Mol. Nutr. Food Res.

2005, 49, 235 238

DOI 10.1002/mnfr.200400050

235

Migration of nitrosamines from rubber products are balloons and condoms harmful to the human health?
Werner Altkofer, Stefan Braune, Kathi Ellendt, Margit Kettl-Grmminger and Gabriele Steiner
Chemisches und Veterinaeruntersuchungsamt Stuttgart, Fellbach, Germany

Studies performed in 2001 and 2003 surveyed the release of carcinogenic nitrosamines and nitrosatable substances from rubber toy balloons by extraction with artificial saliva and gas chromatographythermal energy analysis (GC-TEA). 81% of the 16 in 2001 sampled balloons and 93% of the 14 in 2003 sampled balloons released nitrosamines above the recommended level in Germany of 10 lg per kg material. Furthermore, 32 rubber condom samples collected in 2004 from the German market were surveyed for nitrosamines by determining the amount migrating into an artificial sweat test solution. The levels released from condoms varied from a 10 to 660 lg per kg material (i. e., up to 1.4 lg nitrosamines per condom). In a model calculation, not considering the differences that may exist in the resorption rate, we have calculated that the exposure from condoms may exceed the exposure from food 1.5 3 fold. To our knowledge so far no legal binding legislation exists worldwide concerning nitrosamine migration from toy balloons or condoms.
Keywords: Balloons / Condoms / Migration / Nitrosamines / Nitrosatable substances / Rubber / Received: July 28, 2004; revised: October 8, 2004; accepted: October 20, 2004

1 Introduction
1.1 General aspects It has been known for many years that the exposure of humans to nitrosamines and/or their precursor compounds is associated with high risks. Exposure to nitrosamines in the general population occurs via different sources, e. g., food, cosmetics, tobacco, and commodities [1] here especially rubber products. Balloons and condoms are commonly made of natural rubber. In order to achieve the high elastic properties and necessary strength of the material, the rubber has to be vulcanized. However, additives, such as secondary amines, dithiocarbamates, and thiurams, used in this process, may have an adverse effect on the chemical safety. During vulcanization, dithiocarbamates, thiurams, and some sulfenamides (morpholine derivatives) can be converted to secondary amines. In the presence of nitrosating agents, secondary amines react to the corresponding N-nitrosamines, in short nitrosamines. It also has been shown that these vulcanizing additives can form nitrosamines directly in contact with nitrosating agents [2]. Many

nitrosamines have demonstrated to exhibit carcinogenic activity in animals [3]. The presence of nitrosamines in rubber products has been a concern since many years, but only few current studies have been published concerning the specific chemical safety of balloons [4 7] or condoms [3, 8]. Balloons are often taken into the mouth to inflate them. Children even tend to chew on them. Condoms are intended for close contact with the skin and mucous membranes. Due to this close contact with the human body and especially the mucous membranes, the chemical safety of these products is of great importance. The aim of the present study was to investigate the release of nitrosamines from condoms using artificial sweat test solution, an issue that to our knowledge has not been investigated in other studies so far. The study furthermore aimed at describing the present situation concerning the release of nitrosamines and nitrosatable compounds from balloons into artificial saliva. Nitrosatable substances were analyzed in the migrates of the balloons, since they may be formed in vivo into nitrosamines. In the presence of nitrite (e. g., in saliva from bacterially reduced nitrate from food), nitrosatable substances may react in the gastric juices to the corresponding nitrosamines [6]. The release of nitrosatable substances from condoms was not part of this study.

Correspondence: Kathi Ellendt, Chemisches und Veterinaeruntersuchungsamt Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany E-mail: [email protected] Fax: +49-711-588176

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1.2 Legislation To our knowledge, no legal binding limits are imposed on the migration of nitrosamines from condoms or toy balloons worldwide. Concerning balloons, Germany issued a guideline: Recommendation XXI of the BfR (Federal Institute for Risk Assessment) on commodities based on natural and synthetic rubber [9]. It states, that only up to 10 lg nitrosamines per kg material and 5 lg per dm2 nitrosatable substances should migrate into artificial saliva in 1 h at 408C. As to standards concerning other commodities coming into contact with the mucous membranes, soothers and teats for babies were regulated in the EU after high levels of nitrosamines were discovered in these products. Commission Directive 93/11/EEC [10] states that nitrosamines and nitrosatable substances (nitrosamine precursors) may not be released in detectable amounts from elastomer or rubber teats and soothers (detection limits: 10 lg nitrosamines per kg material and 100 lg nitrosatable substances per kg material). Other countries have also issued standards on nitrosamines in teats and soothers, e. g., Canada has recently lowered the maximum permitted level from 60 to 10 lg per kg total volatile N-nitrosamines (dichloromethane extraction) [11], the USA have set a recommendation of 20 ppb total nitrosamines for pacifiers [12] and 10 ppb for baby bottle nipples [13].

2 Materials and methods

2.1 Sampling In 2001 a total of 16 packages with rubber balloons were randomly sampled in Baden-Wrttemberg (Germany). 14 packages of balloons were sampled in 2003. 32 condom packages, sampled at random on the local market, were surveyed in 2004.

A mixture of nitrosamines consisting of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), and N-nitrosomorpholine (NMOR) was employed as reference standard. N-Nitrosodiisopropylamine (NdiPA) was used as internal standard. The nitrosamines and the internal standard were obtained from Promochem (Wesel, Germany). A Hewlett-Packard 5890 A GC coupled to a thermal energy analyzer (Thermedics, Woburn, USA) was used. A 5 lL aliquot of the extract was injected (splitless) onto a 30 m Stabilwax DB column (0.25 mm ID, 0.5 lm film). The injection port was maintained at 1908C. The initial temperature of the oven was 408C, held for 1.5 min, then programmed at 458C N min1 to 1308C, 88C N min1 to 1708C and 28C N min1 to 1858C. The pyrolysis oven was set to 5008C. Evaluation of raw data was conducted using Chromeleon Version 6.60 software (Dionex, Idstein, Germany). Each analyzed nitrosamine was calculated separately. The individual nitrosamine values were summed up to give the total nitrosamine value. Simulating physiological conditions, the release of nitrosamines from condoms into artificial sweat test solution (4.5 g NaCl, 0.3 g KCl, 0.3 g Na2SO4, 0.4 g NH4Cl, 3.0 g lactic acid, and 0.2 g urea dissolved in 1 L distilled water) according to the German 35 Methode 82.10 [15] (standard for the determination of colorfastness of toys) was determined. Unlike Biaudet et al. [8], the lubricant was not removed, the extraction medium was altered, and migration time was reduced. The entire condom was fastened into a sealable tube and brought into contact by filling 40 mL sweat simulant inside the condom, plus 20 mL on the outside of the condom, and put in a shaking water bath for 1 h at 378C (dynamic migration test). Determination of the released nitrosamines was performed analogously to the balloon samples. Recovery studies were performed for NDEA and NDBA using four spike levels: 37.5, 75, 150, and 375 lg N kg-1 material for each nitrosamine.

2.2 Experimental For the balloons, the release of nitrosamines and nitrosatable substances was determined in a static migration using nitrite containing saliva simulant according to EN 12868 [14] (4.2 g NaHCO3, 0.5 g NaCl, 0.2 g K2CO3, 30 mg NaNO2 dissolved in 1 L distilled water; adjusted to pH 9). The balloons were cut into small pieces. If a package of balloons consisted of various colors, all colors were mixed and a homogeneous sample was prepared. 5 10 g of the homogeneous sample were then contacted with artificial saliva for 1 h at 408C. Determination of the released nitrosamines was performed according to EN 12868 method A (standard for teats and soothers). The nitrosatable substances, were determined as their corresponding nitrosamines listed below after nitrosation as described in EN 12868.

3 Results and discussion

3.1 Nitrosamines in balloons The following nitrosamines were detected in the balloons and condoms: N-dimethylnitrosamine, N-diethylnitrosamines, and N-dibutylnitrosamine. Their structures are shown in Fig. 1. In balloons small amounts (a10 lg N kg1) of N-nitrosomorpholine were additionally detected. Often, a mixture of two or three of these nitrosamines was released from the samples. The detected nitrosamines are very potent carcinogens in animals [21], and thus classified as probably carcinogenic to humans.

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Migration of nitrosamines from balloons and condoms

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Figure 1. Structures of nitrosamines detected in the samples of this study.

ples N-diethylnitrosamine and in 9% of the samples Nnitrosomorpholine was detected. Regarding the vulcanization accelerators, curiously, only dibutlydithiocarbamate was determined in the rubber, while dimethyldithiocarbamate and diethyldithiocarbamate were not detected in any of the samples (experimental data not shown).

3.2 Nitrosamines in condoms

Figure 2. Levels of nitrosamines, expressed in lg per kg material, released from balloons into artificial saliva (contact conditions: 1 h at 408C); maximum recommended level in Germany: 10 lg N-nitrosamines per kg material.

In the samples collected in 2001, nitrosamines released from the balloons ranged from a10 to 380 lg N kg1. 81% of the 16 analyzed balloons were above the German recommendation of 10 lg N kg1. 380 lg N kg1 equal theoretically 0.46 lg nitrosamines per balloon. Obviously no efforts on behalf the manufacturers to minimize the release of nitrosamines were carried out, since the levels did not decrease significantly in the past years. In 2003, only one of the 14 sampled balloons did not exceed the recommended migration level of 10 lg nitrosamines per kg material. The levels ranged from a10 to 220 lg N kg1. Figure 2 shows the levels of nitroamines released in 2003 compared to 2001. As far back as 1991, Majerus and Otteneder [6] criticized high levels of nitrosamines ranging from 10 to 300 lg N kg1 (10 balloons) and appealed to the manufacturers to alter their recipes regarding the vulcanization accelerators. In the samples surveyed in 2001 the concentrations of nitrosatable substances released ranged from a10 to 4300 lg N kg1, while in samples from 2003 levels ranged from a10 to 2500 lg N kg1. This equals a maximum level of nitrosatable substances of 12,4 lg per dm2 balloon in 2001 and 5.2 lg per dm2 in 2003, respectively. In 2001 five samples were found to contain nitrosatable compounds above the German recommended level of 5 lg per dm2, while in 2003 only one sample did not meet this requirement. In a total of 30 samples, the nitrosamines most often detected were N-dimethylnitrosamine (in 97% of the balloons) and N-dibutylnitrosamine (93%). In 34% of the sam-

Up to 660 lg nitrosamines per kg material were released into the artificial sweat. Using the highest level found as a calculation basis, this equals approximately 1.4 lg nitrosamines per condom. In comparison, it is estimated, that the consumer is exposed to 0.2 0.5 lg per day from food, 1.5 6.0 lg per day from tobacco, and a 0.05 lg per day from cosmetics [1]. Thus, in the worst case, if highly contaminated condoms are used daily, the exposure from condoms might exceed the exposure from food by a factor of 1.5 3 per person (considering exposition to 50% of the migrate per person). Encouragingly though, in three samples nitrosamines were not detected. This shows that it is possible to produce condoms without nitrosamine contamination. Figure 3 shows the distribution of nitrosamine levels within the 32 analyzed samples. The limit of quantification (by means of S/N) was on average 10 lg N kg1 for the analyzed nitrosamines. Recoveries determined for four spike levels

Figure 3. Distribution of nitrosamine levels, expressed in lg per kg material, released from condoms into artificial sweat (n = 32 samples; contact conditions: one whole condom for 1 h at 378C in a shaking water bath).

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ranged from 89 to 102% for N-diethylnitrosamine and 76 to 103% for N-dibutylnitrosamine with an average relative standard deviation of 7%. Although the contact time of 1 h is not common practice in the use of a condom, the levels obtained do provide an informative basis. A comparison between short exposure (10 min) and 1 h were performed. While this was a single experiment, the results suggest, that the bulk of nitrosamines are released within the first few minutes, since no significant difference between amounts released within 10 min (260 lg N kg1) or 60 min (263 lg N kg1) were found. As expected, N-dibutylnitrosamine was most commonly found (in 91% of the sample migrates), since the vulcanization accelerator dibutyldithiocarbamate was determined in 88% of eight samples (experimental data not shown). In 34% of these samples N-diethylnitrosamine and in 9% of these samples N-dimethylnitrosamine was released. The example of teats and soothers showed that adoption of restrictive legislation led to modifications in the recipes and to a significant decrease in nitrosamine levels [6, 16 19]. Due to the fact that balloons have not met the BfR Recommendation XXI in recent conducted surveys in Germany and these results were also confirmed by a recently conducted survey of The Netherlands [7], Germany is discussing to impose a legally binding limit for nitrosamines and nitrosatable substances in balloons [19]. A limit for nitrosamines and their precursors might follow for condoms. Nonetheless, due to the high carcinogenic potency of the detected nitrosamines, the aim should be the prevention of exposure to these substances or if that does not succeed, to keep the exposure as low as reasonably achievable (ALARA principle).

[7] Bouma, K., Migration of nitrosamines and nitrosatable substances from balloons. Inspectorate for Health Protection and Veterinary Public Health of The Netherlands Report ND1TOY01/01, January 23, 2002. [8] Biaudet, H., Mouillet, L., Debry, G., Migration of nitrosamines from condoms to physiological secretions. Bull. Environ. Contam. Toxicol. 1997, 59, 847 853. [9] Bundesinstitut fr Risikobewertung (BfR), Recommendation XXI: commodities based on natural and synthetic rubber. 1. 4. 2004. www.bfr.bund.de. [10] Commission Directive 93/11/EEC of March 1993 concerning the release of the N-nitrosamines and N-nitrosatable substances from elstomer or rubber teats and soothers. Official Journal 17/04/1993, L 093, 0037 0038. [11] Department of Justice of Canada, Summary of Hazardous Products (Pacifiers) Regulations; April 30, 2004. http://laws. justice.gc.ca/en/H-3/C.R.C.-c.930/134260.html. [12] U.S. Consumer Product Safety Commission, Summary of the Requirements for pacifiers 16 C.F.R. Part 1511; January 2001. www.cpsc.gov/businfo/regsumpacifier.pdf. [13] U.S. Food and Drug Administration, Sec. 500.450 Volatile NNitrosamines in Rubber Baby Bottle Nipples (CPG 7117.11), March 1995. http://www.fda.gov/ora/compliance_ref/cpg/ cpgfod/cpg500-450.html. [14] European Committee for Standardization, EN 12868 Child use and care articles. Methods for determining the release of N-nitrosamines and N-nitrosatable substances from elastomer or rubber teats and soothers, December 1999. [15] Bundesamt fr Verbraucherschutz und Lebensmittelsicherheit (BVL), 35 Methode 82.10 Prfung von bunten Kinderspielwaren auf Speichel- und Schweiechtheit, Band II/1, Beuth, Berlin, June 1985. [16] Havery, D. C., Fazio, T., Survey of Baby Bottle Rubber Nipples for Volatile N-Nitrosamines J. Assoc. Off. Anal. Chem. 1983, 66, 1500 1503. [17] Sen, N. P., Kushwaha, S. C., Seaman, S. W., Claskson, S. G., Nitrosamines in Baby Bottle Nipples and Pacifiers: Occurrence, Migration, and Effect of Infant Formulas and Fruit Juices on in Vitro Formation of Nitrosamines under Simulated Gastric Conditions. J. Agric. Food Chem. 1985, 33, 428 433. [18] Bouma, K., Teats and Soothers Inspectorate for Health Protection and Veterinary Public Health of The Netherlands Report NDTOY03/01, November 05, 2002. [19] Bundesministerium fr Verbraucherschutz, Ernhrung und Landwirtschaft, press report Mller: Nitrosaminbelastung von Luftballons muss verringert werden, February 17, 2004. www.verbraucherministerium.de. [20] Billedeau, S. M., Thompson. H. C., Miller, B. J., Wind, M. L., Volatile N-Nitrosamines in Infant Pacifiers Sold in the United States as Determined by Gas Chromatography/Thermal Energy Analysis. J. Assoc. Off. Anal. Chem. 1986, 69, 31 34. [21] IARC, Monographs on the evaluation of the carcinogenic risk of chemicals to man. Overall Evaluations of Carcinogenicity. Lyon, France 1987, Suppl. 7.

4 References
[1] Eisenbrand, G., Fuchs, A., Koehl, W., N-Nitroso compounds in cosmetics, household commodities and cutting fluids Eur. J. Cancer 1996, 5, 41 46. [2] Janzowski, C., Hemm, I., Eisenbrand, G., Handbuch Umweltmedizin, VI-4, 20, Suppl. 11/2000, pp. 1 30. [3] Proksch, E., Review Toxocological evaluation of nitrosamines in condoms. Int. J. Hyg. Environ. Health. 2001, 204, 103 110. [4] Bundesinstitut fr Risikobewertung, Nitrosamine in Luftballons, Stellungnahme vom 4. 12. 2003. www.bfr.bund.de. [5] Bundesinstitut fr Risikobewertung, Berwertung von Nitrosaminen in Luftballons, Ergaenzende Stellungnahme vom 26. 3. 2004. www.bfr.bund.de. [6] Majerus, P., Otteneder, H., Nitrosamine in Bedarfsgegenstnden aus Natur- und Synthesekautschuk. Dt. Lebensm.rundschau 1991, 87, 171 176.

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