Genotoxic effects of N-nitrosodicyclohexylamine in isolated human lymphocytes

Article abstract of DOI:10.1007/s002040000205

Dicyclohexylamine × nitrite is classified as an experimental equivocal tumorigenic agent by the National Toxicology Program. Since no genotoxic effects of the substance itself are known, the reported tumorigenic potential of dicyclohexylamine × nitrite could be due to generation of N-nitrosodicyclohexylamine (N-NO-DCHA), which occurs under conditions of use and can be detected in foils that contain dicyclohexylamine × nitrite. Therefore, we investigated possible mutagenic properties of N-NO-DCHA in the Ames test and the cytokinesis-block micronucleus assay with human lymphocytes. Since N-NO-DCHA is not commercially available, the substance was synthesized and purified by thin-layer chromatography. Identity was confirmed by gas chromatography/mass spectroscopy (GC/MS) and ¹H- and ¹³C-NMR. More than 97% purity was achieved. Stability and availability in the solvent were checked by GC/MS. N-NO-DCHA induced micronuclei in isolated human lymphocytes at a dose range of 15-100 μg/ml (= 71.4-476.2 μM), exceeding the base rate significantly at one or two nontoxic concentrations in four out of six experiments. For the Ames test, arochlor-1254-, β-naphthoflavone/phenobarbital- and pyrazole-induced S9-fractions were used with Salmonella typhimurium TA100, TA1535, TA98 and TA104. No effects were seen in the Ames test, with the exception of microcolony induction at doses higher than 250 μg (= 1.2 mmol) N-NO-DCHA/plate using TA104 and 20% arochlor-1254 induced S9 at pH 6.5. In conclusion, N-NO-DCHA was negative in the Ames test using TA98, TA100 and TA1535, inconclusive using TA104, and weakly genotoxic in the in vitro micronucleus test with isolated human lymphocytes. With regard to the tumorigenicity of the majority of nitrosamines, our data underline the necessity of further studies on possible genotoxic effects of N-NO-DCHA.

Full text of DOI:10.1007/s002040000205

Arch Toxicol 82001) 75: 118±122
DOI 10.1007/s002040000205
GENOTOXICITY
Gotz A. Westphal á Michael M. Muller
Claudia Herting á Jurgen Bunger á Ernst Hallier
Genotoxic effects of N-nitrosodicyclohexylamine
in isolated human lymphocytes
Received: 19 October 2000 / Accepted: 4 December 2000 / Published online: 8 March 2001
Ó Springer-Verlag 2001
Abstract Dicyclohexylamine ´ nitrite is classi®ed as an tumorigenicity of the majority of nitrosamines, our data
``experimental equivocal tumorigenic agent'' by the underline the necessity of further studies on possible
National Toxicology Program. Since no genotoxic genotoxic e€ects of N-NO-DCHA.
e€ects of the substance itself are known, the reported
tumorigenic potential of dicyclohexylamine ´ nitrite
could be due to generation of N-nitrosodicyclohexyl-
amine 8N-NO-DCHA), which occurs under conditions
of use and can be detected in foils that contain dic-
yclohexylamine ´ nitrite. Therefore, we investigated
possible mutagenic properties of N-NO-DCHA in the
Ames test and the cytokinesis-block micronucleus assay
with human lymphocytes. Since N-NO-DCHA is not
commercially available, the substance was synthesized
and puri®ed by thin-layer chromatography. Identity was
con®rmed by gas chromatography/mass spectroscopy
Key words N-Nitrosodicyclohexylamine á Ames test á
Cytokinesis-block micronucleus assay
Introduction
Dicyclohexylamine ´ nitrite [CAS 3129-91-7] is used as
an anticorrosive agent. According to the National
Toxicology Program of the USA the substance is an
``experimental equivocal tumorigenic agent'' 8National
Toxicology Program 1991a). Since no data on the
genotoxic e€ects of dicyclohexylamine ´ nitrite are
available, the tumorigenic potential of dicyclohexyl-
amine ´ nitrite might be linked to the generation
of N-nitrosodicyclohexylamine 8N-NO-DCHA), which is
formed by the nitrosation of dicyclohexylamine 8Fig. 1)
8Kabaco€ et al. 1984). Up to 250 mg/kg N-NO-DCHA
can be detected in foils that contain dicyclohexylamine ´
nitrite 8Reinhard et al. 2000). Since the vast majority of
nitrosamines are carcinogenic 8Hecht 1997), this could
as well be the case for N-NO-DCHA.
Nitrosamines may show mutagenic e€ects in in vitro
assays only under certain conditions. Therefore, we used
three nitrosamines as positive controls 8N-nitrosodi-
phenylamine, N-nitrosopyrrolidine and N-nitrosodieth-
ylamine). The structurally closely related nitrosamine
N-nitrosodiphenylamine [CAS 86-30-6] was shown to be
carcinogenic 8at 1,000 or 4,000 ppm) in both sexes of
F344 rats, including induction of transitional-cell carci-
nomas of the urinary bladder, but was not carcinogenic
in B6C3F1 mice of either sex 8National Toxicology
Program 1990, 1991b). However, in vitro studies were
inconclusive or negative 8McGregor 1994) with the ex-
ception of one study where microcolony induction in
Salmonella typhimurium TA104 was seen in the presence
of arochlor-1254-induced S9 at pH 6.5 8Zielenska and
Guttenplan 1988). N-Nitrosopyrrolidine is a potent ex-
perimental carcinogen 8Berger et al. 1987) and can only
1
8GC/MS) and H- and 13C-NMR. More than 97% pu-
rity was achieved. Stability and availability in the solvent
were checked by GC/MS. N-NO-DCHA induced mi-
cronuclei in isolated human lymphocytes at a dose range
of 15±100 lg/ml 8=71.4±476.2 lM), exceeding the base
rate signi®cantly at one or two nontoxic concentrations
in four out of six experiments. For the Ames test, aro-
chlor-1254-, b-naphtho¯avone/phenobarbital- and pyr-
azole-induced S9-fractions were used with Salmonella
typhimurium TA100, TA1535, TA98 and TA104. No
e€ects were seen in the Ames test, with the exception of
microcolony induction at doses higher than 250 lg
8=1.2 mmol) N-NO-DCHA/plate using TA104 and
20% arochlor-1254 induced S9 at pH 6.5. In conclusion,
N-NO-DCHA was negative in the Ames test using
TA98, TA100 and TA1535, inconclusive using TA104,
and weakly genotoxic in the in vitro micronucleus test
with isolated human lymphocytes. With regard to the
M. M. Muller á C. Herting á J. Bunger á E. Hallier
G. A. Westphal 8&)
Department of Occupational Health,
Georg-August-University, Waldweg 37,
37073 Gottingen, Germany
E-mail: gwestph@gwdg.de
Tel.: +49-551-396193
Fax: +49-551-396184

119
Germany) and Giemsa improved 8Gurr/Promochem GmbH,
Wesel, Germany).
Synthesis and puri®cation of N-NO-DCHA
Synthesis was carried out according to a basic procedure for the
formation of nitrosamines 8Organikum 1986). Brie¯y, 48 ml of a
2.5 M sodium nitrite solution 80.12 mol NaNO2) was added slowly
to 20 ml dicyclohexylamine 80.1 mol DCHA) dissolved in 200 ml
10 M sulfuric acid under stirring 8Warning! nitrogen oxides may
Fig. 1 Generation of N-nitrosodicyclohexylamine by the nitro-
sation of dicyclohexylamine
be e€ectively activated to Ames-test-positive metabolites escape. The reaction should be conducted in a fume hood and with
adequate skin and eye protection!). The reaction mixture was con-
by pyrazole-induced rat liver S9 8Burke et al. 1994).
Pyrazole is known to induce CYP2E1 in the rat liver.
N-Nitrosodiethylamine is one of the strongest known
tinuously maintained below 5°C and stirred for 2 hs after the last
addition of sodium nitrite solution. The reaction mixture was al-
lowed to warm to room temperature when nitrogen oxides could
carcinogens 8Lewis et al. 1997) and was proposed to be
metabolized by CYP2E1 as well 8Verna 1996). However,
we could show that N-nitrosodiethylamine can be acti-
vated by b-naphtho¯avone/phenobarbital 8BNF/PB) -
induced S9 to Ames-test-positive metabolites 8Westphal
et al. 2000). Mutagenic e€ects of these nitrosamines can
only be detected using certain strains: N-nitrosopyrr-
olidine and N-nitrosodiethylamine were only mutagenic
using TA1535 and N-nitrosodiphenylamine was only
mutagenic in TA104.
The aim of this study was to evaluate possible mu-
tagenic properties of N-NO-DCHA in the Ames test
under conditions that have been reported to be e€ective
for the above-mentioned nitrosamines. Additionally, we
performed the in vitro cytochalasin the B-block micro-
nucleus test.
escape from the reaction solution. Subsequently, the sulfuric acid
was neutralized with a 10 M sodium hydroxide solution. Unreacted
DCHA crystalized and was ®ltered from the neutralized solution.
The product N-NO-DCHA was extracted from the aqueous solu-
tion using a total of 400 ml dichloromethane. The dried extract was
concentrated to 10 ml in vacuo and 1 ml of this extract was applied
to each preparative tlc plate. These were developed using dichlo-
romethane as a solvent. A strong UV-absorbing band 8Rf=0.32)
containing the desired product was scratched out from the glass
plates. The product was eluted with dichloromethane from the
combined silica fractions of ten plates. After ®ltration the extract
was concentrated to dryness in vacuo, yielding a pale yellow resi-
due. Overall yield was 221.8 mg N-NO-DCHA 81.06%) with a
purity >97%, as judged by gas chromatography/mass spectros-
copy 8GC/MS).
Instrumental analysis
GC/MS analysis was carried out on a Hewlett-Packard GC 6890/
MSD 5973 system 8Agilent Technologies, Waldbronn, Germany)
with splitless injection using
a HP-5 MS capillary column
830 m´250 lm, layer thickness: 0.25 lm; Agilent Technologies,
Waldbronn, Germany) and helium as the carrier gas 8constant
¯ow rate: 0.6 ml/min). A linear temperature gradient of 10°C/min
from 50°C 81-min hold after injection) to 280°C 82-min hold until
return to initial conditions) was applied. Injector and transfer line
temperatures were kept at 250°C. The compound had a retention
time of 16.1 min under these conditions. Mass spectra were
acquired using electron impact 8EI) ionization and the full-scan
mode.
Materials and methods
Chemicals
Synthesis and structure elucidation of N-nitrosodicyclohexylamine
ꢀN-NO-DCHA)
All chemicals used in the synthesis and the stability experiments
were of analytical grade purity and were obtained from Sigma
8Deisenhofen, Germany) or Sigma-Aldrich 8Steinheim, Germany).
Dichloromethane was a product of J.T. Baker 8Griesheim, Ger-
many). Preparative silica gel 60 F254 thin-layer chromatography
8tlc) plates 820´20 cm, 0.25-mm layer thickness) were supplied by
Merck 8Darmstadt, Germany). Water was puri®ed by passage
through an Elix 3 and Milli-Q system 8Millipore, Eschborn,
Germany). This water was used for all aqueous solutions and
bu€ers.
1H- and 13C-nuclear magnetic resonance 8NMR) spectra were
recorded in CD2Cl2 on a Varian VXR 200 instrument 8Varian Inc.,
Darmstadt, Germany) at the Institute of Organic Chemistry,
University of Gottingen.
Characterization of N-NO-DCHA
Mass spectrum 8EI, 70 eV): m/z 8relative abundances and assign-
ments in parentheses) 210 851) [M+], 193 89) [M+ ± OH], 129 843)
[M+ ± C6H9], 111 812) [M+ ± C6H11O], 98 833) [M+ ± C6H10NO],
83 8100) [C6H11], 67 828) [C5H7], 55 898) [C4H7].
1H-NMR 8200 MHz, CD2Cl2): d = 1.5 8m, broad, 20 H, -CH2),
4.15 8m, 1 H, -CH), 5.1 8m, 1 H, -CH).
Mutagenicity testing
13C-NMR 850.3 MHz, CD2Cl2): d = 27.6 8s, C-4¢ ), 27.7 8s, C-
4), 27.8 8s, C-3¢ ), 28.3 8s, C-3), 31.6 8s, C-2¢), 36.7 8s, C-2), 54.4 8s,
C-1¢), 60.6 8s, C-1).
2-Amino¯uorene [CAS 153-78-6], purity ³99.0%, lot 9151564,
2-aminoanthracene [CAS 610±49±1], practical grade, lot 77H1867,
N-nitrosodiethylamine [CAS 55-18-5], purity ³99.0%, analysis
number 194492/153097 and N-nitrosopyrrolidine [CAS 930-55-2],
purity ³99.0%, lot 70475-018 were obtained from Sigma-Aldrich
8Steinheim, Germany). NADP, NAD, glucose 6-phosphate, mi-
tomycin C and cytochalasin B were purchased from Fluka, Sigma-
Aldrich 8Steinheim, Germany).
13C-NMR assignments of N-NO-DCHA are based on data of
previously described 13C-NMR spectra of analogous N-nitro-
soamines 8Kalinowski et al. 1984).
Other materials used were FICOLL, phosphate-bu€ered saline Stability of N-NO-DCHA in dimethyl sulfoxide ꢀDMSO)
8PBS) and phytohemagglutinin 8Biochrom KG, Berlin, Germany),
RPMI 1640 8supplemented with 15% fetal calf serum, 1,5% glu- For the genotoxicity tests N-NO-DCHA was dissolved in DMSO
tamin, penicillin and streptomycin) and arochlor-1254-induced S9 85 lg/ll). Ten microlitres of the freshly prepared solution was
8ICN, Eschwege, Germany), Trypan blue 8Merck, Darmstadt, diluted with 990 ll water and extracted with 5 ml dichloromethane.

120
One microlitre of the dried dichloromethane extract was analysed
by GC/MS. Only a single peak of N-NO-DCHA was detected. Results
Subsequently, the DMSO solution of N-NO-DCHA 85 lg/ll) was
stored for 24 h in a fridge at 4±8°C. Again, a 10-ll aliquot was
N-NO-DCHA was synthesized and characterized by
taken, worked up as described above and analysed by GC/MS. No
signs of decomposition of the synthesized compound upon storage
in DMSO were detected.
GC/MS, H- and 13C-NMR. Probably because of the
1
poor solubility in sulfuric acid of the starting compound
DCHA, only a small yield of N-NO-DCHA 81.06%)
could be recovered from the synthesis. Nevertheless, the
product isolated by preparative tlc showed very high
purity 8>97% as judged by GC/MS). In addition,
stability of the compound in DMSO tested over 24 h at
4±8°C proved to be excellent; thus ful®lling a prerequi-
site for valid genotoxicity testing.
Micronucleus test
The cytokinesis-block micronucleus assay was carried out accord-
ing to Fenech 81993). Human blood was collected by venipuncture
in heparinized tubes, diluted 1:1 in PBS and separated by a FI-
COLL gradient. 1.2´106 cells were seeded in 2.5 ml RPMI each.
Cell viability was checked by the Trypan blue exclusion test.
Lymphocyte stimulation was done by addition of 5 lg/ml phy-
tohemagglutinin and cultivation at 37°C, 100% humidity and 5%
CO2 for 68 h. N-NO-DCHA 8up to 25 lg/ll) was added as a so-
lution in DMSO 8in a volume of 20 ll/2.5 ml each) 24 h after
starting the cultures. Cell division was blocked 44 h after starting
the cultures by adding 4.5 lg/ml cytochalasin B. Mitomycin C was
used as standard mutagen.
Cells were sampled 28 h after addition of cytochalasin B by
centrifugation for 5 min at 175 g and 20°C. The cells were resus-
pended in 3 ml hypotonic saline 80.07 M KCl/0.15 M NaCl) and
collected for 5 min at 175 g and 20°C. The pellet was resuspended
in 3 ml ice-cold methanol/acetic acid 84:1). Finally, the cells were
pelleted for 5 min at 175 g and 4°C, and transferred onto ice-cold
degreased slides. The slides were coded and subsequently stained
with 5% Giemsa solution for 15 min.
Micronucleus test
N-NO-DCHA induced micronuclei in human lympho-
cytes at doses ranging from 5 to 100 lg/ml 8=74.4±
476 lM), exceeding the base rate signi®cantly in four
out of six experiments, apparent from the NDI at one or
two non-toxic concentrations 8Table 1). No pH devia-
tions related to the test substance were observed. N-NO-
DCHA was toxic above 100 lg/ml.
The maximum concentrations of the test article were deter-
mined by evaluation of the nuclear divison index 8NDI) according
to Eastmond and Tucker 81989). The cells were scored according to
the criteria outlined by Fenech 81993). In addition, only cells with
preserved cytoplasm containing not more than ®ve micronuclei
were included in the count. At least six concentrations were tested.
Blood from three di€erent donors was used. For each donor two
independent experiments were performed. Acceptance criteria
for a genotoxic e€ect were: NDI ³1.1, a frequency of micronulei
signi®cantly exceeding the control, and stable pH. Samples were
randomized prior to scoring.
Ames test
In the absence of toxicity, the substance was tested up to
2 mg N-NO-DCHA per plate. This is at least twice the
maximum soluble concentration, which was observed as
a precipitation of the substance at concentrations above
1 mg/500 ll. In experiments involving TA98 and pyr-
azole-induced S9, 500 lg N-NO-DCHA per plate was
slightly toxic. However, due to the limited solubility of
the substance, pronounced bacterial toxicity could not
be achieved.
Ames test
With the exception of microcolony induction above
250 lg/plate 8500 ll preincubation volume respectively)
using TA104 in the presence of arochlor-1254-induced
S9 at pH 6.5, N-NO-DCHA was not active in the Ames
The Ames test was performed according to Maron and Ames
81983) using S. typhimurium TA100, TA98, TA1535 and TA104
with 30-min preincubation. Various S9 preparations were used.
Pyrazole induction was performed according to Tu et al. 81981),
and BNF/PB induction according to Ong et al. 81980). S9-mix was test 8Table 2). The same e€ect was seen with N-nitro-
concentrated up to 20%, supplemented with up to 5 mM NADP
and 25 mM glucose 6-phosphate. Arochlor 1254-induced S9 was
the colonies, reliable scoring was not possible. Addi-
additionally applied at pH 6.5.
sodiphenylamine. However, owing to the small size of
tionally, the evaluation of this e€ect was impaired by
N-NO-DCHA 850 lg/ll) dissolved in DMSO was added
pronounced base-rate deviations of TA104 at pH 6.5.
Microcolony induction was not seen using TA98, TA100
or TA1535.
8maximal 20 ll DMSO per plate). The nitrosamine precipitates at
concentrations above 500 lg/ml N-NO-DCHA. Therefore, in the
absence of toxicity, a maximum of 2 mg N-NO-DCHA per plate
was applied, which is at least twice the maximum solubility.
The compounds N-nitrosopyrrolidine, N-nitrosodiphenyl-
amine, N-nitrosodiethylamine, 2-amino¯uorene and methylme-
thanesulfonate served as positive controls. CYP2E1, 2A1, 1A1,
2B1 and 2B2 expression in the BNF/PB- and the pyrazole-induced
S9-fractions, and the activity of the control mutagens N-nitroso-
pyrrolidine and N-nitrosodiethylamine are described elsewhere
8Westphal et al. 2000). The acceptance criterion for a valid test
was that solvent and positive controls were within the range of the
historical controls. The positive controls for the Salmonella strains
and the various S9 fractions are summarized in the Results
section.
Discussion
No data on the genotoxic e€ects of dicyclohexylamine ´
nitrite are available. Dicyclohexylamine was intensively
tested as a minor metabolite of cyclamate, without
showing genotoxic e€ects 8e.g. review by Bopp et al.
1986; Brusick et al. 1989). This was con®rmed by our
own preliminary experiments, in which we observed no
mutagenic e€ects of dicyclohexylamine ´ nitrite 8data
TA1535, TA98, TA100 and TA104 were kindly provided by the
Bruce Ames Laboratory, Berkeley, Calif., USA.

121
Table 1 Micronuclei 8MN) per 1,000 binucleated cells and nuclear an NDI above 1.1 or a cell count below 1,000 valid cells were not
division index 8NDI) induced by N-NO-DCHA in isolated lym- included owing to toxicity 8tox). Concentrations that were not ex-
phocytes of three di€erent donors 8two independent experiments amined are indicated as not determined 8nd)
each). Samples were randomized prior to scoring. Samples showing
N-NO-DCHA Donor 1
8lg/ml)
Donor 2
Donor 3
Experiment 1
Experiment 2
Experiment 3
Experiment 4
Experiment 5
Experiment 6
MN
NDI
MN
NDI
MN
NDI
MN
NDI
MN
NDI
MN
NDI
0
5
10
15
20
30
40
60
3
nd
5
nd
11*
6
8
4
9
1.6
nd
1.4
nd
1.5
1.7
1.9
1.4
1.4
1.3
8
8
8
11
28***
tox
tox
tox
tox
tox
88
1.5
1.2
1.2
1.4
1.4
tox
tox
tox
tox
tox
2
4
3
8
9*
2
1.6
1.4
1.5
1.5
1.5
1.7
1.4
1.3
nd
4
1
3
7
5
2
2
4
1.6
1.4
1.5
1.5
1.5
1.5
1.4
1.4
nd
7
nd
5
20*
5
24** 1.2
tox
tox
tox
tox
151
1.4
nd
1.5
1.8
1.3
5
nd
9
7
11
1.5
nd
1.3
1.5
1.4
1.3
tox
tox
tox
tox
6
2
tox
tox
tox
tox
tox
tox
tox
tox
162
14**
nd
tox
27
80
100
0.15 lM
nd
tox
23
21***
14
tox
tox
Mitomycin C
*P<0.05, **P<0.005, ***P<=0.0005 8according to the chi-square test)
Table 2 Summary of the Ames
Activation
Strain
N -NO-DCHA
Positive control
test results. Various metabolic
activation systems were used
with four di€erent strains. Each
condition was tested at least
twice
Pyrazole S9
TA1535
TA100
TA98
TA100
TA1535
TA100
TA104
TA100
TA104
Negative
Negative
Negative
Negative
Negative
Negative
Microcoloniesa
Negative
Negative
N-Nitrosopyrrolidine
2-Amino¯uorene
2-Amino¯uorene
2-Amino¯uorene
N-Nitrosodiethylamine
2-Amino¯uorene
N-Nitrosodiphenylamine
Methylmethanesulfonate
Methylmethanesulfonate
b-Naphtho¯avone/
phenobarbital S9
Arochlor-1254 S9
Without activation
aMicrocolony induction was seen using arochlor-1254-induced S9 8ICN) at pH 6.5
not shown). Thus, we investigated whether N-NO- microcolonies. The evaluation was additionally compli-
DCHA, which can originate from dicyclohexylamine ´ cated by strong base-rate deviations using TA104 at pH
nitrite, has genotoxic properties.
6.5. Since isolated positive results with TA104 are not
Weak genotoxic e€ects of N-NO-DCHA were seen known 8Gatehouse et al. 1994), with the exception of the
in the micronucleus test with human lymphocytes ®ndings with N-nitrosodiphenylamine 8Zielenska and
8Table 1). In consideration of the NDI 8NDI >1.1), this Guttenplan 1988), this e€ect might be pH-related rather
e€ect was seen at non-toxic concentrations. The dose than substance-related.
response showed two maxima. The second maximum
N-NO-DCHA could not be activated by pyrazole- or
may be related to toxic e€ects, although the NDI was BNF/PB-induced S9, although we applied highly con-
above 1.1. Reproducibility seems to be limited by the centrated S9-mix. In the case of the positive control N-
poor solubility of N-NO-DCHA and culture conditions. nitrosopyrrolidine, 4% pyrazole-induced S9 is sucient
Nevertheless, a trend towards an elevation was seen in to generate a clear mutagenic response, and 20% BNF/
each valid experiment, signi®cant in four out of six PB-induced S9 85 mM NADP and 25 mM glucose 6-
experiments. This meets the currently accepted re- phosphate) is able to activate N-nitrosodiethylamine
quirements for a positive micronucleus test 8Miller et al. eciently 8Westphal et al. 2000). Thus, either the cy-
1997).
clohexyl residues of N-NO-DCHA seem to be extremely
In the Ames test with TA104, N-NO-DCHA induced stable or else the decomposition products of N-NO-
microcolonies above 250 lg per plate when arochlor- DCHA are not mutagenic.
1254 at pH 6.5 was used. The same e€ect was observed
By analogy to the mechanism discussed for N-nitro-
with N-nitrosodiphenylamine. This probably represents sodiphenylamine 8McGregor 1994), the genotoxic e€ects
an e€ect that has been described for N-nitrosodiphe- of N-NO-DCHA in the in vitro micronucleus test may
nylamine in an earlier report 8Zielenska and Guttenplan be due to transnitrosation reactions. In this case, N-NO-
1988). Concurrent to the disappearance of `normal- DCHA would act as a nitrite carrier. Lucas et al. 81999)
shaped' colonies, the background lawn di€erentiated to demonstrated the formation of nitrite adducts following

122
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Maron DM, Ames BN 81983) Revised methods for the Salmonella
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incubation with purine nucleotides and 2¢-deoxyguano-
sine with N-nitrosoindoles. However, we did not observe
mutagenic e€ects in the Ames test without metabolic
activation.
In conclusion, N-nitrosodicyclohexylamine is weakly
genotoxic in the micronucleus test with human lym-
phocytes, negative in the Ames test with TA100, TA98
and TA1535, and inconclusive in the Ames test with
TA104 with metabolic activation at pH 6.5.
McGregor D 81994) The genetic toxicology of N-nitrosodiphenyl-
amine. Mutat Res 317:195±211
Miller B, Albertini S, Locher F, Thybaud V, Lorge E 81997)
Comparative evaluation of the in vitro micronucleus test and
the in vitro chromosome aberration test: industrial experience.
Mutat Res 392:45±59
Acknowledgements The authors thank Dr. R. Machinek, Institute
of Organic Chemistry, Gottingen for acquiring the 1H and 13C-
NMR spectra and Angelika Monnich for excellent technical
assistance.
National Toxicology Program 81990) United States National
Toxicology Program. Chemical Status Report. NTP Chemtrack
System. Research Triangle Park, N.C. TR-164 Bioassay of N-
Nitrosodiphenylamine for Possible Carcinogenicity [CAS No.
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