3β-hydroxyandrost-5-en-17-one acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No reliable in vitro genetic toxicity study was available for the test substance; therefore a read-across approach with the pharmacologically active ingredient DHEA was used to cover these endpoints.

In vitro gene mutation study in bacteria

2 reliable, K2 published studies were used to investigate the in vitro genetic toxicity of DHEA in bacteria (Watanabe et al., 1998 and FDA report, 2015). Watanabe et al. performed a Bacterial Reverse Mutation Assay according to a method similar to OECD 471 on the strains S. typhimurium TA 102 and TA2638 and on E. coli WP2 uvr A pKM 101 and WP2 pKM 101 with metabolic activation. In the FDA report, the test item was tested on the strains S. typhimurium TA100, TA1535, TA97a, TA102 and WP2 uvrA with and without metabolic activation according to OECD 471. Negative results were obtained in the two experiments.

In vitro chromosome aberration study

In the key study reported in the FDA report (2015), chromosome aberrations of EM-760 were evaluated in human peripheral blood with and without metabolic activation, with an exposure period of 4 or 19h, according to OECD 473. The cells were harvested and evaluated 22 hours after the start of the exposure. The test item was not clastogenic in the conditions described in the test.

In vitro mammalian cell gene mutation assay

In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that T008506 is not mutagenic in the mouse lymphoma L5178Y test system in the absence and presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

other information

Justification for type of information:

Data from the supporting substance Dehydroepiandrosterone is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic index reduced up to 70% at the highest tested dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dehydroepiandrosterone was tested in at least two independent experiments using at least four dose levels in duplicate. The test item was found to be negative. This result is considered relevant for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

Conclusions:

No reliable in vitro cytogenicity study is available for Dehydroepiandrosterone acetate. Therefore, reliable data from the supporting substance Dehydroepiandrosterone (DHEA) is used to cover this endpoint. DHEA did not increase the number of chromosome aberrations, polyploidy or endoreduplication, and was judged as negative.
The same is assumed for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

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

Justification for type of information:

Data from the supporting substance Dehydroepiandrosterone is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97a, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1380 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1380 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dehydroepiandrosterone was tested in a dose range finding and a definitive experiment, using 6 dose levels in triplicate. The test item was found to be negative. This result is considered relevant for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

Conclusions:

No reliable genetic toxicity study is available for Dehydroepiandrosterone acetate. Therefore, reliable data from the supporting substance Dehydroepiandrosterone (DHEA) is used to cover this endpoint. DHEA did not increase the number of revertant colonies of any strains tested, and was judged as negative. The same is assumed for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

Reference 3

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

Justification for type of information:

Data from the supporting substance Dehydroepiandrosterone is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 2638

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli, other: WP2 pKM 101

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dehydroepiandrosterone was tested in at least two replicate experiments using five dose levels and three plates per dose, and each chemical was tested in two laboratories to assess reproducibility. The test item was negative in two replicate experiments performed in each of the two independent laboratories. This result is considered relevant for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

Conclusions:

No reliable genetic toxicity study is available for Dehydroepiandrosterone acetate. Therefore, reliable data from the supporting substance Dehydroepiandrosterone (DHEA) is used to cover this endpoint. DHEA did not increase the number of revertant colonies of any strains tested, and was judged as negative in two replicate experiments performed in each of the two independent laboratories.
The same is assumed for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-22 to 2018-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mouse Lymphoma Assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16GB2547
- Expiration date of the lot/batch: 01 July 2018 (retest date)
- Purity test date: 26 August 2016 (release darte)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not available
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study.

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). Cell density was kept below 1 x 10^6 cells/ml.
- Normal (negative control) cell cycle time: not indicated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/str
eptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10
medium).
Exposure medium:
For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium).
Environmental conditions: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 73 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.3 - 38.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not indicated
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine, 2 x 10-7 M aminopterine and 1.6 x 10-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ßnaphthoflavone)

Test concentrations with justification for top dose:

Upon mixing with exposure medium the test item precipitated at concentrations of 3.13 mg/ml (= 31.3 μg/ml) and above. Based on these solubility findings, DMSO was selected as vehicle and 250 μg/ml was selected as the maximum final concentration for the dose.
Dose range finding test (3h treatment): 16, 31, 63, 125, 250 μg/mL with and without S9-mix
Dose range finding test (24h treatment): 16, 31, 63, 125, 250 μg/mL without S9-mix

The highest concentrations tested in the first mutation experiment were determined by the solubility of the test item in the culture medium (precipitation at 31 µg/mL)
Mutagenicity assay I (3h treatment): 0.12, 0.24, 0.49, 0.98, 2.0, 3.9, 7.8, 15.6, 31.3 μg/mL with and without S9-mix;
Mutagenicity assay II (24h treatment): 0.24, 0.49, 0.98, 2.0, 3.9, 7.8, 15.6, 31.3 μg/mL without S9-mix;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed in Charles River Laboratories Study No. 20153273. The test item was suspended or dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension in the dose range finding test or until the test item had completely dissolved in the mutagenicity tests.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix; at 15 µg/mL (3h treatment period), at 5 µg/mL (24h treatment period)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix; at 7.5 µg/mL (3h treatment period)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used.

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 48 (3h and 24h treatment)
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): selective medium (TFT-selection)

STAIN (for cytogenetic assays): After the inclubation, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well.

NUMBER OF REPLICATIONS:
1 replicate per concentration, the solvent control was tested in duplicate

NUMBER OF CELLS EVALUATED:
Determination of cloning efficiency: One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
Determination of mutation frequency: 9.6 x 10^5 cells/concentration plated (5 x 96-well microtiter plates/concentration, each well containing 2000 cells in selective medium (solvent controls and treatment groups); 9.6 x 10^5 cells/concentration plated (10 x 96-well microtiter plates/concentration), each well containing 1000 cells in selective medium (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (RSG) (see calculations in section "any other information on materials and methods incl. tables")

Rationale for test conditions:

The highest concentration tested should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/ml or 0.01 M (whichever is the lowest).
The highest concentration was determined by the solubility of the test item in the culture medium since the test item was not toxic and difficult to dissolve in aqueous solutions

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

No statistical analysis

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not measured
- Water solubility: no data
- Precipitation: The test item precipitated in the exposure medium at the highest concentration tested (31.3 μg/ml) in the absence and presence of S9-mix

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with test item concentration ranged of 16 to 250 μg/mL in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
3h treatment: In the absence of S9-mix, the relative suspension growth was 3% at the test item concentration of 250 μg/ml compared to the relative suspension growth of the solvent control. The test item precipitated in the exposure medium at 31 μg/ml and above.
In the presence of S9-mix, the relative suspension growth was 45% at the test item concentration of 125 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 250 μg/ml. The test item precipitated in the exposure medium at 31 μg/ml and above.
24h treatment: The relative suspension growth was 25% at the test item concentration of 63 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 125 and 250 μg/ml. The test item precipitated in the exposure medium at 31 μg/ml and above.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix)
functioned properly.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database, except in the first experiment in the presence of S9-mix, in which the mutation frequency of one of the solvent control cultures was just above the acceptability criteria for this assay

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutation Experiment 1
No severe toxicity was observed in the absence and presence of S9-mix. Therefore the lowest dose level of 0.12 μg/ml was not regarded relevant for mutation frequency measurement.
The dose levels selected to measure mutation frequencies in the absence and presence of S9-mix at the TK-locus were: 0.24, 0.49, 0.98, 2.0, 3.9, 7.8, 15.6 and 31.3 μg/ml exposure medium.
The relative total growth (RTG) was not decreased significantly up to and including the highest precipitating dose level

Mutation Experiment 2
No significant toxicity was observed and all dose levels were used for mutation frequency measurement.
The relative total growth (RTG) was not decreased significantly up to and including the highest precipitating dose level.

OTHER: The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 15 and 20 (3 hour treatment) and 67 (24 hour treatment)

Remarks on result:

other:

Remarks:

Mutation experiment 1

Conclusions:

Interpretation of results: negative with and without metabolic activation
In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No reliable in vivo genetic toxicity study was available for the test substance; therefore a read-across approach with the pharmacologically active ingredient DHEA was used to cover this endpoint.

In vivo micronucleus test

A K2 study (FDA report, 2015) was performed to confirm the negative results obtained in vitro after exposure to the pharmacologically active ingredient DHEA. The in vivo mouse bone marrow micronucleus assay followed the OECD 474 and concluded on the absence of clastogenic effects in vivo.

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

Justification for type of information:

Data from the supporting substance Dehydroepiandrosterone is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dehydroepiandrosterone was tested in an in vivo micronucleus test in mouse bone marrow using three dose levels. The test item did not induce micronuclei and was therefore considered negative.
This result is considered relevant for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

Conclusions:

No reliable in vitro mammalian cytogenicity study is available for Dehydroepiandrosterone acetate. Therefore, reliable data from the supporting substance Dehydroepiandrosterone (DHEA) is used to cover this endpoint. DHEA did not increase the number of micronuclei, and was judged as negative.
The same is assumed for Dehydroepiandrosterone acetate. Justification for this read across approach is included in IUCLID section 13.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No reliable in vitro or in vivo genetic toxicity study was available for the test substance to cover the endpoints "in vitro gene mutation study in bacteria" and "in vitro cytogenecity study in mammalian cells"; therefore a read-across approach with the pharmacologically active ingredient DHEA was used to cover these endpoints. A Key study was performed with teh test item to cover the endpoint "in vitro gene mutation study in mammalian cells".

In vitro gene mutation study in bacteria

•Watanabe et al. (K2, 1998) performed a Bacterial Reverse Mutation Assay according to a method similar to OECD 471 on the strains S. typhimurium TA 102 and TA2638 and on E. coliWP2 uvr A pKM 101 and WP2 pKM 101 with metabolic activation, in DMSO as vehicle. The cells were exposed for 48 hours.

The following test concentrations were tested in triplicates, by two different labs:

Lab 1:

- TA102: 0, 156, 313, 625, 1250 and 2500 μg/plate;

- TA2638: 0, 39, 78, 156, 313 and 625 μg/plate;

- WP2/pKM101 and WP2uvrA/pKM101: 0, 313, 625, 1250, 2500 and 5000 μg/plate;

Lab 2:

- TA102, TA2638, WP2/pKM101 and WP2uvrA/pKM101: 0, 313, 625, 1250, 2500 and 5000 μg/plate.

The test item did not increase the number of revertant colonies of any strains tested.

• In the FDA report published in 2015, DHEA was tested on the strains S. typhimurium TA100, TA1535, TA97a, TA102 and WP2 uvrA with and without metabolic activation according to OECD 471. Bacteria were cultured in agar on plates for 52 hrs. Study included three trials with replicates with each trial. Positive control had to have 3 fold increase over vehicle control. The study was considered valid and the test item was not mutagenic to any of the strains tested.

In vitro chromosome aberration study

• In the key study reported in the FDA report (2015), chromosome aberrations of EM-760 were evaluated in human peripheral blood with and without metabolic activation, with an exposure period of 4 or 19h, according to OECD 473. The cells were harvested and evaluated 22 hours after the start of the exposure. The vehicle used was ethanol.

Colcemid, harvested, stained, and metaphase cells were analyzed for the presence of chromosomal aberrations.

The following test concentrations were evaluated for chromosome aberrations:

-S9 (4hr exposure): 138, 175, 200, 225 μg/ml

-S9 (19 hr exposure): 25, 50, 66, 100 μg/ml

+S9 (4 hr exposure): 138, 175, 225; 300 μg/ml

The test item was not clastogenic in the conditions described in the test, which was considered valid.

• A K2 supporting study (Oshiro et al., 1986) was included to support the negative results observed in the FDA report.

An in vitro rat primary hepatocyte unscheduled DNA synthesis assay was conducted following a method similar to OECD 482. The vehicle was DMSO.

The following test concentrations were tested in triplicate: 0, 20, 24, 28, 32, 36 μg/mL.

One-hundred net nuclear counts per three coverslips were obtained from each test compound concentration, the medium and positive controls, and 200 net nuclear counts per six coverslips from the solvent control. DHEA had no detectable genotoxic effect in the rat primary hepatocyte UDS assay.

In vitro mammalian cell gene mutation assay

Gijsbrechts (2018) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (key study, K1).

The test item was dissolved in dimethyl sulfoxide up to a concentration of 25 mg/ml.

In the first experiment, the test item was tested up to concentrations of 31.3 μg/ml in the absence and presence of S9-mix. The treatment period was 3 hours. In the second experiment, the test item was again tested up to concentrations of 31.3 μg/ml in the absence of S9-mix. The treatment period was 24 hours. No toxicity was observed up to and including the highest precipitating dose level of 31.3 μg/ml.

The mutation frequency found in the solvent control cultures was within the range of the the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

In vivo micronucleus test

An In Vivo mouse bone marrow micronucleus assay was conducted in 2006 (FDA report 2015) on CD-1 mice.

5 mice per test group were exposed to DHEA by oral gavage.

The following test concentrations were investigated:

Positive controls: 80 mg/kg, 5 males for the 24h harvest time

Vehicle controls: 0 mg/kg, 5 males for the 24h harvest time and 5 males for the 48 h harvest time

500 mg/kg: 5 males for the 24h harvest time

1000 mg/kg: 5 males for the 24h harvest time

2000 mg/kg: 5 males for the 24h harvest time and 5 males for the 48 h harvest time

According to the criteria described in the report, the study was considered valid.

DHEA was not clastogenic in vivo under the conditions described in the test.

Justification for classification or non-classification

Based on the negative results obtained in the currently available in vitro and in vivo genetic toxicity tests on DHEA, T008506 should not be classified for mutagenicity according to the criteria laid out in the CLP regulation.