Risk assessment of N-nitrosodiethylamine (NDEA) and N-nitrosodiethanolamine (NDELA) in cosmetics.

N-nitrosamines and their precursors found in cosmetics may be carcinogenic in humans. Thus the aim of this study was to carry out risk assessment for N-nitrosamines (N-nitrosodiethanolamine [NDELA], N-nitrosodiethylamine [NDEA]) and amines (triethanolamine [TEA], diethanolamine [DEA]) levels in cosmetics determined using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedures. NDELA and NDEA concentrations were present at levels of "not detected" (N.D.) to 596.5 µg/kg and N.D. to 40.9 µg/kg, respectively. TEA and DEA concentrations ranged from N.D. to 860 µg/kg and N.D. to 26.22 µg/kg, respectively. The nitrite concentration (3-2250 mg/l), number of nitrosating agents to a maximum 5, and pH (3.93-10.09) were also assessed. The impact of N-nitrosamine formation on the levels of TEA, DEA, nitrite, and other nitrosating agents was also examined. N-nitrosamine concentrations correlated with the number of nitrosating agents and nitrite concentrations. Data demonstrated that higher nitrite concentrations and a greater number of nitrosating agents increased NDELA and NDEA yields. Further, the presence of TEA and DEA exerted a significant influence on N-nitrosamine formation. Risk assessments, including the margin of exposure (MOE) and lifetime cancer risk (LCR) for N-nitrosamines and margin of safety (MOS) for amines, were calculated using product type, use pattern, and concentrations. Exposure to maximum amounts of NDELA and NDEA resulted in MOE > 10,000 (based upon the benchmark dose lower confidence limit 10%) and LCR <1 × 10-5, respectively. In addition, TEA and DEA concentrations in cosmetic samples resulted in MOS values >100. Therefore, no apparent safety concerns were associated with cosmetic products containing NDELA, NDEA, TEA, and DEA in this study. However, since amines and nitrosating agents produce carcinogenic nitrosamines, their use in cosmetics needs to be minimized to levels as low as technically feasible.

Formation and inhibition of N-nitrosodiethanolamine in cosmetics under pH, temperature, and fluorescent, ultraviolet, and visual light.

N-nitrosodiethanolamine (NDELA), a type of nitrosamine, is a possible human carcinogen that may form in cosmetic products. The aim of this study was to examine the formation and inhibition of NDELA through chemical reactions of secondary amines including mono-ethanolamine, di-ethanolamine (DEA), and tri-ethanolamine (TEA), and sodium nitrite (SN) under varying conditions such as pH, temperature, and fluorescent, ultraviolet (UV), and visual light (VIS) using liquid chromatography-mass spectroscopy. In a mixture of TEA and SN under acidic conditions pH 2, residual NDELA concentrations rose significantly under various storage conditions in the following order: 50°C > 40°C > UV (2 W/m2) > VIS (4000 lux) > fluorescent light > 25°C > 10°C. In a mixture of DEA and SN under the same acidic pH 2 conditions, NDELA formation was significantly elevated in the following order: UV (2 W/m2) > VIS (4000 lux) > 50°C > 40°C > fluorescent light > 25°C > 10°C. Inhibition of NDELA formation by d-mannitol, vitamin C (Vit C), or vitamin E (Vit E) was determined under varying conditions of pH, temperature, and fluorescent, UV, and VIS. At high concentrations of 100 or 1000 µg/ml, Vit E significantly decreased residual NDELA compared with control levels under acidic pH 2, but not under basic pH 6. Among various antioxidants, Vit E reacted more effectively with many nitrosating agents such as nitrate and nitrite found in cosmetic products. Therefore, to reduce NDELA, it is recommended that cosmetics be stored under cool/amber conditions and that Vit E or Vit C inhibitors of nitrosation be optimally added to cosmetic formulations at concentrations between 100 and 1000 µg/ml.

Genotoxicity assessments of N-nitrosoethylisopropylamine (NEIPA) and N-nitrosodiisopropylamine (NDIPA) in the C57BL/6J mouse.

N-Nitrosamines, known as drug impurities and suspected carcinogens, have drawn significant public concern. In response to drug regulatory needs, the European Medicines Agency (EMA) has previously proposed a carcinogenic potency categorization approach based on the N-nitrosamine α-hydroxylation hypothesis, i.e., that N-nitrosamine mutagenicity increases with the number of α-hydrogen atoms. However, this structure-activity relationship has not been fully tested in vivo. NEIPA (N-nitrosoethylisopropylamine) and NDIPA (N-nitrosodiisopropylamine) are small N-Nitrosamines with similar structures, differing in that the former compound has an additional α-hydrogen atom. In this study, NEIPA and NEIPA doses, 25-100 mg/kg, were administered orally to C57BL/6 J mice for seven consecutive days, and their mutation and DNA damage effects were compared. Compared with NDIPA, the mutagenicity and DNA damage potencies of NEIPA (which contains one more α-hydrogen) were much greater. These differences may be related to their distinct metabolic pathways and target organs. This case study confirms the role of α-hydroxyl modification in the mutagenicity of nitrosamines, with oxidation at the α-hydrogen being a crucial step in the formation of mutagens from N-Nitrosamines, and can inform mutagenicity risk assessment and the formulation of regulatory standards for N-nitrosamine impurities.

On-line solid phase extraction-ultra-high performance liquid chromatography coupled to tandem mass spectrometry for the determination of N-nitrosodiethanolamine in baby shampoo.

N-nitrosodiethanolamine (NDELA) is a carcinogenic contaminant of concern in the cosmetics industry. Contaminated raw material, degradation, reactions of ingredients of the formulation, or migration of packaging material can be responsible for the presence of NDELA in the final product. Liquid chromatography coupled to tandem mass spectrometry is the most widely accepted technique for the quantitation of NDELA in cosmetic products. Still, there is no consensus regarding the sample preparation procedure. The aim of this work was to evaluate the performance of two-dimensional liquid chromatography coupled with tandem mass spectrometry for the determination of NDELA in shampoo. In the first dimension an Oasis HLB SPE-column was used and in the second dimension a CSH C18 column. NDELA-d8 was used as an internal standard. The 2D-LC parameters were optimized by a central composite multivariate design. However, before quantitation, a sample preparation step using solid-phase extraction was necessary to eliminate compounds present in the formulation, especially surfactants that were not compatible with the chromatographic columns. Moreover, the complex matrices and singular compositions of shampoo from different manufacturers required adjustments of the sample preparation procedure for each sample. The limit of quantitation of the method for the determination of NDELA in shampoo was in the range of 5-10 ng g-1. The accuracy of the method at the LOQ (10 ng g-1) was 114 % and the inter-day precision of 15.3 % (n = 9). One sample out of 12 presented an NDELA concentration of 54 ng g-1.

Potassium bromate enhances N-ethyl-N-hydroxyethylnitrosamine-induced kidney carcinogenesis only at high doses in Wistar rats: indication of the existence of an enhancement threshold.

As susceptibility to carcinogens varies considerably among different strains of experimental animals, evaluation of dose-response relationships for genotoxic carcinogen in different strains is indispensable for risk assessment. Potassium bromate (KBrO(3)) is a genotoxic carcinogen inducing kidney cancers at high doses in male F344 rats, but little is known about its carcinogenic effects in other strains of rats. The purpose of the present study was to determine dose-response relationships for carcinogenic effects of KBrO(3) on N-ethyl-N-hydroxyethylnitrosamine (EHEN)-induced kidney carcinogenesis in male Wistar rats. We found that KBrO(3) showed significant enhancement effects on EHEN-induced kidney carcinogenesis at above 250 ppm but not at doses of 125 ppm and below when evaluated in terms of induction of either preneoplastic lesions or tumors in male Wistar rats. Furthermore, KBrO(3) significantly increased the formation of oxidative DNA damage at doses of 125 and above but not at doses of 30 ppm and below in kidneys. These results demonstrated that low doses of KBrO(3) exert no effects on development of EHEN-initiated kidney lesions and induction of oxidative DNA damage. Taking account of previous similar findings in male F344 rats, it is strongly suggested that a threshold dose exists for enhancement effects of KBrO(3) on kidney carcinogenesis in rats.

Monitoring of N-nitrosodiethanolamine in cosmetic products by ion-pair complex liquid chromatography and identification with negative ion electrospray ionization mass spectrometry.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of N-nitrosodiethanolamine (NDELA), which is a non-volatile N-nitrosoamine. In this method, sodium 1-octanesulfonate has been used as an ion complexation agent for NDELA. The resulting complex was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Eurospher-100 C18 column (250 x 4.6 mm, 5 microm), water/acetonitrile (95/5, v/v) and UV detection at 234 nm. The detector response was linear in the range 0.03-10.00 microg mL(-1) and the limit of detection was 0.01 microg mL(-1). The complexation of the NDELA and sodium 1-octanesulfonate was confirmed by negative ion electrospray ionization mass spectrometry at m/z 327 [M-Na](-) and also MS/MS measurements of the resulting fragments. For real sample analyses, NDELA was measured in cosmetic products after clean up by solid-phase extraction (SPE) with C(18) cartridge for water-soluble samples and liquid-liquid extraction (LLE) by methylene chloride for less water-soluble samples. The average values for NDELA recovery by SPE and LLE were 86.9 and 51.8% and relative standard deviations (RSDs%) were 0.9 and 5.6%, respectively. The presented method is easy to use, robust and can be implemented on any reversed-phase HPLC system with UV detection.

Mutagenicity of N-nitrosodiethanolamine in a V79-derived cell line expressing two human biotransformation enzymes.

N-nitrosodiethanolamine (NDELA) has demonstrated carcinogenic activity in various rodent models. However, it is negative or only weakly active in standard in vitro genotoxicity assays. This poor response might be due to the requirement of specific enzymes for its activation. Previous work indicated that cytochrome P450 (CYP) 2E1, alcohol dehydrogenases and sulphotransferases (SULTs) can convert NDELA into reactive metabolites. We report here that NDELA induces concentration-dependent gene mutations (at the hprt locus) in V79-hCYP2E1-hSULT1A1 cells, engineered for expression of human CYP2E1 and human SULT1A1, but is inactive in parental V79 cells. Mutagenicity of NDELA in V79-hCYP2E1-hSULT1A1 cells was abolished by the CYP2E1 inhibitor 1-aminobenzotriazole, but unaffected by the SULT1A1 inhibitor pentachlorophenol. The efficiency and specificity of these inhibitors was demonstrated in gene mutation assays using SULT- and CYP2E1-dependent reference mutagens, 2-nitropropane and N-nitrosodimethylamine, respectively. In this study, it is documented for the first time that NDELA can induce gene mutations in mammalian cells. Whereas human CYP2E1 was required for its activation, human SULT1A1 was not involved either in its activation or its inactivation in our cell model.

Determination of N-nitrosodiethanolamine in cosmetic products by reversed-phase dispersive liquid-liquid microextraction followed by liquid chromatography.

A new analytical method for the determination of N-nitrosodiethanolamine (NDELA), a very harmful compound not allowed in cosmetic products, is presented. The method is based on a new approach of dispersive liquid-liquid microextraction (DLLME) useful for extraction of highly polar compounds, called reversed-phase DLLME (RP-DLLME), followed by liquid chromatography-ultraviolet/visible (LC-UV/Vis) determination. The variables involved in the RP-DLLME process were studied to provide the best enrichment factors. Under the optimized conditions, a mixture of 750µL of acetone (disperser solvent) and 125µL of water (extraction solvent) was rapidly injected into 5mL of toluene sample solution. The extracts were injected into the LC-UV/Vis system using ammonium acetate 0.02M as mobile phase. After chromatographic separation, the eluate passed throughout a photolysis unit in order to convert NDELA to nitrite, and then it was merged with a flow stream of Griess Reagent and passed throughout a post-column reactor at 50°C to derivatize nitrite into an azo-dye, which was finally measured spectrophotometrically at 540nm. The method was successfully validated showing good linearity, an enrichment factor of 31.5±0.9, limits of detection and quantification of 1.1 and 3.6ngmL-1, respectively, and a good repeatability (RSD <8%). Finally, the proposed analytical method was applied to the determination of NDELA in commercial cosmetic samples of different nature, specifically three lipophilic creams and a hydrophilic shower gel, with good relative recovery values (87 - 117%) thus showing that matrix effects are negligible. These results were compared with those obtained by applying the ISO 10130 official method, which uses the same detection approach. It was concluded that a great improvement in the sensitivity was achieved, whereas the use of organochlorine solvents is avoided and therefore it can be considered as a greener approach.

Genotoxicity of glycidamide in comparison to (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and alpha-acetoxy-N-nitroso-diethanolamine in human blood and in mammalian V79-cells.

Genotoxic activity of glycidamide (GA) was investigated in comparison to that of the known carcinogens (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide ((+/-)-BPDE) and alpha-acetoxy-N-nitroso-diethanolamine (alpha-A-NDELA), using the hypoxanthine-phosphoribosyl-transferase (hPRT) gene mutation assay with V79 mammalian cells and modified alkaline single cell gel electrophoresis (alkaline comet assay with and without treatment of cells with formamido-pyrimidine-DNA-glycosylase (FPG)) in lymphocytes from human whole blood. As shown earlier, GA induced significant DNA damage in lymphocytes from treated whole blood at > or = 300 microM (4 h) (Baum et al., Mutat. Res. 2005, 580, 61-69). In the present study, using the alkaline comet assay with FPG treatment, increased formation of DNA strand breaks was observed in lymphocytes treated with GA (10 microM; 4 h). alpha-A-NDELA and (+/-)-BPDE were genotoxic at 10-30 microM (1 h). Genotoxic activity of these compounds was not enhanced after FPG treatment. FPG treatment thus offers an enhanced sensitivity of DNA damage detection for genotoxic compounds with preference for N(7)- resp. N(3)-purine alkylation. In the hPRT assay with V79 cells, mutagenic activity of (+/-)-BPDE became significant at > or = 3 microM (24 h). For alpha-A-NDELA significant activity was observed at greater, not dbl 10 microM (24 h). As previously observed, GA was considerably less effective, inducing significant mutagenicity roughly at about 80-300-fold higher concentrations (800 microM; 24 h) (Baum et al., Mutat. Res. 2005, 580, 61-69).

Etiology of bromate-induced cancer and possible modes of action-studies in Japan.

Renal cell tumors were significantly increased in male and female rats given potassium bromate at 250 and 500 mg/L in drinking water. In at least one other study renal cell tumors were produced in male rats at 125 mg/L. Among male mice given 750 mg/L of potassium bromate, there were no significant differences in renal cell tumors between treated and control groups after 88 weeks on test. In oxidative DNA damage tests 8-oxodeoxyguanosine (8-oxodG also referred to as 8-OH-dG) was induced in DNA in the male rat kidney in 1 week, and in females after 3 weeks at 500 mg/L, and also in both male and female rats at 250 mg/L, but not at 125 mg/L. DNA adducts are considered to be an initial step in the carcinogenesis process, however, the administered doses are not always sufficient to cause mutations, possibly due to DNA repair. In the two-step rat renal carcinogenesis model using N-ethyl-N-hydroxyethylnitrosamine (EHEN) as initiator, promotion activity by potassium bromate was measured using the BrdU labeling index. The promoting activity of bromate in male rats was much greater and extended to doses as low as 60 mg/L in male rats, whereas in females the response was limited to 250 and 500 mg/L. Therefore, it was concluded that the mechanisms contributing to cancer in the male rat were more complex than in the female rat. The accumulation of alpha2mu-globulin in the kidneys of male rats exposed to potassium bromate probably accounts for the greater labeling index in the male rat relative to the female rat. Accumulation of alpha(2mu)-globulin as a result of treatment with chemicals is unique to the male rat and does contribute to carcinogenic responses. Neither humans nor female rats display this response. Nevertheless, bromate must be considered carcinogenic because of the response of the female rats. The better correlation between 8-oxodG formation and tumor response indicates that dose-response information from the female rat would be much more relevant to human risk assessment. The fact that an elevation of BrdU-LI in the kidney of the female rat is consistent with the possibility that cell proliferation observed in female rats resulted from oxidative stress and/or cytotoxic responses in the kidney. Therefore, oxidative stress is most likely the mechanism of interest for cancer risk in humans.

Carcinogenicity of methylated derivatives of N-nitrosodiethylamine and related compounds in Sprague-Dawley rats.

Five nitrosamines, which can be considered alkyl derivatives of N-nitrosodiethylamine, were tested for carcinogenicity by administration to Sprague-Dawley rats in drinking water at approximately equimolar concentrations. N-Nitrosodi-n-propylamine was a potent carcinogen but less so than N-nitrosodiethylamine and gave the same spectrum of tumors. N-Nitrosodiisopropylamine was very much weaker than N-nitrosodiethylamine and induced only tumors of the nasal turbinates in significant incidence. At the doses given, neither N-nitrosodiisobutylamine nor N-nitrosodi-sec-butylamine was significantly carcinogenic. In contrast, the cyclic nitrosamine N-nitrosohexamethyleneimine was equally potent with N-nitrosodiethylamine and gave the same spectrum of tumors in liver, esophagus, and nasal turbinates. The results support the concept that oxidation at the alpha carbon atom of nitrosamines is a significant step in carcinogenesis.

Vinylethylnitrosamine: a potent respiratory carcinogen in Syrian hamsters.

Vinylethylnitrosamine (VEN), an alpha-beta unsaturated analogue of diethylnitrosamine (DEN), which may be formed by the enzymic conversion necessary for carcinogenesis, was synthesized and its biologic effect was examined by sc administration to Syrian hamsters. Distribution studies showed that the maximum amount of unaltered compound was found in various tissues 45 minutes after injection. The chemical was only partially excreted unchanged after 5 hours. Weekly treatment for life resulted in high incidence of malignant respiratory tract neoplasms with short latencies and in tumors of the upper digestive tract and pancreas. The effects of VEN were compared to those of the assumed parent compound, DEN.

Basic lead acetate: promoting effect on the development of renal tubular cell tumors in rats treated with N-ethyl-N-hydroxyethylnitrosamine.

The development of renal tubular cell tumors by the end of experimental week 32 was studied in inbred Wistar male rats fed a diet containing 1,000 or 500 ppm N-ethyl-N-hydroxyethylnitrosamine (EHEN) for 2 weeks and then given 1,00 ppm basic lead acetate (LA) for 20 weeks. A low dose of LA enhanced the development of renal tubular cell tumors in rats treated with EHEN and increased the number and size of the tumors. The incidence of renal tubular cell tumors at the end of week 32 was 50% in rats treated with 1,000 ppm EHEN for 2 weeks and 100% in rats treated with 1,000 ppm EHEN for 2 weeks and then given 1,000 ppm LA for 20 weeks. The incidences of renal tumors of more than 3 mm in diameter were 70% in rats treated with 1,000 ppm EHEN plus LA and 0% in rats treated with EHEN or LA alone. The low dose of LA showed the enhancing effect of the development of renal tubular cell tumors in rats treated with a subthreshold dose of 500 ppm EHEN.

Promotion of 2-(ethylnitrosamino)ethanol-induced renal carcinogenesis in rats by nephrotoxic compounds: positive responses with folic acid, basic lead acetate, and N-(3,5-dichlorophenyl)succinimide but not with 2,3-dibromo-1-propanol phosphate.

The promoting effects of nephrotoxic chemicals, folic acid (FA), N-(3,5-dichlorophenyl)succinimide (NDPS), 2,3-dibromo-1-propanol phosphate (Tris-BP), and basic lead acetate (LAB), on 2-(ethylnitrosamino)ethanol (EHEN)-induced renal carcinogenesis were examined in F344 rats. The rats were treated with 0.1% EHEN in their drinking water for 1 week and then given one of the nephrotoxic chemicals for 35 weeks. FA was injected sc once a week at a dose of 300 mg/kg for the first 8 weeks and thereafter at 100 mg/kg. NDPS, Tris-BP, and LAB were mixed in the diet at concentrations of 0.5, 0.01, and 0.1%, respectively. At week 3 the right kidney was removed to enhance renal neoplasia. Renal cell tumor incidence was significantly increased by both FA and LAB and was slightly increased by NDPS, whereas Tris-BP had no effect. The data show that FA, LAB, and NDPS are promoters of EHEN-induced renal carcinogenesis.

Trisodium nitrilotriacetate monohydrate: promoting effects on the development of renal tubular cell tumors in rats treated with N-ethyl-N-hydroxyethylnitrosamine.

The effect of trisodium nitrilotriacetate monohydrate [N,N-bis(carboxymethyl)glycine trisodium salt] (Na3NTA X H2O) on development of renal tubular cell tumors induced with N-ethyl-N-hydroxyethylnitrosamine [CAS:13147-25-6; 2-(ethylnitrosamino)-ethanol] (EHEN) was studied. Six-week-old male inbred W rats were given a diet containing 1,000 ppm of EHEN for 2 weeks and then a diet containing a high (10,000 ppm) or low (500 ppm) concentration of Na3NTA X H2O for 30 weeks. The rats were killed during week 32. The higher concentration of Na3NTA X H2O enhanced the development of renal tubular cell tumors and increased the number and size of tumors in rats treated with EHEN, but the lower concentration of Na3NTA X H2O did not. The incidence of renal tubular cell tumors in week 32 was 33% in rats treated with 1,000 ppm EHEN for 2 weeks, 100% in rats treated with 1,000 ppm EHEN for 2 weeks plus high Na3NTA X H2O diet for 30 weeks, and 39% in rats treated with 1,000 ppm EHEN for 2 weeks and then given low Na3NTA X H2O diet for 30 weeks. Numbers of atypical cell foci per kidney area (No./cm2) were 17.0 +/- 7.6 in rats treated with EHEN and high Na3NTA X H2O, 7.3 +/- 2.2 in rats treated with EHEN and low Na3NTA X H2O, 3.7 +/- 1.4 in rats treated with EHEN alone, and 1.0 +/- 2.4 in rats treated with high Na3NTA X H2O diet alone. Atypical cell foci retained a tubular pattern and consisted of basophilic cells with a large nucleus or clear cells with a small nucleus.

Penetration of rat skin by N-nitrosodiethanolamine and N-nitrosomorpholine.

N-Nitrosomorpholine (NMOR) and N-nitrosodiethanolamine (NDELA) were painted on the clipped upper dorsal skin of male F344 rats. NDELA was applied undiluted, dissolved in water, and dissolved in cutting oil; NMOR was applied dissolved in water and in ethyl acetate. Aqueous solutions of the nitrosamines were used for gavage. Rats were housed individually. Blood and urine samples were analyzed for nitrosamines by chromatography combined with a Thermal Energy Analyzer. Maximum penetration of NMOR was approximately equal to 34% 2 hours after application of 5 mg to the skin or by gavage; less than 1% appeared in the urine in 24 hours. Skin painting with NDELA in water (20 mg/100 microliters) and in cutting oil (25 mg/25 microliters) yielded small concentrations of NDELA (always < 25 micrograms/ml blood). When 50 mg of undiluted NDELA was painted on the skin, 130 to 220 micrograms/ml of blood was recovered after 1 hour. Administering 50 mg NDELA in water by gavage yielded similar blood concentrations. Maximum skin penetration observed with NDELA was 78% 1 hour after application of 50 mg. From 20 to 30% of the NDELA applied undiluted and by gavage appeared in the urine in 24 hours. Although animals and humans differ, skin exposure to NMOR or NDELA represents a risk due to absorption.

beta-Cyclodextrin: promoting effect on the development of renal tubular cell tumors in rats treated with N-ethyl-N-hydroxyethylnitrosamine.

Injection (sc) of beta-cyclodextrin (beta-C) increased the number and size of renal tubular cell tumors in inbred Wistar (W) rats treated with 1,000 ppm of N-ethyl-N-hydroxyethylnitrosamine (EHEN). The incidence of renal tumors at the end of the 32-week experiment was 50% in rats treated with 1,000 ppm EHEN for 2 weeks and 100% in rats treated with 1,000 ppm EHEN for 2 weeks and then given daily sc injections of beta-C for 1 week. The incidence of renal tumors more than 3 mm in diameter was 70% in rats treated with 1,000 ppm EHEN before beta-C but 0% in rats treated with EHEN alone. In addition, beta-C promoted the development of renal tumors in rats treated with 500 ppm EHEN, which is a subthreshold dose for renal tubular cell tumorigenesis. These results show that beta-C promotes EHEN-induced renal tubular cell tumorigenesis.

DL-Serine: promoting activity on renal tumorigenesis by N-ethyl-N-hydroxyethylnitrosamine in rats.

Subcutaneous injection of DL-serine increased the number and size of renal tubular cell tumors in male W rats treated with 500 or 1,000 ppm N-ethyl-N-hydroxyethylnitrosamine [(EHEN) CAS: 13147-25-6, 2-(ethylnitrosamino)ethanol]. At the end of the 32-week experiment, the incidences of renal tumors were 95% in rats treated with 1,000 ppm EHEN for 2 weeks and then given three sc injections of DL-serine every 2 weeks, 33% in rats treated with 1,000 ppm EHEN for 2 weeks, and 28% in rats treated with 500 ppm EHEN for 2 weeks and then given three sc injections of DL-serine every 2 weeks. No renal tumors were found in rats treated with 500 ppm EHEN alone or given three sc injections of DL-serine alone every 2 weeks.

Effects of route of administration and dose on the carcinogenicity of N-nitrosodiethanolamine in the Syrian golden hamster.

N-Nitrosodiethanolamine was assayed for carcinogenicity in Syrian golden hamsters by s.c. injection, topical application, and oral cavity swabbing. Three groups of 30 hamsters each received 27 weekly s.c. injections of either 500, 170, or 58 mg of N-nitrosodiethanolamine per kg in 0.9% NaCl solution. In the group treated with 500 mg/kg, 19 of 30 animals developed nasal cavity tumors, 7 of 30 had tracheal tumors, and 2 of 30 had tumors of the larynx. Among the animals treated with 170 mg/kg, 7 of 29 presented with nasal cavity tumors and 4 of 29 presented with tracheal tumors. In the group treated with 58 mg/kg, only two tracheal tumors were observed. Acetone solutions of N-nitrosodiethanolamine were applied to the shaved backs of three groups of 30 hamsters, each three times weekly for 36 weeks, at doses of 25, 8, or 2.5 mg; the total doses were the same as in the groups treated by s.c. injection. At the 25-mg dose level, 5 of 30 animals developed nasal cavity tumors and 4 of 30 animals had tumors of the trachea. No skin tumors were observed. The incidence of respiratory tract tumors in the groups treated with 8 or 2.5 mg was not significant compared to controls. The oral cavities of 40 hamsters were swabbed three times weekly for 45 weeks with 20 mg of N-nitrosodiethanolamine; the total dose was the same as the highest doses given by s.c. or topical administration. Seventeen of 38 hamsters had nasal cavity tumors, 6 of 38 developed tracheal tumors, and 1 of 38 presented with a tumor of the larynx. No tumors were observed in the oral cavity. The results of this study demonstrate that N-nitrosodiethanolamine is organospecific for the Syrian golden hamster nasal cavity and trachea and that it induces tumors in these sites at doses lower than previously reported.

Relative carcinogenic effectiveness of derivatives of nitrosodiethylamine in rats.

The carcinogenicity of five derivatives of nitrosodiethylamine was compared with that of the parent compound by p.o. administration to rats. All were less potent than was nitrosodiethylamine. When nitrosobis(2-methoxyethyl)amine and nitrosobis(2-ethoxyethyl)amine were administered at equimolar doses in drinking water, there was a high incidence of liver tumors, but the animals died later than they did after nitrosodiethylamine treatment, which also induced esophageal tumors. Nitrosoiminodipropionitrile and nitrosobis(2,2-diethoxyethyl)amine failed to induce tumors at the same dose level. Nitrosobis(2-chloroethyl)amine was administered in oil by gavage at a dose lower than that of nitrosodiethylamine and produced a much weaker tumor response; 5 of 15 treated rats had forestomach papillomas, and 1 had olfactory adenocarcinoma and no other induced tumors.