Prasterone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-Across to structural analogue & precursor Prasterone enantate:

Ames test (OECD TG 471): negative

TK Assay (OECD TG 490/ 476): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Feb - May 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Prasterone enantate (Androst-5-en-17-one, 3-hydroxy-, (3.beta.)) is the precursor and structural analogue of Prasterone (CAS 53-43-0). In vivo, it is releasing Prasterone into the body.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Method originally contained in TG 476, separated in TG 490 in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver homogenate, derived from male Sprague-Dawley rats pre-treated with
Aroclor 1254, was obtained from MP Biomedicals GmbH, Eschwege, Germany (S9 batch no. 4949H; protein content 38.1 mg/mL; EROD activity: 5256.63 pmoles 7-hydroxyresorufin formed per minute per mg protein). The S9 mix was prepared with 200 mmol/Lglucose-6-phosphate, 32 mmol/L NADP and S9 at a ratio of 1: 1 :2.
Immediately before treatment an appropriate quantity of S9 was thawed and mixed with the cofactors and added to the test cultures at a ratio of 1 :20 representing final concentrations of 2.5 mmol/L glucose-6-phosphate, 0.4 mmol/L NADP and 2.5% S9.

Test concentrations with justification for top dose:

20, 40, 60, 80, 100 µg/ ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

4-nitroquinoline-N-oxide

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 60 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Evaluation of the data does not indicate that prasterone enantate is a mutagen in the mouse lymphoma assay, when tested up to dear cytotoxic and precipitating dose levels in the absence and presence of metabolic activation.

Executive summary:

In the reported study prasterone enantate did not lead to an increase in the number of mutant colonies that exceeded the threshold of twice the mutant colony counts of the corresponding solvent controls, when tested up to the recommended dose levels in the absence or presence of metabolic activation. No relevant shift in the ratio of small and large mutant colonies was determined at any test concentration.
Cytotoxic effects of prasterone enantate could be observed in the nonactivated treatments starting at test concentrations of 60 µg/mL (pulse treatment) and 25 µg/mL (continuous treatment). In the activated treatments only weak cytotoxic effects were induced at the highest test concentration of 120 µg/mL.
Visible precipitates of the test substance in the cell culture medium occurred starting at 80 µg/mL.
The negative and solvent controls (medium and 1 % DMSO) showed spontaneous mutant frequencies in the expected range. The positive controls NQO (0.025 and 0.1 µg/mL) and 3-MC (2.0 µg/mL) induced significant increases in the mutant frequency indicating the sensitivity of the test system.

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Study period:

Feb to June 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Prasterone enantate (Androst-5-en-17-one, 3-hydroxy-, (3.beta.)) is the precursor and structural analogue of Prasterone (CAS 53-43-0). In vivo, it is releasing Prasterone into the body.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98, E.coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

liver S9-mix from Aroclor 1254 -treated rats

Test concentrations with justification for top dose:

Endiol: six concentrations from 0.1 to 5.0 mg/plate
Sodium azide: 5 µg/plate (TA 1535 and TA 100 without S9)
2-Nitrofluorene: 10 µg/plate (TA 1538 and TA 98 without S9)
4-Nitro-o-phenylenediamine: 10 µg/plate (TA 1537 without S9)
Benzo[a]pyrene: 2.5 µg/plate (TA 100 and TA 98 with S9)
Cyclophosphamide: 400 µg/plate (TA 1535 with S9)
2-Aminoanthracene: 5 µg/plate (with S9), 5 µl Ethylmethansulfaonate & 5 µg N-Methyl-N-nitro-N-nitrosoguanidine (E.coli WP2 without S9)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Sodium azide (TA 1535 and TA 100, -S9), 2-Nitrofluorene (TA 1538 and TA 98, -S9), 4-Nitro-o-phenylenediamine (TA 1537, -S9), Benzo[a]pyrene (TA 100 and TA 98, +S9), Cyclophosphamide (TA 1535, +S9), 2-Aminoanthracene (+S9)

Remarks:

5 µl Ethylmethansulfaonate & 5 µg N-Methyl-N-nitro-N-nitrosoguanidine (E.coli WP2 without S9)

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 1538, TA 98, E.coli WP2uvrA

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

Precipitates in the agar were found starting at 2.5 mg/plate onwards.

Conclusions:

Evaluation of the data does not indicate that prasterone enantate possesses a mutagenic potential in the bacterial reverse mutation test (plate incorporation procedure and preincubation modification), including Salmonella typhimurium and Escherichia coli as test organisms, when tested up to a maximum recommended dose level of 5.0 mg/ plate in the absence and presence of a metabolic activation system (S9 mix).

Executive summary:

Prasterone enantate was tested in the Salmonella/microsome test for point-mutagenic effects in doses up to 5000 µg/plate on the five histidine-auxotrophic Salmonella typhimurium LT2 strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 and the E.coli strain WP2uvrA.

No cytotoxic effect was seen. Precipitates in the agar were found starting at 2.5 mg/plate onwards.

There was no evidence for a mutagenic activity of the test item, when tested up to the highest recommended dose level of 5.0 mg/plate in the absence and presence of S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read-across to structural analogue & precursor Prasterone enantate:

MNT in vivo (OECD TG 474): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Study period:

Jan to Mar 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Prasterone enantate (Androst-5-en-17-one, 3-hydroxy-, (3.beta.)) is the precursor and structural analogue of Prasterone (CAS 53-43-0). In vivo, it is releasing Prasterone into the body.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

Sesame oil

Duration of treatment / exposure:

Animals were treated once.

Frequency of treatment:

Single intraperitoneal administration of the test substance or the positive or negative control substance.

Post exposure period:

The animals were sacrificed for bone marrow preparation 24 hours after test substance administration. Additional groups of animals of negative control and high dose group (2000 mg/kg) were sacrificed 48 hours after test substance administration.

Remarks:

Doses / Concentrations:
0, 500, 1000 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 for sacrifice time 24 hours, additionally 5 for sacrifice time 48 hours for control and high dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (administration by gavage)

Tissues and cell types examined:

Bone marrow preparations were examined for the incidence of micronucleated cells per 2000 polychromatic (PCE) and 1000 normochromatic (NCE) erythrocytes per animal.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

At the 24 hours time point male and female mice in the high dose group showed a statistically significant (p < 0.05) decrease in the ratio of PCE/NCE, whereas the decrease was more pronounced in the female animals. This decrease in the ratio of PCE/NCE can be taken as indication of bone marrow depression demonstrating that the test compound has reached the target cells.

Conclusions:

No mutagenic activity of the test item in the described test system.

Executive summary:

A mouse bone marrow micronucleus test (according to OECD 474) was conducted on 5 animals/sex and dose group receiving each a single i.p. injection of the formulated analogue/precursor of Androst-5-en-17-one, 3-hydroxy-, (3.beta.)-, at dosages of 0 (vehicle control), 500, 1000 and 2000 mg/kg bw. Animals were sacrificed 24 hours after test substance administration and bone marrow smears were prepared. Additionally 5 animals were used for vehicle control and high dose (2000 mg/kg bw) group with a sacrifice and sampling time of 48 h after test substance administration.

At the 24 hours time point male and female mice in the high dose group showed a statistically significant (p < 0.05) decrease in the ratio of PCE/NCE, whereas the decrease was more pronounced in the female animals. This decrease in the ratio of PCE/NCE can be taken as indication of bone marrow depression demonstrating that the test compound has reached the target cells. The male and female mice treated with the substance showed at all 3 dose levels ( 0.5, 1.0 and 2.0 g/kg body weight) neither a biologically relevant nor statistically significant increase (p < 0.05) in micronucleated PCE and NCE as compared to the vehicle control at either of the two sampling times, i.e. 24 or 48 hours after a single treatment.

The micronucleus frequencies determined in the vehicle and positive control were within the expected historical range.

From the results obtained, it is concluded that the test substance has no mutagenic activity in the in vivo MNT.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Data on genotoxicity are cited in RTECS database (Jan 2012) for Androst-5-en-17-one, 3-hydroxy-, (3.beta.)-:

In an interlaboratory collaborative study the substance was shown to be negative in an Ames Test using the tester strains S. typhimurium TA 102 and TA 2638 as well as E. coli WP2/pKM101 and WP2 uvrA/pKM101 with metabolic activation and tested up to the recommended highest dose of 5000 µg/plate [Mutation Research. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1967- v. 416, p. 169, 1998 (MUREAV)].

The substance had no detectable genotoxic effect in a rat (male Fischer 344 rats) primary hepatocyte UDS assay when tested in concentrations from 20 -36 µg/ml [Environmental Mutagenesis. (Alan R. Liss, Inc., 41 E. 11th St., New York, NY 10003) V.1 -1979-v.8, p. 461, 1986 (ENMUDM)]

Further results are also cited in RTECS database (Jan 2012), but not used for classification, as they were considered not to be relevant:

In an in vivo-in vitro replicative DNA synthesis (RDS) test the substance was administered to male Fischer 344 rats once orally by gavage at dosages of 500 and 1000 mg/kg. Hepatocytes of these rats were isolated after 24, 39 and 48 h and RDS incidence (= percentage of RDS-positive hepatocytes calculated from data for 2000 hepatocytes/liver) was measured. The substance gave in both dosages positive results in the RDS incidence after 24 and 39 h, with a peak response time of 24 h, and equivocal results after 48 h [Mutation Research. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1967- v. 320, p. 189, 1994 (MUREAV)]. The substance is known to be a peroxisome proliferator. Therefore, the authors itself concluded that the induced replicative DNA synthesis in rat hepatocytes might have been induced by direct growth stimulation.

Positive results for genotoxicity for the substance were obtained from two pre-screen assays, Vitotox^TMand RadarScreen in a validation study with over 250 compounds. The two assays were early screens for mutagenicity and clastogenicity, respectively, and were based on the following principles: ¿The VitotoxTM assay is a bacterial reporter assay in Salmonella typhimurium based on the SOS¿response, and it contains a luciferase gene under control of the recN promoter¿. ¿The RadarScreen assay is a RAD54 promoter-linked beta-galactosidase reporter assay in yeast¿ (Saccharomyces cerevisiae). However, the authors concluded that the positive results were false positives because previously published data (see above: Ames and UDS) revealed no mutagenic or clastogenic potential. [Mutation Research. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1967- v. 676, p. 113, 2009 (MUREAV)].

Moreover, an in vivo mouse micronucleus test with an analogue/precursor of Androst-5-en-17-one, 3-hydroxy-, (3.beta.)- (Enantate), which is known to release in vivo Androst-5-en-17-one, 3-hydroxy-, (3.beta.)-, revealed negative genotoxicity (see chapter "Genetic toxicity in vivo")

Summarizing, based on the available data the substance is considered not to be genotoxic. 

Justification for selection of genetic toxicity endpoint
in vivo data relevant for non-classification

Justification for classification or non-classification

Based on read-across from data for Prasterone enantate the substance has not to be classified for genotoxicity according to Directive 67/548/EEC or Regulation (EC) No. 1272/2008 (CLP).