Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Read across data were available from ‘Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite’ 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 µL/plate. Gene mutation was also negative in the HPRT-V79 mammalian cell mutagenicity test in HPRT cells in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 1000 µg/mL. 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. Based on structural similarity, the registered substance is also considered to have no mutagenic or clastogenic potential.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods and is considered relevant 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, 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).
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 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.
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 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 10-6clonable 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 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 10-6clonable cells in the case of EMS and ranging from 586.36 to 850.00 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 10-6clonable 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)

Additional information

Additional information from genetic toxicity in vitro:

No test data were available for current substance, however read across data were available from '‘Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite’. Justification for read across is provided in a separate document attached in Section 13.

 

Bacterial reverse mutation
In a key in vitro genetic toxicity study, the read across 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, no mutagenic effect was exerted in bacterial strains without and with metabolic activation, therefore no mutagenic potential is expected for registered substance.

 

Mammalian gene mutation
A key study with the read- across 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 read across 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, no mutagenic effect was exerted in mammalian V79 cells without and with metabolic activation, therefore no mutagenic potential is expected for registered substance.

 

Chromosome aberration

A key Micronucleus test with read-across 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 iniectabiliaserved 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 concenrtations.
In conclusion, no chromosomal aberration was exerted in human peripheral lymphocytes without and with metabolic activation, therefore no clastogenic potential is expected for registered substance

 

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 selection of genetic toxicity endpoint
Although the bacterial gene mutation study was selected, the mammalian gene mutation and chromosomal aberration tests are equivalent endpoints.

Justification for classification or non-classification

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.

Categories Display