Registration Dossier
Registration Dossier
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 610-945-6 | CAS number: 53037-34-6
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 08 August 2016 to 22 August 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Oligomerisation reaction products of glyoxal and urea.
- EC Number:
- 610-945-6
- Cas Number:
- 53037-34-6
- Molecular formula:
- Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
- IUPAC Name:
- Oligomerisation reaction products of glyoxal and urea.
- Details on test material:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: provided by the Sponsor
- Expiration date of the lot/batch: 06/11/2016.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: solvent water, maximal concentration in the solvent 94.5 g/l, stability in the solvent 48 hours
Constituent 1
Method
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- other: rfa mutated
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix of liver rats
- Test concentrations with justification for top dose:
- 5000, 1600, 500, 160 and 50 μg/plate. The preliminary study showed no cytotoxicity of the test item at the highest dose tested (5000 μg/plate); therefore this concentrations range was used for the mutagenicity test.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: deionized water. - Justification for choice of solvent/vehicle: A preliminary dissolution test was performed in order to define the most appropriate solvent as well as the maximal concentration tested.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- mitomycin C
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:: - Direct plate incorporation method (Test 1): a first experiment of genotoxicity was conducted, with and without metabolic activation, on the range of concentrations defined by the preliminary study: 2 ml of top agar, 0.1 ml of bacterial inoculum and 0.1 ml of the test item with/without S9-mix (0.5 ml of S9-mix or 0.5 ml buffer) were mixed and poured on the surface of the bottom agar. After solidification, plates were incubated at 37 °C ± 2 °C during 48 to 72 hours. Positive and negative controls were included in the experiment. After the mentioned period, colonies were counted. - Pre-incubation method (Test 2): a second experiment was performed, also with and without metabolic activation, with dose levels defined after analysis of results obtained on the first experiment. This second experiment was performed in order to confirm or for complement results of the first one: 0.1 ml of bacterial inoculum and 0.1 ml of test item with/without S9-mix (0.5 ml of S9-mix or 0.5 ml buffer) were mixed and incubated at 37ºC ± 2 °C to 30 min. After this period, 2 ml of top agar was added and mixed. The mixture was poured on the surface of the bottom agar and plates were incubated at 37 °C ± 2 °C during 48 to 72 hours. Positive and negative controls were included in the experiment. After the mentioned period, colonies were counted.- Cell density at seeding: it was applied 0.1ml of bacterial inoculum containing 10^8-9 cells/ml.DURATION- Preincubation period: about 20-30min at 37ºC (+/-2ºC), only for the Test 2- Exposure duration: 48-72hSELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.NUMBER OF REPLICATIONS: 3DETERMINATION OF CYTOTOXICITYPetri plates were observed and any cytotoxicity sign was reported (bottom bacterial layer reduction). The cytotoxicity intensity on the bottom bacterial layer is evaluated qualitatively on each plate by naked eyes:a. total destruction of the bottom bacterial layer (the revertants development does not occurs in this case).b. moderated destruction of the bottom bacterial layer.In the preliminary experiment is considered the test item as cytotoxic if the spontaneous revertant rate is lower than 0.7 (R < 0.7). The possible destruction of the bottom bacterial layer is also taken into account.OTHER:- Methodology of the preliminary experiment (direct plate incorporation method): it was performed on the strain S. typhimurium TA100 in order to evaluate the cytotoxicity of the test item and to select the range of dose levels for the further experiments: Top agar, buffer, bacterial inoculum and the test item with/without S9-mix were mixed and poured on the surface of the bottom agar. After solidification, plates were incubated at 37 °C ± 2 °C during 48 to 72 hours. Positive and negative controls were included in the experiment. After the mentioned period, colonies were counted.- Method of counting: All colony numbers were determined by a plate reader (Sorcerer, v 2.2)
- Evaluation criteria:
- The test item is considered as mutagenic if at the end of the verifications steps, it has been obtained, in a reproducible way, a relation dose-effect on one or some of 5 strains with and/or without metabolic activation. The mutagenicity is taken into account for a given concentration only when the number of revertants is equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 strains (R >= 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 strains (R >= 3).The test item is considered as non-mutagenic if, in the outcome at the Test 1 and the Test 2, the rate of revertants always remained lower than the double of the rate of spontaneous reversion for all the concentrations of tested product, for TA98, TA100 and TA102 strains (R < 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 strains (R < 3), with and without metabolic activation, and on the condition of having made sure that the absence of mutagen effect is not bound to the toxicity of the experimented concentrations.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- at 5000 μg/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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
- Untreated negative 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
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative 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
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: no sign of precipitate was observed.RANGE-FINDING/SCREENING STUDIES: The preliminary cytotoxicity test performed on the TA100 strain showed no cytotoxicity of the test item at the highest dose tested (5000 μg/plate) (R >= 0.7). The same range of concentrations was used for the Test 1 and 2.HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data:Without S9TA98: range 746.3-1531.3, mean 1095.7, SD 163.5 (n=77)TA100: range 1663.0-3329.7, mean 2131.4, SD 288.4 (n= 80)TA102: range 1737.7-3921.3, mean2737.5, SD 578.3 (n=71)TA1535: range 1659.3-2803.3, mean2154.9, SD 226.7 (n=73)TA1537: range 117.7-1082.0 , mean 333.1, SD 196.0 (n=74) With S9TA98: range 931.0-3686.3 , mean 2047.5, SD755.5 (n=77)TA100: range 923.3-4667.0, mean 2592.7, SD 988.7 (n=76) TA102: range 1955.7-6017.3, mean 3631.8, SD 799.6 (n=72)TA1535: range 151.0-433.7, mean 250.1, SD 76.5 (n=73)TA1537:range 134.7-506.7, mean 275.7, SD 101.6 (n=75)- Negative (solvent/vehicle) historical control data:Without S9TA98: range 16.7-42.0, mean 26.1, SD 4.2 (n=192)TA100: range 49.7-220.3, mean 142.8, SD 43.9 (n=) TA102: range 275.3-461.7, mean 370.9, SD 29.3 (n=183)TA1535: range 10.0-38.0, mean 21.2, SD 5.9 (n=183)TA1537:range 9.3-28.0, mean 17.9, SD 3.8 (n=189)With S9TA98: range 24.7-56.7, mean 34.7, SD 5.3 (n=194)TA100: range 58.3-233.0, mean 164.8, SD 44.4 (n=198) TA102: range 331.7-526.7, mean 433.7, SD 36.2 (n=179)TA1535: range 12.7-38.3, mean 21.5, SD 5.9 (n=182)TA1537:range 12.0-37.0, mean 22.9, SD 4.4 (n=186)The positive controls must show a revertants number equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 (R >= 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 (R >=3).The mean negative controls are within the historical data.All criteria of validity regarding control plates were met.ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxic effect was observed. Ratios 0.6 shown during Test 1 on strain TA1535 at 160 and 50 μg/plate without metabolic activation must not be considered according ratios observed at the upper concentrations.
- Remarks on result:
- other: Test 2
Any other information on results incl. tables
TEST 1: Results for each strain and dose
Concentrations (μg/plate) | Mean values of revertants / Ratio treated – solvent (R) | Salmonella Thypimurium strains | |||||||||
|
| TA 100 | TA 102 | TA1535 | TA 1537 | TA 98 | |||||
|
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
5000 | Mean | 283.3 | 259.3 | 436.7 | 432.0 | 19.3 | 19.0 | 19.0 | 28.3 | 47.3 | 49.0 |
R | 1.5 | 1.3 | 1.1 | 1.0 | 0.8 | 1.1 | 1.1 | 0.9 | 1.3 | 1.3 | |
1600 | Mean | 207.3 | 235.0 | 397.3 | 381.0 | 17.0 | 16.7 | 25.3 | 23.0 | 40.3 | 40.7 |
R | 1.1 | 1.1 | 1.0 | 0.9 | 0.7 | 1.0 | 1.5 | 0.8 | 1.1 | 1.1 | |
500 | Mean | 193.3 | 202.3 | 378.3 | 425.0 | 18.0 | 18.7 | 15.3 | 25.0 | 35.3 | 46.7 |
R | 1.0 | 1.0 | 0.9 | 1.0 | 0.8 | 1.1 | 0.9 | 0.8 | 1.0 | 1.3 | |
160 | Mean | 191.3 | 195.7 | 359.7 | 375.3 | 13.0 | 17.7 | 16.3 | 20.3 | 38.0 | 38.3 |
R | 1.0 | 0.9 | 0.9 | 0.9 | 0.6 | 1.0 | 1.0 | 0.7 | 1.0 | 1.0 | |
50 | Mean | 187.3 | 235.0 | 368.7 | 411.3 | 13.7 | 16.0 | 22.0 | 20.7 | 33.3 | 45.7 |
R | 1.0 | 1.1 | 0.9 | 0.9 | 0.6 | 0.9 | 1.3 | 0.7 | 0.9 | 1.2 | |
PC | Mean | 1909.7 | 4156.3 | 2501.7 | 3854.7 | 2549.7 | 292.7 | 798.7 | 494.7 | 1095.0 | 3646.7 |
| R | 10.1 | 20.0 | 6.2 | 8.8 | 110.9 | 16.9 | 47.0 | 16.3 | 29.6 | 98.6 |
NC (Water) | Mean | 188.3 | 207.3 | 406.7 | 439.7 | 23.0 | 17.3 | 17.0 | 30.3 | 37.0 | 37.0 |
NC(untreated) | Mean | 183.7 | 182.3 | 362.7 | 449.3 | 17.0 | 15.0 | 13.7 | 29.0 | 24.0 | 37.7 |
TEST 2: Results for each strain and dose
Concentrations (μg/plate) | Mean values of revertants / Ratio treated – solvent (R) | Salmonella Thypimurium strains | |||||||||
|
| TA 100 | TA 102 | TA1535 | TA 1537 | TA 98 | |||||
|
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
5000 | Mean | 416.7 | 299.0 | 444.7 | 593.3 | 24.7 | 30.3 | 14.3 | 21.0 | 36.0 | 40.0 |
R | 2.5 | 1.4 | 1.2 | 1.4 | 1.5 | 2.0 | 0.9 | 1.0 | 1.4 | 1.4 | |
1600 | Mean | 195.7 | 248.0 | 397.3 | 489.7 | 19.0 | 17.7 | 19.3 | 14.0 | 28.7 | 34.7 |
R | 1.2 | 1.2 | 1.1 | 1.1 | 1.1 | 1.2 | 1.2 | 0.7 | 1.1 | 1.2 | |
500 | Mean | 172.3 | 197.3 | 382.3 | 455.0 | 17.3 | 20.7 | 13.3 | 18.3 | 18.3 | 33.7 |
R | 1.0 | 0.9 | 1.1 | 1.0 | 1.0 | 1.3 | 0.8 | 0.9 | 0.7 | 1.2 | |
160 | Mean | 154.7 | 179.3 | 359.3 | 439.3 | 14.3 | 17.7 | 18.3 | 17.7 | 20.7 | 28.7 |
R | 0.9 | 0.9 | 1.0 | 1.0 | 0.9 | 1.2 | 1.1 | 0.9 | 0.8 | 1.0 | |
50 | Mean | 176.0 | 187.3 | 368.0 | 434.3 | 13.7 | 16.7 | 16.3 | 24.0 | 23.7 | 42.0 |
R | 1.0 | 0.9 | 1.0 | 1.0 | 0.8 | 1.1 | 1.0 | 1.2 | 0.9 | 1.5 | |
PC | Mean | 1949.7 | 2424.0 | 3026.7 | 3737.7 | 2221.7 | 220.0 | 752.0 | 279.7 | 914.0 | 2116.7 |
| R | 11.5 | 11.6 | 8.3 | 8.6 | 133.3 | 14.3 | 46.0 | 13.5 | 34.7 | 74.7 |
NC (Water) | Mean | 169.3 | 209.0 | 364.0 | 435.3 | 16.7 | 15.3 | 16.3 | 20.7 | 26.3 | 28.3 |
NC(untreated) | Mean | 165.7 | 200.3 | 342.0 | 420.0 | 17.0 | 17.3 | 18.0 | 21.0 | 20.7 | 26.7 |
PC = positive control
NC = negative control
Applicant's summary and conclusion
- Conclusions:
- The test item was found to be mutagenic for strain TA100 under the test conditions of pre-incubation.
- Executive summary:
The aim of this test was to determine the ability of the test item to induce mutation, it was assessed using the bacterial reverse mutation test (Ames test). The test was performed following the OECD Guideline 471 with GLP on five Salmonella typhimurium strains. The test item dilutions were prepared in water for analysis. Firstly, a preliminary cytotoxicity test was performed on S. typhimurium TA100 strain with the following concentrations: 5000, 1600, 500, 160 and 50μg/plate, for triplicate, with and without S9. Plates were incubated at 37ºC for 48-72h and after this period colonies were counted. As the preliminary study showed no cytotoxicity of the test item, this concentrations range was used for the genotoxicity Test 1 (direct method) and Test 2 (pre-incubation method), and positive and negative/solvent controls were included. The same test conditions as the preliminary test were used. The revertant analysis showed no cytotoxic effect. Moreover, it was observed a ratio R higher to the double of the spontaneous rate of reversion during Test 2 for strain TA100 at 5000μg/plate without metabolic activation: R = 2.5. Besides, a dose response was observed during Test 2 for this strain TA100 without metabolic activation, and for strain TA1535 with metabolic activation. In addition, no sign of precipitate was observed. Based on the result of this study, the test item was found to be mutagenic for strain TA100 under the test conditions of pre-incubation.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
