Triclosan is an antibacterial agent found in many household products that is studied for its potential mutagenic and toxic effects. The study tested whether triclosan is mutagenic using two E. coli strains in an assay with and without liver enzyme activation. Results showed triclosan did not induce reverse mutations, indicating it is not mutagenic. However, HPLC analysis found new peaks when triclosan was reacted with liver enzymes, suggesting metabolism produces derivatives. The study supports further testing triclosan's effects at ecological concentrations on aquatic life and humans with significant exposure.

1. Testing Triclosan as a Mutagenic Compound
Mujtaba M. Qureshi
Department of Biochemistry
Rutgers School of Environmental and Biological Sciences
mmq10@scarletmail.rutgers.edu
732-649-9070

2. Abstract
Triclosan, an antibacterial and antifungal agent found in household consumer products
(from toothpaste to soaps), is a chemical studied to form TCDD, a dioxin that has toxicological
effects on the environment, aquatic life, and mammals. We study the effects of triclosan and its
metabolites as potential mutagens on E.coli as our model organism, by observing reverse
mutations as our mutagenic assay. Two strains of E.coli in particular are observed: WPAtrp and
WP2trp urvA. WPAtrp is a mutant that cannot grow without an outside source of tryptophan, and
WP2trp urvA is a hypersensitive version of WPAtrp, where nucleotide excision repair is
eliminated in order to severely reduce the possibility of fixing any mutations. Two positive
controls are used: methylmethanesulfonate and 2-aminoanthracene. Samples are treated with
S9 activation and without S9 activation, in order to test for metabolites. We use the ‘E.coli
Mutagenicity Test Kit’ protocol to choose appropriate triclosan doses and to carry out the
mutagenic assay. We use HPLC (High Performance Liquid Chromatography) to confirm
metabolites production. Using these assays, we fail to observe triclosan as a mutagen. The
study shows the need to further test the effects of ecological concentrations of triclosan on
tetrogenic endpoints rather than mutagenic endpoints on aquatic life, as well as on human life in
countries with significant levels of triclosan in their bodies (China, Vietnam, and more).
Introduction
The toxicology of chemicals found in everyday household items is not always studied in
full detail. One such chemical is triclosan, an antibacterial and antifungal agent found in
household consumer products, from toothpaste to soaps (i.e. hand sanitizers). Research shows
that at least 1500 tons of Triclosan were used in 2015 in China alone (Zhang et al., 2015).
Triclosan has been detected in human urine as well as plasma (Hovander et al., 2002), which
may be due to seafood consumption or due to exposure to household consumer products.
Furthermore, research has indicated triclosan by itself has adverse endocrinological effects in

3. human cancer patients (Gee et al., 2007). It has also been found that Triclosan has the capacity
to undergo chlorination in seawater, forming TCDD (2,3,7,8-tetrachlorodibenzopdioxin), a dioxin
(Kanetoshi et al., 1987). This dioxin has been researched and studied to cause a number of
toxicity endpoints, such as auditory neuropathy in mice for instance (Safe et al., 2016).
Research to understand the toxicity of triclosan on health impacts of organisms and
environmental effects remains limited in the genetic level for a number of toxicological
endpoints. Triclosan in previous studies was found to be a tetragen. In this experiment, we test
to see if triclosan is a mutagenic compound through the use of a mutagenic assay. First, we
perform a spot test through the use of a disc assay to test various concentrations of triclosan in
order to find the appropriate dosing range to cause growth inhibition of bacterium (Escherichia
coli in this experiment). Next, we perform a mutagenic assay modified from the ‘Mutagen
Testing’ protocol (Green at al 1975). The assay tests whether or not a chemical is mutagenic by
observing reverse mutations induced by the chemical in question (triclosan in this experiment) in
modified E.coli strains. These E.coli strains are already mutated to not able to survive without
outside sources of specific nutrients (tryptophan in this case, described more later). If triclosan
causes significant reverse mutations, it will allow these strains to grow in greater quantity by 2 –
2.5 fold (Green et al 1975), affirming that triclosan is a mutagen.
We also use C-18 reverse phase HPLC (High Performance Liquid Chromatography)
column as a separation technique to test for the formation of triclosan metabolites when reacted
with S9 hepatic enzymes (cytochrome P450 and other conjugates). Triclosan forms
monohydroxylated triclosan without S9 activation, which may then further decompose by the
splitting of the ether bond into 2,4-Dichlorophenol and 4-Chlorocatechol. Upon S9 activation,
Triclosan forms 3 products: methyl triclosan, triclosan glucuronide, triclosan sulfate. The
mutagenic assay tests for mutagenicity of triclosan in both with and without S9 activation.
We test two strains in the mutagen assay: WPAtrp and WP2urvA. WPAtrp is an E.coli
strain that exhibits a mutation in the tryptophan operon resulting in lack of tryptophan

4. production. The E.coli thus will not culture/grow without an outside source of tryptophan, which
we do not provide in the assay. WP2urva is an E.coli strain that also carries the mutation in the
tryptophan operon, but it also eliminates nucleotide excision repair by eliminating DNA repair
coding gene urvA. It is thus less likely to reverse the mutation caused by the mutagen, allowing
us to see the effect of the mutagen more strongly. This strain will thus be hypersensitive to
mutagens. Next, we use two positive control treatments: methylmethanesulfonate (MMS) and 2-
aminoanthracene, in order to qualify triclosan-treated samples. MMS is a known direct mutagen
that acts regardless of S9 activation [how]. It methylates the nitrogens within deoxyguanosine
and deoxyadenosine. This leads to replication fork damage. We expect to see a 2 - 2.5 fold
increase of E.coli growth in samples tested by MMS. 2-aminoanthracene is not a direct mutagen
and requires S9 activation. It is a positive control that will act as a mutagen only upon S9
activation, which will confirm that S9 activation is functioning properly in our assay. We expect
to see a 2 – 2.5 fold increase of E.coli growth in samples tested by 2-aminoanthracene.
We hypothesize that triclosan is not a mutagen, and that in both S9 activated samples
and S9- samples, a 2 – 2.5 fold increase will not be observed. All procedures are carried out
using aseptic techniques and clean sterile glassware and solutions.
Methods
The dosing range for E. Coli was found by using a disc assay as taken from the E.Coli
Mutagenicity Test Kit. First, aseptic technique was used to ensure all tools and benches were
sterilized via ethanol. Next, E.coli was cultured on agarose plates overnight at 37 degrees
Celsius. Ten hours later, 4 paper discs (1cm wide) were placed on to each agarose plate. Disc
placement was carefully carried out by spreading out discs as far from each other as possible,
while settling on large E.coli colonies. The control disc was treated with 10uL sterilized water.
Disc 2 was treated with 10uL of 1mg/mL Triclosan. Disc 3 was treated with 10uL of 0.1mg/mL

5. Triclosan. Disc 4 was treated with 10uL of 0.001mg/mL of Triclosan. The plates were incubated
for 48 hours at 37degrees Celsius and then observed for inhibition.
Based on the results from the inhibition, the mutagenic assay was carried out following
the ‘E.Coli Mutagenicity Test Kit’. The assay was carried out in agarose plates using E.coli for
two doses of Triclosan tested on each strain: 0.1mg/mL and 0.01mg/mL. The strains tested
were: wp2trp (strain 1) and wp2trpuvrA (strain 1). There were a total of 4 groups, one group for
strain WPAtrp at dose 0.1mg/mL of triclosan, a second group for strain WPA2trp at dose
0.01mg/mL, a third group for strain WP2trp urvA at dose 0.1mg/mL, and a fourth group for strain
WP2trp urvA at dose 0.01mg/mL. Each group had 7 plates, for a total of 28 plates. Each strain
and dose was tested with triclosan, methylmethanesulfonate, 2-amino anthracene, and
sterilized water as control – once with S9 metabolites and once without. Only 2-
AminoAnthracene isn’t tested without S9. The plates were incubated at 37degrees Celsius for
48h.
Control
(H2O)
Treated
(Triclosan)
MethylMethaneSulfonate 2-
AminoAnthracene
S9+ (10%) 100uL H2O
500uL S9
100uL H2O
500uL S9
100uL strain
100uL
triclosan
100uL H2O
500uL S9
100uL strain
100uL MMS
100uL H2O
500uL S9
100uL strain
100uL 2-AA
S9- 100uL H2O 100uL H2O
100uL strain
100uL
triclosan
100uL H2O
100uL strain
100uL MMS
N/A
Table 1: Solutions shown for each plate within each group.

6. Next, TLC plates were used to test for which dilution factor of methanol with water acts
as the best solvent for triclosan in liquid chromatography. This was done in order to choose the
best solvent to run in the HPLC column. Three dilutions were tested: 20water/80methanol,
50water/50methanol, and 80water/20methanol. Then, all 3 samples (solvent
[20water/80methanol], triclosan without S9, and triclosan with S9) were marked on to the TLC
plates to indicate whether metabolites form or not. The samples were then run on a C-18
reverse phase HPLC column with an 80% methanol and 20% Distilled water phase and the
detector at 254 nM.
Results
The experiment was carried out successfully for the spot test (disc assay), mutagenic
assay (with one exception), TLC plates testing, and the HPLC metabolites readings.
Spot test (Disc Assay) results indicated growth inhibition in some trials for 0.01mg/mL
triclosan treatment as well as 0.1mg/mL triclosan treatment in other trials. The mutagenic assay
was carried out using both concentrations for two separate growths. Results are indicated
below. Controls and triclosan colonies were 50-200 in count, MMS colonies were 340-500, and
2-AA colonies were 50-120. Errors in experiment occurred for group 1 for 2-aminoanthracene
due to poor solubility. DMSO may be a better solvent than H2O. Group 4 had errors, due to re-
heating of the bacteria in hot agar at a temperature above 37 degrees Celsius.
Group 1
0.1 mg/mL dose
WP2trp
Group 2
0.1 mg/mL dose
Wp2trp uvrA
Group 3
0.01mg/mL dose
Wp2trp
Group 4
0.01 mg/mL dose
Wp2trp uvrA
Control 160 colonies 109 colonies 69 colonies 1 colony
Control S9+ 216 colonies 138 colonies 50 colonies 4 colonies

7. Triclosan 148 colonies 136 colonies 56 colonies 3 colonies
Triclosan + S9 135 colonies 112 colonies 43 colonies 50 colonies
MMS 421 colonies 944 colonies 535 colonies 225 colonies
MMS + S9 493 colonies 1048 colonies 341 colonies 12 colonies
2-AA + S9 54 colonies 123 colonies 57 colonies 65 colonies
Table 2: Results from Mutagenic assay of E.coli strains Wp2trp and Wp2trp uvrA, testing two
doses: 0.1mg/mL triclosan and 0.01mg/mL triclosan. Results indicate number of E.coli colonies,
as observed after 48hours of incubation at 37degrees Celsius.
The results from the TLC-solvent test indicated 20water/80methanol to be the best
solvent. TLC was then carried out once again for appearance of triclosan metabolites testing,
and results are indicated below.
Image 1: TLC plates. From left to right: triclosan with S9, triclosan without S9, solvent
(20%water/80%methanol).
HPLC was carried out and the retention times and absorbance units are reported in the
table below.

8. Table 3: The values are from the chromatographs of the samples using a C-18 reverse phase
column with an 80% methanol to 20% Distilled water phase and the detector at 254 nM.
Triclosan was added at 100 uL of a 10 mg/L stock. Retention times and absorbance are shown.
Discussion
Two strains of E.Coli were tested for triclosan mutagenicity, in both S9 activation and
without S9 activation. The results from the mutagen assay indicate that triclosan is not a
mutagenic compound. The positive control MMS indicates an increase in growth greater than 2
– 2.5 fold, showing reverse mutations and thus proving that it is a mutagen. Triclosan shows
almost a 1:1 relationship with the control samples. A 2 – 2.5 fold increase is not seen in both
doses (0.1mg/mL and 0.01mg/mL) of triclosan treatment. Samples treated with 2-AA show no
significant fold increase either. This result for 2-AA may allude to the possibility that S9
activation is not occurring, but when the same samples are run on the HPLC, metabolites are
clearly formed as products in the triclosan+S9 sample. There were errors in solvent used for 2-
AA, so accurate results may not have been observed.
Comparing the triclosan sample and the triclosan+S9 sample, there are 3 new peaks
reported through HPLC. This means that there are at least 3 new compounds present in the
Triclosan+S9 sample, supporting the hypothesis that exposure to S9 resulted in metabolism of

9. triclosan. The likely products/metabolites formed from S9 are: monohydroxylated triclosan,
methyl triclosan, triclosan glucuronide, triclosan sulfate, 2,4-Dichlorophenol, and 4-
Chlorocatechol (Aguillon et al 2010). Retention times in HPLC are determined by solubility with
the solvent. The less soluble, the quicker the compound/metabolite will elute and thus have a
shorter retention time. Based on this, peak 2 which is the least soluble in methanol would be:
triclosan glucuronide. Peak 3 would be triclosan sulfate. Peak 4 would be monohydroxylated
triclosan. Peak 5 is the parent triclosan compound. Peak 6 is even less soluble than the parent
triclosan compound itself, and both 2,4-Dichlorophenol & 4-Chlorocatechol are more soluble, so
peak 6 may be methyl triclosan, which is created from methyl transferase. This would imply that
2,4-Dichlorophenol & 4-Chlorocatechol are found in a mixture with another compound in one of
the earlier peaks. Like monohydroxylated triclosan, they are formed without S9 activation, and
the two compounds are formed from the cleavage of monohydroxylated triclosan in the first
place, so peak 4 is most probably the mixture.
Sample’s peak 4 shows that it was not pure with just the parent triclosan compound.
Peak 4 shows that there was another compound present which we infer it to be
monohydroxylated triclosan and its derivatives, because triclosan itself breaks down into these
compounds without S9 activation, so it would be expected to be observed through HPLC.
In order to ensure that the other peaks are actually due to triclosan metabolizing, we
may use beer-lambert’s law: A=E*C*L. Absorbance values can be substituted into the formula to
calculate for concentrations. Based on the HPLC mAU (mili Absorbance Units) though,
comparing absorbance between the two samples for the parent triclosan peak may be enough
to determine how much of the triclosan reacted (relatively). The parent triclosan absorbance
decreases by half, which leads us to believe that half of the amount of parent triclosan
compound reacted with S9 and formed the metabolites discussed.
This experiment shows conclusive results that neither triclosan nor its S9-activated
metabolites are mutagenic. Other studies also claim that triclosan is not a mutagenic compound,

10. as according to the spot test (Russell 1980). Triclosan has been suggested in some studies to
affect estrogen-dependent cancer growth (Dinwiddie et al., 2014). We push for further research
to study triclosan as a teratogenic compound, not a mutagenic compound. Longer studies for
effects of triclosan on human health, as opposed to animal models, are even more so needed to
give a better understanding on the chemical’s relation to cancer. Additionally, with conflicting
results in studies that show cancer inhibition and others hinting towards tumor inception, the
need for studies in human cancer patients is needed to better clarify what exactly the
relationship is between triclosan, triclosan-derivatives, and cancer-related pathways in humans.
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