Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A registration of norethisterone was already submitted earlier and is public available on the ECHA website. Chapter 7, which is still valid from today's perspective, was amended to fulfill the current information requirements. Consequently the migrated data (IUCLID 5 to IUCLID 6) was kept unchanged and only modified if there was a need for further information and/or to pass the technical completeness check (TCC).

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 mammalian or bacterial test systems in vitro (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
Remarks:
Type of genotoxicity: gene mutation
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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only plate incorporation method applied
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
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:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix)
Species / strain:
S. typhimurium TA 1538
Additional strain 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
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: without metabolic activation: 9-AA, 2-NF, NaN3, with metabolic activation: 2-AA, BP, CP
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Remarks:
precipitation started at 0.75 mg/plate
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No growth inhibition of the background lawn

Conclusions:
Interpretation of results (migrated information):
negative

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

Norethisterone (ZK 5378) 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 intrinsic metabolic activation (liver mix from Aroclor 1254 -treated rat).

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only preincubation method applied
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
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:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix)
Species / strain:
S. typhimurium TA 1538
Additional strain 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
Negative controls:
yes
Solvent 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
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Precipitation started at the test concentration of 0.25 mg/plate. There was no growth inhibition of the background lawn.

Conclusions:
Interpretation of results (migrated information):
negative

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

Norethisterone (ZK 5378) 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) 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)

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key 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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (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:
DMSO or aceton
Negative controls:
yes
Solvent 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:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
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.
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.
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 assys 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
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
CHO cells used in the current investigation were routinely grown in 75cm² Corning plastic flasks in 10 ml of McCoy’s 5a medium supplemented with 10% fetal calf serum and antibiotics consisting of penicillin-G, (100 U/ml) and streptomycin, (100 µg/ml). The cultures were incubated in an atmosphere of 5% CO2 in air. To test the effect of steroid hormones, cultures were set up 24 h prior to the treatment by seeding approx. 1.6 x 10 to 6 cells per flask. Treatments were given by exposing the cultures to test compounds dissolved in DMSO and suspended in the culture medium for 12 h. The control cultures contained 0.5% of DMSO and were incubated under identical conditions. The procedure for the preparation of chromosomes to score for the abnormalities
is described in Experientia, 38 (1982) 845-846.
GLP compliance:
not specified
Type of assay:
other: in vitro cytogenetic/chromosome aberration study
Test material information:
Composition 1
Specific details on test material used for the study:
The test material was procured from Sigma
Species / strain:
lymphocytes: human
Details on mammalian cell lines (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:
DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and 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:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not applicable
Positive controls valid:
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:
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
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
Lymphocyte culturing was carried out on the blood samples obtained from young and healthy male donors (20-25 years age). Whole blood cultures were set up by adding 0.8 ml of blood to 10.0 ml of RPMI- 1640 medium (Flow labs) supplemented with 10% foetal calf serum (Microlab) and 0.1 ml phytohaemagglutinin (Microlab) and were incubated for 72 h at 37°C. Three concentrations of test item were added to different cultures separately for 24, 48 and 72 h. MMS (1X 10 -7 M) and DMSO (5 µl/ml) were added to the simultaneously kept cultures, as positive and negative controls respectively. Each culture was incubated for 72 h. Colcemid (0.01 µl/ml; Gibco) was added to the cultures 2 h prior to harvesting. The slides were prepared, stained and scanned under code as described previously (Bali et al., Environ. Mol. Mutagen., 16 250-254,1990). At least 50-100 well spread metaphases were scanned per treatment for chromatid and chromosome type aberrations.
In the metabolic activation experiments, 30-h old cultures were given 6-h treatment with different concentrations of the test item in the presence of S9 mix. The cells were collected after centrifugation, and the pellet was washed twice with prewarmed
unsupplemented RPMI-1640 medium to remove the drug and S9 mix. Each culture was further incubated for 36 h at 37°C before harvesting. Parallel cultures receiving the same dose of the test item for some treatment duration but without S9 mix were also simultaneously set up for comparison. DMSO (5 µl/ml) and cyclophosphamide (CP; 5 X 10 -7 M) were used as negative and positive controls respectively in these metabolic activation experiments.
GLP compliance:
not specified
Type of assay:
other: in vitro cytogenetic/chromosome aberration study
Test material information:
Composition 1
Specific details on test material used for the study:
Source of the material: Organo India Limited, Calcutta
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
Lymphocyte culturing was carried out on the blood samples obtained from young and healthy male donors (20-25 years age). Whole blood cultures were set up by adding 0.8 ml of blood to 10.0 ml of RPMI- 1640 medium (Flow labs) supplemented with 10% foetal calf serum (Microlab) and 0.1 ml phytohaemagglutinin (Microlab) and were incubated for 72 h at 37°C.
Metabolic activation:
with and without
Metabolic activation system:
No details available
Test concentrations with justification for top dose:
1, 10 and 100 µg/ml (with/without S9 mix)
Vehicle:
DMSO
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and conditions:
The complete information given in the publication is mentioned in the field "Principles of method if other than guideline".
Rationale for test conditions:
The complete information given in the publication is mentioned in the field "Principles of method if other than guideline".
Evaluation criteria:
The complete information given in the publication is mentioned in the field "Principles of method if other than guideline".
Statistics:
Student’s two tailed “t” test was used for calculating the statistical significance.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Conclusions:
positive
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.

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
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
2 mg of each substance was dissolved in 0.2 ml of 95 %. ethanol. This solution was then mixed with 19.8 ml of growth medium (TC 199), filtered through a millipore filtration system, and used as stock solution. 20 ml of heparinized blood was obtained from a female secretary who had no recent exposure to agents known to break chromosomes and who was not using oral contraceptives or other hormones. The blood sample was treated with phytohemagglutin and centrifuged at 500 rpm for 5 min. The plasma containing the lymphocytes was then separated equally into five flasks to which growth medium TC 199 (GIBCO) with penicillin and streptomycin then was added to make final volumes of 10 ml. In each experiment, the substance to be tested was added to make final concentrations of 0.1, 1.0, 10.0, and 100.0 µg/ml, respectively, in four of the five flasks. The fifth flask was cultured without the study substance and served as a control. All flasks were cultured at 37° C for 72 hr. At 90 min prior to harvesting, 0.2 ml of 1 X 10-6 M colchicine was added to each flask. Harvesting was then carried out, and the cells were affixed to slides by an air-dry method. The slides were stained with carbolfuchsin and coded for scoring to eliminate bias. With few exceptions, 200 or more metaphase spreads were scored from each culture. Spreads were scored for gaps (a separation of a portion ofa chromatid without dislocation of the distal fragment); chromatid breaks (a separation of a portion of a chromatid with dislocation of the distal fragment); isochromatid breaks (a separation of a portion of both chromatid arms at similar positions with separation of the distal fragments); and abnormal forms (rings, dicentrics, quadriradials, triradials, 4 N cells, etc). All abnormal cells were photographed and karyotyped.
GLP compliance:
not specified
Type of assay:
other: in vitro cytogenetic/chromosome aberration study
Test material information:
Composition 1
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
20 ml of heparinized blood was obtained from a female secretary who had no recent exposure to agents known to break chromosomes and who was not using oral contraceptives or other hormones. The blood sample was treated with phytohemagglutin and centrifuged at 500 rpm for 5 min. The plasma containing the lymphocytes was then separated equally into five flasks to which growth medium TC 199 (GIBCO) with penicillin and streptomycin then was added to make final volumes of 10 ml.
Metabolic activation:
without
Test concentrations with justification for top dose:
0.1, 1.0, 10.0, and 100.0 µg/ml
Vehicle:
95 %. ethanol
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
no
Details on test system and conditions:
The complete information given in the publication is mentioned in the field "Principles of method if other than guideline".
Rationale for test conditions:
The complete information given in the publication is mentioned in the field "Principles of method if other than guideline".
Evaluation criteria:
The complete information given in the publication is mentioned in the field "Principles of method if other than guideline".
Statistics:
The complete information given in the publication is mentioned in the field "Principles of method if other than guideline".
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
other: poorly growth at the 100.0 µg concentration
Vehicle controls valid:
not specified
Negative controls valid:
not applicable
Positive controls valid:
not applicable
Conclusions:
negative
Executive summary:

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

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:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: taken from Dhillon, 1996
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
other: poorly growth at 100.0 µg concentration
Vehicle controls valid:
not specified
Negative controls valid:
not applicable
Positive controls valid:
not applicable
Remarks on result:
other: taken from Stenchever, 1969
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:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
30 July 1992 to 20. Jan 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well reported GLP, guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Test material information:
Composition 1
Target gene:
not applicable
Species / strain:
hepatocytes: primary cells, female rat
Details on mammalian cell lines (if applicable):
- Type and identity of media: Williams E medium, supplemented (complete medium)
- Properly maintained: yes
Additional strain characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
2 to 20 µg/ml
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO 100-fold diluted with medium
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Species / strain:
hepatocytes: primary male rat cells
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In experiment 1 treatment concentration of 15 µg/ml reduced viability to 32%.

In experiment 2 treatment concentration of 20 µg/ml led to toxic effects.

In the pre-experiment concentations higher than 21.67 µg/ml precipitated in the culture medium.

Conclusions:
Interpretation of results (migrated information):
negative

Norethisterone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS)
Executive summary:

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.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23. Dec 1991 to 27. Apr 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well reported GLP, guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Test material information:
Composition 1
Target gene:
not applicable
Species / strain:
hepatocytes: primary cells, male rat
Details on mammalian cell lines (if applicable):
- Type and identity of media: Williams E medium, supplemented (complete medium)
- Properly maintained: yes
Additional strain characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0.00128 to 100 µg/ml
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO 100-fold diluted with medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Species / strain:
hepatocytes: primary male rat cells
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In experiment 1 treatment concentration of 20 µg/ml induced cell survival to 18%.

In experiment 2 treatment concentration of 32.5 µg/ml reduced viability to 33%.

The top dose of 32.5 µg/ml and four consecutive doses were scored in this experiment. There was very little effects of the test compound on the proportion of cells in S-phase which did not exceed 0.9% in experiment 1 and 0.8% in experiment 2 at any dose tested.

Conclusions:
negative

Norethisterone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS)
Executive summary:

Norethisterone (ZK 5378) did not show any genotoxic potential in two in vitro UDS tests with primary male rat hepatocytes when teste 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.

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

Additional information

A registration of norethisterone was already submitted earlier and is public available on the ECHA website. Chapter 7, which is still valid from today's perspective, was amended to fulfill the current information requirements. Consequently the migrated data (IUCLID 5 to IUCLID 6) was kept unchanged and only modified if there was a need for further information and/or to pass the technical completeness check (TCC).

Norethisterone (ZK 5378) 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 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. typhymurium (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]

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

Additionally, results of genotoxicity studies with norethisterone were cited in RTECS database (Jan 2010):

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; elaborated by the German Committee on Hazardous Substances (AGS) and published by the German Federal Ministry of Labour and Social Affairs, version: 19.04.2016, only available in German, URLhttp://www.baua.de/de/Themen-von-A-Z/Gefahrstoffe/TRGS/Begruendungen- 905-906.html. 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 Directive 67/548/EEC and Regulation (EC) 1272/2008 (CLP) is not required.