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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:
18 August 2017 - 06 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2(R1). Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human use. CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pregnenolone
EC Number:
205-647-4
EC Name:
Pregnenolone
Cas Number:
145-13-1
Molecular formula:
C21H32O2
IUPAC Name:
3-hydroxypregn-5-en-20-one
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white crystalline powder
- Storage conditions: at room temperature
Specific details on test material used for the study:
- Stable at higher temperatures

Method

Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose-range finding test, reported as part of the first experiment (TA100 and WP2uvrA):
Without and with S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
The top dose was selected based on the results of a solubility test (suspension at 16 mg/mL and higher; dilution at 5.12 mg/mL and lower).

First experiment (TA1535, TA1537 and TA98):
Without and with S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
The top dose was selected based on the results of the dose-range finding study.

Second experiment (all tester strains):
Without and with S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
The top dose was selected based on the results of the first experiment.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: according to guidelines.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Remarks:
For details on the positive controls, see table 1 in 'Additional information on materials and methods'
Details on test system and experimental conditions:
The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3 (in both independent experiments)

EXPERIMENTAL DESIGN: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 μg/plate in the first experiment only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
other: TA1535, TA1537, TA100 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Cytotoxicity: in the first experiment, a decreased number of revertants was observed in tester strain TA98 with and without S9 at a test concentration of 5000 μg/plate. No cytotoxicity was observed in the other tester strains at any concentration with and without S9.

In tester strain TA1537 in the absence of S9-mix, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

In the second experiment, no cytotoxicity was observed in any of the tester straind at any of the concentrations tested with and without S9.

- Mutagenicity: in both experiments, no mutagenicity was observed in any of the tester strains at any of the concentrations tested with and without S9.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in both experiments at 512 μg/plate and above (start of the incubation) and at 1600 μg/plate and above (end of the incubation).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: reported as the first experiment

HISTORICAL CONTROL DATA: see table 2 and 3 in 'Any other information on results'
- Results of the solvent control and of the positive control were within the historical data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Any other information on results incl. tables

Table 2 Historical data for the solvent control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 - 36

3 - 32

3 – 20

3 – 23

8 - 41

9 - 55

66 - 161

63 - 160

10 – 59

9 - 69

Mean

11

11

6

7

16

23

105

105

25

31

SD

4

4

3

3

5

7

19

20

7

8

n

2057

2039

1950

1931

2023

2083

2027

2033

1739

1745

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Table 3 Historical data for the positive control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

-

+

-

+

+

-

+

Range

125 - 1381

78 - 1058

55 – 1324

537 – 1848

408 - 2651

93 – 1951

93 - 1359

55 – 1051

410 – 1995

250 - 1977

Mean

839

220

736

908

1330

1128

422

382

1369

929

SD

153

112

331

178

324

484

151

150

310

345

n

2065

1967

1740

2007

2020

1679

1728

1933

1920

2014

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Table 4 Mean number of revertant colonies/3 replicate plates (± S.D.) with oneSalmonella typhimuriumand one Escherichia coli strain in the range-finding test

Without S9-mix

With S9-mix

Dose (µg/plate)  

TA100

WP2uvrA

TA100

WP2uvrA

Positive control

957

±

41

 

1216

±

93

 

612 ±

67

 

522 ±

31

 

Solvent control

107

±

2

 

24

±

7

 

95 ±

10

 

36 ±

10

 

1.7

108

±

18

 

33

±

14

 

95 ±

14

 

37 ±

9

 

5.4

96

±

2

 

24

±

1

 

107 ±

8

 

27 ±

7

 

17

112

±

14

 

29

±

8

 

102 ±

3

 

40 ±

2

 

52

103

±

21

 

26

±

4

 

89 ±

4

 

36 ±

8

 

164

104

±

12

 

28

±

5

 

104 ±

14

 

33 ±

8

 

512

109

±

9

NP

21

±

6

NP

106 ±

7

NP

33 ±

7

NP

1600

92

±

7

SP

18

±

7

SP

84 ±

11

SP

21 ±

1

SP

5000

89

±

2

n MP

23

±

4

n MP

n.a.

 

 

 

 

 

Table 5 Mutagenic response of PRECH-PURCH in the Salmonella typhimurium Reverse Mutation Assay (Experiment 1)

Without S9-mix

 

TA1535

TA1537

TA98

Positive control

827

±

58

 

1353

±

120

 

1105

±

63

 

Solvent control

5

±

2

 

3

±

2

 

14

±

6

 

17

9

±

6

 

7

±

2

 

9

±

4

 

52

8

±

4

 

3

±

1

 

9

±

1

 

164

12

±

4

 

1

±

2

 

12

±

2

 

512

10

±

1

NP

4

±

1

NP

12

±

4

NP

1600

6

±

2

SP

5

±

2

SP

11

±

1

SP

5000

6

±

1

n MP

3

±

1

n MP

6

±

3

n MP

With S9-mix

 

TA1535

TA1537

TA98

Positive control

188

±

7

 

91

±

42

 

904

±

303

 

Solvent control

6

±

2

 

3

±

1

 

26

±

6

 

17

9

±

3

 

6

±

2

 

22

±

4

 

52

9

±

3

 

3

±

2

 

14

±

4

 

164

13

±

4

 

5

±

3

 

15

±

5

 

512

12

±

3

NP

4

±

1

NP

14

±

6

NP

1600

5

±

3

SP

3

±

1

SP

11

±

3

SP

5000

5

±

2

n MP

3

±

1

n MP

8

±

1

n MP

Table 6 Mutagenic response of PRECH-PURCH in the Reverse Mutation Assay (Experiment 2)

Without S9-mix

 

TA1535

TA1537

TA98

TA100

WP2uvrA

Positive control

733

±

57

 

944

±

240

 

1257

±

168

 

689

±

107

 

140

±

33

 

Solvent control

6

±

1

 

8

±

7

 

9

±

2

 

91

±

14

 

17

±

6

 

17

6

±

1

 

7

±

2

 

9

±

2

 

96

±

7

 

19

±

10

 

52

13

±

5

 

6

±

8

 

8

±

3

 

101

±

12

 

18

±

5

 

164

8

±

2

 

10

±

6

 

7

±

3

 

93

±

8

 

14

±

2

 

512

11

±

4

NP

8

±

3

NP

9

±

5

NP

91

±

6

NP

14

±

8

NP

1600

5

±

1

SP

15

±

7

SP

10

±

6

SP

92

±

8

SP

20

±

2

SP

5000

6

±

3

n MP

8

±

2

n MP

10

±

3

n MP

79

±

10

n MP

14

±

5

n MP

With S9-mix

 

TA1535

TA1537

TA98

TA100

WP2uvrA

Positive control

140

±

14

 

135

±

30

 

533

±

33

 

1573

±

37

 

483

±

22

 

Solvent control

10

±

2

 

6

±

2

 

18

±

5

 

94

±

7

 

19

±

4

 

17

9

±

3

 

7

±

4

 

21

±

2

 

90

±

16

 

25

±

5

 

52

9

±

2

 

6

±

2

 

13

±

2

 

93

±

18

 

28

±

2

 

164

27

±

31

 

6

±

2

 

14

±

6

 

89

±

9

 

31

±

4

 

512

12

±

4

NP

9

±

2

NP

18

±

1

NP

94

±

8

NP

22

±

4

NP

1600

11

±

5

SP

13

±

8

SP

20

±

3

SP

91

±

4

SP

27

±

2

SP

5000

9

±

3

n MP

9

±

2

n MP

13

±

6

n MP

94

±

3

n MP

24

±

5

n MP

Definitions:

MP

Moderate Precipitate

NP

No precipitate

SP

Slight Precipitate

n

Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:
The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that PRECH-PURCH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

In an Ames test, performed according to OECD guideline 471 and GLP principles, PRECH-PURCH was assessed for its potential to induce reverse mutations in the histidine locus of Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and at the tryptophan locus of one Escherichia coli strain (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. In both experiments, the five strains were tested up to and including a dose of 5000 μg/plate in the absence and in the presence of a metabolic activation system (S9). At the end of both experiments, the test item precipitated at dose levels of 1600 and 5000 μg/plate. In the first experiment, cytotoxicity was observed in tester strain TA98 with and without S9 at a dose level of 5000 μg/plate. In the other strains, no cytotoxicity was observed at any of the dose levels with and without S9. In the second experiment, no cytotoxicity was observed in any of the strains at any of the dose levels tested, with and without S9. No mutagenicity was observed in both experiments.

Since the results of the solvent and positive controls were within the historical data range, the study was determined to be valid.

Based on the results of this study, it is concluded that PRECH-PURCH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.