Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data from key studies were available for registered substance . Gene mutation was tested to be negative in an Ames bacterial reverse mutation assay in strains TA98, TA100, TA1535 and TA1537 with and without S9 up to cytotoxic concentrations of 25 µg/plate in the absence and presence of metabolic activation. Gene mutation was also negative in the HPRT-V79 mammalian cell mutagenicity test when tested up to cytotoxic concentrations of 1000 µg/mL in the absence and presence of metabolic activation. In the micronucleus test in human peripheral lymphocytes the registered substance tested negative for chromosome aberration when tested up to cytotoxic concentrations of 500 µg/mL in the absence and 1000 µg/mL in the presence of metabolic activation.

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:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
4 strains instead of 5 used
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29th November, 1992
Deviations:
yes
Remarks:
4 strains instead of 5 used
Principles of method if other than guideline:
Four strains instead of five.
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 and TA 100
Additional strain / cell type characteristics:
other: The strains TA98 and TA100 have an additional plasmid (pkM 101) which confers resistance against ampicillin. This plasmid also carries a gene (muc+), which is involved in recA+/lexA- genotypes in the SOS-DNA- repair system.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0.000; 0.780; 1.560; 3.125; 6.250; 12.500; 25.000; 50.000; 100.000; 200.00µL/plate (TA100)
1st Assay (Table II): 0.00; 0.04, 0.20, 1.00, 5.00 and 25.00 µL per plate (TA1535, TA1537, TA98, TA100 all +S9 and –S9)
2nd Assay (Table III): 0.00; 0.30; 0.93; 2.78; 8.33 and 25.00 µL/plate (TA1535, TA1537, TA98, TA100 all +S9 and –S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (Aqua dest.)
- Justification for choice of solvent/vehicle: test substance dispersable in water
Untreated negative controls:
yes
Remarks:
for all strains with as well as without S-9 mix
Negative solvent / vehicle controls:
yes
Remarks:
for all strains with as well as without S-9 mix
Positive controls:
yes
Positive control substance:
other: Na-azide 0.25 µL/plate
Remarks:
TA100 and TA1535 ; -S9
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 ; -S9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 ; -S9
Positive controls:
yes
Positive control substance:
other: 2-aminoantrhacene 0.5 µg/plate
Remarks:
TA100, TA98 ; +S9
Positive controls:
yes
Positive control substance:
other: 2-aminoantrhacene 2.0 µg/plate
Remarks:
TA1535 and TA1537; +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days (at 37°C in the dark)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: other: The criterion for a biologically significant bacteriotoxic effect is either a reduction of the survival of the cells to at least 50% or a reduction of the background growth of auxotrophic cells and the number of revertants.



Evaluation criteria:
In the Ames assay a test compound is considered as mutagenic if there is a dose dependent increase in the number of revertant colonies of one or more strains. For the highest non-toxic concentration an increase should be by a factor of about 2 and deemed to be biologically relevant. Any evidence of mutagenic activity must be reproducible in an independent experiment. In addition statistical evaluation may aid data interpretation.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not in main study, however in the range finding study 25.00µL per plate (highest tested concentration in main study) reduced the survival to 5.3% of the control value.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Preliminary Toxicity Test: 0.000; 0.780; 1.560; 3.125; 6.250; 12.500; 25.000; 50.000; 100.000; 200.00µL/plate (TA100)
The test compound [Trade name] reduced the survival at a concentration of 25 µl per plate to 5.3 % of the control value.
An increase in numbers of his+-revertants over the control value at a concentration of 200 µl per plate was due to histidine-auxotroph cells which were able to grow at reduced cell density.
Therefore the mutagenicity assays were performed in a concentration range from 0.04 to 25.00 µl per plate.

MAIN TEST
Concentrations between 0.04 and 25.00 µl/plate were applied to the bacterial strains TA 1535, TA 1537, TA 98 and TA 100.

No evidence of biologically significant mutagenic activity of [Trade name] was found. The results of the 1st assay and the 2nd assay indicate, that in the tested concentration range with and without metabolic activation no significant increase in the numbers of his+-revertants over the spontaneous values could be detected with the Salmonella tester strains TA 100, TA 1535, TA 98 and TA 1537.

No bacteriotoxic effects were induced at the tested concentrations.

STERILITY CONTROLS
The plates for the sterility control of top agar, S9-mix and test compound showed no growth.

NEGATIVE CONTROLS
In all experiments the control plates without mutagen showed a normal number of spontaneous revertants

POSITIVE CONTROLS
The positive controls demonstrated the sensitivity of the indicator strains and the activity of the metabolizing system.

COMPARISON WITH HISTORICAL CONTROL DATA: yes: Historical Spontaneous Mutation frequencies to Histidine-Protrophy of the Tester-Strains used in Labor L+S AG in 1993.


Remarks on result:
other: all strains/cell types tested
Conclusions:
Evaluation of the results of the two independent experiments did not provide evidence of a mutagenic potency of the test item.
At up to 25.0 µL per plate (cytotoxic concentration), the test item, did not induce biologically relevant increases in revertants in any of the tester strains. This applied to both the activated and the non-activated assay system.
The test item is therefore not found to be genetically active under the aforementioned test conditions.
Executive summary:

The test item containing 39.8% active ingredient was assayed in a bacterial gene mutation assay (Ames test) using the strains TA100, TA1535, TA98 and TA1537. Induced his+-revertants were determined both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9) from Aroclor 1254 induced animals. In two independent mutation experiments, cells were exposed to concentrations of 0.04, 0.20, 1.00, 5.00 and 25.00 (cytotoxic concentration) µL per plate (2nd assay) in the absence and presence of S9. In order to demonstrate the sensitivity of the assay system, positive control agents were used and marked increases in his+-revertants were induced in all tester-strains. In the 1st and 2nd assay the test item induced neither cytotoxicity nor statistically significant increases in histidine-protrophy revertants in any tester-strains with and without metabolic activation in the tested concentration range.

It is thus concluded that the test item did not induce gene mutations in the bacterial mutagenicity assay with and without metabolic activation in vitro when tested under the experimental conditions reported.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 22, 2010
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 male or female 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:
* 5 mL of Chromosome complete culture medium and 1% Penicillin/Streptomycin
* After 48 hours the medium was replaced by 4.95 mL of fresh Ham’s F10 medium with FCS and 50 µL of the vehicle control, the 5 concentrations of the test item or the 2 concentrations of the positive control (Mitomycin C, Colchicine) were added to each culture.
* Afterwards the medium was removed and the cultures were washed two times with Ham’s F10 medium. After addition of 5 mL Chromosome medium containing 5 µg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 10, 25, 100, 250, 1000, 2500 and 4020 µg test item/mL medium
Main test with S9-mix: 125, 250, 500 or 1000 µg test item/mL medium (at 2500µg test item/mL there were no cells of sufficient quality due to cytotoxicity of the test item)
Main test without S9-mix: 62.5, 125, 250 or 500 µg test item/mL medium (at 1000µg test item/mL there were no cells of sufficient quality due to cytotoxicity of the test item)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the other solvents recommended.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.2 µ/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
Experiment 1
- Exposure duration: 4 hours (with and without S9-mix)
- Selection time (if incubation with a selection agent): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
Experiment 2:
- Exposure duration: 4 hours (with S9-mix) or 20 hours (without S9-mix)
- Selection time (if incubation with a selection agent): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (with S9-mix) and 40 hours (without S9-mix)

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

NUMBER OF REPLICATIONS: Duplicate cultures were used for each test item concentration and for the vehicle and positive control cultures.

NUMBER OF CELLS EVALUATED:
The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytokinesis-Biock Proliferation Index, Replicative Index
CBPI = ((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells)) /(Total number of cells)
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
1000 binucleated cells per duplicate cell culture were scored with a phase contrast microscope (optical magnification of 600)to assess the frequency of cells with one, two, or more than two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.
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 vehicle 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.
The assessment was carried out by a comparison of the samples with the positive and the vehicle control.
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.
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).
Key result
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 and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
Osmolality: with a semi-micro osmometer.
The pH value of the test item supplied was measured as 5.62.
No relevant changes in pH of the formulations were noted.
- Effects of osmolality:
No relevant changes in osmolality of the formulations 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 4020 µg test item/mL medium were employed. Pronounced to complete cytotoxicity was noted at concentrations of 1000 µg test item/mL and higher in the experiment with metabolic activation. In the experiment without metabolic activation concentrations of 1000 µg test item/mL and higher caused complete cytotoxicity. Hence, 2500 µg/mL was employed as the top concentration for the mutagenicity tests with metabolic activation and 1000 µg test item/mL in the tests without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 8 or 7 studies (most recent background data, not audited by the QAU-department) are given as follows:
Micronucleus frequency per 1000 cells:
Without metabolic activation(4-h or 20-h exposure)
Untreated control (n = 8)
mean: 4.9
SD: 2.0
range: 1-9
Vehicle control (n = 8)
mean: 7.2
SD: 4.6
range: 1-18
Mitomycin C Positive control (n=7)
mean: 95.8
SD: 66.1
range: 24-286
Colchicine Positive control (n = 7)
mean: 25.4
SD: 10.2
range: 7-43

With metabolic activation (4-h exposure)
Vehicle control (n = 8)
mean: 10.8
SD: 6.2
range: 2-25
Cyclophosphamide Positive control (n = 7)
mean: 60.3
SD: 37.8
range: 20-147

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study cytotoxicity was noted starting at concentrations of 500 or 1000 µg/mL in the experiments without and with metabolic activation, respectively.
Conclusions:
Under the present test conditions, Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite 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 chromosomal damage in the in vitro micronucleus test.
Executive summary:

Test sample of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite was 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: 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 20 hours after the end of exposure. The study was conducted in duplicate.
The test item was diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. 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 4020 µg test item/mL medium were employed. Pronounced to complete cytotoxicity was noted at concentrations of 1000 µg test item/mL and higher in the experiment with metabolic activation. In the experiment without metabolic activation concentrations of 1000 µg test item/mL and higher caused complete cytotoxicity. Hence, 2500 µg/mL was employed as the top concentration for the mutagenicity tests with metabolic activation and 1000 µg test item/mL in the tests without metabolic activation.
In the main study cytotoxicity was noted starting at concentrations of 500 or 1000 µg/mL in the experiments without and with metabolic activation, respectively.
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 the test item at concentrations of 62.5, 125, 250 or 500 µg test item/mL medium (4-h or 20-h exposure) in the absence of metabolic activation ranged from 0.5 to 8.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentrations. The dose level of 1000 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation.
Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were observed: vehicle control: 7.5 or 4.0 micronuclei per 1000 binucleated cells and untreated controls: 8.0 or 4.5 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively). Vehicle and untreated control values fell within acceptation ranges.


Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with the test item at concentrations of 125, 250, 500 or 1000 µg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 3.0 to 8.5 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. The dose level of 2500 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation.
Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were observed: vehicle control: 5.0 micronuclei per 1000 binucleated cells and untreated controls: 5.0 or 6.0 micronuclei per 1000 binucleated cells. Vehicle and untreated control values fell within acceptation ranges.
Under the present test conditions, Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite 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 chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
published in the Official Journal of the European Union L 142, dated May 31, 2008
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, 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 test: 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca. 4600 µg/mL medium)
Main test: 62.5, 125, 250, 500 and 1000 µg solid matter/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was diluted with aqua ad iniectabilia. The test item was not soluble in any of the other solvents recommended.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate dissolved in DMSO
Remarks:
600 and 700 µg EMS/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9,10-dimethyl-1 ,2-benzanthracene dissolved in DMSO
Remarks:
20 and 30 µg DMBA/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.


NUMBER OF CELLS EVALUATED: 1000

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 test item 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.
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).
Key result
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 concentration of 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 and osmolality:
The pH value of the test item supplied was measured to be 5.62.
The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
-pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
-Osmolality: with a semi-micro osmometer .
No relevant changes in the pH or osmolality values of the formulations were noted.
- Water solubility:
The test item was diluted with aqua ad iniectabilia. The test item was not soluble in any of the other solvents recommended.

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 concentrations of 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca 4600 µg/mL medium) were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 1000 µg/mL medium in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 1000 µg solid matter/mL were employed as the top concentration for the mutagenicity tests in the absence and 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 by Bradley et al. (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 mutation frequencies of the solvent controls and the positive controls without and with metabolic activation for the last 58 experiments at LPT (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 106 clonable cells
Without metabolic activation (24-h exposure)
Solvent control (n = 58)
Mean: 14.11
SD: 7.42
Range: 1.30 – 34.80
Positive control (µg/mL) (n = 58) EMS (600)
Mean: 449.1
SD: 444.2
Range: 112.10 - 1708.4
Positive control (µg/mL) (n = 58) EMS (700)
Mean: 468.4
SD: 268.6
Range: 152.0 – 976.9

With metabolic activation(4-h exposure)
Solvent control (n = 58) Mean: 14.88
SD: 8.20
Range: 2.18-38.36
Positive control (µg/mL) (n = 58) DMBA (20)
Mean: 347.1
SD: 241.8
Range: 130.0 – 844.8
Positive control (µg/mL) (n = 58) DMBA (30)
Mean: 563.8
SD: 700.1
Range: 151.3 – 2693.3


ADDITIONAL INFORMATION ON 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 concentration of 1000 µg/mL in the absence and presence of metabolic activation, respectively.
Conclusions:
Under the present test conditions, the active ingredient 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 by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. 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 diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. 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 concentrations of 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca. 4600 µg/mL medium) were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 1000 µg/mL medium in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 1000 µg solid matter/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of metabolic activation.

Main study

Concentrations of 62.5, 125, 250, 500 or 1000 µg solid matter/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 concentration of 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 10.79 and 9.84 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 solid matter/mL culture medium ranged from 5.44 to 10.34 x 10-6 clonable 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 10.93 and 11.61 x 10-6 clonable 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 solid matter/mL culture medium ranged from 4.91 to 12.97 x 10-6 clonable 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 478.40 to 682.86 x 10-6 clonable cells in the case of EMS and ranging from 586.36 to 850.00 x 10-6 clonable 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 10-6 clonable cells for EMS and 130.0 to 2693.3 x 10-6 clonable cells for DMBA.

Under the present test conditions, the active ingredient 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 adverse effect observed (negative)

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

Additional information from genetic toxicity in vitro:  

Bacterial reverse mutation

In a key in vitro genetic toxicity study, the registered test substance containing 39.8% act.ingr. was assayed in a bacterial gene mutation assay (Ames test) using the strains TA100, TA1535, TA98 and TA1537 both in the absence and presence of metabolic activation (Grötsch and Sonnenschein, 1995). In two independent mutation experiments, cells were exposed to concentrations of 0.04, 0.20, 1.00, 5.00 and 25.00 (cytotoxic concentration) µL per plate in the absence and presence of S9. In both assays the test item induced neither cytotoxicity nor statistically significant increases in histidine-protrophy revertants in any tester-strains with and without metabolic activation in the tested concentration range. It was thus concluded that the test item did not induce gene mutations in the bacterial mutagenicity assay with and without metabolic activation in vitro when tested under the experimental conditions reported. In conclusion, negative results were obtained for bacterial mutagenicity up to cytotoxic concentrations in a key study with registered substance.

 

Mammalian gene mutation

A key study with registered substance 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, 2013c). In the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 µg solid matter/mL medium and the undiluted test item (ca. 4600 µg/mL medium) were employed. The test item was diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. Aqua ad iniectabilia served as the vehicle control. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 1000 µg/mL medium in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 1000 µg solid matter/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of metabolic activation Five concentrations 62.5, 125, 250, 500 or 1000 µg solid matter/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 concentration of 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 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.  In conclusion, negative results were obtained for mammalian mutagenicity in a key study with registered substance tested up to cytotoxic concentrations.

 

Chromosome aberration

A key Micronucleus test with registered substance was conducted in human peripheral lymphocytes both in presence (4 hours) and in absence (4 and 20 hours) of metabolic activation (Flügge, 2013d). In the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4020 µg test item /mL medium were employed. The test item was diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. Aqua ad iniectabilia served as the vehicle control. Pronounced to complete cytotoxicity was noted at concentrations of 1000 µg test item/mL and higher in the experiment with metabolic activation. In the experiment without metabolic activation concentrations of 1000 µg test item/mL and higher caused complete cytotoxicity. Hence, 2500 µg/mL was employed as the top concentration for the mutagenicity tests with metabolic activation and 1000 µg test item/mL in the tests without metabolic activation. In the main study cytotoxicity was noted starting at concentrations of 500 or 1000 µg/mL in the experiments without and with metabolic activation, respectively. Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation. The micronucleus frequencies of cultures treated with the test item at concentrations of 62.5, 125, 250 or 500 µg test item/mL medium (4-h or 20-h exposure) in the absence of metabolic activation ranged from 0.5 to 8.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentrations. The dose level of 1000 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation. Vehicle and untreated control values fell within acceptation ranges. The micronucleus frequencies of cultures treated with the test item at concentrations of 125, 250, 500 or 1000 µg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 3.0 to 8.5 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. The dose level of 2500 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation. Vehicle and untreated control values fell within acceptation ranges. Under the present test conditions, Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite 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 chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.  In conclusion, negative results were obtained for chromosome aberration in a key study with registered substance tested up to cytotoxic concentrations.

 

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 (No. 1272/2008 of 16 December 2008), the test item does not have to be classified and has no obligatory labelling requirement for genetic toxicity.