Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells.

The bifunctional alkylating agents epichlorohydrin (ECH) and diepoxybutane (DEB) have been linked to increased cancer risks in industrial workers. These compounds react with DNA and proteins, leading to genotoxic effects. We used the comet assay to monitor formation of cross-links in HL-60 cells treated with ECH, DEB, and the structurally related anti-cancer drug mechlorethamine (HN2). We report a time- and dose-dependent cytotoxicity that correlated with cross-linking activity, following the order HN2 > DEB > ECH. The rate of cross-link repair also varied with drug, with ECH-induced lesions the fastest to repair. High drug doses led to the formation of saturating amounts of HN2 cross-links that were repaired inefficiently. DEB and ECH produced fewer overall cross-links, but some were also resistant to repair. These persistent cross-links may activate cell-cycle arrest to allow repair of damage, with prolonged arrest triggering apoptosis. Quantitative reverse transcription polymerase chain reaction experiments revealed that treatment of HL-60 cells with DEB and ECH results in up-regulation of several genes involved in the intrinsic (mitochondrial) apoptosis pathway, including BAX, BAK1, CASP-9, APAF-1, and BCL-2. These findings contribute to our understanding of the principles underlying the carcinogenic potentials of these xenobiotics.

Formation of epichlorohydrin, a known rodent carcinogen, following oral administration of 1,3-dichloro-2-propanol in rats.

The observed toxicity and carcinogenicity of 1,3-dichloro-2-propanol (DCP) in rodents is thought to be due to the formation of reactive metabolites, epichlorohydrin (ECH) and dichloroacetone (DCA). However, there is no direct evidence for the formation of these metabolites from exposure to DCP in rodents due to the challenges of measuring these reactive intermediates directly in vivo. The objective of this work was to investigate the metabolism of DCP to ECH and DCA in vivo by first developing a sensitive analytical method in a suitable biological matrix and analyzing samples from rats administered DCP. DCA reacted rapidly in vitro in rat blood, plasma, and liver homogenate, precluding its detection. Because ECH rapidly disappeared in liver homogenate, but was relatively long-lived in plasma and blood in vitro, blood was selected for analysis of this metabolite. Following a single oral dose of 50 mg/kg DCP in male or female Harlan Sprague-Dawley rats, ECH was detected in blood with a maximum concentration reached at ≤13.7 min. ECH was cleared rapidly with a half-life of ca. 33 and 48 min in males and females, respectively. Following a single oral dose of 25 mg/kg ECH in male and female rats, the elimination half-life of ECH was ca. 34 and 20 min, respectively; the oral bioavailability of ECH was low (males, 5.2%; females, 2.1%), suggesting extensive first pass metabolism of ECH following oral administration. The area under the concentration vs time curve for ECH following oral administration of DCP and intravenous administration of ECH was used to estimate the percent of the DCP dose converted to ECH in rats. On the basis of this analysis, we concluded that in male and female rats following oral administration of 50 mg/kg DCP, ≥1.26% or ≥1.78% of the administered dose was metabolized to ECH, respectively.

Spectral shift supported epichlorohydrin toxicity and the protective role of sage.

In this study, the toxicity of epichlorohydrin, a chemical intermediate, was investigated by using Allium cepa L. test material as a bio-indicator. In addition, the protective role of sage leaf extract (Slex) against this toxicity was investigated. Toxicity was handled with the help of physiological (germination percentage, root elongation, and weight gain), cytogenetic (mitotic index = MI, micronucleus = MN, and chromosomal abnormalities = CAs), biochemical (malondialdehyde = MDA, superoxide dismutase = SOD, and catalase = CAT), and anatomical (root meristem cell damages) parameters. A. cepa bulbs were divided into 6 groups (1 control, 5 applications). The bulbs in the control group were treated with tap water, and the bulbs in the application group were treated with epichlorohydrin at a dose of 100 mg/L and Slex at two different doses (190 mg/L and 380 mg/L) and germinated. Germination process was continued uninterruptedly for 72 h in all groups. At the end of the period, physiological parameter measurements were carried out in the bulbs. In addition, root tips were collected and made ready for cytogenetic, biochemical, and anatomical measurements and microscopic observations. As a result, exposure to epichlorohydrin caused statistically significant (p < 0.05) decreases in germination percentage, root length, weight gain, and MI, and statistically significant (p<0.05) increases in MN frequency, CA numbers, MDA level, SOD, and CAT enzyme activities. Epichlorohydrin exposure induced CAs such as fragment, sticky chromosome, unequal distribution of chromatin, reverse polarization, and disordered mitosis in root meristem cells. The toxicity of epichlorohydrin was due to its interaction with DNA, and this interaction was confirmed by the spectral shift in the DNA spectrum. In addition, epichlorohydrin caused anatomical damages such as epidermis cell damage, cortex cell damage, thickening of the cortex cell wall, and flattened cell nuclei in root meristem cells. The application of Slex together with epichlorohydrin decreased the toxicity of epichlorohydrin and again caused statistically significant (p < 0.05) improvements in the values of all the parameters examined. In other words, germination percentage, root length, weight gain, and MI increased again and MN frequency, CAs numbers, MDA level, SOD, and CAT enzyme activities decreased. It was determined that this improvement was even more pronounced at 380 mg/L dose of Slex. As a result, it was determined that epichlorohydrin caused multiple-toxicity for the investigated indicator organism, and Slex had a reducing role in this toxicity. For this reason, Slex should be included in the daily diet as an antioxidant beverage in order to protect from the toxicity of chemical agents exposed in daily life or to reduce their effects.

One-generation reproductive toxicity study of epichlorohydrin in Sprague-Dawley rats.

This study was conducted to evaluate the potential reproductive toxicity of epichlorohydrin in a one-generation reproduction toxicity study in compliance with OECD Test Guideline 415. Twenty-four male and female rats per group were given epichlorohydrin by gavage at 0, 3.3, 10, and 30 mg/kg/day. Males were dosed for 10 weeks prior to and during mating. Females were dosed from 2 weeks before mating to day 21 of lactation. At 30 mg/kg, an increase in the incidence of clinical signs (i.e., nasal discharge, soft feces, depression, and piloerection), gross necropsy findings (i.e., cystic pustule of the epididymidis and enlargement of the kidney) and the weights of heart, liver, and epididymidis, a decrease in male fertility, and an increased incidence of histopathological changes of the testis, epididymidis, and kidney were observed. At 10 mg/kg, decreased male fertility and increased kidney weight and incidence of histopathological changes of the epididymidis were found. There was a slight, but nonsignificant, reduction in the male fertility index at the dose of 3.3 mg/ kg. Under these experimental conditions, the lowest-observed-adverse-effect level of epichlorohydrin was 3.3 mg/kg/day for parent animals and their offspring. The absolute toxic dose for parent animals and their offspring was estimated to be 10 mg/kg/day.

Induction of oxidative stress in the epididymis of rats after subchronic exposure to epichlorohydrin.

This study investigated the effects of epichlorohydrin (ECH) on spermatogenesis and antioxidant system in rats. An increase in the incidence of clinical signs, gross pathology and histopathology findings in the epididymidis, and sperm abnormalities and a decrease in the testicular spermatid counts, epididymal sperm counts, and sperm motility were observed at 30 mg/kg/day. Oxidative stress in the epididymal tissue was detected at > or =3.3 mg/kg/day. The results show that graded doses of ECH elicit depletion of antioxidant defense system and that the adverse effects on male reproductive function in ECH-treated rats may be due to the induction of oxidative stress.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA.

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Inhalation carcinogenicity of epichlorohydrin in noninbred Sprague-Dawley rats.

Inhalation exposure experiments with the direct-acting alkylating agent epichlorohydrin (ECH) were done on noninbred male Sprague-Dawley rats. Single 6-hour exposure to ECH and follow-up for 14 days showed the median lethal concentration to be about 360 ppm. Further inhalation experiments were done with 6-hour exposure 5 days/week. A short-term 30-exposure regimen with 100 ppm ECH produced malignant squamous cell carcinomas of the nasal cavity in 15 of 140 rats and respiratory tract papillomas in 3 rats. Among 100 rats, lifetime exposure to 30 ppm yielded 1 malignant squamous carcinoma of the nasal cavity plus 1 nasal papilloma. No nasal or respiratory tract tumors were produced by lifetime exposure of 100 rats to 10 ppm. As controls, 100 air-treated and 50 untreated rats were used. A dose-rate effect was observed for ECH inasmuch as 30-day exposure to 100 ppm (3,000 ppm-days) produced 15 cancers in comparison to the 1 cancer from the lifetime exposure to 30 ppm (8,700 ppm-days) and no cancers from lifetime exposure to 10 ppm (2,500 ppm-days).

Carcinogenicity study of trichloroethylene, with and without epoxide stabilizers, in mice.

Previous analytical studies of industrial samples of trichloroethylene (TRI) have revealed the presence of mutagenic and carcinogenic epoxides which, it was proposed, might be responsible for the carcinogenicity of such samples, as demonstrated with mice in other laboratories. To test this hypothesis, Swiss mice (ICR/HA) of both sexes, bred and kept in SPF conditions, were dosed daily with TRI in corn oil by gavage (males: 2.4 g/kg, females: 1.8 g/kg) with or without the addition of epichlorohydrin (EPC, 0.8%, w/w), 1,2-epoxybutane (BO, 0,8%), or EPC + BO (0.25% + 0,25%) for 18 months. The ensuing observation period terminated at 106 weeks (from start of experiment). Gross and microscopic examination of all organs revealed a statistically significant increase in the incidence of forestomach papillomas and carcinomas after EPC-, BO-, and (EPC + BO)-stabilized samples of TRI, but not after pure, amine base-stabilized TRI. This type of tumor is believed to be induced by the direct alkylating epoxides epichlorohydrin and epoxybutane, whose industrial use in stabilizing chlorinated aliphatic hydrocarbons should be discontinued. No other significant increase in tumor incidences was found. Again, this study does not support the suggestion that trichloroethylene itself is carcinogenic under realistic exposure conditions.

Conditions affecting the mutagenicity of trichloroethylene in Salmonella.

Trichloroethylene (TCE) is a high production volume chemical frequently stabilized with oxiranes. These oxiranes may be responsible for the mutagenic activity of TCE in Salmonella, which has been occasionally, but not consistently, reported. High purity and oxirane-stabilized TCE samples were tested for their mutagenic potential in Salmonella typhimurium strains TA 1535, TA 98, and TA 100. Stabilized TCE was tested using a preincubation protocol up to a dose level of 10,000 micrograms per plate, but no mutagenic response was observed in either the presence or absence of a supplementary metabolic activation system (S9 mix) derived from Aroclor 1254-induced male rat liver. TCE without oxirane stabilizers also was nonmutagenic when tested in a vapor delivery system at nominal concentrations of up to 20% and using S9 mix derived from either rat or hamster. TCE containing 0.5-0.6% 1,2-epoxybutane did induce mutagenic responses from strains TA 1535 and TA 100 in the presence and absence of S9 mix. The lowest effective dose was about 0.63% in TA 1535 in the absence of S9 mix. Vapor-phase tests with 1,2-epoxybutane showed that an atmospheric concentration of 0.009% could induce 12-fold and 3-fold increases, respectively, in strains TA 1535 and TA 100. These increases would account for the mutagenic activity of the stabilized TCE sample. Epichlorohydrin (another commonly used stabilizer) induced similar increases in mutant numbers at an atmospheric concentration of 0.0009%. The absence of a significant response caused by unstabilized TCE in the presence of S9 mix is probably due to a lack of assay sensitivity, since chloral, a metabolite of TCE, is a mutagen in TA 100 [Haworth et al.: Environ Mutagen [Supplement 1] 5:3-142, 1983].

Propylene oxide and epichlorohydrin induce DNA strand breaks in human diploid fibroblasts.

The induction of DNA strand breaks in human diploid fibroblasts (VH-10) was demonstrated after in vitro exposure with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH). Alkaline DNA unwinding (ADU), pulsed field gel electropharosis (PFGE), and the comet assay were used to measure DNA single. (SSBs) and double-strand breaks (DSBs). A dose-dependent increase of DNA strand breaks, measured by ADU, was observed in the dose range 2.5-20 mMh of PO and 0.25-2 mMh of ECH. The dose-response of ECH was about five times higher compared with that of PO (211 vs. 41 SSBs. 100 Mbp-1.mMh-1). The induction rates of DSBs, measured by PFGE, were found to be 18 times higher for ECH compared to PO (4.8 and 0.27 DSBs.100 Mbp-1.mMh-1 for ECH and PO, respectively). Using these two methods, the SSBs/ DSBs ratio was estimated to be 148 for PO and 44 for ECH. The data obtained by the comet assay also demonstrated a dose-dependent ability of PO and ECH to induce DNA damage. It was found that ECH was about six times more effective as an inducer of DNA strand breaks compared to PO (200 and 32x100 Mbp-1.mMh-1 for ECH and PO, respectively). The SSBs/DSBs ratios calculated using comet assay and PFGE data were 125 for ECH and 41 for PO. In addition, ECH is about 10 times more toxic than PO with respect to survival. These properties of ECH can at least in part be explained by its higher chemical reactivity connected with a higher rate of DNA alkylation.

Correlation of sperm motion parameters with fertility in rats treated subchronically with epichlorohydrin.

We investigated the relationship between fertility and sperm motin endpoints in rats treated subchronically with the male reproductive toxicant, epichlorohydrin (ECH). Male rats were given ECH orally for 23 days at dosages of 0, 6.25, 12.5, or 25 mg/kg/day. They were mated twice (at 19 and 22 days) to estimate fertility by (1) detection of fertilized ova (presence of sperm head and tail or two pronuclei) 18 hours after mating and by (2) counting implants on day 14 of gestation. Both indices showed dose-related reductions (P less than 0.001). Motion parameters of cauda epididymal sperm were assessed using the CellSoft computer-assisted sperm motion analysis (CASA) system after the rats were asphyxiated on day 25. Curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement were reduced in a dose-related manner. The fertility indices, percent fertilized ova, and percent implantation on day 14 of gestation were correlated significantly (r = 0.68; P = 0.0001). The following motion parameters were also correlated significantly with fertility (P less than 0.0003; r1 = percent fertilized ova and r2 = percent implantation): linearity (r1 = 0.42; r2 = 0.40), amplitude of lateral head displacement (r1 = 0.54; r2 = 0.48), curvilinear velocity (r1 = 0.53; r2 = 0.50), straight-line velocity (r1 = 0.55; r2 = 0.50), and percent motile sperm (r1 = 0.42; r2 = 0.32). These results suggest a relationship between toxicant-induced reductions in sperm motion and fertility.

Carcinogenicity study with epichlorohydrin (CEP) by gavage in rats.

Weanling Wistar rats of both sexes were given epichlorohydrin by gastric intubation for 2 years, 5 times a week at dosages of 0, 2, and 10 mg/kg body weight. Mortality and body weight gain were recorded and histopathological examination for tumours was carried out; after 1 year also haematology was performed. Towards the end of the study a slight dose-related increase in mortality was observed in males, along with a decrease in mean body weight in the survivors. At pathological examination a high incidence (100% for females, 81% for males) of squamous cell carcinomas of low-grade malignancy was observed in the forestomach of animals at risk (greater than 18 months) from the 10 mg/kg group. In the 2 mg/kg group forestomach tumours were found at a lower incidence (7% for females, 14% for males), whereas this tumour was not found in control animals. Other tumours diagnosed in this study occurred at background level and were not influenced by treatment.

Reaction of epichlorohydrin with 2'-deoxynucleosides: characterization of adducts.

Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance and thus has the potential for human exposure in the workplace. It has been shown to be genotoxic in several systems and is a compound capable of reacting with biological nucleophiles. This study details the products formed from the reaction of ECH with 2'-deoxynucleosides at pH 7 and 37 degrees C for 6 h. Reaction with 2'-deoxyguanosine yielded 7-(3-chloro-2-hydroxypropyl) guanine (7-CHP-Gua) resulting from alkylation at N-7 of 2'-deoxyguanosine followed by depurination. Two unusual adducts were also partially characterized which resulted from further reaction of 7-CHP-Gua with another molecule of ECH to yield 1,7-bis(3-chloro-2-hydroxypropyl)guanine (1,7-bis-CHP-Gua) which could then cyclize with the exocyclic amino group to yield 1,N2-(2-hydroxypropano)-7-(3-chloro-2-hydroxypropyl) guanine (1,N2-HP-7-CHP-Gua). Reaction with 2'-deoxyadenosine gave only one product, namely 1,N6-(2-hydroxypropano)-2'-deoxyadenosine (1,N6-HP-dAdo). The reaction of 2'-deoxythymidine with ECH also yielded one product which was identified as 3-(3-chloro-2-hydroxypropyl)-2'-deoxythymidine (3-CHP-dThd). A 3-(3-chloro-2-hydroxypropyl)-2'-deoxyuridine (3-CHP-dUrd) product was isolated from the reaction of ECH with 2'-deoxycytidine. This product most likely resulted from the deamination of an initially formed 3-(3-chloro-2-hydroxypropyl) -2'-deoxycytidine (3-CHP-dCyd), a phenomenon which we have previously reported to occur during the reaction of 2'-deoxycytidine with other aliphatic epoxides. Evidence is also presented that 3-CHP-dUrd is converted to 3-(2,3-dihydroxypropyl)-2'deoxyuridine (3-DHP-dUrd) under physiological conditions, with a half-life of 213 h. Reaction of ECH with calf thymus DNA (pH 7.0, 37 degrees C, 3 h) resulted in the formation of 7-CHP-Gua (200 nmol/mg DNA.

Comparison of propylene oxide and epichlorohydrin effects in two transformation tests (C3H/10T1/2 and SHE cells).

The neoplastic cell transformation induced by propylene oxide (PO) and epichlorohydrin (ECH) was studied in two in vitro assays, mouse embryo fibroblasts (C3H/10T1/2) and Syrian hamster embryo (SHE) cells. In C3H/10T1/2 cells treated with PO (2.5-10 mM), the transformation frequencies were enhanced about 2-4 times in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with the transformation frequencies in the absence of TPA. In SHE cells, an even higher increase (about 6-9 times) was reached at concentrations of 2.5-20 mM. The presence of TPA strongly influenced the ability of ECH to induce the morphological transformation at low-moderate concentrations (0.25-1 mM). At the highest concentrations applied, 1 mM in C3H/10T1/2 cells and 0.5 mM in SHE cells, 41- and 4-fold increases, respectively, were observed. In C3H/10T1/2 cells, the rad-equivalence (rad/mMh) of PO and ECH in the presence of TPA was calculated to be 36 +/- 8 and 296 +/- 65 (mean +/- S.E.), respectively.

Mutagenic activity of some clastogenic chemicals at the hypoxanthine guanine phosphoribosyl transferase locus of Chinese hamster ovary cells.

4 presumptive clastogens (caffeine, dimethyl sulfoxide, diethylstilbestrol and p,p'-DDE) and 4 chemicals thought to induce chromosomal mutations in L5178Y mouse lymphoma cells (procarbazine X HCl, epichlorohydrin, hycanthone and iodomethane) were tested in the CHO/HGPRT gene mutation assay for the induction of 6-thioguanine-resistant ( 6TGR ) mutants. Of the clastogens, p,p'-DDE was mutagenic at several concentrations and diethylstilbestrol (DES) increased the 6TGR mutant frequency over control levels at the 6.7 and 8.0 micrograms/ml doses, but the results for DES were neither convincing nor significant. Caffeine was not mutagenic although at very high concentrations (6667-8000 micrograms/ml) there was a slight elevation in mutant frequency over background. This was probably due to a selective effect of caffeine against the HGPRT+ phenotype, for 2 different HGPRT- cell lines were refractory to the toxic effects of caffeine at the highest test level (8000 micrograms/ml). All 4 'chromosomal mutagens' produced dose-related increases in mutant frequencies at the HGPRT locus of these CHO cells. 6TGR colonies were generally uniform in size when normal precautions were taken to prevent the formation of satellite (secondary) colonies. Excepting DES, dimethyl sulfoxide, and caffeine, these data demonstrate that 5 of 8 clastogenic chemicals reproducibly induce mutations at the HGPRT locus of CHO cells which lack the small colony-forming potential of 3.7.2C L5178Y cells.

Genetic toxicology of epichlorohydrin: a review.

Epichlorohydrin (ECH) is one of the more commercially important aliphatic epoxides used extensively as an industrial intermediate, a laboratory reagent, and as an insecticide. It is a volatile, colourless liquid with an ethereal odour. It behaves as an alkylating agent. Reports have shown it to cause the respiratory and dermal toxicity in animals and humans. It has also been reported to be carcinogenic in experimental models. Thus, the wide-spread use of this aliphatic epoxide is of great concern in human health problem. The purpose of this paper is to critically review and update the mutagenic and clastogenic effects of ECH based on available literature.

Micronucleated reticulocyte induction by ethylating agents in mice.

Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.

Industrial mutagens and potential mutagens I. Halogenated aliphatic derivatives.

The halogenated aliphatic hydrocarbons represent one of the most important categories of industrial chemicals from a consideration of volume, use categories, environmental and toxicological considerations and hence most importantly, potential population risk. The major halocarbons reviewed, primarily in terms of their occurrence, utility, stability, distribution, and levels of exposure as well as their metabolism, carcinogenicity and mutagenicity included: vinylchloride, vinylidene chloride, trichloroethylene, perchloroethylene, ethylene dichloride, ethylene dibromide, chloroprene, chloroform, carbon tetrachloride, fluorocarbons (trichlorofluoromethane and dichlorodifluoromethane), epichlorohydrin, halohydrins (2-chloro- and 2-bromoethanol) and haloethers (bis(chloromethyl); chloromethyl'-methyl; bis(2-chloroethyl)-and bis(2-chloroisopropyl)ether. In many instances, data were not available to assess world production, populations at risk and degrees of exposure. With the exception of vinylchloride, vinylidene chloride, epichlorohydrin and 2-halo ethanols, there is an acknowledged paucity of definitive mutagenicity data concerning the majority of halogenated hydrocarbons. Their ubiquitous distribution, and in a number of cases, their carcinogenicity both in man and animals, dictates the urgent need to more exhaustively investigate their potential mutagenicity.

The effect of deuterium labeling on the genotoxicity of N-nitrosodimethylamine, epichlorohydrin and dimethyl sulfate.

Deuterated and non-deuterated N-nitrosodimethylamine, epichlorohydrin and dimethyl sulfate were evaluated for the ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. Non-deuterated nitrosodimethylamine induced twice the amount of DNA-strand breaks as the deuterated form. No evidence of a deuterium isotope effect was seen for the direct-acting alkylating agents epichlorohydrin and dimethyl sulfate.

Genotoxicity, metabolism and blood kinetics of epichlorohydrin in mice.

Epichlorohydrin (ECH), a direct mutagen in vitro, did not induce chromosomal aberrations in bone-marrow cells of CD1 mice given single oral doses of 50 and 200 mg/kg in water. The ECH diol derivative (3-chloro-1,2-propanediol) was tested in vitro by a forward-mutation assay on the yeast Schizosaccharomyces pombe and showed a weak but significant mutagenic effect. The failure of ECH to induce mutagenic effects appears to be due to the rapid metabolic clearance of the compound in vivo. ECH blood kinetics at both doses, and at the same time the concentration of the diol, were determined. ECH rapidly disappeared from mouse blood, being no longer detectable 20 min after treatment. In contrast, 3-chloro-1,2-propanediol was measurable up to 5 h after dosage. No difference was observed in the kinetic and metabolic behavior of ECH after single and repeated doses (50 and 200 mg/kg/day for 7 days). When 3-chloro-1,2-propanediol was tested, neither glutathione depletion nor epoxide hydrolase inhibition (evaluated with both styrene-7,8-oxide and ECH as substrates) could be detected in mouse liver. Finally, no difference in ECH blood kinetics or metabolism were observed in experiments in which the compound was administered (200 mg/kg) intraperitoneally in water or orally as a solution in dimethyl sulfoxide.