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EC number: 500-105-6 | CAS number: 39423-51-3 1 - 6.5 moles propoxylated
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Four reliable studies were available for this endpoint (Klimisch 1
studies): 2 studies performed according to OECD guideline 471 (one key
and one supporting study), 1 study performed according to OECD guideline
476, and 1 performed according to OECD guideline 482. None of these
studies observed any genotoxic effect.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- No data
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: J-98
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: no data
- Stability under test conditions: no apparent change in the physical state of the test or control articles during the assay
OTHER SPECIFICS:
- Name of test material (as cited in study report): 4236-44-30 - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1538
- Details on mammalian cell type (if applicable):
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 fraction of rat liver homogenate obtained from Arcolor 1254-treated Sprague Dawley rats
- Test concentrations with justification for top dose:
- 12, 40, 120, 400 and 1200 µg/plate
- Vehicle / solvent:
- -solvent(s) used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without metabolic activation
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- sodium azide
- Remarks:
- TA1535 and TA100=10 µg/ml
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without metabolic activation
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- TA1538 and TA98=50 µg/ml
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without metabolic activation
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537=1500 µg/ml
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without metabolic activation
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- other: 2-anthramine
- Remarks:
- in all strains=50 µg/ml
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation);
DURATION
- Preincubation period: 48h
- Exposure duration: 48-72h
SELECTION AGENT (mutation assays): histidine and biotin
NUMBER OF REPLICATIONS:
positive and negative controls: in triplicate
treated groups: in duplicate
tester strains: treated with five levels of the test compound
DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition is tetsted at the following concentrations: 10,000, 3333, 1000, 333 and 100 µg/plate with strains TA1538 and TA100 ( in duplicate).
- Evaluation criteria:
- revertant colonies are counted (Artek Counter Model 880)
- positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies
- negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: dose levels for the Preliminary Toxicity Screen were 10,000, 3333, 1000, 333 and 100 µg/plate.
Tester Strains TA1538 and TA100 showed complete inhibition at 10,000 ug/plate, excessive inhibition at 3333 µg/plate and moderate inhibition a t 1000 µg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA: all positive and negative controls were within acceptable range of mean historical data
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- The results for the test article 4236-44-30, Order# 5-98, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 1200, 400, 120, 40 and 12 µg/plate.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- No data
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- 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.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Experimental Toxicology and Ecology
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Abl. Nr. 85-4113
- Date of production: November 16, 2000
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: was guaranteed by the sponsor
OTHER SPECIFICS:
- Name of test material (as cited in study report): Polyetheramin T 403; Substance No.: 01/0618-1
- Analytical purity: 93.9 %
- Homogeneity: Homogeneous by visual inspection - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix prepared from Sprague Dawley rat livers after Aroclor 1254 activation
- Test concentrations with justification for top dose:
- 0, 22, 110, 550, 2750, 5500 µg/plate (SPT)
0, 4, 20, 100, 500, 1500 µg/plate (SPT)
0, 10 ,30 ,100, 300, 1000 µg/plate (PIT) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: see details below
- Details on test system and experimental conditions:
- Standard plate test
Test tubes containing 2 ml portions of soft agar kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation) or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.
Preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx.30 seconds.
Both Tests
In each experiment 3 Test plates per dose per control used. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.
Positive Controls:
with S-9 mix: 2.5 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535
60 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strain E. coli WP2 uvrA
without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537
5 µg 4-nitroquinoline-N-oxide (4-NQO) (dissolved in DMSO) for the strain E. coli WP2 uvrA
The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed. - Evaluation criteria:
- Positive results
- doubling of the spontaneous mutation rate (control)
- dose response relationship
- reproducibility of the result - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Thus, under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic agent in a bacterial reverse mutation test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2009-10-27 to 2009-12-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- -stability of the test article was not reported
- GLP compliance:
- yes
- Remarks:
- Study conducted in compliance with the US EPA GLP Standards 40 CFR, and the OECD Principle of GLP (C97)186/Final
- Type of assay:
- mammalian cell gene mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 8H306
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: the stability of the test article has not been determined by testing facility or the Sponsor.
OTHER SPECIFICS:
- Name of test material (as cited in study report): JEFFAMINE T-403
- Analytical purity: 100% - Target gene:
- hypoxanthine-guanine phosphoribosyl transferase locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: medium suplemented with hypoxanthine, aminopterin and thymidine (HAT); F12FBS5-Hx is Ham's F12 medium without hypoxanthine suplemented with 5% dialyzed FBS, 100 units pencilin/mL, 100 ug streptomycin/mL and 2mM L-glutamine/mL; Cloning medium: for selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate dish in F12FBS5-Hx containing 10 uM 6-thioguanine (TG, 2-amino-6-mercaptopurine). For clonong efficiency determinations at the time of selection, 100 cells/60mm dish were plated in triplicate in medium without TG.
- Properly maintained:yes
- Periodically checked for Mycoplasma contamination:no
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: the CHO-K1-BHR cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14h, and a cloning efficiency of usually greater than 80%
- Metabolic activation:
- with and without
- Metabolic activation system:
- Arclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- 500, 1000, 2000, 3000, 3500, 4000 and 4400 ug/mL (with and without S9 activation)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: sterile distilled water was chosen by the Sponsor. The test article was soluble in sterile distilled water at a concentration of 44 mg/mL, the maximum concentration prepared for the preliminary toxicity assay - Untreated negative controls:
- yes
- Remarks:
- medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation (-S9) Migrated to IUCLID6: EMS (ethyl methanesulfonate) was used at a stock concentration of 20 uL/mL, for a final concentration of 0.2 uL/mL
- Untreated negative controls:
- yes
- Remarks:
- medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with metabolic activation (+S9) Migrated to IUCLID6: B(a)P (Benzo(a)pyrene: was used at a stock concentration of 400 ug/mL, for a final concentration of 4 ug/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 5h
- Expression time (cells in growth medium): 18-24h
- Selection time (if incubation with a selection agent): 7-10 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable
SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): 10% aqueous Giemsa
NUMBER OF REPLICATIONS:2
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable
OTHER:no data - Evaluation criteria:
- All conclusions were based on scientific judgment, however, the following criteria are presented as a guide to interpretation of the data: -the test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least tow consecutive concentrations showing mutant frquencies of > 40 mutants per 1E -6 clonable cells; - if a single point above 40 mutants per 1E -6 clonable cells was observed at the highest concentration, the test article was considered suspect; - if no culture exhibited a mutant frequency of > 40 mutants 1E-6 clonable cells, the test article was considered negative
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- 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:
- Effects of osmolality: the osmolity of the solvent control was 264 mmol/kg and the osmolality of the highest concentration, 4400 ug/mL , was 269 mmol/kg
- Evaporation from medium: no data
- Water solubility: the test article was soluble in sterile distilled water at a concentration of 44 mg/mL, the maximum concentration prepared for the preliminary toxicity assay
- Precipitation:no test article precipitate at any concentration in treatment medium
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: Preliminary toxicity assay: the preliminary toxicity assay was used to established the optimal concentrations for the mutagenesis assay and consisted of evaluation of test article effect on cloning efficiency. In the preliminary toxicity assay, the maximum concentration of JEFFAMINE T-403 tested was 4400 ug/mL (10 mM). Selection of concentrations for the mutagenesis assay was based on the cloning efficiency relative to the solvent control. Substantial toxicity, i.e., cloning efficiency ¿50% of the solvent control, was observed at 4400 ug/mL with and without S9 activation. Based on these findings, the concentrations chosen for the mutagenesis assay arnged from 150 to 3000 ug/mL for both the non-activated and S9-activated cultures.
COMPARISON WITH HISTORICAL CONTROL DATA:
The stability of the negative and positive control articles and their mixtures was demonstated by acceptable results that met the criteria for a valid test. The historical control data are presented in Appendix II -see att.background material
ADDITIONAL INFORMATION ON CYTOTOXICITY:
no data - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Under the conditions of this study, the test article was concluded to be negative in the CHO/HGPRT Mutation Assay, with and without metabolic activation.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1988-03-29 to 1988-05-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- comet assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 88-006
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the glass bottle receieved from the Sponsor
OTHER SPECIFICS:
- Name of test material (as cited in study report): 5601-96-1
- Analytical purity: 97 % - Species / strain / cell type:
- hepatocytes: obtained from male F344 rats
- Details on mammalian cell type (if applicable):
- - Type and identity of media: WME containing 10% calf serum
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 0.167, 0.5, 1.67, 5.0, 16.7, 50, 167, 500, 1667 and 5000 ug/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: Solvent was chosen based on the suggestion of the sponsor. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionized water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-acetamidofluorene- 1.0 E-7 M
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 18-20 hours (10 uCi/ml of 3H-thymidine, specific activity of 50-80 Ci/mM, was added during exposure)
NUMBER OF REPLICATIONS: 3 coverslips per group
NUMBER OF CELLS EVALUATED: A total of 150 cells/group were counted
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was evaluated by visual inspection
OTHER: After exposure, the cultures were washed three times with phosphate buffered saline by aspiration. The coverslips were swelled in 1 % sodium citrate for 10-15 minutes and fixed in three 10 minute changes of 100 % ethanol:glacial acetic acid (3:1). The coverslips were air dried and mounted cell surface up on glass slides with permount. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored in light proof slide boxes containing desiccant for one week. Seven days later, autoradiographs were developed. - Evaluation criteria:
- The test substance was reported positive when the minimum net grain count of 3 per nuclei was consistently observed in triplicate wells. Where possible a dose response profile should have been observed. Due to the need to initially screen the test substance over a wide range of doses, an adequate dose response may have not been attained, and the test substance may have been classified as a "suspect" genotoxicant. To resolve the genotoxic potential of the test substance, the sponsor may have chosen to initiate a second experiment with dose levels closely bracketing the positive response which resulted in classifying the substance as a "suspect" genotoxicant.
- Key result
- Species / strain:
- hepatocytes: rat
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values of net nuclear grain counts were within the acceptable range of mean historical data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: The highest dose level scored in the assay was 16.7 ug/ml due to excessive toxicity observed at the higher dose levels. In addition to the 16.7 ug/ml dose level, 0.5, 1.67 and 5.0 ug/ml dose levels were also scored.
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- The results for the test substance 5601-96-1 were negative in the Rat Hepatocyte Primary Culture/DNA Repair Test under the conditions of the assay, without metabolic activation. These findings were based upon the inability of the test substance to produce a mean net nuclear grain count of three or greater than the vehicle control mean net nuclear grain count at dose levels up to the highest non-toxic dose level of 16.7 ug/ml.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Referenceopen allclose all
Toxicity:
A bacteriotoxic effect (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 500 µg -1,500 µg/plate onward.
In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed depending on the strain and test conditions at doses >= 300 µg/plate.
Solubility: No precipitation of the test substance was found.
Mutagenicity:
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 S-9 mix or after the addition of a metabolizing system.
Mutagenesis Assay
Relative cloning efficiency was 81% and 14 % at the highest concentration cloned in the non-activated and S9 -activated systems, respectively. Non of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 10^6 clonable cells.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
One reliable study was available for this endpoint (Klimisch 2). This
study was performed according to OECD guideline 474 and did not observe
any genotoxicity.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1988-04-25 to 1988-05-25
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The OECD Guideline 474 states that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment. 6 Animals died minutes after administrating the dose in the definitive study. However, these animals were replaced.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Principles of method if other than guideline:
- The OECD Guideline 474 states: that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment.
Also, the likelihood that the test substance reach the general circulation or the target tissue has not been discussed. - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Order #88-006, recieved on March 25, 1988
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: no apparent change in its physical state
OTHER SPECIFICS:
- Name of test material (as cited in study report): 5601-96-1 - Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 29-37g, females: 26-32g
- Assigned to test groups randomly: yes, randomized by bodyweight, assigned to groups by use of a random number table
- Fasting period before study: no
- Housing: max 5 per cage (according to sex and dose group)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 % humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark, 12 hrs light
IN-LIFE DATES: From: April 1988 To: May 1988 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: deionized water
- Details on exposure:
- one single intraperitoneal dose per mouse
- Duration of treatment / exposure:
- 1 single dose
- Frequency of treatment:
- once
- Post exposure period:
- test substance: 30, 48 and 72h
positive control: 30h
negative control: 48h - Dose / conc.:
- 2.5 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- triethylenemelamine
- Route of administration: intraperitoneal
- Doses / concentrations: 0.5 mg/kg - Tissues and cell types examined:
- bone marrow of femur
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
based on dose range finding study with following doses: 50, 100, 250, 500 and 1000 mg/kg bw .
Pharmacotoxic signs were observed in the 100 mg/kg group and at higher dose levels evaluated. Mortality occurred in the 250 mg/kg bw group (1/4). All animals but one, died within 2 hours post-dose in the 500 and 1000 mg/kg bw dose groups. Therefore, 150 mg/kg bw was selected as dose.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All mice were sacrified by cervical dislocation.
DETAILS OF SLIDE PREPARATION:
One end of each femur (one proximal, one distal to the iliac end) was opened carefully with scissors until a small opening to the marrow canal became visible. A 1 ml tuberculin syringe filled with approx. 0.2 ml fetal bovine serum was inserted into the marrow cavity and the marrow was gently flushed (to assure maximum dispersion) into a 5ml round bottom culture tube containing 1.0 ml of fetal bovine serum. Each femur was flushed with fetal bovine serum until all the marrow was out and the bone appeared almost translucent.
The suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed leaving a small amount of fetal bovine serum with the remaining cell pellet. The pellet was resuspended with a pasteur pipette to assure a homogenous mixture.
A small drop of the cell suspension was spread immediately onto an ethanol pre-cleaned glass slide. The smears were quickly dried on a slide warmer set at approx. 56°C, dipped in absolute methanol (2sec) and air dried.
Stained with Giemsa.
METHOD OF ANALYSIS:
Slides were screened for good preparation: i.e. well spread, undamaged, perfectly stained.
1000 polychromatic erythrocytes (PCE)(immature) per animal were scored for the presence of micronuclei, as well as for the number of micronucleated normochromatic erythrocytes (NCE)(mature) present in the optical field containing these 1000 PCE.
Data are expressed as the total number of mean percentage of micronucleated PCE for 10000 PCE per group (1000 PCE per mouse, whenever possible). An additional 1000 erythrocytes (PCE and NCE) were also scored per mouse and the proportion was expressed as the PCE/NCE ratio.
OTHER: Slides were coded randomly by study number and number designation. The code was kept on a separate sheet until the slides were evaluated. Following evaluation, slides were decoded. Coding of the slides was carried out by an individual not involved in the actual scoring. - Evaluation criteria:
- If the spontaneous rate of micronuclei in the PCE is less than 0.5% and the positive control is statistically significantly greater (p<0.05) than the spontaneous rate and at least seven animals per group survived the treatment, the results are deemed acceptable.
- Statistics:
- - one-tailed t-tests were used to make pairwise comparisons between each treatment group and its concurrent vehicle control for statistically significant increases in the number of micronucleated PCE.
- the proportion of PCE per 1000 erytrocytes per animal were evaluated by pairwise two-tailed t-test after an arcsin transformation was performed.
All comparisons were made for each sacrifice time separately comparing treated groups versus the vehicle control group. - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- decreased activity, abnormal gait, body drop and decreased body tone, six animals were dead within minutes of dose administration
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.75 - 50 mg/kg
- Clinical signs of toxicity in test animals:
2.5 mg/kg: decreased activity, body drop and abnormal gait (immediately after treatment), no signs (24h), decreased body tone (most animals at 48h), body drop, abnormal gait (1 female, 48h), piloerection, decreased body tone (all, 72h)
1 mg/kg: no signs (immediately to 24h after treatment), decreased body tone (most animals, at 48 and 72h after administration)
0.75 mg/kg: no signs (immediately to 24h after treatment), piloerection, decreased body tone, abnormal gait (all males, 48h), decreased body tone (all, 72h).
2.5 mg/kg was selected for the definitive study
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
8/10000 (30h), 13/10000 (48h), 10/10000 (72h), 11/10000 (neg control), 342/10000 (pos control)
- Ratio of PCE/NCE (for Micronucleus assay):
1.248 (30h), 1.639 (48h), 1.358 (72h), 1.246 (neg control), 0.821 (pos control)
- Appropriateness of dose levels and route: dose level seems appropriate and was based on the range finding study. However, six animals (4 males, 2 females) were dead within minutes of dose administration, indicating that the dose (2.5 mg/kg) was near or at the maximum tolerated dose. Death occurred in 2 males of the 30h group, 2 males of the 48h group, 2 females of the 72h group. Necroscopy for all six mice did not reveal visible lesions. Mice were replaced so that there were 10 mice per dose group.
- Statistical evaluation: OK.
results of positive control are statistically different from results of negative control (induction of micronuclei and PCE/NCE ratio)
number of induced micronuclei is not statistically different between the test substance and the negative control (at none of the sacrifice times).
PCE/NCE ratio was significantly increased for the 48h treated group as compared to the vehicle control. The significance of this positive result is undetermined. - Conclusions:
- The test article, administered in single intraperitoneal dose at 2.5 mg/kg bw, did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro:
Ames test according to OECD guideline 471 (Pharmakon Research International, 1982): The results for the test substance were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 1200, 400, 120, 40 and 12 µg/plate. Cytotoxicity was not determined for Salmonella strains TA1535, TA1537, TA98 and TA100, but was observed for test strains TA1538 and TA100. Positive and solvent controls were tested simultaneously and were within acceptable range of mean historical data.
CHO/HGPRT Mutation Assay according to OECD guideline 476 (BioReliance, 2010): under the conditions of this study, the test substance was concluded to be negative. Cytotoxicity was observed, however no further details were available. Negative, vehicle and positive control data were tested simultaneously and the stability of these controls and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.
Rat Hepatocyte Primary Culture/DNA Repair Test according to OECD guideline 482: Under the conditions of the assay the substance was found negative without metabolic activation. These findings were based upon the inability of the test substance to produce a mean net nuclear grain count of three or greater than the vehicle control mean net nuclear grain count at dose levels up to the highest non-toxic dose level of 16.7 ug/ml. Positive and solvent controls were tested simultaneously and their values of net nuclear grain counts were within the acceptable range of mean historical data.
Genetic toxicity in vivo:
Micronucleus test according to OECD guideline 474 (San Sebastian, 1988): The test substance, administered in single intraperitoneal dose at 2.5 mg/kg bw, did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. Following toxicity was observed: decreased activity, abnormal gait, body drop and decreased body tone, six animals were dead within minutes of dose administration. Vehicle and positive controls were valid.
Justification for classification or non-classification
Based on the available data and the criteria of the CLP Regulation, the substance should not be classified for mutagenicity.
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.

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