaturated acids was found to increase with an increasing number of double bonds. Saturated fatty acids of a chain length of 5 to 18 carbon atoms were completely ineffective. The varied efficiencies of different unsaturated fatty acids indicate that unsaturation per se was not the basis of the nutritional requirement and indicate certain acids that would be useful in further studies of the role of unsaturated acids in cell function.