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EC number: 280-055-7 | CAS number: 82864-58-2
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From April 25th to June 6th, 2007
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Remarks:
- Source study has reliability 1. Details on the read across are available in section 13.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 007
- Report date:
- 2007
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 Jul 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 19 May 2000
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Similar Substance 01
- IUPAC Name:
- Similar Substance 01
- Test material form:
- solid
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- The Salmonella strains were obtained from KNOLL Aktiengesellschaft on 30 Oct 1989. The Escherichia coli strain was obtained from Merck on 09 Sep 1991.The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa), UV sensitivity (Δ uvrB) and ampicillin resistance (R factor plasmid).E. coli WP2 uvrA is checked for UV sensitivity.Histidine and tryptophan auxotrophy is automatically checked in each experiment via the spontaneous rate.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor-induced rat liver S9 mix
- Test concentrations with justification for top dose:
- 1st Experiment - standard plate test: 0, 23, 115, 575, 2875 and 5750 μg/plate
2nd Experiment - preincubation test: 0, 23, 115, 575, 2875 and 5750 μg/plate for TA 1535, TA100, TA 98, E.coli; 0, 12, 60, 300, 1500 and 3000 μg/plate for TA 1537.
3rd Experiment - preincubation test: 0, 2, 10, 50, 250 and 500 μg/plate for TA 1535, TA 100; 0, 0.4, 2, 10, 50 and 100 μg/plate for TA 1537, TA 98. - Vehicle / solvent:
- - Vehicle used: DMSO
- Test item preparation: to achieve a solution in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. All test substance formulations were prepared immediately before administration.
- Justification for choice of vehicle: due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without S9 mix only contains the vehicle
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylendiamine (NOPD)
- Details on test system and experimental conditions:
- BACTERIA CULTURES
For testing, deep-frozen (-70 °C to -80 °C) bacterial cultures are thawed at room temperature, and 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g/l Difco nutrient broth + 5 g/l NaCl) and incubated in the shaking water bath at 37 °C for about 12 - 16 hours. As a rule, a germ density of > 10^8 bacteria/ml is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
METHOD OF APPLICATION: standard plate incorporation and preincubation
STANDARD PLATE TEST - 1st Experiment
Salmonella typhimurium
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % [w/v] agar + 0.6 % [w/v] NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle (negative control)
0.1 ml fresh bacterial culture 0.5 ml S9 mix (with metabolic activation) or 0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar: 980 ml purified water 20 ml Vogel-Bonner E medium 15 g Difco bacto agar 20 g D-glucose, monohydrate.After incubation at 37 °C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.
Escherichia coli
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % [w/v] agar + 0.6 % [w/v] NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle (negative control)
0.1 ml fresh bacterial culture
0.5 ml S9 mix (with metabolic activation) or 0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 ml solution B (agar)
100 ml solution A (saline solution)
8 ml solution C (glucose solution)
10 mlsolution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.
Replicates: 3 test plates per dose or per control.
PREINCUBATION TEST - 2nd and 3rd Experiment
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.Replicates: 3 test plates per dose or per control.
TITER DETERMINATION
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.In the standard plate test, 0.1 ml of the overnight cultures is diluted to 10^-6 in each case.
Test tubes containing 2-ml portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order: 0.1 ml vehicle (without and with test substance), 0.1 ml fresh bacterial culture (dilution: 10^-6), 0.5 ml S9 mix
In the preincubation test, 0.1 ml of the overnight cultures is diluted to 10^-6 in each case.0.1 ml vehicle (with and without test substance), 0.1 ml bacterial suspension and 0.5 ml S9 mix are incubated at 37 °C for about 20 minutes using a shaker. Subsequently, 2 ml of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Evaluatrion: the titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
EXOGENOUS METABOLIC ACTIVATION
S9 fraction
The S9 fraction is prepared according to Ames et al. (1, 2). At least 5 male Sprague-Dawley rats (200 - 300 g; Charles River Laboratories Germany GmbH) receive a single intraperitoneal injection of 500 mg per kg body weight Aroclor 1254 (as a 200 mg/ml solution in corn oil) 5 days before sacrifice.
During this time, the animals are housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24 °C and a relative humidity of 30 - 70 %. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours).
Standardized pelleted feed and tap water from bottles are available ad libitum. Five days after administration, the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of +4 °C). The livers are weighed and washed in a weight-equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4 °C, 5 ml portions of the supernatant (so-called S9 fraction) are stored at -70 °C to -80 °C.
S9 mix
The S9 mix is prepared freshly prior to each experiment for this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 1 volume of S9 fraction is mixed with 9 volumes of S9 supplement (cofactors). This preparation, the so-called S9 mix, is kept on ice until used. The concentrations of the cofactors in the S9 mix are: MgCl2 8 mM KCl 33 mM Glucose-6-phosphate 5 mM NADP 4 mM Phosphate buffer (pH 7.4) 15 mMThe phosphate buffer is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4 : 1.To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.
DETERMINATION OF CYTOTOXICITY
oxicity detected by a- decrease in the number of revertants- clearing or diminution of the background lawn (= reduced his- or trp- background growth)- reduction in the titeris recorded for all test groups both with and without S9 mix in all experiments.
SOLUBILITY
Precipitation of the test material is recorded. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation. - Evaluation criteria:
- The test chemical is considered positive in this assay if the following criteria are met: a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.A test substance is generally considered non-mutagenic in this test if: the number of revertants for all tester strains were within the historical negative controlrange under all experimental conditions in at least two experiments carried out independently of each other.
Results and discussion
Test results
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- bacteriotoxic effect depending on the strain and test conditions
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MUTAGENICITY
The test substance did not lead to an increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay).
TOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 575 μg/plate onward.In the preincubation assay strong bacteriotoxicity (reduced his- background growth, decrease in the number of his+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 10 μg/plate onward using the Salmonella strains.
For the strain E. coli WP2uvrA weak bacteriotoxicity (reduced trp- background growth, decrease in the number of trp+ revertants) was observed at 5750 μg/plate in the presence of S9 mix only. Besides a clear reduction in the titer was observed from 2875 μg/plate onward in the presence of S9 mix.
SOLUBILITY
Test substance precipitation was found from about 250 μg/plate onward with and without S9 mix.
CONTROLS
Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria.In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.
Any other information on results incl. tables
Standard plate test
Dose | Without metabolic activation | With metabolic activation | |
Mean (SD) | Mean (SD) | ||
DMSO | 17 (3) | 20 (3) | |
TA 1535 | 23 µg | 20 (4) | 19 (6) |
115 µg | 19 (2) | 17 (2) | |
575 µg* | 16 (4) | 17 (8) | |
2875 µg * | 17 (2) | 15 (2) | |
5750 µg * | 19 (2) | 12 (2) | |
MNNG | 5.0 µg | 908 (38) | |
2-AA | 2.5 µg | 118 (19) | |
DMSO | 106 (4) | 117 (6) | |
TA 100 | 23 µg | 113 (12) | 110 (5) |
115 µg | 118 (7) | 110 (3) | |
575 µg* | 106 (13) | 112 (11) | |
2875 µg * | 108 (5) | 108 (4) | |
5750 µg * | 101 (2) | 95 (5) | |
MNNG | 5.0 µg | 1110 (160) | |
2-AA | 2.5 µg | 1118 (88) | |
DMSO | 12(4) | 11 (1) | |
TA 1537 | 23 µg | 8 (1) | 10 (2) |
115 µg | 9 (1) | 8 (3) | |
575 µg* | 9 (2) | 12 (2) | |
2875 µg * | 5 (2) | 8 (1) | |
5750 µg * | 4 (3) | 7 (2) | |
AAC | 100 µg | 376 (29) | |
2-AA | 2.5 µg | 154 (15) | |
DMSO | 28 (3) | 41 (2) | |
TA 98 | 23 µg | 28 (2) | 37 (5) |
115 µg | 31 (2) | 31 (4) | |
575 µg* | 29 (3) | 29 (3) | |
2875 µg * | 37 (6) | 30 (3) | |
5750 µg * | 28 (4) | 24 (5) | |
NOPD | 10 µg | 629 (38) | |
2-AA | 2.5 µg | 644 (52) | |
DMSO | 36 (2) | 47 (3) | |
E. Coli WP2 uvrA | 23 µg | 30 (6) | 47 (3) |
115 µg | 32 (3) | 42 (7) | |
575 µg* | 31 (4) | 44 (7) | |
2875 µg * | 34 (4) | 38 (8) | |
5750 µg * | 35 (4) | 42 (3) | |
4-NQO | 5 µg | 684 (81) | |
2-AA | 60 µg | 264 (13) |
* Precipitation
PREINCUBATION TEST - 01
Dose | Without metabolic activation | With metabolic activation | |
Mean (SD) | Mean (SD) | ||
DMSO | 16 (2) | 16 (3) | |
TA 1535 | 23 µg | 12 (2) | 14 (3) |
115 µg | 12 (4) | 11 (2) | |
575 µg* | 7 (2) | 9 (3) | |
2875 µg * | - | - | |
5750 µg * | - | - | |
MNNG | 5.0 µg | 733 (34) | |
2-AA | 2.5 µg | 124 (14) | |
DMSO | 102 (12) | 117 (13) | |
TA 100 | 23 µg | 89 (7) | 112 (6) |
115 µg | 77 (8) | 106 (7) | |
575 µg* | 32 (11) | 83 (15) | |
2875 µg * | - | 62 (22) | |
5750 µg * | - | - | |
MNNG | 5.0 µg | 716 (25) | |
2-AA | 2.5 µg | 778 (69) | |
DMSO | 8 (3) | 8 (1) | |
TA 1537 | 12 µg | 4 (2) | 9 (4) |
60 µg | 5 (1) | 7 (4) | |
300 µg* | 3 (2) | 3 (1) | |
1500 µg * | 3 (1) | 3 (2) | |
3000 µg * | - | - | |
AAC | 100 µg | 450 (22) | |
2-AA | 2.5 µg | 124 (14) | |
DMSO | 0 µg | 29 (6) | 40 (4) |
TA 98 | 23 µg | 28 (4) | 29 (2) |
115 µg | 18 (2) | 23 (3) | |
575 µg* | 13 (2) | 14 (3) | |
2875 µg * | - | 6 (2) | |
5750 µg * | - | - | |
NOPD | 10 µg | 648 (29) | |
2-AA | 2.5 µg | 547 (26) | |
DMSO | 37 (4) | 37 (3) | |
E. Coli WP2 uvrA | 23 µg | 37 (7) | 36 (3) |
115 µg | 36 (9) | 35 (5) | |
575 µg* | 38 (4) | 37 (4) | |
2875 µg * | 33 (6) | 30 (4) | |
5750 µg * | 29 (2) | 15 (4) | |
4-NQO | 5.0 µg | 547 (26) | |
2-AA | 60 µg | 247 (29) |
* Precipitation
PREINCUBATION TEST - 02
Dose | Without metabolic activation | With metabolic activation | |
Mean (SD) | Mean (SD) | ||
DMSO | 14 (2) | 15 (3) | |
TA 1535 | 2 µg | 15 (5) | 14 (4) |
10 µg | 13 (2) | 13 (3) | |
50 µg | 13 (4) | 14 (2) | |
250 µg * | 11 (2) | 11 (1) | |
500 µg * | 9 (4) | 9 (3) | |
MNNG | 5.0 µg | 556 (31) | |
2-AA | 2.5 µg | 146 (19) | |
DMSO | 100 (6) | 104 (10) | |
TA 100 | 2 µg | 97 (10) | 100 (5) |
10 µg | 99 (9) | 95 (8) | |
50 µg | 106 (4) | 98 (5) | |
250 µg * | 80 (14) | 76 (6) | |
500 µg * | 55 (18) | 94 (8) | |
MNNG | 5.0 µg | 723 (46) | |
2-AA | 2.5 µg | 763 (106) | |
DMSO | 8 (2) | 9 (1) | |
TA 1537 | 0.4 µg | 9 (2) | 8 (3) |
2 µg | 9 (3) | 9 (2) | |
10 µg | 6 (3) | 5 (2) | |
50 µg | 6 (1) | 6 (2) | |
100 µg | 3 (2) | 5 (3) | |
AAC | 100 µg | 429 (20) | |
2-AA | 2.5 µg | 115 (8) | |
DMSO | 26 (1) | 29 (3) | |
TA 98 | 0.4 µg | 23 (8) | 28 (5) |
2 µg | 26 (3) | 27 (2) | |
10 µg | 20 (4) | 22 (2) | |
50 µg | 19 (5) | 21 (4) | |
100 µg | 12 (3) | 19 (3) | |
NOPD | 10 µg | 503 (61) | |
2-AA | 2.5 µg | 543 (13) |
Applicant's summary and conclusion
- Conclusions:
- Under the experimental conditions, test item is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation
- Executive summary:
The substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The test was performed according to the OECD guideline 471. Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA were assessed in three experiments carried out independently of each other (standard plate test and preincubation assay) with and without metabolic activation (Aroclor-induced rat liver S9 mix).
Precipitation of the test substance was found from about 250 μg/plate onward with and without S9 mix.
A bacteriotoxic effect was observed depending on the strain and test conditions from about 575 μg/plate onward in the standard plate test and from about 10 μg/plate onward in the preincubation test.
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.
Conclusion
The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions.
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