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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted by the Council on July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(N-ethylanilino)ethanol
EC Number:
202-160-9
EC Name:
2-(N-ethylanilino)ethanol
Cas Number:
92-50-2
Molecular formula:
C10H15NO
IUPAC Name:
2-(N-Ethylanilino)ethanol
Test material form:
liquid - solid: mixture of
Details on test material:
At room temperature, the substance can be either a primrose yellow solid or a liquid.
Specific details on test material used for the study:
Batch/Lot Number 18052904
Analysed Concentration 99.47 %
(Refer Certificate of Analysis in APPENDIX 9)
Manufactured by Binhai Henglian Chemicals Co., Ltd.
Supplied to JRF by Aceto France SAS
Date of Manufacture May 29, 2018
Date of Expiry May 28, 2019
Other characteristics Water soluble in: 2000 mg/L
Appearance Primrose yellow solid/yellowish liquid
*At room temperature, the phase of the substance can be either solid or liquid
Storage Condition (at JRF):
- Storage Temperature : Kept at 0 – 30 0C, Away from light and humidity
- Storage Container :In original container as supplied by the Sponsor
- Storage Location : Test Item Control Office, JRF

Method

Target gene:
This test evaluates the test item for its ability to induce reverse mutations in the tester strains and restore the functional capability of the bacteria to synthesize an essential amino acid. In this case, the revertant bacteria are detected by their ability to grow in the absence of histidine, which is required by the Salmonella typhimurium tester strains.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
Initial Toxicity-Mutation Test: 5, 1.5, 0.5, 0.15, 0.05, 0.015, 0.005, 0.0015 µL/plate
Confirmatory Mutation Test: 0.16, 0.31, 0.63, 1.25, 2.5 and 5 µL/plate
Vehicle / solvent:
dimethyl sulfoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other:
Details on test system and experimental conditions:
Source: 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.

S9 fraction: buffered and supplemented with the essential co-factors β-NADP and Glucose-6-phosphate to form the “S9 mix”. This mix is added to the top agar in this activated assay. The S9 fractions procured from Dr. G.P. Meshram, Nagpur, and (Lot N° MWR/ARI/S9F/01/17 Sept 2017) was used in the study.

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). The flasks were then incubated at 37 ± 1 °C in an orbital shaking incubator (120 rpm) for 15 h up to the early stationary or late exponential growth phase. After the incubation period, the culture flasks were removed from the orbital shaking incubator. The cultures were aseptically diluted with oxoid nutrient broth (ONB) and the optical density was measured at 660 nm using a spectrophotometer (Model 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 this study

Genotype Confirmation Test:
The genotype of the tester strain was confirmed for all the strains regularly (once in a month). The tester strains of Salmonella typhimurium were tested for histidine dependence, biotin dependence, histidine and biotin dependence, rfa mutation, uvrB mutation through sensitivity to ultraviolet light and the R-factor resistance for ampicillin and tetracycline.

Initial Toxicity-Mutation Test:
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 0.2 M phosphate buffer was added in the absence of the metabolic activation system and 500 µL of 5% v/v S9 mix was added in the presence of the metabolic activation system. Volume of 100 µL of the relevant stock solution of test item, DMSO and relevant positive control were used for treatment, as a negative control and as a positive 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. Duplicate sets of plates were maintained for each concentration of test item, positive control and negative control. The petri plates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the state of background bacterial lawn inhibition and reduction in the number of colonies.

Confirmatory Mutation Test:
Plates were maintained in triplicate for each test item concentration and for the positive control and negative control during both assays. The number of revertant colonies was recorded after the 48 h incubation period. Treatment with 2-aminoanthracene in the absence of metabolic activation was performed for tester strain TA100 in both the initial toxicity-mutation assay and confirmatory mutation assay to verify the efficiency of the S9 fraction used.
Rationale for test conditions:
See details on test system and conditions.
Evaluation criteria:
A result is considered positive if a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Strains TA1535, 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 TA98, 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 was used as an aid in the evaluation of the dose response.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not judged as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.
Statistics:
Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Initial Toxicity-Mutation Test

Normal growth, in the background lawn, was observed, up to tested concentration of 5µL/plate in all tester strains, both in the absence and presence(5% v/v S9 mix)of the metabolic activation. Results revealed that there was no positive mutagenic effect in tester strains, up to the tested concentration of 5 µL/plate of2-(N-ethylanilino)ethanolin the absence and presence of the metabolic activation(5% v/v S9 mix), when compared with that of the negative control.Statistical analysis did not reveal any significant effect. Hence,0.16, 0.31, 0.63, 1.25, 2.5, and 5 µL/plate concentration levels of2-(N- ethylanilino)ethanolwere selected for the confirmatory mutation test in the absence and presence of the metabolic activation system(10% v/v s9 mix)for tester strains.

Confirmatory Mutation Test

Normal growth, in the background lawn, was observed, up to tested concentration of 5 µL/plate both in the absence and presence of the metabolic activation system (10% v/v S9 mix) in tester strains.Results revealed that there was no positive mutagenic effect in tester strains TA1537, TA1535, TA98, TA100, and TA102, at the tested concentration levels0.16, 0.31, 0.63, 1.25, 2.5 and 5 µL/plateof2-(N-ethylanilino)ethanol,intheabsence and presence of metabolic activation system (10% v/v S9 mix), when compared with the concurrent negative control. Statistical analysis did not reveal any significant effect.

A. Mean  Count of His+Revertant Colonies in Negative Control, Positive Controls Treatment Plates in the Absence ofMetabolic Activation(Initial Toxicity-Mutation Test)

Concentration of

2-(N- Ethylanilino)Ethanol (µL/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)

8.50

0.71

17.00

1.41

24.00

5.66

133.50

6.36

226.00

5.66

0.0015

7.00

0.00

18.50

0.71

24.00

7.07

140.00

7.07

228.00

21.21

0.005

8.00

2.83

17.50

0.71

25.50

7.78

140.00

15.56

228.50

16.26

0.015

7.50

0.71

16.00

4.24

24.00

9.90

140.50

3.54

228.00

1.41

0.05

11.00

1.41

17.50

3.54

22.00

5.66

129.00

2.83

237.00

12.73

0.15

9.50

0.71

14.50

0.71

28.00

5.66

140.50

3.54

233.00

24.04

0.5

10.50

0.71

19.00

2.83

25.00

2.83

127.00

5.66

236.50

9.19

1.5

7.50

0.71

19.50

2.12

23.50

7.78

141.50

4.95

225.50

4.95

5

8.50

2.12

18.00

0.00

27.50

6.36

129.00

8.49

230.50

19.09

PC

399.50

2.12

468.00

39.60

641.50

50.20

861.00

39.60

1062.00

14.14

2Aa

-

-

-

-

-

-

143.50

7.78

-

-

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (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 (0.5µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),- = Not Applicable.

B. Mean Count of His+Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence ofMetabolic Activation(Initial Toxicity-Mutation Test)

 

Concentration of

2-(N- Ethylanilino)Ethanol (µL/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)

7.50

0.71

21.50

4.95

26.50

7.78

140.50

4.95

248.00

11.31

0.0015

6.50

2.12

23.00

1.41

29.00

8.49

131.50

6.36

240.00

9.90

0.005

6.50

0.71

19.50

2.12

26.50

6.36

129.50

9.19

256.00

2.83

0.015

7.50

2.12

20.00

1.41

28.00

5.66

129.50

6.36

237.00

5.66

0.05

7.00

1.41

20.50

6.36

27.00

5.66

129.00

11.31

250.50

4.95

0.15

6.50

0.71

20.00

4.24

27.00

7.07

130.00

8.49

231.50

4.95

0.5

9.00

1.41

22.50

7.78

25.00

9.90

132.50

21.92

247.00

15.56

1.5

10.00

0.00

17.50

2.12

27.00

5.66

131.50

10.61

248.50

10.61

5

7.50

0.71

20.00

1.41

28.50

9.19

135.00

18.38

242.00

25.46

PC-2Aa

264.00

0.00

552.00

46.67

755.00

25.46

945.00

24.04

1160.50

33.23

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

C. Mean Count of His+Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Confirmatory Mutation Test)

Concentration of

2-(N- Ethylanilino)Ethanol (µL/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)

10.33

3.21

19.00

3.61

27.33

2.31

135.33

19.09

226.67

23.71

0.16

9.67

1.53

18.33

2.08

26.00

2.00

143.67

10.21

233.67

34.20

0.31

9.33

3.06

18.67

2.08

27.33

4.73

116.67

15.31

234.67

12.42

0.63

10.67

1.53

20.00

4.00

29.33

2.08

135.00

14.93

224.00

15.10

1.25

9.00

1.00

20.33

2.52

28.33

1.53

129.67

23.01

229.00

7.94

2.5

12.00

2.65

20.33

1.15

26.00

3.00

120.00

12.12

229.67

11.59

5

8.67

0.58

19.33

3.79

29.33

2.31

125.33

7.02

226.67

17.39

PC

296.33

39.68

419.00

65.37

561.67

54.85

821.33

70.27

1054.33

28.54

2Aa

-

-

-

-

-

-

122.67

5.51

-

-

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide,PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (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

D. Mean Count of His+Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence ofMetabolic Activation(Confirmatory Mutation Test)

Concentration of

2-(N- Ethylanilino)Ethanol (µL/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)

11.67

2.52

18.67

2.52

27.00

1.00

135.33

0.58

227.67

13.65

0.16

10.00

2.65

18.67

2.89

27.67

1.53

128.00

23.52

217.33

14.01

0.31

10.67

3.79

19.00

3.00

27.67

1.53

132.33

14.57

236.67

11.02

0.63

11.33

2.08

16.00

2.00

25.67

2.52

129.67

4.62

227.67

17.95

1.25

11.33

1.53

21.33

2.08

29.00

5.57

130.33

11.59

227.00

3.61

2.5

10.33

2.08

20.33

5.51

28.67

3.21

130.67

12.42

225.33

14.01

5

9.00

1.00

17.67

1.53

29.00

2.65

131.33

11.68

220.00

16.09

PC-2Aa

329.33

48.95

506.67

70.73

665.67

40.70

898.67

57.49

1053.00

99.98

Key:  SD = Standard Deviation, NC = Negative Control,DMSO = Dimethyl sulfoxide,PC = Positive Control, 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 results of this study, it is concluded that 2-(N-ethylanilino)ethanol is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100, and TA102 when tested under the specified experimental conditions.