Varaprasad Bobbarala et al.

/ Journal of Pharmacy Research 2009, 2(9),1540-1541

Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
Mutagenicity Study of Butyl Methoxy Dibenzoylmethane by Using Salmonella
typhimurium
Somasekhar Penumajji 1, Varaprasad Bobbarala2*, K. Chandrasekhar Naidu 3
1
Vivimed labs Limited, 2nd, 4th Floor, Veeranag towers, Habsiguda, Hyderabad, A.P.
2For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam-17, A. P,

3Department of Botany, Andhra University, Visakhapatnam-530003, A.P. India.

Received on: 20-05-2009; Accepted on:15-07-2009

ABSTRACT
Reverse mutation assay in Salmonella typhimurium was performed to test mutagenic potential of butyl methoxy dibenzoylmethane using
test strains of TA 97a, TA 98, TA 100, TA102 and TA 1537. The bacterial cells were exposed to the test chemical at doses of 5, 10, 50, 100, 250
and 500ug/plate with and without metabolic activation system. The exposed bacteria were plated onto minimal glucose agar medium
supplemented with L-Histidine. The histidine revertants were counted and compared with those in the solvent control group. The concurrent
positive controls maintained showed the sensitivity of the assay with or without metabolic activation. Two replicates of the entire experiment
were conducted. There was no evidence of mutagenicity at any dose level of butyl methoxy dibenzoylmethane in any of the strains of S.
typhymurium with or without metabolic activation, as evident from both the experiments. It is therefore concluded that butyl methoxy
dibenzoylmethane is non-mutagenic in strains of TA 97a, TA 98, TA 100, TA102 and TA 1537 of S. typhymurium.

Keywords: Avobenzone, Butyl Methoxy Dibenzoylmethane, Salmonella typhymurium,

INTRODUCTION
Avobenzone (Butyl Methoxy Dibenzoylmethane) is an oil soluble
ingredient used in sunscreen products to absorb the full spectrum of
UV-A rays. It is a dibenzoylmethane derivative. Its ability to absorb
ultraviolet light over a wider range of wavelengths than many organic
sunscreen agents has led to its use in many commercial preparations
marketed as broad spectrum sunscreens.
increase its photostability [5]. According to some studies, the most
effective sunscreens contain avobenzone and titanium dioxide [6, 7].
Avobenzone can degrade faster in light in combination with mineral
UV absorbers like zinc oxide and titanium dioxide, though with the
right coating of the mineral particles this reaction can be reduced [8].
A manganese doped titanium dioxide may be better than undoped
titanium dioxide to improve avobenzone’s stability [9]. The objective
of the study is to evaluate the mutagenic potential of the test article
and/or its metabolites to induce reverse mutations at the histidine
locus in the genome of several strains of S. typhimurium.

MATERIALS AND METHODS

The selection of the strain viz. TA 97a, TA 98, TA 100, TA102
Figure 1: Structure of Avobenzone and TA 1537 for the assay is made in recommendation by the supplier
viz. Prof. B. N. Ames, California, USA at whose laboratory these tester
Avobenzone was patented in 1973 and was approved in the strains are developed.
EU in 1978. It was approved worldwide by FDA in 1988. Avobenzone Strain His- LPs Repair pKM101 Nature of
has been shown to degrade significantly in light, resulting in less mutation Mutation
protection over time [1, 2, 3]. The UV-A light in a day of sunlight in a TA 97a his O1242 rfa S uvrB + +4 near CCC
(frameshift)
temperate climate is sufficient to break down most of the compound. TA 98 his D 3052 rfa S uvrB + -1 near CCC
Data presented to the food and drug administration by the cosmetic, (frameshift)
toiletry and fragrance association indicates a -36% change in TA 100 his G 46 rfa S uvrB + AT?GC
(Base pair substitution)
avobenzone’s UV absorbance following one hour of exposure to TA 102 PAQ1 his rfa - + GC?AT
sunlight [4]. Complexing avobenzone with cyclodextrins may also G 428/s his (Base pair substitution)
TA 1537 his D 3052 - S uvrB - -1 near CCC
(frameshift)
*Corresponding author. All the strains except TA102 are defective in DNA repair
Dr. Varaprasad Bobbarala capacity (uvrB) and have a defective lipopolysaccharide barrier on
Scientist In-Charge,
For U Biosciences/IMMA Labs, the cell wall (rfa). These two properties confer extra sensitivity to
A/4A, Park lane Residency, East point colony, DNA damage and also greater permeability of large molecules into the
Visakhapatnam, A.P-530017, India. cell. These strains also contain resistance transfer factor (plasmid
Tel.: + 91-9949129539 pKM 101). This factor which confers resistance to tetracycline. The
E-mail: varaprasadphd@rediffmail.com

Journal of Pharmacy Research Vol.2.Issue 9.September 2009 1540-1541

 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(9),1540-1541
strains are tested routinely for cell membrane permeability and when- the treatment groups was compared with the number of revertants in
ever applicable for ampicillin and tetracycline resistance. All the strains the respective solvent control group. The mutagenic activity of the
are stored in liquid nitrogen cylinder (-160ºC), in cryocans. For use in test chemical was assessed by applying the following criteria. A test
the assay, subcultures were grown overnight in nutrient broth at 37ºC. chemical is considered as mutagenic if treatment with the test chemi-
This culture provided approximately 1-3 x 10-9 cells/mL which was cal produces an increase in the number of revertant colonies at least
accessed by cell count. twice compared to those in the solvent controls with evidence of
The culture is maintained at temperature of 37ºC and the positive dose relationship, in two separate experiments in any bacte-
period of incubation is for 48 to 72 hours and the groups were seven, rial strain either with or without S-9 mix. A test chemical is considered
and the number of cultures per group are 24 (6 cultures/strain), the non-mutagenic, if it does not induce significant increase in the num-
bacterial strains were cultured in nutrient broth. Vogel Bonner minimal ber of revertants and does not show any dose response relationship.
medium E was chosen as the selective medium. The top agar con- In two separate experiments, with any bacterial strain either with or
tained 0.6% agar, 0.5% NaCl and 0.05mM histidine-biotin (Maron and without S-9 mix.
Arnes, 1983). Lyophilised S-9 vials were obtained from Molecular The mean number of histidine revertants at all the doses i.e.,
Toxicology Inc., U.S.A and maintained at -20ºC at INTOX. This S-9 5, 10, 50, 100, 250 and 500ug/plate with and without metabolic activation
was prepared by ‘Aroclor 1254’ as the liver enzyme inducer in Sprague in all the strains were compared with the respective solvent controls.
Dawley rats, as labelled on the vials. The S-9 mix was prepared imme- It was observed that in two separate experiments, Butyl Methoxy
diately prior to its use in experimental procedure. The microsomal Dibenzoyl methane at any of the dose levels did not induce significant
enzyme reaction mixture contained following components for 10mL. increase in the number of histidine revertants in any of the tester
S-9 fraction 1.0mL, 0.4M MgCl2 -1.65M KCl salt solution 0.2mL, 1.0M strains. Also there was no indication of dose response relationship.
G-6-P, 0.1M NADP, 0.2M Phosphate buffer pH7.4 about 5.0mL, dis- The concurrent positive control articles, for each strain, induced a
tilled water 3.35mL. significantly high number of histidine revertants in both the
Plates containing bacterial culture and top agar supplemented experiments.
with histidine to check spontaneous revertants was used as negative CONCLUSION
control. Plates containing bacterial culture, DMSO in top agar supple- The comparison of mean number of histidine revertants in
mented with histidine to check the solvent effect was used as a sol- the treated and the control groups, during both the experiments,
vent control. Positive control is without s-9 mix (Methyl methane showed no significant difference. Under the conditions of this study
sulfonate 1ul/pate for TA100 and TA102 strains. 4-nitroquinoline-1- no evidence of mutagenicity was observed 5, 10, 50, 100, 250 and
oxide (4-NQNO) 0.5ug/plate for TA97A, TA98 and TA1537 strains) 500ug/plate doses of Butyl Methoxy Dibenzoylmethane in S .
and with s-9 mix (2 Aminofluorene (2AF) 10ug/plate for TA 97a, TA98, typhimurium cells. It is therefore, concluded that Butyl Methoxy
TA100 and TA1537 strains. Danthron 30ug/plate for TA102 strain). Dibenzoylmethane is non-mutagenic in strains TA97a, TA98, TA100,
The dose range finding was performed using the tester strain TA100, TA102 and TA1537 of S. typhimurium.
both with and without metabolic activation. The test chemical was REFERENCES
checked for cytotoxicity, detected by substantial reduction in the 1. Chatelain E, Gabard B. (September 2001). “Photostabilization of
number of revertant colonies and/or absence or thinning of back- Butyl methoxydibenzoylmethane (Avobenzone) and Ethylhexyl
methoxycinnamate by Bis-ethylhexyloxyphenol methoxyphenyl
ground lawn. During the range finding study, it was revealed that the triazine (Tinosorb S), a new UV broadband filter”. Photochem
test article was cytotoxic while 500ug was not. Hence the dose viz. 5, Photobiol 74(3): 401–406.
10, 50, 100, 250 and 500ug/plate were selected for the assay. 2. Tarras-Wahlberg N, Stenhagen G, Larko O, Rosen A, Wennberg AM,
Wennerstrom O (October 1999). “Changes in ultraviolet absorption
Overnight cultures were prepared by transferring a colony of sunscreens after ultraviolet irradiation”. J Invest Dermatol 113(4):
from appropriate master plate to 25ml of nutrient broth. Inoculated 547–553.
cultures were kept for 16h at 37ºC temperature and 4h in shaker incu- 3. Wetz F, Routaboul C, Denis A, Rico-Lattes I (Mar-Apr 2005). “A new
long-chain UV absorber derived from 4-tert-butyl-4'-
bator to achieve cell density about 109 cells/ml. The cultures were methoxydibenzoylmethane: absorbance stability under solar
harvested on checking the cell count. Plating procedure is done with- irradiation”. J Cosmet Sci 56(2): 135–148.
4. CTFA letter re: Tentative Final Monograph for OTC Sunscreen
out metabolic and with metabolic activation. Without metabolic acti- 5. Scalia S, Simeoni S, Barbieri A, Sostero S (November 2002). “Influence
vation cell suspension of a tester strain (0.1ml) and 10 microlitre of of hydroxypropyl-beta-cyclodextrin on photo-induced free radical
solvent or freshly prepared test chemical were added to 2ml of top production by the sunscreen agent, butyl-methoxydibenzoylmethane.”.
J Pharm Pharmacol 54(11): 1553–1558.
agar with L-Histidine. These mixtures were overlaid onto the hard- 6. Warwick L. Morison, M.D. (March 11, 2004.). “Photosensitivity”.
ened surface of 25ml minimal glucose agar plates. The plates were The New England Journal of Medicine 350: 1111–1117.
7. Sunscreen Drug Products for Over-the-Counter use; Marketing Status
inverted and incubated at 37ºC for 48h. Each dose was tested in trip- of Products Containing Avobenzone; Enforcement Policy (PDF). US
licates. Each experiment was repeated once to check the reproducibil- Food and Drug Administration. 1997-04-30.23354.
ity of the results. For metabolic activation the procedure remains the 8. h t t p : / / w w w . f d a . g o v / c d e r / o t c m o n o g r a p h s / S u n s c r e e n /
sunscreen_avobenzone_enforc_policy_19970420.pdf. Retrieved on
same except that 0.5ml of S-9 mix was added to the top agar. 2007-06-03.
RESULTS 9. Stability Study of Avobenzone with Inorganic Sunscreens, Kobo
The plates were check for sterility. The plates were observed Products Poster, 2001, Online version
10. Wakefield G, Lipscomb S, Holland E, Knowland J (July 2004). The
for a uniform lawn of auxotrophs and the colonies for hisitidine rever- effects of manganese doping on UVA absorption and free radical
tants as the prototrophs. Histidine revertant colonies at each test generation of micronised titanium dioxide and its consequences for
point were counted. The mean number of histidine revertants for all the photostability of UVA absorbing organic sunscreen components.
Photochem Photobiol Sci 3(7): 648–52.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.2.Issue 9.September 2009 1540-1541