Registration Dossier

Administrative data

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 in different Salmonella typhimurium strains with and without metabolic activation up to 5000 µg/plate. 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 125 µg and 1000 µg/mL, respectively. Finally, in a key in vitro Micronucleus study in human peripheral lymphocytes, no chromosome damage was observed in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 125 µg/mL (4 and 20h exposure time) and up to 500 µg/mL (4h exposure), respectively.

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 methods, therefore the study is considered relevant, adequate and reliable for classification.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
Test concentrations with justification for top dose:
Main tests: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
In a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test, ten concentrations (0.316, 1.0, 3.16. 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg act. ingr. per plate) were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.
Hence, 5000 µg act. ingr./plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection (aqua ad iniectabilia)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide in aqua ad iniectabilia
Remarks:
(10 µg/plate) TA100 and TA1535 without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene in DMSO
Remarks:
(10 µg/plate) TA98 without S9
Negative solvent / vehicle controls:
yes
Positive control substance:
other: 9-amino-acridine in ethanol, abs.
Remarks:
(100 µg/plate) TA 1537 without S9
Negative solvent / vehicle controls:
yes
Positive control substance:
other: Mitomycin C in DMSO
Remarks:
(10 µg/plate) TA 102 without S9
Negative solvent / vehicle controls:
yes
Positive control substance:
other: Benzo(a)pyrene in DMSO
Remarks:
(10 µg/plate) TA 98, TA 102 and TA 1537 with S9
Negative solvent / vehicle controls:
yes
Positive control substance:
other: 2-amino-anthracene in DMSO
Remarks:
(2 µg/plate) TA 100, TA 1535 with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) & preincubation

DURATION
- Preincubation period: 20 min (2nd experiment)
- Exposure duration: 48-72 h (1st and 2nd experiment)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate (3 plates/concentration and strain)

DETERMINATION OF CYTOTOXICITY
- Method: other: a reduction in the number of spontaneous revertants, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures
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
- at one or more concentrations the number of revertants is reproducibly increased in at least one strain with or without metabolic activation. A 2-fold increase in comparison to the solvent control is regarded as being relevant for a positive response in the strains TA 98, TA 100 and TA 102. For the strains TA 1535 and TA 1537 a 3-fold increase represents a biological relevant effect. The Mann and Whitney test (p ≤ 0.05) may be used to determine statistical significance.
Or
- a concentration-related increase of the revertants is observed. The Spearman's rank correlation coefficient may be applied.
- 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.

Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.
Statistics:
The Mann and Whitney test (p ≤ 0.05) may be used to determine statistical significance.
The Spearman's rank correlation coefficient may be applied.
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
Remarks:
No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]¬esters, disodium salts 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:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts 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 (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 substance was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. The vehicle served as the negative control.

Preliminary test

The test substance was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations ranging from 0.316 to 5000 µg act.ingr./plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.

Hence, 5000 µg act.ingr./plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 3.16 to 5000 µg act.ingr./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 µg act.ingr./plate in all test strains.

No increase in revertant colony numbers as compared with control counts was observed up to a concentration of 5000 µg act.ingr./plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies 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 substance tested up to a concentration of 5000 µg act.ingr./plate, caused no mutagenic effect in theSalmonella typhimuriumstrains 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 methods, therefore the study is considered relevant, adequate and reliable for classification.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
* fresh Ham’s F10 medium
* chromosome medium with 5 µg/mL Cytochalasin B
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary experiment: 10, 25, 100, 250, 1000, 2500 and 4358 µg active ingredient/mL medium
First And Second MainTest (4 h and 20-h exposure) without S9: 7.81, 15.63, 31.3, 62.5 or 125 µg/mL medium
First and Second Main Test (4h exposure) with S9: 7.81, 15.63, 31.3, 62.5 or 125 µg/mL medium
Third Main Test (4 h exposure) without S9: 31.3, 62.5, 125, 250 or 500 µg /mL
Third Main Test (4h exposure) with S9: 31.3, 62.5, 125, 250 or 500 µg /mL
The concentrations employed were chosen based on the results of a preliminary cytotoxicity study. Cytotoxicity was noted starting at a concentration of 100 µg active ingredient/mL in the preliminary experiment without and with metabolic activation.
Hence, 125 µg active ingredient/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure).
In a third experiment without and with metabolic activation (4-hour exposure) 500 µg active ingredient/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg active ingredient/mL had not resulted in sufficient clear-cut cytotoxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiol. saline (aqua ad iniectabilia)
Untreated negative controls:
yes
Remarks:
untreated medium
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
True negative controls:
yes
Positive controls:
yes
Remarks:
clastogen
Positive control substance:
mitomycin C
Remarks:
0.2 µg/mL without S9
Positive controls:
yes
Remarks:
clastogen
Positive control substance:
cyclophosphamide
Remarks:
20 µg/mL with S9
Positive controls:
yes
Remarks:
aneugen
Positive control substance:
other: Colchicine
Remarks:
0.02 µg/mL without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4h (with and without S9)
Experiment 2: 4h (with S9) and 20 h (without S9)
Experiment 3: 4h (with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells):
28 h (Experiment 1with and without S9; Experiment 2 with S9 and Experiment 3 with and without S9)
44h (Eperiment 2 without S9)

SPINDLE INHIBITOR (cytogenetic assays): 5µg/mL Cytochalasin B
STAIN (for cytogenetic assays): 10% Giemsa.

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000/concentration

DETERMINATION OF CYTOTOXICITY
- Method: other: Treatment of cultures with cytoB, and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and ensures that only cells that divided during or after treatment are scored.

Evaluation criteria:
Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and solvent control cultures were determined. Individual culture data were provided.
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.
There is no requirement for verification by additional testing of a clear positive or negative response.
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.
Although most experiments give clearly positive or negative results, in some cases the data set would preclude making a definite judgement about the activity of the test item. These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated.
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[3]) 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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
125 µg/mL were employed as the top concentration for the first and second experiment, 500 µg/mL were employed in the third experiment. It was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut 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 negative 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 (pH range: 7.66 – 7.73).

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the test item tested up to cytotoxic concentrations, 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 any chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant damage.


Executive summary:

Test samples of the test item 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: two experiments with an exposure time of 4 hours and two different concentration ranges and one experiment with an exposure time of 20 hours. The experiment with S9 mix was carried out threefold with one exposure time of 4 hours employing two different concentration ranges. The harvesting time was 24 hours after the end of exposure. Each treatment was conducted in duplicate.

The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. The vehicle aqua ad iniectabilia served as the vehicle control.

Preliminary experiment

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 10, 25, 100, 250, 1000, 2500 and 4358 µg active ingredient/mL medium were employed. Cytotoxicity was noted starting at a concentration of 100 µg test item/mL in the experiment without and with metabolic activation.

Hence, 125 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). In a third experiment without and with metabolic activation (4-hour exposure) 500 µg/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut cytotoxicity.

Main study

In the main study cytotoxicity was noted starting at a concentration of 125 µg 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. Positive controls induced significant increases in micronuclei in both experiments with/without metabolic activation.

 

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

The micronucleus frequencies of cultures treated with the test item at concentrations of 7.81, 15.63, 31.3, 62.5 or 125 µg active ingredient/mL medium in the first and second experiment (4 h and 20-h exposure) or 31.3, 62.5, 125, 250 or 500 µg active ingredient/mL in the third experiment (4-hour exposure) in the absence of metabolic activation ranged from 3.0 to 8.0 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control (in this test: vehicle control: 8.0, 6.0 or 6.5 micronuclei per 1000 binucleated cells, untreated controls: 5.5, 4.5 or 5.5 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 the test item at concentrations of 7.81, 15.63, 31.3, 62.5 or 125 µg active ingredient/mL medium in the first and second experiment or 31.3, 62.5, 125, 250 or 500 µg active ingredient/mL in the third experiment in the presence of metabolic activation ranged from 3.0 to 9.5 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control (in this test: vehicle control: 4.5 or 9.0 micronuclei per 1000 binucleated cells, untreated controls: 5.0, 6.0 or 4.5 micronuclei per 1000 binucleated cells).

Under the present test conditions, the test item tested up to cytotoxic concentrations, 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 any chromosomal damage 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 methods, therefore the study is considered relevant, adequate and reliable for classification.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
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, by using the HOECHST stain 33258.
- The spontaneous mutation rate was continuously monitored.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
preliminary cytotoxicity experiment without and with metabolic activation: 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium
The concentrations employed in the main study were chosen based on the results of a preliminary cytotoxicity study. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 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.
mutagenicity test without metabolic activation: 7.81,15.63, 31.3, 62.5 or 125 µg/mL medium
mutagenicity test with metabolic activation: 62.5, 125, 250, 500 or 1000 µg/mL medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was completely dissolved in aqua ad iniectabilia
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate in DMSO
Remarks:
600 and 700 µg/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9,10-dimethyl-1,2-benzanthracene in DMSO
Remarks:
20 and 30 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 (1st 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: other: relative plating efficiency was determined for each dose to obtain an accurate measure of the toxic effect of the chemical.
Evaluation criteria:
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.
Statistics:
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).
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 125 or 1000 µg/mL in the absence and presence of metabolic activation, respectively.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Along with toxicity, changes in the pH of the test solutions was assessed. The pH was measured at the highest test item treatment level.
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 in the medium were noted.
- Effects of osmolality:
Along with toxicity, the Osmolality of the test solutions was assessed. Osmolality determination were carried out in test solutions without target cells both in the presence and absence of metabolic activation. The Osmolality of the highest test item treatment condition, lowest precipitating test item level and the highest soluble test item level in test solution was measured.
- Precipitation:
Along with toxicity, the ability of the test item to cause precipitation in the test solution was assessed.


RANGE-FINDING/SCREENING STUDIES:
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 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 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 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 10 6 survivors in S9 activation solvent controls (BRADLEY, M. O., B. BHUYAN, M. C. FRANCIS, R. LANGENBACH, A. PETER¬SON and E. HUBERMANN. Mutagenesis by chemical agents in V79 Chinese hamster cells: a report and analysis of the literature. A report of the Gene-Tox Program. Mutation Research 87, 81 - 142 (1981)).
The background data obtained at LPT are given at the end of chapter 'Results and Discussion'. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Conclusions:
Interpretation of results:
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 tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. Aqua ad iniectabilia served as the 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 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 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 7.81,15.63, 31.3, 62.5 or 125 and 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 125 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 15.47 and 17.27 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 7.81, 15.63, 31.3, 62.5 or 125µg test item/mL culture medium ranged from 3.20 to 12.62x 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 14.29 and 14.32 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, 500 or 1000 µg test item/mL culture medium ranged from 7.33 to 13.13 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 307.73 to 830.00 x 10-6clonable cells in the case of EMS and ranging from 319.22 to 909.47 x 10-6clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPTranges 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 10-6clonable 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)

Additional information

Additional information from genetic toxicity in vitro:  

Registered substance was examined for bacterial and mammalian gene mutation as well as for chromosomal aberration by means of a liquid test item containing 41.5% active ingredient.

 

Bacterial mutagenicity

In a key test for bacterial mutation, 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments were tested without and with metabolic activation with test item dissolved in aqua ad iniectabilia based on a correction factor of 2.41 for active ingredient (Flügge, 2013a). In a preliminary test, ten concentrations ranging from 0.316 to 5000 µg act.ingr./plate were tested without metabolic activation in strain TA 100. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. In the main study, six concentrations ranging from 3.16 to 5000 µg act.ingr./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 without and with metabolic activation up to the top concentration of 5000 µg act.ingr./plate in all test strains. Under the present test conditions the test item tested up to a concentration of 5000 µg act.ingr./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 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 the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 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 7.81,15.63, 31.3, 62.5 or 125 and 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 125 or 1000µg/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 were 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. 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.

 

Chromosome aberration

A key in vitro micronucleus test (Flügge, 2013c) was conducted using human peripheral lymphocytes both in the presence and absence of metabolic activation, employing 2 exposure times (4 hours and 20 hours). The experiment with S9 mix was carried out threefold with one exposure time of 4 hours employing two different concentration ranges. The harvesting time was 24 hours after the end of exposure. The test item was completely dissolved in aqua ad iniectabilia, which also served as the vehicle control. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only.

Based on a preliminary experiment, cytotoxicity was noted starting at a concentration of 100 µg test item/mL in the experiment without and with metabolic activation. Hence, 125 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). In a third experiment without and with metabolic activation (4-hour exposure) 500 µg/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut cytotoxicity.

In the main study cytotoxicity was noted starting at a concentration of 125 µg 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. Positive controls induced significant increases in micronuclei in both experiments with/without metabolic activation.

Both in the tests without metabolic activation (4- and 20-hour exposure) and with metabolic activation (4 -h exposure), there was no increase in micronuclei up to the cytotoxic concentrations when compared to control. Under the present test conditions, the test item tested up to cytotoxic concentrations, 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 any chromosomal damage 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 CLP Guidance (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.