Britesmile.co.za

Genotoxic Assessment of BriteSmile Whitening Procedure Gel
in Salmonella typhimurium
NATHOO, S.A. and MARSHALL, M.V.
Oral Health Clinical Services, Inc., Piscataway, NJ and and HESS, Inc., Richmond, TX
Negative Controls
Hydrogen Peroxide Concentrations in Test Substance Dilutions
Abstract
Negative control plates were run concurrently with each assay.Three plates containing only The spontaneous reversion frequencies as well as positive and negative controls for tester minimal media and tester strains served as medium controls and three minimal media plates strain TA100 were acceptable in the initial and repeat bioassays. Toxicity was seen with with 50 µL of Aroclor-induced rat liver homogenate (S9) and tester strains served as S9 BriteSmile Whitening Procedure Gel in the absence of S9 at 5,000 ?g/plate. Because O b j e c t i v e : To determine the potential for BriteSmile Whitening Procedure Gel
controls. Plates containing 100 µL of water and tester strains were used as solvent controls.
A range-finding study was conducted in tester strains TA98 and TA100 at levels of 0.001, BriteSmile Whitening Procedure Gel never exceeded twice background at any of the dose ( B riteSmile Gel) to induce mutations in tester strains of Salmonella typhimu ri u m .
0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 mg BriteSmile gel/plate (Table 1); all additions of test levels examined (Figure 1), it was not mutagenic in tester strain TA100 in the presence or BriteSmile Gel contains 15% hydrogen peroxide (HP), and it is used in conjunction with Positive Controls
substance and controls were made in 100 µL volumes. The positive and negative controls absence of S9 in the plate incorporation assay.
a visible wavelength light source to whiten teeth. BriteSmile Gel is formulated with a were within acceptable ranges for tester strains TA98 and TA100. Because toxicity was not light-activated component to reduce the contact time needed for tooth bleaching. Inclusion In the presence of S9, 2-aminoanthracene (2.5 µg/plate) was used in the range-finding with observed in tester strain TA98 at 5 mg/plate, this dose level was selected as the highest of a light activated component enables a lower HP concentration to be used to provide TA98 and TA100 and in the definitive test with TA97a, TA98, TA100, and TA1535. Danthron dose level of BriteSmile Whitening Procedure Gel for definitive testing in the presence and tooth whitening. Methods: To assess the mutagenic potential of BriteSmile Gel, five tester
(100 µg/plate) was used in TA102 in the presence of S9. In the absence of S9, ICR-191 The spontaneous reversion frequencies as well as positive and negative controls for tester strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102, and TA1535) were (1.0 µg/plate) was used in TA 9 7 a ; 2-nitrofluorene (5 µg/plate) was used for the ra n g e - f i n d i n g strain TA102 were acceptable in the initial and repeat bioassays. In tester strain TA102, no exposed to BriteSmile Gel at levels up to 5 mg/plate in the presence and absence of an and definitive study in TA 9 8 ; NaN3 (2 µg/plate) was used for the range-finding and definitive Initial and Repeat Bioassays
toxicity was seen with BriteSmile Whitening Procedure Gel in the presence or absence of external metabolic activation source (S9, an Aroclor-induced rat liver homogenate).
test with TA100 and in the definitive test in TA1535; mitomycin-c (0.25 µg/plate) was used S9 at dose levels up to 5,000 ?g/plate. The number of revertant colonies exceeded twice Results: No mutagenic activity was seen in any of the five tester strains in the presence of Overview
background with the test substance in the absence of S9, but not in the presence of S9.
S9, and no mutagenic activity was seen in TA97a, TA98, TA100, and TA1535 in the Range-finding Assay
The threshold value for a positive response was exceeded when concentrations of hy d r o g e n absence of S9. A weak mutagenic response was seen in TA102, a tester strain that is Initial and repeat plate incorporation bioassays were conducted in tester strains TA97a, peroxide in BriteSmile Gel exceeded 86 ?g/plate. Because BriteSmile Whitening Procedure sensitive to oxidative mutagens. For comparison, aqueous hydrogen peroxide was also TA98, TA100, TA102, and TA1535 at levels of 0.03, 0.08, 0.1, 0.3, 0.8, 1, and 5 mg/plate in A range-finding test was performed prior to the definitive bioassay to determine appropriate Gel exceeded twice back ground in tester strain TA102 in the absence of S9 in the included in TA102 without S9. HP was mutagenic (exceeded twice background) at 0.15 the presence and absence of S9. All additions of test substance BriteSmile Whitening dose levels of the test substance to be used in definitive bioassays. The test substance was plate incorp o ration assay, it can be considered weakly mutagenic (Figure 2). The slopes and 0.2 mg/plate. B riteSmile Gel was also weakly mutagenic at similar levels of HP without Procedure Gel were made in 0.1 mL of water.
analyzed in duplicate at eight dose levels for toxicity at various concentrations up to of the linear portions of the dose-response curves were 347.0 revertants/?g HP and H y d ro gen Pe roxide T i t r a t i o n
S9. These results are similar to other published results that show a weak mutagenic 5 mg/plate. Toxicity was defined as a decrease in the number of revertant colonies per plate response for HP in tester strain TA102 in the absence of S9. Conclusion: The biological
423.4 revertants/?g HP, respectively, for the initial and repeat assays of BriteSmile Gel.
and/or by a thinning or disappearance of the bacterial lawn, or by the appearance of pinhead relevance of this positive response is questionable as intracellular and extracellular The spontaneous reversion frequencies as well as positive and negative controls for tester For comparison, aqueous hydrogen peroxide was analyzed for mutagenic activity in defense mechanisms are present in the oral cavity that decompose HP. The response of tester strain TA102 in the absence of S9 at levels up to 200 ?g/plate. A weak mutagenic BriteSmile Gel is more appropriately compared to tests performed in the presence of S9 strain TA97a were acceptable in the initial and repeat bioassays. There was consistent evi- Definitive Assay Performance
response was observed, which is consistent with literature reports on the mu t a g e n i c in which no mutagenic activity was detected. S u p p o rted by Bri t e S m i l e, Inc., Wa l nut Creek, CA.
dence of toxicity in the initial and repeat assays in the absence of S9 at 5,000 ?g/plate. The Assays were performed within a hood in subdued lighting. After overnight incubation, tester mutagenic response of BriteSmile Whitening Procedure Gel never exceeded twice back- p o t e ntial of aqueous hydrogen peroxide (Figure 3). The threshold value for a positive strains were refrigerated; S9 homogenate and cofactor solutions were mixed at a ratio at ground at any of the dose levels examined (Figure 1). Therefore, BriteSmile Whitening response was exceeded with hydrogen peroxide concentrations above 100 ?g/plate. The 1 mL reconstituted S9 homogenate to 9 mL cofactor solution; and top agar was melted and Procedure Gel was not mutagenic in tester strain TA97a in the presence or absence of S9 slopes of the linear portions of the dose-response curves were 430.4 revertants/?g HP and Materials & Methods
maintained at 42-45°C. Dose selection was determined from the range-finding study.
400.5 revertants/?g HP for the initial and repeat assays, respectively.
Dilutions were arranged so that the test substance was delivered in 100 µL water. The testsubstance and tester strain were added to sterile tubes containing 2 mL top agar (-S9) or Determination of Hydrogen Peroxide Levels
0.5 mL S9/cofactor mix was added prior to test substance and tester strain (+S9); and the mixture was vortexed and poured onto MGA plates. After solidifying, plates were inverted The spontaneous reversion frequencies as well as positive and negative controls for tester The spontaneous reversion frequencies as well as positive and negative controls for tester Hydrogen peroxide content was determined by titration with KMnO4 according to and incubated 48-72 hr at 37±2°C. An independent repeat bioassay was conducted to confirm strain TA98 were acceptable in the initial and repeat bioassays. Toxicity was observed in the strain TA1535 were acceptable in the initial and repeat bioassays. Toxicity was observed at procedures published in the USP. Briefly, a 20-ml volume of 2.0 N H2SO4 was added to a results of the initial study. After 48-72 hr, plates were removed from the incubator, and initial and repeat assays at 5,000 ?g/plate in the absence of S9. Because BriteSmile 5,000 ?g/plate in the absence of S9. Because test substance BriteSmile Whitening 20-ml sample, and this mixture was titrated with 0.1N KMnO4 until a slight pink color r eve rtant colonies on each plate were counted manually or with an automatic colony counter.
Whitening Procedure Gel never exceeded twice background at any of the dose levels exam- Procedure Gel never exceeded three times background at any of the dose levels examined remained in the solution after addition of KMnO4. Each ml of 0.1N KMnO4 is equivalent to The background bacterial lawn was evaluated for toxicity and precipitate formation, and the ined (Figure 1), it was not mutagenic in tester strain TA98 in the presence or absence of S9 (Figure 1), it was not mutagenic in tester strain TA1535 in the presence or absence of S9 1.7005 mg H2O2, and the following formula was used to determine peroxide content: mean nu ber of revertant colonies for all replicates was determined.
Conclusions
% H2O2 = (ml KMnO4 titrant) x 0.1N x 1.7005 mg H2O2/Sample weight (g)
Bacterial Cultures
• There was no evidence of an increase in the number of revertant colonies that Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102, and TA1535 were exceeded twice background in tester strains TA97a, TA98, TA100, and TA1535 at grown in Oxoid Nutrient Broth #2 for approximately 16 hr at 37±2° C with agitation at dose levels up to 5 mg/plate in the presence or absence of a metabolic activation 300±25 rpm. For each mutagenicity assay, confirmation of phenotypic markers was also performed. The presence of the rfa wall mutation in TA97a, TA98, TA100, TA102, andTA1535 was confirmed by demonstrating sensitivity to crystal violet. For UV sensitivity, • In tester strain TA102, the number of reve rtant colonies did exceed twice back gr o u n d bacteria were irradiated at 7,000 µJoules. TA102 is relatively insensitive to UV; all othertester strains are sensitive to UV. The presence of the pKM101 plasmid in tester strains at dose levels of0.8, 1, and 5 mg/plate in the absence of metabolic activation, but TA97a, TA98, TA100, and TA102 was confirmed by resistance to ampicillin. Tester strain not in the presence of metabolic activation.
TA1535 lacks the ampicillin resistance plasmid, and it was sensitive to ampicillin.The presenceof the pAQ1 plasmid in TA102 was demonstrated by growth in tetracycline; tester strains • The lack of a mutagenic response indicates BriteSmile Whitening Procedure Gel TA97a, TA98, TA100, and TA1535 were sensitive to tetracycline. Tester strains grew on is nongenotoxic (non-mutagenic) in tester strains TA97a, TA98, TA100, and plates containing 0.0005% histidine and 3 µM biotin, but not on plates that lacked histidine.
TA1535 in a plate incorporation assay.
General Information - Plating
• BriteSmile Whitening Procedure Gel is weakly genotoxic (mutagenic) in tester Minimal media (MGA) plates containing reduced glucose levels (0.4%) were purchasedfrom Molecular Toxicology, Inc. (Boone, NC) and stored at room temperature. Sterile 1X strain TA102 only in the absence of external metabolic activation.
NADP cofactor mix was diluted with reconstituted lyophilized Aroclor-induced rat liverhomogenate (S9, Molecular Toxicology, Inc. Boone, NC) at a ratio of 1 mL S9 to 9 mL 1X • Based on these results, BriteSmile Whitening Procedure Gel is not likely to be igure 1: Mutagenic Response of BriteSmile Gel in Tester Strains TA97a, TA98, TA100, and
Figure 2: Mutagenic Response of BriteSmile Gel in Tester Strain TA102 Without S9
Figure 3: Mutagenic Response of Aqueous Hydrogen Peroxide in Tester Strain TA102
NADP cofactor mix immediately prior to use.
carcinogenic in animals or in humans.
TA1535 Without S9
Without S9
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