Limitations of urinary mutagen assays for monitoring occupational exposure to antineoplastic drugs

The sensitivity of the Salmonella reversion test of Ames as a screen for accidental absorption of 17 antineoplastic agents by drug handlers was evaluated. Dilutions of each drug were added to agar inoculated with each of two Salmonella typhimurium strains (TA98 and TA100); control plates contained no test drug. Colonies were counted after incubation at 36 degrees C for 48 hours. The drugs were tested in the presence of a liver preparation to provide metabolic activation of mutagenicity. Urine samples collected from patients after doses of three mutagenic drugs were extracted and tested with the Ames test. For 11 of the 17 drug solutions, no mutagenic activity was seen, but many of these 11 were toxic to the organisms. The most highly mutagenic drugs were doxorubicin and cisplatin, with mechlorethamine, carmustine, dacarbazine, and cyclophosphamide exhibiting less mutagenic activity. Urine from patients treated with doxorubicin or cyclophosphamide showed mutagenicity, but the results suggested that the quantity of these drugs that would have to be absorbed to produce a definite reaction in urine is unlikely to be achieved by drug handlers who use standard precautions. Because of its lack of sensitivity and the potential effects of environmental and dietary factors on the results, this bacterial mutagenicity test should not be used routinely for detection of accidental absorption of antineoplastic drugs.     

Occupational exposure to antineoplastic drugs in four Italian health care settings

Exposure assessment of health care workers to antineoplastic drugs (ADs) is still an open issue since new, critical, and emerging factors may put pharmacists who prepare hazardous drugs or nurses who administer anti cancer agents to an increased risk of developing adverse health effects. Overall, eight pharmacies and nine patient areas have been surveyed in this study. Wipe and pad samples were experienced during the surveillance program in four Italian health care settings. Urine samples were collected from workers handling ADs. Cyclophosphamide (CP), ifosfamide (IF), and gemcitabine (GEM) were detected in all the work environments by using a LC-MS/MS method-based capable of analysing all the three drugs simultaneously. In total, 54% of wipe samples were positive for at least one drug and 19% of pad samples were shown to be contaminated by cyclophosphamide. Pharmacies were generally more contaminated than patient areas with the exception of one site where a nurse had an acute exposure during the cleaning-up of an hazardous drug solution spill. In total, 22 urine samples collected from pharmacists and 78 urine samples from nurses had no detectable concentrations of any antineoplastic drugs. Despite the adherence to the recommended safety practices residue contamination on surfaces and floors has continued to be assessed in all the investigated sites.     

Comparative studies of the mutagenicity of urine from smokers and non-smokers on a controlled non-mutagenic diet

Measuring the mutagenicity of urine is widely viewed as a means of evaluating human exposure to potentially genotoxic materials. Diet and cigarette smoking have both been reported to affect the mutagenicity of human urine, but the relationship between smoking status and the expression of diet-related urinary mutagenicity is unknown. It has been reported that some promutagens are more active in in vitro assays when tested in the presence of urine from smokers than when tested in the presence of urine from non-smokers. We aimed to determine whether the differences in urinary mutagenicity between smokers and non-smokers result from increased urinary mutagenicity from dietary heterocyclic amine mutagens in smokers compared with non-smokers. Groups of smokers and non-smokers (6-12) were given identical diets, previously shown to be low in heterocyclic amines and very low in mutagenicity. The diet consisted exclusively of raw food and of food cooked in boiling water. After a 2-day dietary stabilization period, 24-hour urine samples were collected for three consecutive days. The regimen was repeated in the following week. For comparison, both groups were also placed on a "western" diet, consisting of a variety of foods prepared by several cooking methods, designed to reflect what a typical United States family might consume. Urine was concentrated using XAD-2 resin and then assayed for mutagenic activity in the Ames test. The urine of smokers was significantly more mutagenic than that of non-smokers when on both the raw/boiled and the "western" diets. These results indicate that the increased urinary mutagenicity observed in smokers compared with non-smokers is not due to enhanced mutagenicity of diet-related heterocyclic amine mutagens in the urine of smokers.     

Mutagenic activity of major mammalian metabolites of cyclophosphamide toward several genes of Escherichia coli.

Representative intermediates of the major mammalian metabolic pathway of cyclophosphamide, a drug that is not mutagenic as such unless it is metabolically activated, were assayed for their direct mutagenic activity toward a bacterial indicator strain, Escherichia coli 343/113. The compounds tested were 4-hydroperoxycyclophosphamide and the two urinary metabolites, carboxyphosphamide and 4-ketocyclophosphamide. Further, the degradation products of 4-hydroxycyclophosphamide, phosphoramide mustard, acrolein, and nornitrogen mustard, were also tested. The mutagenicity test systems were those used previously to demonstrate the liver enzyme-mediated mutagenic activity of cyclophosphamide in E. coli 343/113: stationary cell suspensions were treated for 180 min at 37 degrees C with different concentrations of the compound under test; the induction of forward mutations from 5-methyltryptophan sensitivity to resistance (MTR) and from galRs18 to gal+ as well as back mutations from arg56 to arg+ was measured by plating aliquots of the treated bacterial population on different selective mutation media. Except for acrolein, all cyclophosphamide metabolites tested are directly mutagenic toward E. coli 343/113. With all substances the highest induced mutation frequency is that of arg+ mutations, followed by gal+ and MTR mutations; this indicates that mostly base-pair substitution type mutations are induced. The mutagenic potential, however, differs greatly between compounds at concentrations between 0.1 and 20mM. The results show that the first step in the mammalian biodegradation of cyclophosphamide gives rise to compounds that are directly mutagenic, and that this mutagenicity is retained and even enhanced through all further metabolic steps to produce the compound of highest mutagenicity, nornitrogen mustard.     

Evaluation of genotoxic risk of handling cytostatic drugs in clinical pharmacy practice

The genotoxic risk of handling antineoplastic drugs was evaluated in fifteen women preparing chemotherapeutics in the Pharmacy Department of the University Hospital Maastricht. Twenty nurses of the same hospital, who were not exposed to cytostatics, acted as controls. Endogenous exposure to antineoplastic drugs was assessed by determination of urine mutagenicity, as well as by analysis of urinary methotrexate levels. As genotoxicological end-points, sister chromatid exchanges and hypoxanthine guanine phosphoribosyl transferase locus point mutations were studied in peripheral lymphocytes obtained via venous puncture. No differences in urine mutagenic activity, in sister chromatid exchange frequencies and in hypoxanthine guanine phosphoribosyl transferase point mutation frequencies between exposed and non-exposed groups were detected. Higher sister chromatid exchange frequency was observed in smokers as compared to non-smokers.     

Using a closed-system protective device to reduce personnel exposure to antineoplastic agents.

Surface contamination with and personnel exposure to antineoplastic agents before and after the implementation of a closed-system protective device were studied. Samples were collected before and six months after implementation of PhaSeal, a closed-system device for limiting exposure to antineoplastic agents during preparation and administration. Personnel exposure was evaluated by collecting 24-hour urine samples from pharmacists, pharmacy technicians, and nurses working full-time in a chemotherapy drug infusion center and pharmacy. Surface contamination was assessed by wiping potentially exposed surfaces. Both types of samples were analyzed for cyclophosphamide and ifosfamide by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. All 17 wipe samples collected before implementation of PhaSeal had detectable levels of cyclophosphamide, and 11 were positive for ifosfamide. Six months after system implementation, 7 of 21 wipe samples had detectable levels of cyclophosphamide and 15 were positive for ifosfamide. Of eight employees who provided urine samples, six were positive for cyclophosphamide and two for ifosfamide before implementation, and none were positive for either drug after implementation. The PhaSeal system appeared to reduce exposure of health care personnel to cyclophosphamide and ifosfamide.     

Mutagenicity of human urine after the consumption of fried salted salmon

Mutagenicity in the urine of four non-smoking individuals who had eaten salted salmon cooked at home for both lunch and supper was monitored by means of Salmonella/microsome mutagenicity tests. Extracts from fresh and salted salmon had the same level of mutagenicity after being cooked for 10 min at 200 degrees C, but no activity was detected before cooking. Salmonella strains TA98 and TA1538 were equally sensitive to the mutagens and required metabolic activation. No mutagenicity was shown with TA100 and TA1535. Urine samples were tested using a concentrate prepared by means of an XAD-2 resin column. Mutagenicity was detected mainly in urine excreted during 4-5 hr after the ingestion of cooked salmon, but only weak mutagenicity, or none at all, was detected in the urine after the ingestion of vegetables. The levels of urinary mutagenicity due to salmon consumption were not affected when cabbage was eaten simultaneously. The excretion of mutagenic substances was completed within about 20 hr, and there were almost no mutagens in the urine 24 hr after the ingestion of cooked salmon.     

Biological monitoring of hospital personnel occupationally exposed to antineoplastic agents

To detect trace amounts of urinary cyclophosphamide (CP), ifosfamide (IF) and methotrexate (MTX), sensitive and specific high-performance liquid chromatography/ tandem mass spectrometry (HPLC-MS/MS) procedures, incorporating either liquid-liquid (for CP and IF), or solid-phase, extraction (for MTX) have been developed. Urinary platinum (Pt) was also detected using inductively coupled plasma-mass spectrometry (ICP-MS). These methods showed acceptable imprecision and inaccuracy. The limit of detection (LOD) was 50 ng/l for CP and IF, 200 ng/l for MTX and 1 ng/l for Pt. Biomonitoring was performed on two consecutive days on nine subjects preparing, and seven administering, antineoplastic drugs. Urine was collected at the beginning, at the end and during the work shift. Eighteen urine samples were positive for CP (range: 50-10031 ng/l), whereas IF was detected in one subject only (153 ng/l). LOD was never exceeded for MTX. In urine samples from nurses and pharmacy technicians, Pt was detected in three subjects (range 920-1300 ng/l). These findings were compared with the results from a previous survey carried out in the same hospital when different work practices were in use. The proposed methods are simple, fast and reliable and can be used to identify exposure of hospital personnel handling antineoplastic drugs.     

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.     

Drugs hazardous to healthcare workers. Evaluation of methods for monitoring occupational exposure to cytostatic drugs.

We review the literature concerning possible health risks for individuals (e.g. healthcare workers and pharmaceutical plant employees) occupationally exposed to cytostatic drugs. Cytostatic drugs possess toxic properties and may therefore cause mutagenic, carcinogenic and teratogenic effects. Hence, individuals handling these drugs in the course of their employment may face health risks. For this reason, it is important to monitor occupational exposure to these drugs. An overview of exposure monitoring methods is presented and their value is discussed. Most studies involve nonselective methods for biological monitoring and biological effect monitoring, such as the urinary mutagenicity assay and analysis of chromosomal aberrations and sister-chromatid exchanges in peripheral blood lymphocytes. The disadvantages of these biological methods are that their sensitivity is low and it cannot be proved beyond any doubt that the results found were caused by occupational exposure to cytostatic drugs. For occupational health services it is important to have sensitive and specific methods for monitoring exposure to cytostatic drugs. One of the most promising methods seems to be the determination of cyclophosphamide in urine using gas chromatography-tandem mass spectrometry. Several studies have demonstrated exposure to cyclophosphamide and other cytostatic drugs, even when protective measures were taken and safety guidelines were followed. To estimate the magnitude of any health effects arising from this exposure, we calculated the risk of cancer due to occupational exposure to cyclophosphamide on the basis of available human and animal dose-response data and the amounts of cyclophosphamide found in urine. The initial results show an extra cancer risk for pharmacy technicians and nurses.     

Exposure of hospital workers to airborne antineoplastic agents

Practices for handling antineoplastic drugs were surveyed, and ambient-air sampling for four antineoplastic agents was conducted in outpatient oncology clinics. A questionnaire was administered in 1981 to the nurse or pharmacist in charge of drug preparation at 10 hospital oncology clinics. At three sites, air samples were collected during working hours in medication-preparation rooms and nearby offices. The air-sampling pumps contained filters at breathing-zone height; room air was drawn through each filter for 40 hours. Extracts from the filters were assayed by high-performance liquid chromatography (HPLC) for fluorouracil and cyclophosphamide in seven sets of samples and methotrexate and doxorubicin in five sets of samples. Mass spectrometry (MS) was used to confirm detection of fluorouracil. Total use of each monitored drug was recorded at each site. Nine clinics had no ventilation hood, and drugs were prepared by nurses in eight clinics. Routine use of gloves (three clinics) and masks (one clinic) was uncommon, and wastes were disposed of in uncovered receptacles in four of the clinics. Eating and drinking occurred in seven of the preparation rooms. At the main air-sampling site, fluorouracil (0.12-82.26 ng/cu m) was detected in air during 200 of the 320 hours monitored. Cyclophosphamide (370 ng/cu m) was present during 80 hours. In the two other sites, fluorouracil was detected by HPLC but not confirmed by MS, and no cyclophosphamide was detected. No detectable amounts of methotrexate and doxorubicin were present. Fluorouracil was the most frequently used drug, and cyclophosphamide was second. Results suggest that personnel handling antineoplastic drugs are subject to potential systemic absorption of these agents by inhalation.     

Analysis of mutagenic activity in human urine after concentration on different resins and high-performance liquid chromatography

Smokers' urine was tested for mutagenic activity on Salmonella typhimurium strain TA1538 with metabolic activation after adsorption on different resins and desorption with organic solvents. The amounts of XAD-2 were 1.25 and 5 g/100 ml urine, the amounts of alumina, cyanopropyl and C18 were all 5 g/100 ml and extrelut 80 g/100 ml. Adsorbed organic chemicals were eluted with acetone from XAD-2, with dichloromethane from extrelut and with a series of solvents from the other resins (hexane, toluene, dichloromethane and methanol). All columns gave similar results, with the exception of extrelut, which had poor recovery of mutagenic activity. Higher resin/urine ratios and sequences of columns gave better results. The organic eluates from XAD-2 columns loaded with the urine of patients treated with cyclophosphamide and melphalan were mutagenic on strain TA1535 with S9, and some mutagenic activity was also detectable in the aqueous eluate. Cisplatin was adsorbed on XAD-2, C18 and extrelut, but was eluted only from extrelut using dimethylformamide as a solvent. Smokers' urine was separated into several fractions with high-performance liquid chromatography, using C-18 columns with a series of solutions of 2.5 mM phosphoric acid and acetone or with a gradient of methanol. Several fractions containing dissolved organic compounds and no histidine were mutagenic with metabolic activation, but the overall mutagenic activity was still lower than the one detected with one-step chromatography on XAD-2. Using XAD-2 resins with a high ratio of resin to urine still seems to be the method of choice for studying urinary mutagenicity.     

Mutagenicity studies of urine and faecal samples from rats treated orally with the food colourings brown FK and Red 2G

Urine and faecal extracts from rats given Brown FK or Red 2G orally (800 mg/kg body weight) were investigated for mutagenicity. Extracts were subjected to liquid fluctuation and plate incorporation assays with Salmonella typhimurium strains TA98 and TA100 in the presence and absence of liver microsomes and/or a β-glucuronidase-sulphatase preparation. Urine from Red 2G-treated rats only exhibited direct activity when coloured fractions from polyamide-column concentrates were tested with TA100. All other urines, as well as aqueous and ether faecal extracts from animals receiving either colouring, were no more mutagenic than the respective control extracts obtained from the same animals prior to dosing.     

Oral absorption,metabolism and excretion of 1-phenoxy-2-propanol in rats

1. This study was designed to determine the absorption, metabolism and excretion of 1-phenoxy-2-propanol in Fischer 344 rats following oral administration in an effort to bridge data with other propylene glycol ethers.

2. Rats were administered a single oral dose of 10 or 100?mg?kg^{?1 14}C-1-phenoxy-2-propanol as a suspension in 0.5% methyl cellulose ether in water (w/w). Urine was collected at 0–12, 12–24 and 24–48?h and faeces at 0–24 and 24–48?h post-dosing and the radioactivity was determined. Urine samples were pooled by time point and dose level and analysed for metabolites using LC/ESI/MS and LC/ESI/MS/MS.

3. The administered doses were rapidly absorbed from the gastrointestinal tract and excreted. The major route of excretion was via the urine, accounting for 93 ± 5% of the low and 96 ± 3% of the high dose. Most of the urinary excretion of radioactivity occurred within 12?h after dosing; 85 ± 2% of the low and 90 ± 1% of the high dose. Total faecal excretion remained 4. Rapid oral absorption, metabolism and urinary excretion of 1-phenoxy-2-propanol in rats were similar to other propylene glycol ethers.     

Absence of mutagenicity of coralyne and related antileukemic agents: structural comparison with the potent carcinogen 7,12-dimethylbenz[a]anthracene.

The structural similarity between antileukemic alkaloid coralyne and the carcinogenic and antineoplastic hydrocarbon 7, 12-dimethylbenz[a]anthracene, as well as the similarity between the antileukemic alkaloid nitidine and the carcinogenic hydrocarbon 5-methylchrysene, prompted a mutagenicity evaluation of coralyne sulfoacetate, nitidine chloride, the 8-ethyl homolog of coralyne, nitidine methosulfate, and the tetramethoxy analog of nitidine by the Ames method against the histidine-auxotroph strains of Salmonella typhimurium TA-1537, TA-1538, TA-98, and TA-100; 7,12-dimethylbenz[a]anthracene was used as a reference standard. The mutagenicity of these antileukemic compounds was either completely eliminated or drastically reduced, but the mutagenic response was generally high for 7,12-dimethylbenz[a]anthracene. The results suggest that the presence of a quaternary nitrogen atom and alkoxy groups could be important in alleviating the mutagenicity of the parent mutagenic and carcinogenic hydrocarbons.     

The handling of antineoplastic drugs in a major cancer center

It has long been known that many commonly used antineoplastic agents are carcinogenic. Yet most health care professionals take few precautions, if any, when handling these drugs. Recent findings suggest a possible hazard to personnel as evidenced by increased mutagenicity of urine of nurses exposed to anticancer drugs during preparation and administration of doses. Although more study is needed to determine the significance of these data, it would seem prudent to take measures to prevent any unnecessary exposure to these drugs by those preparing and administering them. The policies and procedures at the Memorial Sloan-Kettering Cancer Center require that chemotherapeutic drugs be prepared in a vertical laminar flow containment hood by personnel wearing sterile disposable gloves. Chemotherapeutic agents are specially labeled to ensure segregated disposal of waste, which is subsequently incinerated.     

Amplification of doxorubicin mutagenicity by cupric ion.

There is a presumption that copper and anthracycline drugs will interact with DNA to produce genotoxicity. This is of concern because serum copper levels are increased in certain neoplastic diseases. To test the interaction, it was determined if the metal ion could alter the mutagenesis of doxorubicin and related drugs in the Salmonella microsome test. In the standard form of the test, doxorubicin was strongly mutagenic against frameshift-sensitive strain TA98. When cupric acetate was added with doxorubicin it amplified the mutagenesis of the antineoplastic with an increase of approximately 19% in peak mutagenic values. This apparent "chemoactivation" was evaluated by additional applications of the Salmonella test. Preincubation of cupric acetate, drug, and bacteria (+/- rat liver S9 fraction) also resulted in a copper-amplifying effect. In the preincubation method copper produced a drug concentration-related increase of more than 700% in the mutagenicity of doxorubicin. This large an increase occurred without S9 in the test. The effects observed in TA98 were not seen with TA102, a strain sensitive to oxidation mechanisms. Copper amplification in the mutagenicity of a positive control, aflatoxin B1, was also observed with TA98 but these effects were not seen with the chelator, EDTA, the antifolate antineoplastic drug, methotrexate, or a test negative amino acid, methionine. Results point to a direct frameshift mechanism to explain the increase in mutagenicity with copper. Amplification of mutagenicity found in this study provides initial experimental support that anthracycline-metal ion-DNA associations might contribute to genotoxicity as has been inferred in the literature.     

Effect of liver enzyme inducers on metabolite excretion in rats treated with 1-nitropyrene

Non-induced and phenobarbital (PB) or methylcholanthrene (MC) pretreated rats were injected with 1-nitropyrene (1-NP). Mutagenic activity of urine and feces samples were compared by the Salmonella/microsome assay. The highest, indirect-acting mutagenicity was associated with urines from MC-induced rats; HPLC analysis of organic extracts of urine samples showed that the differences in mutagenic response can be ascribed to different amounts of hydroxy derivatives of N-acetylaminopyrene excreted. Monohydroxy derivatives of 1-NP, being detected in the HPLC profiles of urine from PB-induced rats only, could be responsible of the higher direct-acting mutagenic activity of these samples as compared to urine from non-induced or MC-induced rats. The excretion rate of aminopyrene, the main metabolite of 1-NP identified in rat feces samples, was not affected by inducer pretreatment.     

Oral absorption, metabolism and excretion of 1-phenoxy-2-propanol in rats

1. This study was designed to determine the absorption, metabolism and excretion of 1-phenoxy-2-propanol in Fischer 344 rats following oral administration in an effort to bridge data with other propylene glycol ethers. 2. Rats were administered a single oral dose of 10 or 100 mg kg(-1) 14C-1-phenoxy-2-propanol as a suspension in 0.5% methyl cellulose ether in water (w/w). Urine was collected at 0-12, 12-24 and 24-48 h and faeces at 0-24 and 24-48 h post-dosing and the radioactivity was determined. Urine samples were pooled by time point and dose level and analysed for metabolites using LC/ESI/MS and LC/ESI/MS/MS. 3. The administered doses were rapidly absorbed from the gastrointestinal tract and excreted. The major route of excretion was via the urine, accounting for 93 +/- 5% of the low and 96 +/- 3% of the high dose. Most of the urinary excretion of radioactivity occurred within 12 h after dosing; 85 +/- 2% of the low and 90 +/- 1% of the high dose. Total faecal excretion remained < 10%. Rats eliminated the entire administered dose within 48 h after dosing; recovery of the administered dose ranged from 100 to 106%. Metabolites tentatively identified in urine were conjugates of phenol (sulphate, glutathione) with very low levels (< 2%) of hydroquinone (glucuronide), conjugates of parent compound (glucuronide, sulphate) and a ring-hydroxylated metabolite of parent. There was no free parent compound or phenol in non-acid-hydrolysed urine. In acid-hydrolysed urine, 61% of the dose was identified as phenol and 13% as 1-phenoxy-2-propanol. Although the parent compound was stable to acid hydrolysis, some of the phenol in acid hydrolysed urine may have arisen from degradation of acid-labile metabolite(s) as well as hydrolysis of phenol conjugates. 4. Rapid oral absorption, metabolism and urinary excretion of 1-phenoxy-2-propanol in rats were similar to other propylene glycol ethers.     

Mutagenic activity analysis of body fluids in the first phase of clinical drug trials

On the example of the model drugs metronidazole and ornidazole, a possible use of an analysis of mutagenic activity of blood and urine in the first stage of clinical testing of drugs is demonstrated. Mutagenicity of blood and urine after metronidazole administration in the dose of 1 and 2 g was examined in 3 experiments with 6 volunteers, the presence of mutagenic drugs in urine in the dose of 1 g was observed till 48 h, in the dose of 2 g till 72 h after administration. Besides base-substitution mutations also frameschif mutations were detected, but with considerable individual variability. Mutagenic activity in blood and urine after the administration of 1 g of ornidazole was investigated in the 1st experiment in 3, in further two experiments in 8 volunteers. It was characterized by a two-peak increase in mutagenicity in blood and urine in the intervals of 1-6 h and 48-72 h. Saturation of 1 g of ascorbic acid daily in the course of 1 week prior to the administration of ornidazole was manifested by a decrease in mutagenic activity in blood and increased excretion of mutagenic drugs in urine.