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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: WoE approch, result: positive


 

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:
April 16-25, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only one strain tested (to reproduce result from previous study), documentation limited
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
only one strain was tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his-operon
Species / strain / cell type:
S. typhimurium TA 1537
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
0, 4, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment)
0, 20, 100, 500, 2500, 3750 and 5000 µg/plate (2nd experiment)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
a dose-related increase in the number of revertants was observed, but this was only exceeding the critical factor of 2 in the second experiment.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The test substance is mutagenic in the bacterial reverse mutation assay.
Executive summary:

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with one Salmonella typhimirium strain (TA 1537). The following doses were tested:


0, 4, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment)


0, 20, 100, 500, 2500, 3750 and 5000 µg/plate (2nd experiment)


the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a dose-related increase in the number of revertants was also observed, but this was only exceeding the critical factor of 2 in the second experiment. However, based on the result of the study, test item is considered to be mutagenic in bacterial cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only 4 bacterial strains tested
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
6 doses from 4 to 5000 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (range-finding study (Experiment 1) and main experiment (Experiment 2))

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and microscopical inspection of the bacterial background lawn

The results of both experiments (exp.1 and 2) were used for determining the mutagenic properties of the test substance.
Evaluation criteria:
A chemical is considered to have a positive response, if it
-produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
-induces a dose-related increase in the mean number of revertants per plate
The results must be reproducible.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
In this assay the test substance was considered to be mutagenic.
Executive summary:

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). Six test concentration from 4 – 5000 µg/plate were used. In the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a slight increase was also observed, but only in one strain. Therefore the test item was considered to be mutagenic in bacterial cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 5 to 7, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
only 4 strains tested
GLP compliance:
yes
Remarks:
KoreaGLP
Type of assay:
bacterial reverse mutation assay
Target gene:
his-operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
experiment I: 0, 8, 40, 200, 1000, 5000 µg/plate
experiment II: 6.9,20.6, 61.7, 185.2, 555.6, 1666.7 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (range-finding study (Experiment 1) and main experiment (Experiment 2))

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates (experiment I), duplicate (experiment I)

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and microscopical inspection of the bacterial background lawn

The results of both experiments (exp.1 and 2) were used for determining the mutagenic properties of the test substance.
Evaluation criteria:
Results are judged as positive if the number of colony increases with dose-response and reaches 2 times that of the control, or if there is statistically significant increase in the number of colonies in any of the tester strains.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the presence of S9-mix, there was significant increase in the number of revertants in strain TA 1537, which reached up to 8 times that of the control and was reproducible.
Conclusions:
The test item was mutagenic in the bacterial reverse mutation assay.
Executive summary:

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). The following doses were tested:


experiment I: 0, 8, 40, 200, 1000, 5000 µg/plate


experiment II: 6.9,20.6, 61.7, 185.2, 555.6, 1666.7 µg/plate


In the presence of S9-mix, there was significant increase in the number of revertants in strain TA 1537, which reached up to 8 times that of the control and was reproducible. Therefore the test item was considered to be mutagenic in bacterial cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance was not clastogenic in the in vivo mammalian erythrocyte micronucleus test (reference 7.6.2-1).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1st, 1996 to July 18th, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
other: SHOE:NMRI
Details on species / strain selection:
The mouse is the recommended test species for this assay.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tierzucht Schönwalde GmbH i.G., Schönwalde, Germany
- Age at study initiation: appr. 8 weeks
- Weight at study initiation: males mean: 36.8 g, females mean: 29.8 g
- Housing: five animals per cage
- Diet: ad libitum rat/mice diet ssniff (ssniff GmbH, Soest, Germany)
- Water: tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 50+/-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: sesame oil

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was suspended in sesame oil. A magnetic stirrer was used to keep the preparation homogenous until administration.
Duration of treatment / exposure:
one treatment
Frequency of treatment:
one oral gavage treatment
Post exposure period:
Animals were killed 12, 24 or 48 h after dosing.
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
killing times: 12, 24 or 48 h, 5 males and 5 females each
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
killing times: 12, 24 or 48 h, 5 males and 5 females each
No. of animals per sex per dose:
5 per killing time
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
prelinimary toxicity study

DETAILS OF SLIDE PREPARATION:
A suspension of bone marow (taken from the femorae) and fetal bovine serum was formed and centrifuged for 5 minutes at app. 1200 rpm. The supernatant was discarded. The sediment was smeared on a clean slide. Staining was performed with Giemsa solution.

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded.
Evaluation criteria:
A substance is considered positive if there is a significant increase in the numbe rof micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurent negative control group.
Statistics:
Wilcoxon-test (one-sided)
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No mortality and no clinical signs of toxicity were observed. No macroscopic findings were recorded at dissection.

Please refer to the attached background material.

Conclusions:
The test item was not clastogenic in vivo under the conditions of the present study.
Executive summary:

The test substance was tested in an in vivo micronucleus test according to OECD Guideline 474. The test item was suspended in sesame oil and administered once via gavage at doses of 0 and 2000 mg/kg bw to male and female NMRI mice. The animals were killed either after 12, 24 or 48 h (5/sex/killing time). Endoxan (cyclophosphamide) was used as positive control at a dose of 50 mg/kg bw. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values. The positive control induced a marked statistically significant increase in the number of polychromatic cells with micronuclei. The ratio of polychromatic erythrocytes to normocytes was not changes to a significant extent. Under the conditions of the study, th test item is not mutagenic in this test.

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

Additional information

Ames test, reference 7.6.1-1


The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). Six test concentration from 4 – 5000 µg/plate were used. In the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a slight increase was also observed, but only in one strain. Therefore the test item was considered to be mutagenic in bacterial cells.


 


Ames test, reference 7.6.1-2


The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with one Salmonella typhimirium strain (TA 1537). The following doses were tested: 0, 4, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment); 0, 20, 100, 500, 2500, 3750 and 5000 µg/plate (2nd experiment), the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a dose-related increase in the number of revertants was also observed, but this was only exceeding the critical factor of 2 in the second experiment. However, based on the result of the study, test item is considered to be mutagenic in bacterial cells.


 


Ames test, reference 7.6.1-3


The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). The following doses were tested: experiment I: 0, 8, 40, 200, 1000, 5000 µg/plate; experiment II: 6.9,20.6, 61.7, 185.2, 555.6, 1666.7 µg/plate. In the presence of S9-mix, there was significant increase in the number of revertants in strain TA 1537, which reached up to 8 times that of the control and was reproducible. Therefore the test item was considered to be mutagenic in bacterial cells.


 


In vivo-study: Micronucleus assay, reference 7.6.2-1


The test substance was tested in an in vivo micronucleus test according to OECD Guideline 474. The test item was suspended in sesame oil and administered once via gavage at doses of 0 and 2000 mg/kg bw to male and female NMRI mice. The animals were killed either after 12, 24 or 48 h (5/sex/killing time). Endoxan (cyclophosphamide) was used as positive control at a dose of 50 mg/kg bw. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values. The positive control induced a marked statistically significant increase in the number of polychromatic cells with micronuclei. The ratio of polychromatic erythrocytes to normocytes was not changes to a significant extent. Under the conditions of the study, the test item is not mutagenic in this test.


Based on these results and additionally taking into account a structurally similar substance (1-Naphthalenesulfonic acid, 6-diazo-5,6-dihydro-5-oxo-, 4-benzoyl-1,2,3-benzenetriyl ester, CAS 5610-94-6), a genotoxic potential can be excluded. This substance was tested according to OECD guidelines 471, 473 and 476, and no clastogenic or mutagenic potential was revealed.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not considered to be classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.