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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key Ames test no increase in mutations were observed up to tested concentrations of 5000 µg/plate in 5 differentSalmonella typhimuriumstrains with and without metabolic activation. In a key mammalian gene mutation test in HPRT cells, the test item did not induce mutations in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 250 and 1000 µg/mL, respectively. In a keyin vitroMicronucleus study in human peripheral lymphocytes, no chromosome damage was observed with and without metabolic activation when tested up to cytotoxic concentration of 1000 µg/mL.

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:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid test guidelines, therefore it is considered to be relevant, adequate and reliable for classification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Cytotoxicity test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate
Mutagenicity test: 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate (plate incorporation and preincubation test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide in aqua ad iniectabilia
Remarks:
(10 µg/plate):TA 1535, TA 100, without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-Nitro-fluorene in DMSO
Remarks:
(10 µg/plate):TA 98, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9-Amino-acridine in ethanol, abs.
Remarks:
(100 µg/plate): TA 1537, without S9-mix
Positive controls:
yes
Positive control substance:
other: Mitomycin C in DMSO
Remarks:
( 10 µg/plate): TA 102, without S9-mix
Positive controls:
yes
Positive control substance:
other: Benzo(a)pyrene in DMSO
Remarks:
( 10 µg/plate): TA 98, TA 102, TA 1537, with S9-mix
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene in DMSO
Remarks:
(2-4 µg/plate): TA 100, TA 1535, with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
1st independent experiment : in agar (plate incorporation);
2nd independent experiment : preincubation.

DURATION
1st independent experiment :
- Exposure duration: 48 h to 72 h
2nd independent experiment :
- Preincubation period: 20 min
- Exposure duration: 48 h to 72 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity is evidenced by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or by the degree of survival of the treated cultures.
Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a sparse background lawn.

Evaluation criteria:
The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA 98, TA 100 and TA 102 and 3-fold of the vehicle control for TA 1535 and TA 1537 in both independent experiments;
Or
- a concentration-related increase of the revertants is observed (The Spearman’s rank correlation coefficient) .
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
number of revertants compared with the vehicle control (p ≤ 0.05, U-test according to MANN and WHITNEY)
concentration-related increase of the revertants ((Spearman’s rank correlation coefficient)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: other: all strains tested in mutagenicity test, TA 100 without S9-mix in cytotoxicity test
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The test item was examined in the 5 Salmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.91 was used as the supplied test item contains only 34.40% active matter. Aqua ad iniectabilia was used as vehicle control.
The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µgtest item/plate in all test strains.
No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to aconcentrationof 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid test guidelines, therefore it is considered to be relevant, adequate and reliable for classification.
Qualifier:
according to guideline
Guideline:
other: OECD 487
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: Human peripheral blood was obtained by venipuncture from young (approximately 18- 35 years of age), healthy, non-smoking individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels.
Details on mammalian cell type (if applicable):
- Type and identity of media:
Ham’s F10 supplemented with 10% fetal calf serum (FCS) and 1% Penicillin/Streptomycin: initiation and maintenance
Ham's F10: after 24 hours
addition of chromosome medium with 5 µg/mL Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Cytotoxicity (preliminary) test: 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL medium
Main test (with and without S9): 125, 250, 500 or 1000 µg/mL medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.2 µg/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: Colchicine
Remarks:
0.02 µg/mL without S9-mix
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
20 µg/mL with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9-mix : 4 hours or 20 hours; with S9-mix: 4 hours
- Selection time (if incubation with a selection agent): 24hours (after adding Cytochalasin B)
- Fixation time (start of exposure up to fixation or harvest of cells):28 hours(in the experiments with and without S9 and 4h exposure) or 44 hours (in the experiments without S9 and 20h exposure)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): 10% Giemsa.


NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED:
1000 binucleated cells per duplicate cell culture were scored to assess the frequency of cells with one, two or more than two micronuclei (cytotoxicity testing).
The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration) (mutagenicity testing).


DETERMINATION OF CYTOTOXICITY
- Method: other: Cytokinesis-Block Proliferation Index (CBPI)


Evaluation criteria:
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response.
A positive result from the in vitro micronucleus test indicates that the test item induces chromosome damage or damage to the cell division apparatus.
Negative results indicate that, under the test conditions used, the test substance does not induce chromosome breaks and/or gain or loss in cultured mammalian cells.
Equivocal results may be clarified by analysis of another 1000 cells from all the cultures to avoid loss of blinding. If this approach does not resolve the result, further testing would be necessary. Modification of study parameters over an extended or narrowed range of conditions, as appropriate, would be considered in follow-up experiments. Study parameters that might be modified include the test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions.
Statistics:
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971[1]) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).
Species / strain:
lymphocytes: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the vehicle control and the test item formulations in the medium were determined employing a digital pH meter type WTW pH 525 (series no. 51039051). No changes in the pH values were noted.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL medium were employed. Cytotoxicity and haemolysis were noted starting at a concentration of 1000 µg active ingredient/mL.
Hence, 1000 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the test item tested up to a cytotoxic concentration of 1000 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant damage.
Executive summary:

Test samples of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after the end of exposure. The study was conducted in duplicate.

The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.91 was used as the supplied test item contains only 34.40% active matter. Aqua ad iniectabilia was used as vehicle control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL medium were employed. Cytotoxicity and haemolysis were noted starting at a concentration of 1000 µg active ingredient/mL.

Hence, 1000 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the main study cytotoxicity and haemolysis were noted at the top concentration of 1000 µg of active ingredient/mL in the experimentswithout and with metabolic activation.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation.

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with active ingredient at concentrations of 125, 250, 500 or 1000 µg/mL medium (4h and 20-h exposure) in the absence of metabolic activation ranged from 8.0 to 16.5 micronuclei per 1000 binucleated cells. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487,(in this test: vehicle control: 9.5 or 14.0 micronuclei per 1000 binucleated cells, untreated controls: 7.5 or 16.5 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively)).

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with active ingredient at concentrations of 125, 250, 500 or 1000 µg/mL in the presence of metabolic activation ranged from 11.0 to 21.0 micronuceli per 1000 binucleated cells. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 -25 micronuclei per 1000 binucleated cells according to OECD 487, (in this test: vehicle control: 11.5 or 16.0 micronuclei per 1000 binucleated cells, untreated controls: 12.0 or 11.0 micronuclei per 1000 binucleated cells).

Under the present test conditions, the test item tested up to a cytotoxic concentration of 1000µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test.

In the same test, Mitomycin C and cyclophosphamide induced significant damage.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid test guidelines, therefore it is considered to be relevant, adequate and reliable for classification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus at the X-chromosome
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
*V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS. Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2.
* For subculturing, a trypsin (0.05%)-EDTA (ethylenediamine¬tetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
* Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. The cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes. The spontaneous mutation rate was continuously monitored.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate dissolved in dimethyl sulfoxide (DMSO).
Remarks:
600 and 700 µg EMS/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9,1 0-dimethyl-1,2-benzanthracene(DMBA) dissolved in dimethyl sulfoxide (DMSO)
Remarks:
20 and 30 µg DMBA/mL, with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: Without S9-mix:4 hours (1 st experiment) or 24 hours (2nd experiment); With S9-mix: 4 hours
- Expression time (cells in growth medium): until day 8 with one subcultivation on day 5
- Selection time (if incubation with a selection agent): about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).

SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL);

NUMBER OF REPLICATIONS:
cytotoxicity: triplicate
mutagenicity: for selection of mutants 5 replicate plates; for the estimation of plating efficiencies (PE) 3 replicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: plating efficientcy

Evaluation criteria:
So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here (see UKEMS guidelines for discussion). Our experience has shown that the following pre¬determined descriptive criteria are the most useful for interpretation of the results:
-lf in both independent experiments solvent and positive controls show results within the norm and if the test compound does not increase the mutation, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1 000 000 cells per condition have been evaluated, the compound is considered as negative in the test.
-In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the compound is considered as positive in the test.
Equivocal results, if applicable are clarified by further testing, in agreement with Sponsor and Study Monitor.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 250 or 1000 µg/mL in the absence and presence of metabolic activation, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in the pH values in the medium were noted.
- Effects of osmolality: No relevant changes in osmolality of the formulations were noted.

RANGE-FINDING/SCREENING STUDIES:
The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary study cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 250 or 1000 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 250 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency of the cultures treated with concentrations of 15.63, 31.3, 62.5, 125 or 250 µg test item/mL culture medium without metabolic activation ranged from 7.77 to 15.29 x 10 6 clonable cells. These results are within the normal range of the vehicle controls.The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 or 1000 µg test item/mL culture medium with metabolic activation ranged from 8.41 to 16.25 x 10 6 clonable cells. These results are within the normal range of the vehicle controls.
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10 6 survivors in non-activation solvent controls and 6 to 46 per 106 survivors in S9 activation solvent controls. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls EMS (600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation frequency.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.
Executive summary:

The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.91 was used as the supplied test item contains only 34.40% active matter. Aqua ad iniectabilia was used as vehicle control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary study cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 250 or 1000 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 250 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

Main study

Five concentrations 15.63, 31.3, 62.5, 125 or 250 or 62.5, 125, 250, 500 or 1000 µg test item/mL were selected for the experiments without and with metabolic activation, respectively.

Cytotoxicity

In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 250 or 1000 µg/mL in the absence and presence of metabolic activation, respectively.

Experiments without metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 7.93 and 15.00 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 15.63, 31.3, 62.5, 125 or 250 µg test item/mL culture medium ranged from 7.77 to 15.29 x 106clonable cells. These results are within the normal range of the vehicle controls.

Experiments with metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 11.47 and 11.55 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250 or 1000 µg test item/mL culture medium ranged from 8.41 to 16.25 x 106clonable cells. These results are within the normal range of the vehicle controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 463.23 to 511.43 x 10-6clonable cells in the case of EMS and ranging from 430.67 to 990.67 x 10-6clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6clonable cells for EMS and 130.0 to 2693.3 x 106clonable cells for DMBA.

Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans. 

Additional information

Bacterial mutagenicity

A key study was conducted wiht registered substance. A liquid test item containing 34.4% active ingredient was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (Flügge, 2013a). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in aqua ad iniectabilia ; a correction factor of 2.91 was used. In a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test, ten concentrations of 0.316 up to 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate, hence six concentrations of 31.6, 100, 316, 1000, 3160 and 5000µg test item/plate were employed in the plate incorporation test and in the preincubation test without and with metabolic activation. No signs of cytotoxicity were noted in the plate incorporation and preincubation test. No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

In conclusion, the registered substance tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Mammalian mutagenicity

A key study was conducted with registered substance. A liquid test item containing 34.4% active ingredient was tested in cultured mammalian cells (V79, genetic marker HPRT) both in the presence (4 hours) and absence (4 and 24 hours) of metabolic activation (Flügge, 2013b). In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 50000 µg/mL medium were employed. Cytotoxicity was noted in form of decreased plating efficiency starting at concentrations of 250 or 1000 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 250 µg test item/mL was employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation. Five concentrations 15.63, 31.3, 62.5, 125 or 250 or 62.5, 125, 250, 500 or 1000 µg test item/mL were selected for the experiments without and with metabolic activation, respectively. In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2)was noted in the first and second experiments at the top concentrations 250 or 1000 µg/mL in the absence and presence of metabolic activation, respectively. Both in the experiments with and without metabolic activation, the mutation frequencies of treated cell cultures within the normal range of the vehicle controls. The positive controls caused a pronounced increase in the mutation frequencies, indicating the validity of this test system.

In conclusion, the registered substance tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects. 

Chromosome aberration

A key study was conducted with registered substance. A liquid test item containing 34.4% active ingredient was assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation (Flügge,2013c).The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after the end of exposure. The study was conducted in duplicate.The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.91 was used as the supplied test item contains only 34.40% active matter.The concentrations employed were chosen based on the results of a cytotoxicity study, showing cytotoxicity and haemolysis at a concentration of 1000 µg active ingredient/mL.Hence, 1000 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.In the main study cytotoxicity and haemolysis were noted at the top concentration of 1000 µg of active ingredient/mL in the experiments without and with metabolic activation.Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation. In the main test without metabolic activation (4- and 20-hour exposure), the micronucleus frequencies of cultures treated with active ingredient at concentrations of 125, 250, 500 or 1000 µg/mL medium (4-h and 20-h exposure) ranged from 8.0 to 16.5 micronuclei per 1000 binucleated cells and treated groups did not differ from controls. In the main test with metabolic activation (4-hour exposure), the micronucleus frequencies of cultures treated with active ingredient at concentrations of 125, 250, 500 or 1000 µg/mL medium ranged from 11.0 to 21.0 micronuclei per 1000 binucleated cells and treated groups were als not different from controls.

In conclusion, the registered substance tested up to a cytotoxic concentration of 1000 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant damage.


Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial and mammalian mutagenicity and chromosomal aberration. Based on the available results, there were no indications of mutagenicity or genotoxicity, and no further testing is needed. The substance can be considered to have no mutagenic or genotoxic potential.


Justification for classification or non-classification

Based on these results and according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for genetic toxicity.