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EC number: 205-647-4 | CAS number: 145-13-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 017
- 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
- 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
Constituent 1
- 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.
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