Evaluation of the chronic toxicity and oncogenicity of N,N-diethyl-m-toluamide (DEET).

Chronic toxicity and/or oncogenicity studies were conducted in rats, mice, and dogs with the insect repellent DEET. DEET was mixed in the diet and administered to CD rats for two years at concentrations that corresponded to dosage levels of 10, 30 or 100 mg/kg/day for males and 30, 100, or 400 mg/kg/day for females; to CD-1 mice for 18 months at dosage levels of 250, 500, or 1000 mg/kg/day; and to dogs for one year, via gelatin capsules, at dosage levels of 30, 100, or 400 mg/kg/day. In the rodent studies, each group consisted of 60 animals of each sex, and two concurrent independent control groups, each containing 60 animals/sex were included in each study. Each group in the dog study consisted of four male and four female dogs and one control group was included in the study. Treatment-related effects were observed at the highest dose level in all three studies. For rats, the effects included decreases in body weight and food consumption and an increase in serum cholesterol in females only. In mice, the effects observed were decreases in body weight and food consumption in both sexes. The effects observed in dogs included increased incidences of emesis and ptyalism, and levels of transient reduction in hemoglobin and hematocrit, increased alkaline phosphatase (males only), decreased cholesterol, and increased potassium. One male dog in the high-dose group also exhibited ataxia, tremors, abnormal head movements, and/or convulsions on several occasions during the study. The highest no-observed-effect levels (NO-ELs) for rats, mice and dogs were determined to be 100, 500, and 100 mg/kg/day, respectively. No specific target organ toxicity or oncogenicity was observed in any of the studies.

Absorption, distribution, metabolism, and excretion of N,N-diethyl-M-toluamide in the rat.

This study was conducted to evaluate the pharmacokinetic parameters of absorption, distribution, metabolism, and excretion (ADME) of the personal insect repellent N,N-diethyl-m-toluamide (DEET) after oral or dermal administration of [14C]DEET in the rat. Six experiments were conducted using separate groups, each consisting of five male and five female rats. Three experiments involved the determination of ADME patterns after oral administration of [14C]DEET as: 1) a single low dose (100 mg DEET/kg body weight); 2) a single high dose (500 mg DEET/kg body weight); and 3) a repeated low dose (100 mg DEET/kg body weight daily for 14 days). A fourth experiment involved the determination of ADME patterns after dermal administration of [14C]DEET at a single low dose of 100 mg DEET/kg body weight. In these four experiments, urine and feces were collected over a 7-day posttreatment period, after which time the animals were euthanized and selected tissues and organs were harvested. Urine, feces, and tissues were analyzed for total 14C content. The major urinary metabolites were identified, and the urinary metabolic profile for each dosage regimen was determined. The remaining two experiments examined the distribution of radioactivity in tissues of animals euthanized at peak 14C blood levels after receiving a single oral low dose or a dermal low dose. In the three experiments designed to determine the ADME patterns of DEET after oral administration, 85-91% of the administered radioactivity was found in the urine and 3-5% was found in the feces. The overall quantitative pattern of excretion of radioactivity into the urine and feces was similar for males and females in the three groups; however, the rate at which the radioactivity was excreted into the urine differed noticeably between individual oral dosing regimens. The fastest rate was observed in the repeated oral low-dose group, followed by the single oral low-dose and the single oral high-dose groups. In the group of rats that received the dermal low dose, 74-78% of the administered dose was found in the urine and 4-7% was found in the feces. An additional 6.5% was found on the surface of the skin at the application site or in association with the occlusive enclosure. The rate of absorption and subsequent excretion of administered radioactivity into the urine and feces was much slower after dermal administration than after all oral dosing regimens. Total tissue residues of 14C activity at 7 days ranged from 0.15 to 0.67% of the administered dose for all dosage regimens. At peak 14C blood levels, the percentages of administered dose reaching the systemic circulation and total 14C tissue residues were significantly higher in the group of animals administered [14C]DEET orally vs. the animals administered [14C]DEET by the dermal route of administration. In both cases, the only tissues with 14C residues consistently higher than that of plasma were the liver, kidney, and fat. HPLC analysis of urine from rats in the ADME phase of the study showed that DEET was metabolized completely in all treatment groups, with little or no parent compound excreted in the urine. Two major urinary metabolites were identified by mass spectroscopy. In both metabolites, the aromatic methyl substituent in the DEET molecule was oxidized to a carboxylic acid moiety. One of the metabolites also had undergone N-dealkylation of an ethyl substituent on the amide moiety.

Absorption, metabolism, and excretion of N,N-diethyl-m-toluamide following dermal application to human volunteers.

The absorption, metabolism, and excretion of N,N-diethyl-m-toluamide (DEET) in male human volunteers following dermal application of [14C]DEET was studied. DEET was applied to two groups of six volunteers either as the undiluted technical grade material or as a 15% solution in ethanol. The material was applied over a 4 x 6-cm area on the volar surface of the forearm and was left in contact with the skin for 8 hr, then rinsed off the skin. Application sites also were tape stripped at 1, 23, and 45 hr after rinsing. Serial blood samples and all urine and feces were collected for 5 days after application. Aliquots of these materials were analyzed for total radioactivity in order to define absorption and excretion patterns. Urine samples also were analyzed by HPLC to characterize the metabolic profile and/or to identify metabolites. Absorption of DEET as evidenced by plasma radioactivity occurred within 2 hr after dose application. Elimination of radioactivity from plasma was rapid and quantifiable levels of radioactivity were observed in plasma for only 4 hr after the end of the 8-hr exposure period. Urine was the principal route of excretion of radioactivity and accounted for an average of 5.61 and 8.33% of the applied dose in the undiluted DEET and 15% DEET in ethanol groups, respectively. Excretion of radioactivity in the feces was less than 0.08% of the applied dose in both groups. DEET did not accumulate in the superficial layers of the skin as evidenced by low amounts of radioactivity in the tape strippings.(ABSTRACT TRUNCATED AT 250 WORDS)

Teratologic evaluations of N,N-diethyl-m-toluamide (DEET) in rats and rabbits.

The potential for DEET to produce developmental toxicity was evaluated in Charles River CD rats and New Zealand White rabbits. Rats were administered undiluted DEET by gavage on Gestational Days (gd) 6-15 at dosage levels of 0, 125, 250, and 750 mg/kg/day. Rabbits were administered undiluted DEET by gavage on gd 6-18 at dosage levels of 0, 30, 100, and 325 mg/kg/day. Group sizes were 25 females per group for rats and 16 females per group for rabbits. Control rats and rabbits were administered corn oil at the same dosage volumes administered in the high-dose DEET groups. In rats, maternal toxicity in the form of clinical signs including two deaths and depressed body weight and food consumption was observed at the high-dose level of 750 mg/kg/day. Rat fetal body weights per litter also were reduced at 750 mg/kg/day. In rabbits, maternal toxicity in the form of depressed body weight and food consumption was observed at the high-dose level of 325 mg/kg/day. No maternal toxicity was observed at the low- or mid-dose groups for rats or rabbits. With the exception of the reduced fetal weights in rats at 750 mg/kg, there was no evidence of fetal toxicity, no effects on any of the gestational parameters, nor were there any treatment-related increases in external, visceral, or skeletal variations or malformations in the offspring from the rats and rabbits from these studies.

Neurotoxicity evaluation of N,N-diethyl-m-toluamide (DEET) in rats.

The neurotoxic potential of N,N-diethyl-m-toluamide (DEET) was evaluated following acute oral administration or following multigeneration plus chronic dietary administration to the rat. For the acute study, rats were administered undiluted DEET at dose levels of 50, 200, or 500 mg/kg by gavage. A dose level of 500 mg/kg was considered to be the highest practical dose that could be evaluated in this study based upon observations of overt toxicity at 500 mg/kg and mortality at 1000 mg/kg in a dose range-finding study. The two measures of neurotoxicity evaluated in the acute study were functional observational battery (FOB) and motor activity measurements. An apparent treatment-related effect in thermal response time (increased) was noted for both sexes 1 hr after dosing at the 500 mg/kg dose level. A questionable effect on rearing activity (decreased) also was noted at the same dose level. For the multigeneration plus chronic dietary administration study, rats were administered DEET at dietary concentrations of 0, 500, 2000, or 5000 ppm continuously over two generations and then chronically for 9 months. A dietary concentration of 5000 ppm meets the criteria for a maximum tolerated dose (MTD) based on traditional chronic toxicology assessments. Evaluations included FOB, motor activity, discriminative acquisition and reversal in an M-maze, acoustic startle habituation, passive avoidance acquisition and retention, and microscopic examination of central and peripheral nervous tissue. The only effect that was considered to be possibly treatment-related was a slight increase in exploratory locomotor activity at the 5000 ppm dose level. Based on the results of these studies, the nervous system does not appear to be a selective target when DEET is administered to rats either as a single oral dose at high dose levels or chronically at the MTD.

Absorption, distribution and excretion of the colour fraction of Caramel Colour IV in the rat.

Caramel Colour IV prepared from [U-14C]glucose was ultrafiltered in order to isolate the high molecular weight colour fraction (HMCF). The colour fraction that was non-permeable to a 10,000-Da porosity membrane, contained 84% of the colour, 22% of the solids and 24% of the radioactivity of the [14C]Caramel Colour IV. The absorption, distribution and excretion of [14C]HMCF were evaluated in male rats after administration of single or multiple oral doses of the material at a dosage level of 2.5 g/kg body weight. Rats on the multiple oral dosage regimen were given unlabelled HMCF in their drinking water for 13 days before the administration of a bolus dose of [14C]HMCF on day 14. On both dosage regimens, the predominant route of excretion was by way of the faeces. Less than 3% of the administered radioactivity was excreted in the urine and only a negligible amount was found in the expired air. More than 99% of the administered radioactivity was excreted within 96 hr. The principal tissues in which radioactivity was found were the mesenteric lymph nodes, liver, kidney and tissues of the gastro-intestinal tract. No major differences were observed in the absorption, distribution or excretion patterns between the single and multiple oral dose regimens.

Comparative bladder tumor promoting activity of sodium saccharin, sodium ascorbate, related acids, and calcium salts in rats.

Sodium saccharin and sodium ascorbate are known to promote urinary bladder carcinogenesis in rats following initiation with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or N-butyl-N-(4-hydroxybutyl) nitrosamine. Sodium salts of other organic acids have also been shown to be bladder tumor promoters. In addition, these substances increase urothelial proliferation in short term assays in rats when fed at high doses. When they have been tested, the acid forms of these salts are without either promoting or cell proliferative inducing activity. The following experiment was designed to compare the tumor promoting activity of various forms of saccharin and to evaluate the role in promotion of urinary sodium, calcium, and pH as well as other factors. Twenty groups of 40 male F344 rats, 5 weeks of age, were fed either FANFT or control diet during a 6-week initiation phase followed by feeding of a test compound for 72 weeks in the second phase. The chemicals were administered to the first 18 groups in Agway Prolab 3200 diet and the last 2 groups were fed NIH-07 diet. The treatments were as follows: (a) FANFT----5% sodium saccharin (NaS); (b) FANFT----3% NaS; (c) FANFT----5.2% calcium saccharin (CaS); (d) FANFT----3.12% CaS; (e) FANFT----4.21% acid saccharin (S); (f) FANFT----2.53% S; (g) FANFT----5% sodium ascorbate; (h) FANFT----4.44% ascorbic acid; (i) FANFT----5% NaS plus 1.15% CaCO3; (j) FANFT----5.2% CaS plus 1.34% NaCl; (k) FANFT----5% NaS plus 1.23% NH4Cl; (l) FANFT----1.15% CaCO3; (m) FANFT----1.34% NaCl; (n) FANFT----control; (o) control----5% NaS; (p) control----5.2% CaS; (q) control----4.21% S; (r) Control----control; (s) FANFT----5% NaS (NIH-07 diet); (t) FANFT----control (NIH-07 diet). NaS, CaS and S without prior FANFT administration were without tumorigenic activity. NaS was found to have tumor promoting activity, showing a positive response at the 5 and 3% dose levels, with significantly greater activity at the higher dose. CaS had slight tumor promoting activity but without a dose response, and S showed no tumor promoting activity. In addition, NaCl showed weak tumor promoting activity, but CaCO3 was without activity. NH4Cl completely inhibited the tumor promoting activity of NaS when concurrently administered with it. NaCl administered with CaS or CaCO3 administered with NaS showed activity similar to that of NaS. Sodium ascorbate was also shown to have tumor promoting activity, with slightly less activity than NaS. Ascorbic acid showed no tumor promoting activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative analysis of the proliferative response of the rat urinary bladder to sodium saccharin by light and scanning electron microscopy and autoradiography.

Three methods used to detect proliferative changes in the rat urothelium, light microscopy, scanning electron microscopy, and autoradiography, were compared for their sensitivity in detecting changes produced by administration of sodium saccharin. Weanling male F344 rats were fed sodium saccharin as 0, 3, 5, or 7.5% of the diet, and the bladders were evaluated after 4, 7, and 10 wks of feeding. Light microscopic changes and an increase in labeling index were seen at all time points in rats fed 7.5% sodium saccharin, but not at the lower doses. A slight increase in labeling index was also observed at 10 wks in the 5.0% group. Scanning electron microscopic changes were evident as early as 4 wks with increasing severity at the 3, 5, and 7.5% doses. This study demonstrates that the hyperplastic response of the urothelium to sodium saccharin administration varies with dose and time, and that observation by scanning electron microscopy is the most sensitive of the three methods evaluated for detecting these changes.

Evaluation of the dose response and in utero exposure to saccharin in the rat.

A two-generation bioassay on sodium saccharin (NaS), involving 2500 second-generation male rats, was designed to determine the dose response for urinary bladder tumours in male rats and to evaluate other changes possibly related to the occurrence of the tumours. Six treatment groups (125-700 rats/group) were fed dietary levels of NaS ranging from 1.0 to 7.5%. To evaluate the role of in utero exposure, two additional groups were exposed to NaS either only during gestation via dams fed diet containing 5.0% NaS or for a single generation beginning at birth. In the latter group, the nursing dams were placed on an NaS diet immediately after giving birth and their offspring were weaned onto diets containing 5.0% NaS. A third additional group, included to evaluate the specificity of NaS and the role of excess sodium in the occurrence of urinary bladder tumours, was fed diet containing sodium hippurate (NaH) for two generations--5.0% NaH to the first generation and to the second until 8 wk old, and subsequently 3.0% because of unexpected toxicity. A clear dose response for urinary bladder tumours was observed in the second-generation NaS-treated male rats. The steep slope of the dose-response curve indicated a rapid decline in tumour incidence with decreasing dose. The 1.0% dietary level (fed to 700 rats) was considered to be a no-effect level for bladder tumours. The only other treatment-related pathological changes were an increase in urinary bladder weight in rats fed greater than or equal to 3.0% and an increase in mineralization of the kidneys with greater than or equal to 1.0%. Several physiological effects were seen in the NaS-treated groups showing an increase in bladder tumours (i.e. those fed greater than or equal to 3.0%). Some changes, e.g. depressed growth and increased water consumption, were indicative of a general disturbance of these rats, but analysis of body-weight, food-consumption, compound-consumption and water-consumption data revealed no correlations within any dose group between these quantitative data and the occurrence of bladder tumours. Other changes indicative of the compromised situations of the rats fed high dietary levels of NaS were anaemia in weanling rats fed 5.0 or 7.5% and a reduction in litter size at dietary levels greater than or equal to 3.0%. Changes in urine volume and urine osmolality were highly correlated with the occurrence of the urinary bladder tumours.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of high dietary levels of sodium saccharin on mineral and water balance and related parameters in rats.

The effects of sodium saccharin (NaS) treatment on mineral and water balance and a number of related parameters were studied over a 10-day period in 7-month-old Charles River CD rats. Eight groups of 10 male and 10 female rats were studied. In four of the groups the rats were the F1 offspring of rats that had been exposed to NaS at 1, 3, 5 or 7.5% in the diet and the offspring were treated with the same dietary levels of NaS as their parents. Prior treatment in two other groups was modified in order to evaluate the role of in utero exposure to NaS on the study parameters: rats in one group were only exposed in utero via dams fed diets containing 5% NaS while treatment in the other group did not include in utero exposure, but was started at birth via dams fed diets containing NaS and continued at a dietary concentration of 5% NaS. Second-generation rats in another group were fed diets containing 5% sodium hippurate (NaH), a compound with a number of physical and chemical properties similar to those of NaS; this group was included in order to evaluate the specificity of NaS and/or the effect of sodium on the study parameters. A group of untreated rats served as controls. Treatment-related effects were observed in most study parameters. In addition, a number of differences between male and female rats in baseline values and/or in response to NaS administration were observed. With increasing dietary levels of NaS body weights decreased, but there were increases in water consumption, faecal water content, and caecal weights. NaS treatment resulted in increased urine volume and decreased urine osmolality, changes in urine mineral concentrations (increased sodium, decreased potassium and zinc) and increases in fresh and dry bladder weights, bladder-tissue hydration, and mineral concentrations (sodium, potassium, magnesium and zinc) in bladder tissue. The parameters in which clear sex-related differences in baseline values were observed were body weight, food and water consumption, urine volume, urine osmolality, fresh bladder mass, bladder-tissue hydration and the concentrations of sodium, potassium, magnesium and zinc in the bladder tissue. With the exception of urine osmolality, the values were higher in females. Differences between males and females in response to treatment were observed for NaS consumption (increased in females), caecal weight (increased in females), NaS concentration in the urine (increased in males), and the concentration of sodium, potassium, magnesium and zinc in the bladder tissue (increased in males).(ABSTRACT TRUNCATED AT 400 WORDS)