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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Norethisterone is a synthetic sex hormone and active ingredient of approved drugs since several decades. Norethisterone belongs to the category “steroidal progestins” and has progestogenic properties resembling those of the naturally occuring progesterone but is a more potent inhibitor of ovulation. Apart from the data on norethisterone, information from its ester derivates (norethisterone enanthate and acetate) can be used for characterization of the biological activity of progestin, because both esters are rapidly cleaved within the mammalian organism and thus, norethisterone is the systemically active metabolite irrespective of the form which is administered.


 


The genotoxicity of progestogenic hormones have been tested in a number of in house mammalian or bacterial test systems in vitro (cf. Lang & Reimann, Environ Molec Mutagen 21, 272 -304 (1993); Reimann et al., Environ Molec Mutagen 28, 133 -44 (1996)). Overall, norethisterone and/or other members of the category "steroidal progestins" did not induce gene mutations in these assays. Chromosome aberration tests yielded contradictory results for norethisterone and its ester derivate (norethisterone acetate).  As (1) norethisterone does not directly interact with DNA (2) clastogenic effects were not reproducible between different laboratories and typically occurred at high, unphysiological concentrations, available in vitro data do not indicate that norethisterone possesses a relevant genotoxic potential.


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10. Oct to 20. Oct 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983;
The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system
Deviations:
yes
Remarks:
only plate incorporation method applied
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The solutions were prepared immediately before the start of the test.
Target gene:
Histidine gene locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix)
Test concentrations with justification for top dose:
0.025 to 1 mg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without metabolic activation: 9-AA, 2-NF, NaN3, with metabolic activation: 2-AA, BP, CP
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- number of revertants
Evaluation criteria:
A positive response was considered if at least 5 rng/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the spontaneous was higher than 2-fold.
Also a dose dependent increase in the number of revertants was considered to indicate a mutagenic effect.
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
Remarks:
precipitation started at 0.75 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
precipitation started at 0.75 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
precipitation started at 0.75 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation started at 0.75 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
precipitation started at 0.75 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitates in the agar were found starting at 0.75 mg/plate.

Ames test:
- Signs of toxicity: No growth inhibition of the background lawn could be observed.

None of the five tester strains showed increased reversion to prototrophy in assays with with the test item at the concentrations tested between 0.025 and 1 mg/plate, either in the presence or absence of S 9 mix.

The counts recorded on appropriate negative control plates confirmed the characteristically spontaneous reversion rates of the tester strains.

The total colony counts of the bacterial culture confirmed the viability and high cell density of the cultures of the individual strains.

Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains.
Conclusions:
Interpretation of results: negative

Norethisterone is not mutagen in this study with and without metabolic activation
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 1538, TA 98 of S. typhimurium  were exposed to Norethisterone (ZK 5378) in DMSO at concentrations of 0.025, 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mg/plate in the presence and absence of mammalian metabolic activation (S9 liver mix from Aroclor 1254 -treated rat) in the plate incorporation assay. 

The test item was tested up to precipitating concentrations; Precipitates in the agar were found starting at 0.75 mg/plate. There was no evidence of induced mutant colonies over background.

No growth inhibition of the background lawn could be observed.

The positive controls induced the appropriate responses in the corresponding strains.  

In conclusion, Norethisterone did not show any mutagenic potential in a bacterial reverse mutation assay with S. typhymurium (TA 1535, TA 100, TA 1537, TA 1538, TA 98) when tested up to 1.0 mg/plate in the absense and presense of metabolic activation.

 

 

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12. Nov to 5. Dec 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983;
The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system
Deviations:
yes
Remarks:
only preincubation method applied
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The solutions were prepared immediately before the start of the test.
Target gene:
Histidin gene locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix)
Test concentrations with justification for top dose:
0.025 to 1 mg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without metabolic activation: 9-AA, 2-NF, NaN3, with metabolic activation: 2-AA, BP, CP
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- preincubation (60 min at 37°C)


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition


METHODS FOR MEASUREMENTS OF GENOTOXICIY
number od revertants
Evaluation criteria:
A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants eompared to the spontaneous was higher than 2-fold. Also a dose dependent increase in the number of revertants was eonsidered to indieate a mutagenic effect.
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
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The counts recorded on appropriate negative control plates confirmed the characteristically spontaneous reversion rates of the tester strains.
The total colony counts of the bacterial culture confirmed the viability and high cell density of the cultures of the individual strains.

Appropriate positive control chemicals induced marked increases in revertant colony nurnbers with all strains.

Precipitation started at the test concentration of 0.25 mg/plate. There was no growth inhibition of the background lawn.
Conclusions:
Interpretation of results: negative

Norethisterone is negative in the Ames test with and without metabolic activation after preincubation
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 1538, TA 98 of S. typhimurium  were exposed to Norethisterone (ZK 5378) in DMSO at concentrations of 0.025, 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mg/plate in the presence and absence of mammalian metabolic activation (S9 liver mix from Aroclor 1254 -treated rat) in the preincubation assay. 

 

The test item was tested up to precipitating concentrations; Precipitates in the agar were found starting at 0.25 mg/plate. There was no evidence of induced mutant colonies over background.

No growth inhibition of the background lawn could be observed.

The positive controls induced the appropriate responses in the corresponding strains.  

 

In conclusion, Norethisterone did not show any mutagenic potential in a bacterial reverse mutation assay with S. typhymurium (TA 1535, TA 100, TA 1537, TA 1538, TA 98) when tested up to 1.0 mg/plate in the absense and presense of metabolic activation.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
supplied by Prof. Miltenburger, Darmstadt, Germany
Metabolic activation:
with and without
Metabolic activation system:
From male Sprague-Dawley rats pretreated with Aroclor 1254 (purchased from Organon Teknika Co. (Durham, NC))
Test concentrations with justification for top dose:
At least four concentrations of the test substance without and with S9 mix are scored for mutant colonies. The highest dose chosen for evaluation should be clearly toxic, i.e.. it should cause a reduction of the plating efficiency (cell surviva]) or correspond to the substance's solubility limit (precipitates in the culture). Nontoxic compounds will be tested up to 10-² M or 5-10 mg/ml.- (in the absence or presence of liver cells).
Vehicle / solvent:
DMSO or aceton
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: EMS, MNNG (without metabolic activation); DMBA (with S9 mix)
Evaluation criteria:
So far. no satisfactory mathematical methods are available for statistical analysis of mammalian cell mutagenicity experiments such as those performed here. Our experience has shown that the following predetermined descriptive criteria are the most useful for interpretation of the results. An evaluation is made only after a repeat experiment has been carried out. The evaluation of the results is performed as follows: (1)the test substance is classified as mutagenic if it induces with one of the test substance concentrations, reproducibly, a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment; (2) the test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency;
such an evaluation may be considered independently of the enhancement factor for induced mutants; however, in a case-by-case evaluation both decisions depend on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range found in this laboratory, a seemingly concentrationrelated increase in the mutations or a factor of three or even more within this range may be regarded as being irrelevant.
When considerable variations in the results occur, clarificationis sought in additional experiments.
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
not applicable
Positive controls validity:
valid
Additional information on results:
The mutagenicity results and data of nine progestins (cyproterone acetate, dehydrospirorenone, gestodene, gestonorone caproate, levonorgestrel, norethisterone, norethisterone acetate, norethisterone enanthate, norethynodrel) are reported. All sex steroids were investigated using the Ames Test and two sex steroids were also studied in the HPRT test with V79 cells. For all assays evaluation of the data indicates that any of the progestins was able to induce gene mutations wether in the absence or the presence of S9 mix. On the basis of these findings, it seems justifiable to extrapolate that sex steroids in general possess obviously no mutagenic potential detectable in gene-mutation assays in vitro.
Remarks on result:
other:
Remarks:
The mutagenicity results and data of 17 sex steroids are reported. Nine of them are progestins (cyproterone acetate, dehydrospirorenone, gestodene, gestonorone caproate, levonorgestrel, norethisterone, norethisterone acetate, norethisterone enanthate, norethynodrel). All progestogenic steroid hormones were investigated using the Ames Test and two of them (Cyproterone Acetate; Dehydrospirorenone) were also studied in the HPRT test with V79 cells. For all assays evaluation of the data indicates that any of the progestins was able to induce gene mutations wether in the absence or the presence of S9 mix. On the basis of these findings, it seems justifiable to extrapolate that sex steroids in general possess obviously no mutagenic potential detectable in gene-mutation assays in vitro.
Conclusions:
The mutagenicity results and data of nine progestins (cyproterone acetate, dehydrospirorenone, gestodene, gestonorone caproate, levonorgestrel, norethisterone, norethisterone acetate, norethisterone enanthate, norethynodrel) are reported. All sex steroids were investigated using the Ames Test and two sex steroids were also studied in the HPRT test with V79 cells. For all assays evaluation of the data indicates that any of the progestins was able to induce gene mutations wether in the absence or the presence of S9 mix. On the basis of these findings, it seems justifiable to extrapolate that sex steroids in general possess obviously no mutagenic potential detectable in gene-mutation assays in vitro.
Executive summary:

2 of 9 progestins did not induce substantial and reproducible dose dependent increase of the mutation frequency in a mammalian cell gene mutation assay (HPRT) according to OECD TG 476. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Therefore, the substances were considered to be non-mutagenic in the specified test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: test procedure well documented, but only the overall result is mentioend without showing the data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
The test material was procured from Sigma
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Lymphocyte cultures were set up by adding 0.5 ml of whole blood from two adult and healthy female donors (who were occupationally not exposed to mutagens) to 4.5 ml of RPMI-1640 medium (Gibco) supplemented with 15% fetal calf serum (Gibco) and antibiotic–antimycotic (100×lyophilised, 10,000 units/ml penicillin sodium, 10,000 g/ml streptomycin sulfate, 25g/ml amphotericin B; Gibco). Lymphocytes were stimulated to divide by adding 0.1 ml of phytohaemagglutinin-M (PHA-M; Gibco). The cultures were incubated at 37◦C and 5% CO2 for 72 h in dark.
Metabolic activation:
with and without
Metabolic activation system:
Swiss albino healthy rats (Wistar strain) were given 0.1% of phenobarbitone (1 mg/ml) in drinking water for 1 week.The S9 mix was freshly prepared as per the standard procedures of Maron and Ames [Mutat. Res. 113 (1983) 173–215].
Test concentrations with justification for top dose:
at a final concentration of 20, 40 and 75 µg/ml (with/without S9 mix)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
The test item was added for 24, 48 and 72 h duration by adding the steroid after 0, 24 and 48 h in reverse order after the initiation of cultures. In the metabolic activation experiments, 48 h old cultures were given treatment with the test item, along with S9 mix (0.8 ml).
After 6 h of incubation the cells were collected by centrifugation and the pellets were washed twice in pre-warmed medium (37◦C) to remove the drug and S9 mix, and reincubated for 24 h in fresh medium supplemented with antibiotics and fetal calf serum.
Parallel cultures receiving same concentrations of the drug for similar treatment duration but without S9 mix were simultaneously set for comparison. Colchicine (0.20 µg/ml; Microlab) was added to the cultures 2.5 h prior to harvesting. The cells were collected by centrifugation (10 min; 1200 rpm), hypotonic treatment (0.075M KCl) was given for 10–12 min at 37◦C and the cells recollected by centrifugation were fixed in methanol:acetic acid (3:1).
Evaluation criteria:
A total of 300 well-spread metaphases were analysed per treatment per duration for all types of chromatid and chromosome type of aberrations. In case of cultures with metabolic activation, only 200 well-spread metaphases were analysed.
Statistics:
Student’s two tailed “t” test was used for calculating the statistical significance. The level of significance was tested from standard statistical tables of Fisher and Yates
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Remarks on result:
other: data not shown in details
Conclusions:
negative
Executive summary:

The effect of synthetic progestin-norethisterone on human lymphocyte chromosomes in vitro was studied. Norethisterone was found not to induce chromosomal abnormalities with and without metabolic activation under the conditions of the the study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Norethisterone is a synthetic steroidal progestin, first synthesized in the 1950s, that is used in oral form in preparations for the field of gynecological therapy. Norethisterone has progestogenic properties resembling those of the naturally occuring progesterone but is a more potent inhibitor of ovulation. Apart from the data on norethisterone experiments with its ester derivate norethisterone enanthate can be used for characterization of the biological activity of the progestin, because the ester is rapidly cleaved within the mammalian organism and thus, norethisterone is the systemically active metabolite irrespective of the form which is administered.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: poorly growth at 100.0 µg concentration
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: taken from Dhillon, 1996
Conclusions:
contradictory
Executive summary:

A significant increase in aberration frequencies has been observed at all doses and at all treatment durations (except at dose 1.0 µg/ml; 24 h and 48 h treatment. However, 6 h treatment with the drug in the presence of S9 mix, induced a significant increase in aberration frequency at the highest doses (10.0 and 100.0 µg/ml) as compared to the results obtained without metabolic activation. In human lymphocyte cultures, both chromatid and chromosomal type aberrations were observed. However, the frequency of chromatid type aberrations was higher than the chromosomal type.

Norethisterone acetate was evaluted in 72-hr human leukocytes cultures for its effects on chromosomal integrity. No significant chromosomal abnormalities were found.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

For Norethisterone and its esters Norethisterone acetate and Norethisterone enantate several in vitro and in vivo studies on genotoxicity are available (see compilation below).


 


Studies with Norethisterone


Norethisterone (ZK 5378) did not show any mutagenic potential in a bacterial reverse mutation assay with S. typhimurium (TA 1535, TA 100, TA 1537, TA 1538, TA 98) when tested up to 1.0 mg/plate in the absense and presense of intrinsic metabolic activation (liver mix from Aroclor 1254 -treated rat) [Schering AG, Report No. 7347; 1986 -11 -04]


 


Norethisterone (ZK 5378) did not show any mutagenic potential in a bacterial reverse mutation assay with S. typhimurium (TA 1535, TA 100, TA 1537, TA 1538, TA 98) after preincubation when tested up to 1.0 mg/plate in the absense and presense of intrinsic metabolic activation (liver mix from Aroclor 1254 -treated rats) [Schering AG, Report No. 7520; 1987 -03 -09]


 


Norethisterone (ZK 5378) did not show any genotoxic potential in two in vitro UDS tests with primary male rat hepatocytes when tested up to the toxic concentration (experiment 1: 20 ug/ml; experiment 2: 32.5 ug/ml). Furthermore, no biologically relevant increase in the number of S-phase cells were seen. [Schering AG, Report No. A194; 1992 -09 -18]


 


Norethisterone (ZK 5378) did not show any genotoxic potential in two in vitro UDS tests with primary female rat hepatocytes when tested up to the toxic concentration (experiment 1: up to 15 ug/ml; experiment 2: up to 20 ug/ml). Furthermore, no biologically relevant increase in the number of S-phase cells were seen. [Schering AG, Report No. A442; 1993 -03 -15]


 


The genotoxic potential of oral contraceptive steroids such as norethindrone was investigated in short-term rat hepatocyte cultures by measurement of unscheduled DNA synthesis.


Norethindrone caused a small dose-dependent increase in unscheduled DNA synthesis in male rat hepatocytes as judged by the incorporation of [methyl-3H]thymidine into DNA.


This was assessed either by liquid scintillation counting following isolation of DNA or by autoradiography. No increase in unscheduled DNA synthesis could be detected in female rat hepatocytes treated with norethindrone. In male rat hepatocyte cultures there was a dose-dependent increase in radioactive incorporation into DNA determined after a 16 h incubation period.


Pre-treatment of male rats with phenobarbitone prior to hepatocyte preparation decreased the norethindrone mediated unscheduled DNA synthesis relative to control hepatocyte cultures while 3-methylcholanthrene pre-treatment had little effect.


Unscheduled DNA synthesis in norethindrone treated control male rat hepatocytes was reduced by the mixed function oxidase inhibitors SKF 525A or metyrapone. In 24- or 52-hour old hepatocyte cultures in which the cytochrome P-450 content was lower than in freshly prepared cells, or in a hepatocyte-derived cell line lacking cytochrome P-450, unscheduled DNA synthesis due to norethindrone was either decreased or abolished. Structure activity studies showed that only steroids containing a 17a-ethynyl substituent caused an increase in unscheduled DNA synthesis. [Blakey, Carcinogenesis 6(8), 1201-1205, 1985]


 


The effect of norethisterone on human lymphocyte chromosomes in vitro was studied. Norethisterone was found not to induce sister chromatic exchange with and without metabolic activation under the conditions of the study. [Ahmad, Mutation Research 494, 13-20, 2001]


 


Norethisterone was tested for its ability to induce DNA repair synthesis in primary cultures of rat and human hepatocytes. The substance was tested in cultures from two male and two female donors of each species. Rat and human hepatocytes were exposed for 20 h to sub-toxic concentrations of 1, 2, 5, 10, 20, and 50 µM, and DNA repair induction was measured by quantitative autoradiography.


At the 50 µM concentration the relative cell viability ranged from 85 to 94% in rat hepatocytes and from 96 to 99% in human hepatocytes. In terms of DNA repair induction, contrasting results were obtained in rat hepatocytes; the response was frankly positive in one male rat, inconclusive in one female rat due to marked decrease of cytoplasmic labelling, and frankly negative in the other two rats. In human hepatocytes, a frankly negative response was obtained in one male and one female donor, whereas the responses of the other two donors were inconclusive.


It was concluded, that sex steroids differ for their ability to induce DNA repair, and that their genotoxicity may be: (i) different in rat and human hepatocytes, (ii) dependent on the sex of the donor, and (iii) affected by inter-individual variability. [Martelli, Mutat Res 536, 69-78, 2003]


 


In this neutral comet assay in RKO and CHO cells no increase in mean tail moment 4 h after treatment with norethindrone at 1, 10 or 100 mg/ml as compared with DMSO-treated control cells was detected.


The neutral comet assay is more specific for detecting DSBs, in contrast to single-strand breaks and alkali-labile sites, but less sensitive than the alkaline version of the comet assay, additionally, the alkaline comet assay was applied to detect any damage induced by norethindrone. No increase in mean tail moment in the alkaline comet assay was detected, however, the data are not shown in the publication. [Gallmaier, Carcinogenesis vol.26 no.10 pp.1811–1820, 2005]


 


Cytogenetic studies were performed to determine the clastogenic potential of norethindrone. RKO and CHO cells were treated with norethindrone at 10 and 100 µg/ml for 24 h. Subsequently, 50 metaphases of each sample were analyzed for breakage and other structural abnormalities.


The criteria were defined as follows. The test was considered negative when the fraction of aberrant cells was <5%, inconclusive, when >5% but <10%, and positive, when 10% or more. Results between 10 and 20% were considered weakly positive.


In this assay, norethindrone was negative for chromosome aberrations in RKO cells (0% at 10 µg/ml, 2% at 100 µg/ml), but weakly positive in CHO cells (8% at 10 µg/ml, 12% at 100 µg/ml). [Gallmaier, Carcinogenesis vol.26 no.10 pp.1811–1820, 2005]


 


Norethisterone was assessed for their in vivo genotoxic effect on the bone marrow cells of Swiss albino mice. The chromosomal aberration assay and the micronucleus test were employed for the study.


Dose-response analysis was carried out for the above doses 24 h after the final feeding (15 d; 0.3, 1.5, 3.0, 6.0, 12.0, 18.0, 24.0 and 30.0 mg/kg body weight). Time-response studies were done using mice fed 3.0 mg/kg/day, 6, 12, 24, 48, 96 h and 1, 2 and 3 weeks after the final feeding.


No treatment-related effect on the rate of micronuclei was observed after any of the doses. In addition, no increase of the incidence of chromosome breaks or chromosomal translocations which were designated as "classical" chromosomal aberrations were noted.


However, the incidence of so-called "non-classical" chromosomal aberrations such as "stickiness" and "pulverisations" was increased from 3.0 mg/kg/day onwards. Similar findings were said to occur in studies on the natural steroid progesterone. These "non-classical" findings weretransientwith a maximum at 12 and 24 hours after the final treatment and a sharp decline thereafter. A possible drug interaction with the nucleoproteins was discussed as one possible underlying mechanism which should account for the absence of a concomitant effect on the rate of micronuclei. [Shyama, Mutation Research, 300 (1993) 215-221]


 


Norethisterone has been examined for the ability to induce the formation of micronuclei in the liver of female rats. In the micronucleus assay, carried out in rats given a single p.o. dose of 100 mg/kg 3 days before partial hepatectomy and sacrificed for cell sampling 2 days later, the frequency of micronucleated hepatocytes was 2.2 fold higher than in controls, but not statistically significant.


In the liver foci assay, performed to evaluate the tumor initiating activity of p.o. dosing with 100 mg/kg once a week for 6 successive weeks, the values of the number and area of g-glutamyltranspeptidase-positive foci were, as compared to controls, 15.9- and 100-fold higher; the increase of area produced was statistically significant. These results suggest that Norethisterone might be biotransformed in the liver into reactive species and thus behave as a weak genotoxic agent. [Martelli, Mutat Res 419, 33-41, 1998]


 


Joosten et al. (Toxicology Letters 151, 113 -134 (2004) provides an overview of the genotoxicity of hormonal steroids in general and in particular for norethisterone.


 


Studies with Norethisterone acetate


Norethisterone acetate was unable to induce any significant dose-related increase/decrease in the mean number of His + revertants/plate both with and without S9 mix. However, at the highest doses (5E03 and 1E04 µg/plate), there was a decrease in the number of His + revertants. [Dhillon, Mutation Research 367, 1-10, 1996]


 


Norethisterone acetate was evaluated in 72-hr human leukocytes cultures for its effects on chromosomal integrity. No significant chromosomal abnormalities were found. [Stenchever, Obstet. Gynecol. 34, 249-251, 1969]


 


A significant increase in aberration frequencies has been observed at all doses and at all treatment durations (except at dose 1.0 µg/ml; 24 h and 48 h treatment. However, 6 h treatment with the drug in the presence of S9 mix, induced a significant increase in aberration frequency at the highest doses (10.0 and 100.0 µg/ml) as compared to the results obtained without metabolic activation. In human lymphocyte cultures, both chromatid and chromosomal type aberrations were observed. However, the frequency of chromatid type aberrations was higher than the chromosomal type. [Dhillon, Mutation Research 367, 1-10, 1996]


 


Norethisterone acetate caused a dose- and duration-related significant increase in the frequencies of the sister chromatid exchanges in human lymphocyte cultures both with and without S9 mix. [Dhillon, Mutation Research 367, 1-10, 1996]


 


The genotoxicity of the norethisterone acetate was evaluated in an in vivo micronucleus assay in male Swiss mice. Three different doses (100, 1000 and 10000 µg/kg body weight) of of the test item were injected (single i.p. injection) in three separate groups of mice, respectively. After 30 h, the animals were killed and bone marrow smears were prepared.


The highest doses of the norethisterone acetate (1E03 and 1E04 mg/kg body weight) induced a significant increase in the number of micronucleated PCEs as compared


to the negative controls. [Dhillon, Mutation Research 367, 1-10, 1996]


 


The genotoxicity of the norethisterone acetate was evaluated in an in vivo sister chromatid exchange assay in male Swiss mice. Three different doses of norethisterone acetate (100, 1000 and 10000 µg/kg body weight) were injected to different groups of mice. 24 h later, colchicine (6.0 mg/kg body weight; for 2 h) was given as a single i.p. injection. The animals were then killed and the bone marrow smears were prepared.


The highest doses of the norethisterone acetate (1E03 and 1E04 mg/kg body weight) induced a significant increase in the number of sister chromatid exchanges as compared to the negative controls. [Dhillon, Mutation Research 367, 1-10, 1996]


 


Studies with Norethisterone enantate


Norethisterone enanthate was examined for mutagenic activity in five histidine-dependent strains of Salmonella typhimurium (TA 1535 and TA 100 for detection of base-pair substitutions, TA 1537, TA 1538 and TA 98 for detection of frame-shift mutations) using the direct plate incorporation procedure developed by Ames et al.


The studies, which were conducted in the absence and presence of an extrinsic metabolizing system derived from Aroclor 1254-induced rat liver (S 9 mix), employed a range of norethisterone enanthate concentrations from 0.1 to 5 mg per plate.


Negative controls and positive controls with known mutagens produced the expected numbers of revertant colonies.


None of the five tester strains showed increased reversion to prototrophy with norethisterone enanthate at the concentrations tested, either in the absence or presence of S 9 mix.


Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 2.5 mg/plate.


Evaluation of the data does not indicate that norethisterone enanthate is a mutagen in the Ames Salmonella/microsome test. [Report no. 9747, Lang & Schmitt, 1991-09-26]


 


Norethisterone enanthate was examined for mutagenic activity in five histidine-dependent strains of Salmonella typhimurium (TA 1535 and TA 100 for detection of base-pair substitutions, TA 1537, TA 1538 and TA 98 for detection of frame-shift mutations) using the modified Ames test (preincubation for 60 min at 37°C).


The studies, which were conducted in the absence and presence of an extrinsic metabolizing system derived from Aroclor 1254-induced rat liver (S9 mix), employed a range of norethisterone enanthate concentrations from 0.1 to 5 mg per plate.


Negative controls and positive controls with known mutagens produced the expected numbers of revertant colonies. None of the five tester strains showed increased reversion to prototrophy with norethisterone enanthate at the concentrations tested, either in the absence or presence of S 9 mix.


Precipitates in the agar were found starting at 2.5 mg/plate. Growth inhibition of the background 1awn was not observed.


Evaluation of the data does not indicate that norethisterone enanthate is a mutagen in the Ames Salmonella/microsome test using the preincubation modification. This is in agreement with the results of an earlier study using the direct plate incorporation procedure. [Report no. 9708, Lang & Schmitt, 1991-11-25]


 


 


 


Overall, the available in vitro and in vivo studies on genetic toxicity does not lead to the conclusion that a genotoxic potential is present. The German TRGS 905 (Technische Richtlinien für Gefahrstoffe/Technical Rule for Hazardous Substances 905, published by the German Federal Institute for Occupational Safety and Health, last update 13.03.2020) discusses the endpoint genotoxicity for Norethisterone and its esters among various gestagenic steroid hormones. There, for the group of gestagens no convincing indication for a relevant mutagenic potential is seen and consequently no classification for mutagenicity proposed.


 


In addition, an in vivo mutagenicity test of the Dominant-Iethal assay type with Norethisterone acetate in mice revealed no evidence of a mutagenic potential (Schering report 4586 (3959), dated 3. Feb. 1981).


 


Additionally, results of genotoxicity studies with norethisterone were cited in RTECS database (accessed 04/2021):


 


In cells culture (mouse, domestic, mammalian) mutation was seen at 10 ug/L (mouse) and 100 ug/L (domestic, mammalian); cytogenicity was seen at 100 ug/L (mammalian) [American Journal of Obstetrics and Gynecology. (C.V. Mosby Co., 11830 Westline Industrial Dr., St. Louis, MO 63146) V.1- 1920- v. 120, p. 390, 1974 (AJOGAH)]


 


In rat liver cells unscheduled DNA synthesis appeared at 50 umol/L [Carcinogenesis (London). (Oxford Univ. Press, Pinkhill House, Southfield Road, Eynsham, Oxford OX8 1JJ, UK) V.1- 1980- v. 6, p. 1201, 1985 (CRNGDP)]


 


Mice were orally exposed to norethisterone over 15 days. The subsequent cytogenetic test was positive at 45 mg/kg [Mutation Research. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1964- v. 300, p. 215, 1993 (MUREAV)]


 


Rat and human liver cells showed DNA repair after 20 hours exposure to 5 umol/L and 0.05 mmol/L, respectively.


[Mutation Research. (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands) V.1- 1964- v. 536, p. 69, 2003 (MUREAV)]


 


Human lymphocytes developed DNA inhibition at 50 umol/L [Proceedings of the Society for Experimental Biology and Medicine. (Academic Press, Inc., 1 E. First St., Duluth, MN 55802) V.1- 1903/04- v. 146, p. 401, 1974 (PSEBAA)]


 


In hamster lung cells cytogenicity was seen at 100 mg/L [Yakuri to Chiryo. Pharmacology and Therapeutics. (Raifu Saiensu Shuppan K.K., 2-5-13, Yaesu, Chuo-ku, Tokyo 104, Japan) V.1- 1972- v. 19(Suppl 4), p. S1065, 1991 (YACHDS)]

Justification for classification or non-classification

In a number of publications opposing results were reported. However, norethisterone was also tested in standard genotoxicity tests and did not show any genotoxic potential.


Thus, there is no sufficient evidence available to classify norethisterone as genotoxic.


No self classification for norethisterone is recommended according to Regulation (EC) No.1272/2008 (CLP)


The non-classification is in accordance with German legislation for classification of steroid hormones. The German Committee on Hazardous Substances (AGS) recommended for the group of progestin/progesteron ("Gestagene") classification as:


Toxicity to reproduction - Fertility: Category 1A


Toxicity to reproduction - Development: Category 1B


Carcinogenicity: Category 2


See Technical Rule for Hazardous Substances 905 (Technische Richtlinien für Gefahrstoffe/Technical Rule for Hazardous Substances 905, published by the German Federal Institute for Occupational Safety and Health, last update 13.03.2020). The associated documentation and justification for grouping steroid hormones and their classification was published in 09/1999. Norethisterone is mentioned in attachment 2 on page 17. Norethisterone is not classified as genotoxic according to the German legislation (TRGS-905). Classification according to Regulation (EC) 1272/2008 (CLP) is not required.