Pregnenolone

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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 results

Test results 1

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

Test results 2

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.