Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Acetic acid, oxo-, sodium salt, reaction products with 2-aminoethanol and phenol, sodium hydroxide and iron trichloride (EC 948-113-2)
- Name of the substance for which the testing proposal will be used: Acetic acid, oxo-, sodium salt, reaction products with 2-aminoethanol and phenol, sodium hydroxide and iron trichloride (EC 948-113-2)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: none
- Available non-GLP studies: none
- Historical human data: none
- (Q)SAR: Results from QSAR Toolbox are not conclusive on the potential mutagenicity of Acetic acid, oxo-, sodium salt, reaction products with 2-aminoethanol and phenol, sodium hydroxide and iron trichloride (MEAHA-Fe).
- In vitro methods:
1) in vitro gene mutation study in bacteria (OECD guideline 471): Doses (5 000, 1 500, 500, 150 and 50 µg/plate) prepared from solutions of the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation
2) in vitro Mammalian Cell Micronucleus Test (OECD Guideline 487): aqueous solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride induce a statistically significant increase in the number of micronuclei in comparison with corresponding negative controls both in the absence and in the presence of metabolic activation.
3) in vitro gene mutation study in mammalian cells (OECD Guideline 490): solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the absence and in the presence of metabolic activation.
- Weight of evidence: not conclusive: Acetic acid, oxo-, sodium salt, reaction products with 2-aminoethanol and phenol, sodium hydroxide and iron trichloride yields positive results in both an in vitro mammalian micronucleus test (OECD 487) and an in vitro mammalian cell gene mutation assay (OECD 490). These observations are however not confirmed by chemical structure activity relationships to known germ cell mutagens. In addition, the structural analogues Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts (FeNa-EDDHA, CAS 84539-55-9, EC 283-044-5) and Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts (FeNa-EDDHMA, CAS 84539-53-7, EC 283-041-9) do not show positive results in in vitro mammalian mutagenicity assays (according to OECD 476 and 473).
- Grouping and read-across: No in vivo studies are available for the structural analogue Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts (FeNa-EDDHA, CAS 84539-55-9, EC 283-044-5). A negative in vivo micronucleus assay is available for the structural analogue Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts (FeNa-EDDHMA, CAS 84539-53-7, EC 283-041-9). Due to the different outcome of the in vitro studies between MEAHA-Fe and its structural analogues FeNa-EDDHA and FeNa-EDDHMA, read-across from the available in vivo micronucleus study for FeNa-EDDHMA is considered not fully justified to fill this data requirement for MEAHA-Fe.
- Substance-tailored exposure driven testing: not applicable. The substance is used as a fertiliser (wide dispersive use)
- Approaches in addition to above: not applicable
- Other reasons: not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Section 8.4 of Column 2 of Annex IX or the REACH Regulation states that "If there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant."
Therefore, this test proposal is triggered by the positive in vitro mammalian studies (In vitro Mammalian Cell Micronucleus Test (OECD Guideline 487) and in vitro gene mutation study in mammalian cells (OECD Guideline 490)) for the same substance and the lack of in vivo studies for this substance. Due to the different outcome of the in vitro studies between MEAHA-Fe and its structural analogues FeNa-EDDHA and FeNa-EDDHMA, read-across from the available in vivo micronucleus study for FeNa-EDDHMA is considered not fully justified to fill this data requirement for MEAHA-Fe.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
A standard in in vivo mammalian erythrocyte micronucleus assay according to OECD Guideline 474 is proposed.
Cross-referenceopen allclose all
Reason / purpose:
other: in vitro study with same substance
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Batch no: TROD7BB06
Target gene:
thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse lymphoma L5178Y TK+/-cells (ATCC-CRL-9518) purchased from ATCC (American Type Culture Collection-Rockeville, MD 20852 – USA) have been used successfully in “in vitro” experiments for many years. These cells are characterized by their high proliferation rate (10 h – 12 h doubling time of the stock cultures) and their cloning efficiency, usually more than 50 %. They possess a nearly diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the thymidine kinase (TK) locus which allows to detect mutation events at the TK-locus.
Cells from the cell bank stored at- 80°C are systematically checked to be free from mycoplasma contamination (LEMI operating procedure MB05/02).
To prevent background arising from spontaneous mutation, cells lacking TK have to be eliminated by culturing them in a culture medium (Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAXTM – I) supplemented with 10% (v/v) of inactivated horse serum containing HMTG (Cole et al10): 15 μg/mL hypoxanthine, 0.3 μg/mL methotrexate, 9 μg/mL thymidine, 22.5 μg/mL glycine.
After 24 hours incubation at 37° C in a humidified atmosphere containing 5% (v/v) CO2, the culture is centrifuged (200 x G, 10 min) in order to eliminate methotrexate, and the cell pellet is suspended in medium, without methotrexate, containing HTG (hypoxanthine, thymidine and glycine) and incubated at 37° C in a humidified atmosphere containing 5% (v/v) CO2 for 1 day to 3 days.
Cleaned cells are stored at -80°C. Each cell batch is checked free from mycoplasma contamination. Thawed cultures are maintained in complete culture medium (CCM).
Metabolic activation:
with and without
Metabolic activation system:
S9-mix 2.5% v/v
Test concentrations with justification for top dose:
320- 800- 1400- 2000- 3500 and 5000 μg/mL
Vehicle / solvent:
water
Negative solvent / vehicle controls:
yes
Remarks:
Culture medium DMEN, GlutaMAX
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Mouse lymphoma L5178Y TK+/-cells (ATCC-CRL-9518) purchased from ATCC (American Type Culture Collection-Rockeville, MD 20852 – USA) have been used successfully in “in vitro” experiments for many years. These cells are characterized by their high proliferation rate (10 h – 12 h doubling time of the stock cultures) and their cloning efficiency, usually more than 50 %. They possess a nearly diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the thymidine kinase (TK) locus which allows to detect mutation events at the TK-locus.
Cells from the cell bank stored at- 80°C are systematically checked to be free from mycoplasma contamination (LEMI operating procedure MB05/02).
Evaluation criteria:
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated the Global Evaluation Factor (GEF), which is based on the analysis of the distribution of the negative control MF data from participating laboratories. For the microwell version of the MLA the GEF is 126 x 10-6.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related. The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data should be evaluated by expert judgement and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions [e.g. concentration spacing to increase the probability of attaining data points within the 10-20 % RTG range, using other metabolic activation conditions (i.e. S9 concentration or S9 origin) and duration of treatment] could be useful.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic effects observed at largest test concentration (5000 µg/ml) without metabolic activity. All other doses with and without metabolic activity are compatible with the acceptability criteria.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
1) In the absence of metabolic activation – 4 hours treatment

a) An increase in the mutant frequency is observed.
Moreover, the GEF (Global Evaluating Factor) was calculated in these experimental conditions, since the GEF was recommended by the OECD 490 to help in evaluating the test results (Moore et al12 13 14). The GEF is applied as follows: if the negative control mutant frequency (MF) in a microwell experiment is 100 x 10−6, then one of the treatment groups must have a MF of at least [100+126 (the microwell GEF) = 226] x 10-6 in order to meet the GEF criterion for a positive call.
The above criteria, is met for three concentrations tested 1400 - 2000 and 3500 μg/ml, in the absence of metabolic activation for the short exposure time. The measured MF for these three concentrations is 330.2 - 577.5 and 2000.6 x 10-6.

b) Analysis of the size of colonies shows an increase in induced small colonies from 2 to 1251, and an increase in induced large colony for any concentration tested from 12 to 443

2) In the presence of metabolic activation – 3 hours treatment

a) In the presence of 2.5 % S9-mix, an increase in the mutant frequency is observed.
The above criteria (described in §11.3.1. a), is met for two concentrations tested 3500 and 5000 μg/ml, in the presence of metabolic activation. The measured MF for these two concentration is 270.0 and 318.4 x 10-6 falls above GEF criterion of [126+102.5] 228.5x 10-6

b) Analysis of the size of colonies shows an increase in induced small colonies (Number of induced mutants: from 32 to 166) and in induced large colony (Number of induced mutants: from 16 to 54 for any concentration tested
Conclusions:
In the framework of OECD 490 under the described experimental conditions, solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 provided by TRADE Corporation International SA induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the absence and in the presence of metabolic activation (2.5% S9-mix).
Executive summary:

Two aqueous solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 were tested for their capacity to induce mutagenic activity in L5178Y Mouse Lymphoma cells. No long-term treatment has been conducted, only short-term treatment without or with metabolic activation was carried out according to the acceptability criteria of OECD 490.

320- 800- 1400- 2000- 3500 and 5000 μg/mL of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 were evaluated in contact with the cells in the absence of a metabolic activation system.

320- 800- 1400- 2000- 3500 and 5000 μg/mL of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 were evaluated in contact with the cells in the presence of metabolic activation. (S9-mix 2.5 % (v/v)).

For short-term treatment without or with metabolic activation studies, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of colonies compared to negative controls. These results validate the assays.

In the absence and in presence of the metabolic activation system (S9-mix 2.5 % (v/v)) a concentration-related increase in the mutant frequency was measured in presence of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 .

Conclusion:

In the framework of OECD 490 under the described experimental conditions, solutions obtained from Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 provided by TRADE Corporation International SA induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the absence and in the presence of metabolic activation (2.5% S9-mix).

Reason / purpose:
other: in vitro study with same substance
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21/05/2019 - 02/07/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Batch TROD7BB06
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Origin: ATCC CCL 61, ECACC 85051005
Caryotype: stable
Chromosome modal number: 20
Mycoplasma research: Negative (30.05.2018)
Cell cycle: 17h30
Passage number: 18 (assay 1) - 23 (assay 2)
Maintenance of cell cultures: Mc Coy’s + 10 % FCS
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
assay 1: 224 to 5000 µg/mL
assay 2: 89.6 to 5000 µg/mL
Vehicle / solvent:
water
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine (migrated information)
Details on test system and experimental conditions:
Without metabolic activation:
Short-term treatment (Assay 1): After 4 hours incubation with the test item at 37° C in a humidified atmosphere containing 5 % (v/v) CO2, the culture medium is discarded and the cells washed twice with culture medium. 5 mL of fresh complete culture medium are added and the cells incubated at 37° C in a humidified atmosphere containing 5 % (v/v) CO2. Incubation time is about 1.5 to 2 normal cell cycle lengths after the beginning of the treatment.

Long-term treatment (Assay 2): the cells are incubated with the test item about 1.5 to 2 normal cell cycle lengths at 37° C in a humidified atmosphere containing 5 % (v/v) CO2.

With metabolic activation:
Short-term treatment (Assay 1): After 4 hours incubation at 37° C in a humidified atmosphere containing 5 % (v/v) CO2, the culture medium is discarded and the cells layer washed twice with culture medium. 5 mL of fresh complete culture medium are added and the cells layer incubated at 37° C in a humidified atmosphere containing 5 % (v/v) CO2. Incubation time is about 1.5 to 2 normal cell cycle lengths after the beginning of the treatment.

At the end of the incubation period of about 1.5 to 2 normal cell cycle lengths, cells are harvested as follows:
– culture medium is removed
– cells layer washed once with PBS
– cells are detached (about 2 minutes at 37°C) using 0.5 mL trypsin (0.05 % (w/v) in Hank's balanced solution
Ca2+ and Mg2+ free supplemented with 1 mM EDTA)
– then 4.5 mL of Mc Coy’s supplemented with 5 % (v/v) Fetal Calf Serum (FCS) are added
– 200 μL of cell suspension and 200 μL of trypan blue solution at 0.2 % (w/v) in 0.15 M NaCl are added (incubation for 2 minutes).
– thereafter the living cells are counted using an haemocytometer (Malassez cell - LEMI SOP n°MB08/023).
– the remaining cell suspension is centrifuged (200 g, 6 min)
– hypotonic shock (KCl 0.075 M) at 37° C for 3 minutes
– fixation (1hour to 1 night) using the Carnoy mixture (methanol: acetic acid, 3:1)
– spread on coded microscope slides
– stained using Giemsa stain according to the LEMI SOP n°MB08/023.

Cells are analyzed under a microscope (magnification x1 000) for the detection of micronuclei.
Evaluation criteria:
Relative Increase in Cell Count (RICC)
The RICC corresponds to the relative increase in the number of cells in exposed cultures versus increase in non-treated cultures, a ratio expressed as a percentage.
RICC = Increase in number of cells in treated cultures (final – starting) / Increase in number of cells in control cultures (final – starting) x 100
RICC reduction = 100 - RICC

- “Starting” corresponding to the cell number before incubation (= pre-incubation control).
- For positive controls, RICC must be not less than 50%.
- The maximum concentration to be used for micronuclei interpretation is based on cytotoxicity, the highest concentration should aim to achieve 55 ± 5 % cytotoxicity (i.e. reduction in RICC to 45 ± 5 % of the concurrent negative control).

Detection of micronuclei (Assays n°1 and n°2)
Micronucleus frequency is analysed in at least 2 000 cells (1 000 cells per culture, 2 cultures per concentration) for minimum 3 concentrations of the test item (inducing less than 50% of RICC reduction) and controls.
A micronucleus will be taken into account if it collects the following conditions: - micronucleus should be inside the cytoplasm of cell
- micronucleus is morphologically identical to but smaller than the main nucleus (diameter ≤ 1/3 of the diameter of main nucleus)
- micronucleus should not be retracted
- micronucleus should have round or oval shaped
- micronucleus should not be linked or connected to the main nucleus
- micronucleus may touch main nucleus but twice membranes should be clearly distinct and intact - it may have several micronuclei inside a cell.
The number of cells presenting one, or more, micronuclei is considered as a direct response and evaluated statistically using the 2 test.
The results of cultures treated with different concentrations of the test item and results of positive control are considered significant if P <0.05 comparing to absolute negative control (or solvent control, if any, for the test item).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Absolute negative control
The percentage of cells with micronuclei is equal to 2.0 % for assay 1 and 1.6 % for assay 2 in the absence of metabolic activation and equal to 1.7 % in the presence of metabolic activation.

Positive controls
• Without metabolic activation: Mitomycin C positive control significantly increases the percentage of cells with micronuclei compared to absolute negative control (P < 0.001). This percentage is 18.3 % for assay 1 and 13.8 % for assay 2.
• Without metabolic activation: Colchicin positive control significantly increases the percentage of cells with micronuclei compared to absolute negative control (P < 0.001). This percentage is 15.0 % for assay 2.
• With metabolic activation: Cyclophosphamid positive control significantly increases the percentage of cells with micronuclei compared to absolute negative control (P < 0.001). This percentage is equal to 20.1 % for assay 1.

Test item
Assay n°1 (short-term treatment) without metabolic activation
The solution at 5 000 μg/mL of test item does significantly increase the percentage of cells with micronuclei compared to absolute negative control (P < 0.001). This percentage is 7.1 %.
The solution at 3 500 μg/mL of test item does significantly increase the percentage of cells with micronuclei compared to absolute negative control (P < 0.001). This percentage is 5.0 %.
The solution at 2 000 μg/mL of test item does not significantly increase the percentage of cells with micronuclei. This percentage is equal to 2.9 %.

Assay n°1 (short-term treatment) with metabolic activation
The solution at 3 500 μg/mL of test item does significantly increase the percentage of cells with micronuclei compared to absolute negative control (P < 0.001). This percentage is 4.2 %.
The solution at 2 000 μg/mL of test item does significantly increase the percentage of cells with micronuclei compared to absolute negative control (P < 0.01). This percentage is 3.2 %.
The solution at 1 400 μg/mL of test item does significantly increase the percentage of cells with micronuclei compared to absolute negative control (P < 0.05). This percentage is 2.9 %.

Assay n°2 (long-term treatment) without metabolic activation
The solution at 800 μg/mL of test item does significantly increase the percentage of cells with micronuclei compared to absolute negative control (P < 0.001). This percentage is 3.6 %.
The solution at 560 μg/mL of test item does not significantly increase the percentage of cells with micronuclei. This percentage is equal to 2.3 %.
The solution at 320 μg/mL of test item does not significantly increase the percentage of cells with micronuclei. This percentage is equal to 1.9 %.
Some concentrations of the solution exhibit a statistically significant increase compared with the concurrent negative control and a concentration-reponse relationship is observable. The test item is considered clastogenic and/or aneugenic in this test system (CHO cells).
Conclusions:
According to the criteria of conclusion of the study protocol and OECD 487, aqueous solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 (LEMI Code: LM-19/0083) provided by TRADE Corporation International SA, are considered clastogenic and / or aneugenic in the test system used (CHO cells) in the conditions of the assay
Executive summary:

Aqueous solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) were tested for their ability to induce in vitro micronuclei in cultured CHO cells. This study was carried out in the absence and presence of metabolic activation.

For assay 1, CHO cells were exposed 4 h to test item in the absence or presence of metabolic activation (S9-mix 1.1% final (v/v)).

For assay 2, CHO cells were exposed between about 1.5 and 2 times the normal cell cycle to test item in absence of metabolic activation.

For the two assays, positive and negative controls were carried out in parallel. Both assays’ positive controls induced a statistically significant increase in the number of micronuclei in comparison with corresponding negative controls. The values of negative and positive controls do not show a significant difference with the historical experimental values of the laboratory. Negative controls and positive controls validate the two assays.

According to the criteria of conclusion of the study protocol and OECD 487, aqueous solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 (LEMI Code: LM-19/0083) provided by TRADE Corporation International SA, are considered clastogenic and / or aneugenic in the test system used (CHO cells) in the conditions of the assay.

Reason / purpose:
other: in vitro study with same substance
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20/05/2019 - 20/06/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch TROD7BB06
Target gene:
The term reverse mutation implies the mutation of Salmonella typhimurium strains "histidine-dependent" into "histidine-independent" Salmonella typhimurium strains, and the mutation "tryptophane-dependent" Escherichia coli strain into "tryptophane-independent" Escherichia coli, induced either by the substitution of base pairs, or by the addition, or deletion, of one, or more, DNA base pairs.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Results show no bacteriostatic activity in presence of the doses from 50 to 5000 µg test item/plate. Therefore, the test item was tested at the following doses: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Anthramine, cis-Platinum (II) Diammine Dichloride, 7,12-Dimethyl-benz(a)anthracene
Details on test system and experimental conditions:
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1.9 E09 bacteria/mL and 0.1 mL (in aqueous or oily vehicle) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer were successively added to 2 mL of overlay agar, maintained at 45° C, containing 10 % (v/v) of a L Histidine-D-Biotine solution (0.5 mM).

Escherichia coli strain: in a test tube 0.1 mL of the bacterial suspension containing 1.9 E09 bacteria/mL and and 0.1 mL (in aqueous or oily vehicle) of each dilution of the original solution and 0.5 mL of phosphate buffer were successively added to 2 mL of overlay agar maintained at 45° C, containing 5% (v/v) of nutrient broth n° 2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/mL.

Plates were incubated at 37° C over a 48-72-hour period. The number of revertant colonies per plate was counted.
Evaluation criteria:
The following validity criteria were checked to validate each experiment:

• the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
• the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
• the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
• the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
• Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.

The result of the test is considered positive if a dose-reponse relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.

All results must be confirmed in an independent experiment.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
• There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory (Table 11).
• There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5 000, 1 500, 500, 150 and 50 µg/plate) without and with metabolic activation in (Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA ) (pKM 101).
• Results are confirmed in an independent experiment.
Conclusions:
Doses (5 000, 1 500, 500, 150 and 50 µg/plate) prepared from solutions of the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 (LEMI code: LM 19/0083) provided by TRADE Corporation International SA, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guideline n° 471.
Executive summary:

Aqueous solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvrA¯)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out.

 

For assay n° 1, various concentrations were put in contactwith the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)). 

 

For assay n° 2, various concentrations were put in contactwith the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)). 

 

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory).

 

These results validate the two tests.

 

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (5 000, 1 500, 500, 150 and 50 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯A) (pKM 101).

Doses (5 000, 1 500, 500, 150 and 50 µg/plate) prepared from solutions of the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06  (LEMI code: LM 19/0083) provided by TRADE Corporation International SA, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guideline n° 471.

Data source

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: granular

Results and discussion

Applicant's summary and conclusion