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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 03, 2014 to April 17, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione
EC Number:
935-606-2
Cas Number:
1329658-14-1
Molecular formula:
C17H8O4
IUPAC Name:
5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione
Test material form:
solid: particulate/powder
Details on test material:
Short Name: PETA
Long Name: NNEXAMITE™ A56 (PETA)
Chemical Name: Phenylacetylene modified trimellitic anhydride
Lot Number: NEX-X61-A02
CAS Number: 1329658-14-1
Description: Yellow powder
Purity: >99 %
Molecular weight: 276.24
Manufacture date: 04 September 2013
Expiry date: 01 May 2014
Storage conditions: Room temperature (15-25 °C), protected from humidity
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety
Specific details on test material used for the study:
Not further details specified in the study report.

Method

Target gene:
Histidene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The Salmonella typhimurium strains used in this study were mutants derived from Salmonella typhimurium LT2. The strains used in the study were obtained from Molecular Toxicology Inc., 157 Industrial Park Dr. Boone, NC 28607, U.S.A.
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Not specified
Metabolic activation:
with and without
Metabolic activation system:
Unlike mammals, bacteria lack the necessary oxidative enzyme systems for metabolising foreign compounds to electrophilic metabolites capable of reacting with DNA. Sometimes these foreign compounds, when reacting with a mammalian enzyme system, yield mutagenic metabolic products. In order to test these indirectly acting mutagens, a metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction is used. Activity of batch of S9 used in this experiment was characterized by testing a selected pre-mutagen, benzo(a)pyrene, with S. typhimurium TAI00. The S9 fraction is buffered and supplemented with the essential co-factors 13-NADP and Glucose-6-phosphate to form the "S9 mix". This mix is added to the top agar in this activated assay. The S9 fraction procured from D.R.D.O., Nagapur was used in the study.
Test concentrations with justification for top dose:
Cytotoxicity Test: Ten different concentrations, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate were tested for cytotoxicity.
Based on the results of the cytotoxicity test, the test concentrations of78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate of PETA both in the absence and presence (5% v/v S9 mix) of metabolic activation were selected for Trial I.
Trial II was conducted with modified concentrations i.e., 25.6, 64, 160,400, 1000 and 2500 μg/plate in the absence and presence of metabolic activation (S9 concentration was increased to 10% v/v).
Vehicle / solvent:
Solubility and precipitation tests were performed prior to the cytotoxicity test. The test item was insoluble in sterile distilled water (Stock A), while found to be soluble in dimethyl sulfoxide (Stock B). Therefore, dimethyl sulfoxide was selected as the vehicle for treatment.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Solubility Test
Solubility and precipitation tests were performed prior to the cytotoxicity test. The test item was insoluble in sterile distilled water (Stock A), while found to be soluble in dimethyl sulfoxide (Stock B). Therefore, dimethyl sulfoxide was selected as the vehicle for treatment. A volume of 100 μL of the test item from stock B was added to 2 mL of top agar, to assess the precipitation. Slight precipitation was observed at the concentration of 5000 μg/plate. Hence, 5000 μg/plate was selected as the highest concentration to be tested for cytotoxicity, both in the absence and presence (5% v/v S9 mix) of the metabolic activation system.

Cell Viability Test
Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N° 2 (Oxoid Unipath, England). The flasks were then incubated at 37.0 ± 1 °C in an orbital shaking incubator (120 rpm) for 15 h up to an early stationary or late exponential phase.
After the incubation period, the culture flasks were removed from the orbital shaking incubator. Aseptically, the cultures were diluted with oxoid nutrient broth (ONB) and the optical density was measured at 660 nm using a Spectrophotometer (Visiscan 167, manufactured by Systronics).
Oxoid nutrient broth was used as the control blank. Cell viability of the tester strains was determined prior to treatment. The optical density of the cultures was found to be in the acceptable range and so they were used for the study.

Genotype Confirmation Test
Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N° 2 (Oxoid Unipath, England). The flasks were then incubated at 37.0 ± 1°C in an orbital shaking incubator for 15 h up to an early stationary or late exponential phase. The genotype of the tester strains was confirmed once in a month as per the following procedures:

Test for Histidine Requirement
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto a Minimal Glucose Agar (MGA) plate containing an excess of biotin (0.5%). The plates were incubated at 37 ± I °C and examined after 48 h incubation.

Test for Biotin Requirement
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate containing an excess of histidine (0.01%). The plates were incubated at 37 ± I °C and examined after 48 h incubation.

Test for Histidine and Biotin Requirement
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (with histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate without histidine and biotin. The plates were incubated at 37 ± 1 °C and examined after 48 h incubation.

Test for rfa Mutation
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto nutrient agar plate. After the solidification of the top agar, a sterile paper disc dipped in 1 mg/mL solution of crystal violet was transferred to the plates, using sterile forceps. The plates were then incubated at 37 ± 1 °C and examined for the zone of inhibition around the impregnated paper disc after 48 h incubation.

Test for uvrB Mutation
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (with histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate. After the solidification of the top agar, half of the plate was covered with sterilized aluminium foil. Plates were exposed to 15W germicidal lamp (UV radiation) at a distance of33 cm. Non R-factor strains (TA1537, TA1535) were exposed for 6 seconds and R-factor strains (TA98, TAlO0 and TA102) for 8 seconds. The plates were then incubated at 3 7 ± 1 °C and examined after 48 h incubation.

Test for R-factor Resistance (Ampicillin Resistance)
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate supplemented with histidine (0.5%), biotin (0.01%) and ampicillin (24 μg/mL). The plates were incubated at 37 ± 1 °C and examined after 48 h.

Test for R-factor Resistance (Tetracycline Resistance)
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate supplemented with histidine (0.5%), biotin (0.01%) and tetracycline (2 μg/mL). The plates were incubated at 37 ± 1 °C and examined after 48 h.

Cytotoxicity Test
Before commencing the mutagenicity study, PETA was tested for cytotoxicity, to Salmonella typhimurium tester strain TA 100. The experiment was conducted both in the absence and presence of metabolic activation system (5% v/v S9 mix).
A stock solution of 50000 μg/mL of PET A was prepared by dissolving 100 mg (100.04 mg i.e., 100 mg) of test item in dimethyl sulfoxide (stock A) and made up to 2 mL.
Further stock solution of concentrations, viz., 25000 (stock B), 12500 (stock C), 6250 (stock D), 3125 (stock E), 1562.5 (stock F), 781.25 (stock G), 390.63 (stock H), 195.31 (stock I) and 97.66 μg/mL (stock J) were prepared by serial dilution method. Ten different concentrations, viz., 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate were tested for cytotoxicity. Volumes of 100 μL ofrelevant stock solutions A - J were used to obtain the required test concentrations for treatment both in the absence and presence (5% v/v S9 mix) of the metabolic activation system.
Tubes containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45 ± 2 °C. A volume of 500 μL of 5% v/v S9 mix was added in the presence of metabolic activation system and 500 μL of 0.2 M phosphate buffer was added in the absence of metabolic activation system. A volume of 100 μL of the relevant concentration of stock solution of the test item and dimethyl sulfoxide was used for treatment and as a negative control, respectively. Finally 100 μL of bacterial culture was added to the tubes and mixed. Cultures used were checked for cell viability prior to testing. This treatment mixture was poured on an MGA plates and allowed to solidify.
Triplicate sets were maintained for each concentration of PET A and negative control. The petriplates were incubated at 37 ± 1 °C for 48 hours and then treated plates were examined to assess the state of background bacterial growth inhibition and reduction in number of colonies.

Mutagenicity Test
The mutagenicity test was conducted as two independent experiments. In both the trials, the treatment was performed both in the absence and presence of metabolic activation system (5% v/v and I 0% v/v S9 mix in Trial I and II, respectively). The treatments were performed by plate incorporation technique as described in the cytotoxicity test. Plates were maintained in triplicates for each test concentrations of PET A, negative and positive controls.

Trial I [In the Absence and Presence of 5% v/v S9 Mix]
The tester strains were exposed to the test concentrations of78.125, 156.25, 312.5, 625, 1250 and 2500 μg/plate of PET A in the absence and presence (5% v/v S9 mix) of metabolic activation system. The first stock solution (stock A) of the test item was prepared by dissolving 250 mg (250.02 mg i.e., 250 mg)of PETA in dimethyl sulfoxide and made up to 10 mL (25000 μg/mL). For the treatment, a volume of 5 mL of stock A was added to 5 mL dimethyl sulfoxide to obtain 12500 μ/mL (stock B). Further stock solutions viz., 6250 (stock C), 3125 (stock D), 1562.5 (stock E) and 781.25 μg/mL (stock F) were prepared by serial 2 fold dilution. Volumes of 100 μL of relevant stock solutions A-F were used to obtain the required test concentrations for treatment in the absence and presence (5% v/v S9 mix) of the metabolic activation system respectively.

Trial II [In the Absence and Presence of 10% v/v S9 Mix]
A second trial was conducted to confirm the negative results obtained in Trial I. In Trial II, the concentration spacing was modified using a factor of 2.5 and the concentration of S9 mix was increased to 10% v/v. The tester strains were exposed to the test concentrations of 25.6, 64, 160, 400, 1000 and 2500 μg/plate in the absence and presence (I 0% v/v S9 mix) of metabolic activation. Volumes of 100 μL of relevant stock solutions were used to obtain the required test concentrations.
Plates were maintained in triplicates for each test concentration of PET A, negative and positive controls during both the trials. The number of revertant colonies was recorded after 48 h incubation period.
Treatment with 2-aminoanthracene in the absence of metabolic activation were also performed for tester strain TA 100 in both the trials to verify the efficiency of the S9 fraction used in the study.
Rationale for test conditions:
This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and TA102, which are known for their reliability and reproducibility in a short term mutagenicity assay and are also recommended by the OECD and other guidelines.
Evaluation criteria:
Once criteria for a valid assay are met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were: there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the absence or presence of the metabolic activation system.
Biological relevance of the results was considered first:
Strains TA98, TA1535, and TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.

Strain TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean negative control value.
Statistical analysis (i.e. Simple linear regression analysis) was used as an aid in the evaluation of dose response.
A response that did not meet all three of the above criteria (magnitude, concentration responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the first trial were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing and metabolic activation.
Statistics:
Simple linear regression analysis was performed for TA1537, TA1535, TA98, TAl00 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Negative Control
The results of the study indicate that the values of negative control in all strains were within historical range of respective strains.

Positive Controls
2-Aminoanthracene was used as the positive control in the presence of metabolic activation for all the tester strains during both the trials. Large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation. The batch of S9 used in this study was characterised with benzo(a)pyrene that requires metabolic activation by microsomal enzymes by supplier. Benzo(a)pyrene exhibited clear increase in the number of revertants when compared with the concurrent negative control which demonstrated the efficiency of S9 used in this study.
Positive controls exhibited a clear increase in the number of revertants when compared with the concurrent negative controls and were within the range of historical limits. This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.
Increase in revertants were not observed in tester strain TA 100 (Trial I and II) treated with 2-aminoanthracene in the absence of metabolic activation but clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Cytotoxicity Test
Cytotoxicity is characterised by inhibition of the background bacterial lawn and/or reduction in the number of revertant colonies. Complete inhibition of the background lawn with reduction in revertant colonies (83-85 % reduction) was observed at the tested concentration of 5000 μg/plate both in the absence and presence of the metabolic activation system while partial inhibition of the background lawn with reduction in revertant colonies ( 40-42 % reduction) was observed at the tested concentration of 2500 μg/plate both in the absence and presence of metabolic activation system. Normal growth was observed up to the dose level of 1250 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix).
Hence, 2500 μg/plate of PET A was selected as the highest concentration to be tested in the mutagenicity test in the absence and presence of metabolic activation system, for all the tester strains.

Mutagenicity Test
Trial I [In the Absence and Presence of 5% v/v S9 mix]
Partial inhibition of the bacterial background lawn was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. Reduction in the number of colonies was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system in strains TA1535 (53.61% and 51.38%), TA98 (50.02% and 59.56%), TAlO0 (40.25% and 46.90%) and in strain TA102 (15.90% and 17.53%) while reduction in the number of revertant colonies was not observed at the tested concentration of 2500 μg/plate only in the absence and presence of metabolic action system in the strain TA1537. Normal growth was observed up to the dose of 1250 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in the tester strains.
A positive increase in the number of revertant colonies was not observed in any of the tester strains at the tested concentration of 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate in the absence and presence (5% v/v S9 mix) of metabolic activation, when compared with the concurrent negative control.
Results revealed that there was no positive mutagenic effect in tester strains TA1537, TA1535, TA98, TA100 and TA102 at the tested concentration of78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate in the absence and presence (5% v/v S9 mix) of metabolic activation, when compared with the concurrent negative control. Statistical analysis did not reveal any significant effect.

Trial II [In the Absence and Presence of 10% v/v S9 mix]
Partial inhibition of the bacterial background lawn was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system (10% v/v S9 mix) in all the tester strains. Reduction in the number of colonies was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system in strains TA1535 (46.56% and 37.51%), TA98 (33.87% and 45.21%), TA100 (34.10% and 34.97%) and in strain TA102 (18.69% and 18.23%) while reduction in the number of revertant colonies was observed at the tested concentration of 2500 μg/plate only in the presence of the metabolic activation system in the strain TA1537 (49.95%). Normal growth was observed up to the dose of 1000 μg/plate both in the absence and presence of the metabolic activation system (10% v/v S9 mix) in the tester strains.
A positive increase in the number of revertant colonies was not observed in any of the tester strains at the tested concentration of 25.6, 64, 160, 400, 1000 and 2500 μg/plate in the absence and presence (10% v/v S9 mix) of metabolic activation, when compared with the concurrent negative control.
Results revealed that there was no positive mutagenic effect in tester strains TA1537, TA1535, TA98, TAl00 and TA102 both in the absence and presence (10% v/v S9 mix) of metabolic activation at any of the tested concentrations when compared with the concurrent negative control. Statistical analysis did not reveal any significant effect.

Any other information on results incl. tables

Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Trial I)

Concentration of PETA (µg/plate)

His+ Revertant Colonies/Plate (Absence of Metabolic Activation)

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

7.00

1.00

23.00

4.00

24.67

3.51

133.33

11.50

224.33

9.50

78.125

5.67

1.53

21.33

6.51

23.00

3.61

138.67

10.07

216.33

8.50

156.25

6.33

1.53

20.33

3.51

22.67

2.08

133.0

10.15

227.00

12.53

312.5

6.33

1.53

21.33

2.52

23.33

3.06

131.67

10.50

210.67

8.96

625

6.33

3.21

17.67

2.08

25.33

5.03

138.00

11.53

222.00

18.68

1250

6.67

1.53

20.67

2.08

27.33

4.04

137.33

6.03

217.00

10.54

2500

6.67

2.08

10.67

2.52

12.33

2.08

79.67

7.37

188.67

4.73

PC

329.00

28.93

375.33

133.55

552.33

93.30

885.00

163.94

1095.33

174.40

PC-2Aa

-

-

-

-

-

-

136.33

11.50

-

-

Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not applicable.

 

Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Presence of Metabolic Activation (Trial I)

Concentration of PETA (µg/plate)

His+ Revertant Colonies/Plate (Presence of Metabolic Activation [5% v/v S9 mix])

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

9.00

1.00

24.00

4.00

31.33

2.52

134.33

8.50

230.00

16.64

78.125

7.00

1.00

23.33

3.21

26.33

3.21

134.00

6.56

221.00

8.54

156.25

8.33

1.53

20.67

3.79

27.67

4.51

131.00

6.00

233.00

14.11

312.5

7.33

1.53

22.67

5.69

30.00

3.61

130.67

10.79

226.00

13.75

625

8.00

1.00

24.67

3.06

26.00

3.61

136.00

6.56

222.67

18.15

1250

7.67

1.53

22.00

4.00

26.67

3.21

129.00

9.85

230.33

19.76

2500

8.00

2.65

11.67

3.79

12.67

2.08

71.33

7.51

189.67

6.43

PC-2Aa

401.67

90.67

428.67

83.29

806.67

145.95

1291.00

346.22

1031.33

100.85

Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100).

 

Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Trial II)

Concentration of PETA (µg/plate)

His+ Revertant Colonies/Plate (Absence of Metabolic Activation)

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

6.33

1.15

19.33

2.08

20.67

1.53

131.00

7.81

224.67

12.50

25.6

6.33

3.06

20.67

3.06

20.00

2.00

135.00

7.55

227.33

7.23

64

5.67

0.58

18.00

1.00

21.67

2.52

136.00

4.00

224.33

13.87

160

6.00

3.46

17.33

0.58

23.00

2.65

135.33

2.89

223.00

6.00

400

3.33

0.58

20.00

1.00

22.67

2.52

131.67

10.60

225.33

6.51

1000

4.67

0.58

18.00

1.00

20.33

1.15

127.33

3.06

222.33

14.01

2500

5.33

2.08

10.33

2.52

13.67

1.53

86.33

8.74

182.67

9.29

PC

247.67

18.23

234.00

15.10

335.00

38.30

758.67

58.50

1204.67

131.50

PC-2Aa

-

-

-

-

-

-

134.00

6.56

-

-

Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not applicable.

 

Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Presence of Metabolic Activation (Trial II)

Concentration of PETA (µg/plate)

His+ Revertant Colonies/Plate (Presence of Metabolic Activation [10% v/v S9 mix])

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

9.33

2.08

21.33

1.53

24.33

2.08

135.33

10.21

230.33

14.64

25.6

6.67

2.08

21.67

4.04

23.00

4.58

134.00

10.15

230.33

10.07

64

7.00

2.00

21.33

1.53

24.67

1.53

133.00

9.00

222.33

13.05

160

7.33

0.58

20.33

2.08

24.33

3.06

137.00

7.55

233.33

13.05

400

7.33

2.31

18.00

1.00

22.00

1.73

134.33

6.66

233.67

8.50

1000

5.67

0.58

19.33

1.53

22.00

2.65

134.00

6.56

228.67

12.50

2500

4.67

2.08

13.33

4.16

13.33

1.53

88.00

10.54

188.33

11.02

PC-2Aa

271.33

25.79

290.00

23.00

435.00

48.28

954.33

97.29

1456.33

107.75

Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100).

 

Individual Plate Count (Trial I)

Absence of Metabolic Activation

Concentration of PETA (µg/plate)

Number of Revertant Colonies

TA1537

TA1535

TA98

TA100

TA102

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

NC (DMSO)

8

6

7

23

27

19

21

28

25

145

122

133

234

215

224

78.125

4

6

7

28

21

15

26

19

24

148

128

140

208

225

216

156.25

5

8

6

20

17

24

22

25

21

122

142

135

239

228

214

312.5

8

5

6

19

21

24

20

24

26

121

142

132

206

221

205

625

10

4

5

16

20

17

20

30

26

139

149

126

225

239

202

1250

8

7

5

23

19

20

28

31

23

138

131

143

228

207

216

2500

6

9

5

13

8

11

13

14

10

74

77

88

194

187

185

PC

341

296

350

340

523

263

445

614

598

735

860

1060

946

1053

1287

PC-2Aa

-

-

-

-

-

-

-

-

-

136

125

148

-

-

-

 

Presence of Metabolic Activation (5% v/v S9 mix)

Concentration of PETA (µg/plate)

Number of Revertant Colonies

TA1537

TA1535

TA98

TA100

TA102

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

NC (DMSO)

10

8

9

28

24

20

29

31

34

126

143

134

223

249

218

78.125

7

8

6

22

27

21

24

30

25

133

128

141

222

212

229

156.25

7

10

8

18

25

19

23

32

28

137

131

125

235

218

246

312.5

9

7

6

29

18

21

27

34

29

143

123

126

229

238

211

625

8

9

7

24

22

28

23

25

30

135

143

130

242

206

220

1250

6

9

8

18

22

26

28

29

23

121

126

140

248

209

234

2500

7

11

6

9

16

10

12

15

11

64

71

79

197

185

187

PC-2Aa

318

389

498

370

392

524

783

963

674

1684

1031

1158

926

1041

1127

Key: R = Replicate, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100), - = Not applicable.

 

Individual Plate Count (Trial II)

Absence of Metabolic Activation

Concentration of PETA (µg/plate)

Number of Revertant Colonies

TA1537

TA1535

TA98

TA100

TA102

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

NC (DMSO)

7

5

7

20

21

17

22

19

21

135

136

122

237

225

212

25.6

9

7

3

24

20

18

22

18

20

143

134

128

219

232

231

64

6

5

6

19

18

17

22

19

24

132

136

140

209

228

236

160

4

4

10

17

18

17

20

25

24

137

132

137

229

217

223

400

3

4

3

19

20

21

23

20

25

130

122

143

225

219

232

1000

4

5

5

18

19

17

19

21

21

128

124

130

211

238

218

2500

7

3

6

10

8

13

14

12

15

79

84

96

180

175

193

PC

251

264

228

218

236

248

302

326

377

700

759

817

1205

1073

1336

PC-2Aa

-

-

-

-

-

-

-

-

-

133

141

128

-

-

-

 

Presence of Metabolic Activation (10% v/v S9 mix)

Concentration of PETA (µg/plate)

Number of Revertant Colonies

TA1537

TA1535

TA98

TA100

TA102

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

NC (DMSO)

10

11

7

21

23

20

25

22

26

131

128

147

246

217

228

25.6

5

9

6

26

21

18

28

22

19

125

145

132

221

241

229

64

5

7

9

23

21

20

23

25

26

124

133

142

222

211

234

160

7

8

7

18

21

22

25

21

27

144

138

129

221

232

247

400

6

6

10

19

18

17

23

20

23

136

127

140

242

234

225

1000

5

6

6

18

19

21

23

24

19

133

128

141

229

216

241

2500

7

4

3

18

10

12

13

12

15

98

89

77

189

177

199

PC-2Aa

250

264

300

264

313

290

486

390

429

963

853

1047

1471

1342

1556

Key: R = Replicate, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100), - = Not applicable.

 

Results of Cytotoxicity Test (TA100) in Absence and presence of Metabolic Activation

Concentration of PETA (µg/plate)

In the Absence of Metabolic Activation

In the Presence (5% v/v S9 mix) if Metabolic Activation

R1

R2

R3

R1

R2

R3

NC (DMSO)

NI (137)

NI (122)

NI (140)

NI (130)

NI (123)

NI (148)

9.77

NI (127)

NI (136)

NI (139)

NI (127)

NI (143)

NI (133)

19.53

NI (142)

NI (135)

NI (123)

NI (141)

NI (125)

NI (136)

39.06

NI (133)

NI (123)

NI (141)

NI (139)

NI (129)

NI (144)

78.13

NI (129)

NI (138)

NI (137)

NI (143)

NI (134)

NI (123)

156.25

NI (144)

NI (133)

NI (128)

NI (140)

NI (127)

NI (130)

312.5

NI (139)

NI (143)

NI 9125)

NI (137)

NI (128)

NI (144)

625

NI (137)

NI (125)

NI (141)

NI (133)

NI (141)

NI (127)

1250

NI (129)

NI (139)

NI (137)

NI (129)

NI (134)

NI (141)

2500

PT (71)

PI (79)

PI (83)

PI (74)

PI (85)

PI (80)

5000

I (17)

I (21)

I (23)

I (19)

I (27)

I (22)

Key: DMSO = Dimethyl sulfoxide, I = Inhibition, NC = Negative control, NI = No inhibition, PI = Partial inhibition, R = Replicate

Note: Number of revertant colonies observed/plate are provided in parentheses

 

AMES TEST – HISTORICAL CONTROL DATA (November 2012 to February 2014)

Negative Control (Distilled Water) without S9 Mix

Strain

TA1537

TA1535

TA98

TA100

TA102

Mean Revertants per Plate

7.62

18.07

24.57

143.71

236.28

Standard Deviation

2.34

3.37

4.22

10.98

15.72

Maximum

14

30

34

175

298

Minimum

2

9

14

107

201

Negative Control (Distilled Water) with S9 Mix

Mean Revertants per Plate

8.35

20.22

26.14

147.00

241.53

Standard Deviation

2.49

3.70

4.57

11.72

13.95

Maximum

15

31

36

207

297

Minimum

3

9

13

104

202

Negative Control (Dimethyl Sulfoxide) without S9 Mix

Mean Revertants per Plate

7.39

18.44

24.65

142.26

233.99

Standard Deviation

2.30

3.43

4.10

10.15

13.35

Maximum

13

37

36

168

268

Minimum

3

9

12

115

184

Negative Control (Dimethyl Sulfoxide) with S9 Mix

Mean Revertants per Plate

8.34

20.33

26.21

146.25

241.84

Standard Deviation

2.44

3.41

4.12

12.11

14.57

Maximum

14

35

35

178

296

Minimum

2

8

16

109

201

Positive Control without S9 Mix

Mean Revertants per Plate

324.78

426.99

567.00

939.15

1139.80

Standard Deviation

172.50

160.32

179.90

164.06

193.54

Maximum

1587

1151

1487

1782

2116

Minimum

86

185

188

491

677

Positive Control with S9 Mix

Mean Revertants per Plate

342.77

469.93

684.25

1035.23

1253.88

Standard Deviation

153.84

163.98

283.92

182.42

250.30

Maximum

1355

1181

1999

1893

2520

Minimum

138

161

224

420

670

Negative control

Dimethyl sulfoxide

Positive controls in the absence of metabolic activation

TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)

Positive control in the presence of metabolic activation

2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)

Applicant's summary and conclusion

Conclusions:
From the results of the study, it is concluded that PETA is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified conditions.
Executive summary:

This study was performed to evaluate the mutagenic activity of PETA (Supplied by Nexam Chemical AB, Sweden) by the bacterial reverse mutation test, using five histidine deficient (his-) mutant tester strains of Salmonella typhimurium, TA1537, TA1535, TA98, TA100 and TA102. The method followed was as per the guidelines of the OECD No. 471 (July 1997).

 

The treatments were performed by the plate incorporation technique both in the absence and presence of metabolic activation (S9 mix). The S9 mix of 5% and 10% v/v consisted of an S9 fraction (Aroclor 1254 induced rat liver homogenate) supplemented with cofactors.

 

Before conducting the mutagenicity test, PETA was evaluated for cytotoxicity in tester strain TA100, both in the absence and presence of S9 mix (5% v/v S9 mix). Complete inhibition of the background lawn with reduction in revertant colonies (83-85% reduction) was observed at the tested concentration of 5000 μg/plate both in the absence and presence of the metabolic activation system while partial inhibition of the background lawn with reduction in revertant colonies (40-42% reduction) was observed at the tested concentration of 2500 μg/plate both in the absence and presence of metabolic activation system. Normal growth was observed up to the dose level of 1250 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix).

 

Hence, 2500 μg/plate of PETA was selected as the highest concentration to be tested in the mutagenicity test both in the absence and presence of a metabolic activation system, for all the tester strains.

 

Based on the results of the cytotoxicity test, the test concentrations of 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate of PETA both in the absence and presence (5% v/v S9 mix) of metabolic activation were selected for Trial I. Trial I did not show any positive mutagenic response when compared to the negative control at any of the tested concentrations. Trial II was conducted to confirm the negative results of Trial I with modified concentrations i.e., 25.6, 64, 160,400, 1000 and 2500 μg/plate in the absence and presence of metabolic activation (S9 concentration was increased to 10% v/v). A positive mutagenic response was not observed in Trial II confirming the results of Trial I. The efficiency of the test system was demonstrated by a clear increase in revertant colonies observed with the positive controls both in the absence and presence of metabolic activation.

 

From the results of this study, under the specified experimental conditions, PETA is concluded to be non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium.