Cholest-5-en-3-β-yl acetate

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:

19 February 2018 - 22 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

31 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 (Into Operation June 2012)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cholest-5-en-3-β-yl acetate

EC Number:

210-066-4

EC Name:

Cholest-5-en-3-β-yl acetate

Cas Number:

604-35-3

Molecular formula:

C29H48O2

IUPAC Name:

(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylheptan-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-yl acetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White crystalline powder
- Storage condition: At room temperature

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 5.4, 17, 52, 164 and 512 µg/plate
Experiment 2:
Without and with S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 µg/plate
Experiment 3:
Without and with S9-mix: 512 and 1600 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hexane
- Justification for choice of solvent/vehicle:
A solubility test was performed based on visual assessment. The test item was observed to be insoluble in Milli-Q water, DMSO, ethanol and acetone but formed a clear colorless solution in hexane at a concentration of 100 mg/mL. Test item concentrations were used within 2 hours after preparation.

Controls

Controls 1

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, 2.5 µg in DMSO for TA1537 (Direct plate assay)

Remarks:

Without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA) in DMSO, 1 µg for TA98 and TA100 (Direct plate assay), 2.5 µg for TA1535 and TA1537, 5 µg for TA100 (Pre-incubation assay), 15 µg for WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), preincubation

DURATION
- Preincubation: 30 minutes
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

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

Positive controls validity:

valid

Test results 4

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

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: Precipitation of Cholesteryl acetate on the plates was observed at the start of the incubation period at concentrations of 1600 µg/plate and upwards and at 164 µg/plate and above at the end of the incubation period, except in tester strains TA98 (absence and presence of S9-mix) and TA1535 (presence of S9-mix) where precipitate at the end of the incubation period was observed at concentrations of 52 µg/plate and upwards.
Experiment 2: Precipitation of Cholesteryl acetate on the plates was observed at the start of the incubation period at the concentration of 164 µg/plate and no precipitate was observed at the end of the incubation period.
Experiment 3: Precipitate was observed in all tester strains at concentrations 512 and upwards and 1600 µg/plate in the absence and presence of S9-mix, respectively.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 125 – 1248 73 – 1206 55 – 1353 54 – 1051 365 – 1995 250 – 1977
Mean 846 219 787 353 1406 887
SD 146 119 345 162 258 349
n 2348 2229 2003 2234 2200 2276

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1848 408 - 2651 93 – 1951 111 - 1359
Mean 901 1232 1094 437
SD 168 343 477 149
n 2335 2327 2021 2085

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Oct 2015 and Oct 2017.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 - 27 3 – 20 3 – 23 8 - 41 8 - 55 63 – 176 54 - 160 10 – 59 9 - 69
Mean 10 11 6 7 16 23 108 107 25 32
SD 3 4 2 3 5 7 19 20 7 8
n 2356 2336 2264 2235 2319 2360 2341 2336 2075 2078

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Oct 2015 and Oct 2017.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 1600 µg/plate.

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD 471 guideline and GLP principles, Cholesteryl acetate was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD 471 guideline and GLP principles.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay. In the first mutation experiment, the test item was tested up to concentrations of 512 µg/plate in the strains TA1535, TA1537 and TA98. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 164 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at any of the dose levels tested. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Since in the second mutation assay no toxicity and no precipitate on the plates was observed up to the top dose of 164 µg/plate in all tester strains in the absence and presence of S9-mix, an additional experiment was performed. 

In this third mutation experiment, the test item was tested at a concentration range of 512 to 1600 µg/plate in the absence and presence of S9-mix. Precipitate was observed in all tester strains at concentrations 512 µg/plate and upwards and 1600 µg/plate in the absence and presence of S9-mix, respectively. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Cholesteryl acetate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.