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 cytotoxic concentrations of 316 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 39.06 or 156.3 µg test item/mL without and with metabolic activation. In a key in vitro Micronucleus study in human peripheral lymphocytes, the test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9), respectively, revealed no indications of chromosomal damage in the in vitro micronucleus test.

Link to relevant study records

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Endpoint:
in vitro cytogenicity / micronucleus study
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 testing guidelines, therefore it is considered relevant, adequate and reliable for classification.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 22, 2010
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 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:
* Ham’s F10 supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin (culture establishment)
* Ham’s F10 medium with FCS
* Chromosome medium
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
-Preliminary test: 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium
In this preliminary experiment without and with metabolic activation cytotoxicity was noted starting at a concentration of 156.3 μg test item/mL. Hence, 156.3 μg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.
-Main test: 9.77, 19.53, 39.1, 78.1 or 156.3 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
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.2 µg/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: colchicine
Remarks:
0.02 µg/mL, without S9-mix
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
20 µg/mL, with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
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); 40 hours (without S9-mix)

SPINDLE INHIBITOR (cytogenetic assays): 5 µg/mL Cytochalasin B
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: 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.

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytokinesis-Block Proliferation Index
The Cytokinesis-Block Proliferation Index (CBPI) indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells))
CBPI = -------------------------------------------------------------------------------
(Total number of cells)
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Evaluation criteria:
1000 binucleated cells per duplicate cell culture were scored 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.
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, using a chi-square test corrected for continuity according to YATES as recommended by the UKEMS guidelines.
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.
Statistics:
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971[1]) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality:
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 pH or osmolality of the formulations were noted.
- Water solubility:
The test item was completely dissolved in aqua ad iniectabilia.
- Precipitation:
Any possible test item precipitation was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary experiment without and with metabolic activation test item concentrations of 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. Cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL were employed as the top concentration for the mutagenicity tests without and with 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
Standard deviation: 2.0
Range: 1-9
Vehicle control (n = 8):
Mean: 7.2
Standard deviation: 4.6
Range: 1-18
Positive control: Mitomycin C (n = 7):
Mean: 95.8
Standard deviation: 66.1
Range: 24-286
Positive control: Colchicine (n = 7):
Mean: 25.4
Standard deviation: 10.2
Range: 7-43
With metabolic activation (4-h exposure):
Vehicle control (n = 8):
Mean: 10.8
Standard deviation: 6.2
Range: 2-25
Positive control: Cyclophosphamide (n = 7):
Mean: 60.3
Standard deviation: 37.8
Range: 20-147


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

Under the present test conditions, the test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of 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.
Executive summary:

Test sample of Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts 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 completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. 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 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. Cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL was employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the main study cytotoxicity was noted at the top concentration of 156.3 µg/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.

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

The micronucleus frequencies of cultures treated with the test item at concentrations of 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4 h and 20-h exposure) in the absence of metabolic activation ranged from 5.5 to 15.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 6.0 or 12.0 micronuclei per 1000 binucleated cells, untreated controls: 4.0 or 9.0 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 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4-h exposure) in the presence of metabolic activation ranged from 6.0 to 18.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 3.5 or 13.0 micronuclei per 1000 binucleated cells, untreated controls: 4.5 or 8.5 micronuclei per 1000 binucleated cells). Vehicle and untreated control values fell within acceptation ranges.

 Under the present test conditions, the test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of 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.

Endpoint:
in vitro gene mutation study in mammalian cells
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 testing guidelines, therefore it is considered relevant, adequate and reliable for classification.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
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. 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.
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes. 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 test: 19.53, 39.06, 78.13, 156.3, 312.5, 625, and 1250 µg test item/mL
Main test without S9-mix: 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL
Main test with S9-mix: 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 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
The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.

DURATION
- Preincubation period: 24 hours
- 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: cytotoxicity test) or 12 days (6-thioguanine plates: mutagenicity test).

SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL);
STAIN (cytogenetic assays): After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted.

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 is determined for each dose to obtain an accurate measure of the toxic effect of the chemical.
Evaluation criteria:
So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here (see UKEMS guidelines for discussion). Following pre¬determined descriptive criteria are used for interpretation of the results:
-lf in both independent experiments solvent and positive controls show results within the norm and if the test compound does not increase the mutation, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1 000 000 cells per condition have been evaluated, the compound is considered as negative in the test.
-In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the compound is considered as positive in the test.
Equivocal results, if applicable are clarified by further testing, in agreement with Sponsor and Study Monitor.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in the pH values in the medium were noted.
- Effects of osmolality: No relevant changes in osmolality of the formulations were noted.

RANGE-FINDING/SCREENING STUDIES:
- In the preliminary study cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 39.06 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.
- The next higher concentrations resulted in complete cytotoxicity in the preliminary experiment. In addition, the number of mutants was also already considerable decreased in the main experiments at the highest employed concentrations pointing to general pronounced cytotoxicity. Finding a higher concentration with viable and evaluable cells/mutants was therefore not considered realistic.
- 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 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106 survivors in non-activation solvent controls and 6 to 46 per 106 survivors in S9 activation solvent controls.
The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium without metabolic activation ranged from 3.78 to 15.74 x 10-6 clonable cells. These results are within the normal range of the vehicle controls.
The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture mediumwith metabolic activation ranged from 4.44 to 13.04 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 475.00 to 780.00 x 10-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 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 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA.



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 1.05 was used to correct for the purity of the test item.  The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary study test item concentrations of 19.53, 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL medium were employed . Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 39.06 µg test item/mL was employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.

Main study

Five concentrations 2.44, 4.88, 9.77, 19.53 or 39.06 and 9.77, 19.53, 39.06, 78.13 or 156.3 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 39.06 or 156.3 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 14.35 and 16.67 x 10-6 clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture medium ranged from 4.44 to 13.04 x 106 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 17.87 and 16.05 x 10-6 clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium ranged from 3.78 to 15.74 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 475.00 to 780.00 x 10-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 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 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells for EMS and 130.0 to 2693.3 x 10-6 clonable 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:
in vitro gene mutation study in bacteria
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 testing guidelines, therefore it is considered relevant, adequate and reliable for classification.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guideline S2 (R1): 'Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
Version / remarks:
Current step 4 version dated November 9, 2011
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-mix
Test concentrations with justification for top dose:
-cytotoxicity test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate
The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at concentrations of 316 μg/plate and higher. Hence, 316 μg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
-mutagenicity test: 1.0, 3.16, 10.0, 31.6, 100 and 316 µg test item/plate (plate incorporation and preincubation)
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
Positive controls:
yes
Positive control substance:
other: Sodium azide in aqua ad iniectabilia
Remarks:
(10 µg/plate):TA 1535, TA 100, without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-Nitro-fluorene in DMSO
Remarks:
(10 µg/plate): TA 98, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9-Amino-acridine in ethanol, abs.
Remarks:
(100 µg/plate): TA 1537, without S9-mix
Positive controls:
yes
Positive control substance:
other: Mitomycin C in DMSO
Remarks:
( 10 µg/plate): TA 102, without S9-mix
Positive controls:
yes
Positive control substance:
other: Benzo(a)pyrene in DMSO
Remarks:
( 10 µg/plate): TA 98, TA 102, TA 1537, with S9-mix
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene in DMSO
Remarks:
(2-4 µg/plate): TA 100, TA 1535, with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
1st independent experiment : in agar (plate incorporation);
2nd independent experiment : preincubation.

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

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity is evidenced by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or by the degree of survival of treatment cultures.
Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a sparse background lawn.
Evaluation criteria:
The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA 98, TA 100 and TA 102 and 3-fold of the vehicle control for TA 1535 and TA 1537 in both independent experiments;
Or
- a concentration-related increase of the revertants is observed (Spearman’s rank correlation coefficient).
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considerd as non-mutagenic in the AMES test.
Statistics:
number of revertants compared with the vehicle control (p ≤ 0.05, U-test according to MANN and WHITNEY)
concentration-related increase of the revertants (Spearman’s rank correlation coefficient)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 316 µg/plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: mutagenicity: all strains tested; cytotoxicity TA 100 without S9-mix
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The test item was examined in the 5 Salmonella 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 item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. Aqua ad iniectabilia was used as vehicle control.

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants)was noted at concentrations of 316 µg/plate and higher. Hence, 316 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Six concentrations of 1.0, 3.16, 10.0, 31.6, 100 and 316 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 316 µgtest item/plate in all test strains.
No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 316 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial mutagenicity

- In a supporting study, the test item containing >=90% active ingredient was tested for mutagenic activity in the bacterial tester strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98 with and without metabolic activation (Wallat, 1986). Concentrations tested were 8, 40, 200, 1000 and 5000 µg per plate in a first and second test. The test substance did not induce reverse mutations in the presence and absence of metabolic activation in all tested strains.

- A key study was further conducted with test item containing >95% active ingredient in 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (Flügge, 2013c). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. In a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test, ten concentrations of 0.316 up to 5000 µg test item/plate were tested. Cytotoxicity was noted at concentrations of 316 µg/plate and higher. Hence, 316 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. Six concentrations of 1.0, 3.16, 10.0, 31.6, 100 and 316 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 316 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). Under the present test conditions the test item tested up to a cytotoxic concentration of 316 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.

 

In conclusion, negative results were obtained for bacterial mutagenicity in a key study with registered substance tested in 5 strains with and without metabolic activation.

 

Mammalian mutagenicity

Test item containing >=95% active ingredient was tested in cultured mammalian cells (V79, genetic marker HPRT) both in the presence (4 hours) and absence (4 and 24 hours) of metabolic activation (key study; Flügge, 2013d). The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. Aqua ad iniectabilia served as the vehicle control.

Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 and 9.77, 19.53, 39.06, 78.13 or 156.3 µg act.ingr./mL were selected for the main 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 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively. Both in the experiments with and without metabolic activation, the mutation frequencies of treated cell cultures within the normal range of the vehicle controls. The positive controls caused a pronounced increase in the mutation frequencies, indicating the validity of this test system. 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 the registered substance tested in V79 cells with genetic marker HPRT.

 

Chromosome aberration

Test item containing >95% active ingredient 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 (key study; Flügge, 2013e). 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 test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. Aqua ad iniectabilia served as the vehicle control. In the preliminary experiment , cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation. In the main study cytotoxicity was noted at the top concentration of 156.3 µg/mL in the experiments without and with metabolic activation. 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. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Under the present test conditions, the test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. In conclusion, negative results were obtained for chromosome aberration in a key study with registered substance tested in an in vitro Micronucleus test in human peripheral lymphocytes.

 

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  the bacterial gene mutation, mammalian gene mutation and chromosomal aberration tests are equivalent key endpoints.

Justification for classification or non-classification

As there was no indication for genotoxic potential, classification for genotoxicity is not warranted according to CLP (No. 1272/2008 of 16 December 2008).