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EC number: 274-157-0 | CAS number: 69851-61-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Mutagenic effects - bacterial: OECD 471; Ames study with read-across substance. Negative. Reliability = 2.
Mutagenic effects - mammalian: OECD 476; HPRT study in Chinese Hamster V79 cells. Negative. Reliability = 1.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- The test substance was concluded to be negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. No indications of mutagenicity were observed at any dose level in any tester strain when tested up to 10000 μg/plate in the absence or presence of S9. The assay was conducted prior to the 1997 adaptation of the current OECD 471, and did not include an E.coli WP2 or S. typhimurium TA102 tester strain. These strains are known to specifically detect certain oxidising mutagens, cross-linking agents and hydrazines that other Salmonella tester strains may not be sensitive to. However, based on the physico-chemical characteristics of the test substance, the inclusion of a fifth tester strain would not have changed the clearly negative outcome of the Ames assay. In addition, there was no indication of in vitro or in vivo mutagenicity for any other genetic toxicology endpoint.
- Justification for type of information:
- Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction from liver of rats induced with Aroclor 1254 and solution of Co-factors
- Test concentrations with justification for top dose:
- Experiment I: 20, 78, 313, 1250 and 5000 µg/0.1 mL
Repeat Experiment: 500, 1000, 2000, 4000 and 8000 µg/0.1 mL
Concentrations were based on results from a preliminary toxicity test carried out with strain TA100 without activation at concentrations ranging from 0.08 to 5000 µg/0.1 mL. - Vehicle / solvent:
- - Vehicle used: Ethanol
- Untreated negative controls:
- yes
- Remarks:
- ethanol
- Positive control substance:
- 4-nitroquinoline-N-oxide
- sodium azide
- cyclophosphamide
- other: 2-aminoanthracene (TA98, TA100, and TA1537 with S9); daunorubicin-HCl (TA100 without S9); 9(5)-aminoacridine hydrochloride monohydrate (TA1537 without S9)
- Details on test system and experimental conditions:
- In the experiments with and without the addition of microsomal activation mixture three petri dishes were prepared per strain per group (i.e., per concentration or per control group). The plates were incubated for about 48 hours at 37 ± 1.5°C in darkness.
- Evaluation criteria:
- The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains:TA 98, TA 1535 and TA 1537,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable. - Statistics:
- Mean values and standard deviation for each bacterial strain and each concentration were calculated.
- Key result
- Species / strain:
- other: S. typhimurium TA98, TA100, TA1535, TA1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- At the concentrations of 313 μg/0.1 mL and above the substance precipitated in soft agar
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Negative with and without metabolic activation upto 8000 µg/0.1 mL.
- Executive summary:
The study was conducted according to OECD Guideline 471 to determine mutagenic effects of the test substance on histidine-auxotrophic mutants of Salmonella typhimurium.
The investigations were performed on strains TA98, TA100, TA1535 and TA1537 without and with microsomal activation with concentrations of 20, 78, 313, 1250 and 5000 µg/0.1 mL in the first and 500, 1000, 2000, 4000 and 8000 µg/0.1 mL in the repeat experiment. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed without and with microsomal activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No evidence of the induction of point mutations by test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.
- 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
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- To clarify equivocal results an independent repetition experiment (without metabolic activation) with closer spaced concentrations and double cell cultures was performed on sponsor’s request.
- GLP compliance:
- yes
- Type of assay:
- other: HPRT assay in Chinese Hamster V79 cells
- Target gene:
- HPRT locus using V79 cells of the Chinese Hamster
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH stock cultures
- Cell cycle length, doubling time or proliferation index:12 - 14 h doubling time; high cloning efficiency of untreated cells, usually more than 50%
- Methods for maintenance in cell culture if applicable: The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank at the testing facility.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stored over liquid nitrogen (vapour phase); minimal essential medium (MEM)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (via PCR)
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver (S9)
- Test concentrations with justification for top dose:
- Toxicity test: 1, 2.5, 5, 10, 25, 50, 100, 250 µg/mL
Experiment I (without activation): 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100 and 200 µg/mL
Experiment I (with activation): 0.5, 1, 2.5, 5, 10, 25, 50, and 100 µg/mL
Experiment I repeat assay (without activation): 10, 25, 50, 60, 70, 80, 90 and 100 µg/mL
The selection of the concentrations was based on data from the pre-experiments. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the nature of the test item, it was not possible to prepare a solution of the test item with an appropriate solvent and with the recommended concentration of 2 mg/mL. By lowering the highest concentration to 0.25 mg/mL and based on the results of the solubility test, the best suited vehicle was DMSO (1% v/v). - Untreated negative controls:
- yes
- Remarks:
- treatment medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Seeding of the Cultures: Prior to use, cultures were cleansed of pre-existing cells. Two or three day-old exponentially growing stock cultures (more than 50% confluent) were trypsinised at 37°C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.05%. Approximately 10-20e6 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (MEM supplemented with 10% FBS) in a culture flask, respectively.
Treatment: Approximately 24 hours after seeding the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 hours exposure the cultures (single cell culture for experiment I and double cell culture for the repetition of experiment I without metabolic activation) were checked for precipitation and the treatment medium containing the test item was removed. The cells were washed twice with PBS, trypsinised and counted with a cell counter. During the following expression period most of the cells were subcultured in complete culture medium (MEM supplemented with 10% FBS) in a sufficient number of cells (at least 2e6 cells per treatment group). In addition, for determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6 - 7 days) colonies were fixed with methanol, stained with Giemsa and counted. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment.
Selection: At the end of the expression period (i.e. after 7 to 9 days) for selection the mutants, about 4e5 cells for each treatment group were seeded in cell culture petri dishes (diameter 90 mm) with selective medium containing 11 µg/mL 6-thioguanine (TG) for further incubation (additional 9 - 11 days). The cloning efficiencies (CE) were determined in parallel to the selection of mutants. For each treatment group two 25 cm2 flasks were seeded with approximately 200 cells in complete culture medium to determine the cloning efficiencies after additional 6 - 8 days. After incubation for an appropriate time (9 - 11 days for mutant frequency and 6 - 8 days for CE) colonies were fixed with methanol, stained with Giemsa and counted. The mutant frequency was calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.
Assessment of Cytotoxicity and Mutant Frequency: Cytotoxicity was evaluated by relative survival (RS). The cloning efficiency (CE) of cells plated immediately after treatment was adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative / solvent controls (assigned a survival of 100%). The mutant frequency (MF) is the cloning efficiency of mutant colonies in selective medium divided by the cloning efficiency in non-selective medium measured for the same culture at the time of selection - Evaluation criteria:
- A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. - Statistics:
- The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Exp. I (with and without activation): no growth inhibition observed. Exp. I repeat (without activation): relative survival <70%; relative survival for all concentrations except highest was within range 50-63% (slightly below non-toxic section)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Precipitation of the test item was noted in the pre- and main experiment at concentration 100 µg/mL. In the repetition of experiment I precipitation occurred at concentration 80 µg/mL.
For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed.
DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. - Conclusions:
- Under the experimental conditions reported, the test item is non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.
- Executive summary:
The test substance was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster in accordance with OECD Guideline 476. The selection of the concentrations was based on data from the pre-experiments. Experiment I (with and without metabolic activation) and repetition experiment I (without metabolic activation) were performed as a 4 h short-term exposure assay. In consultation with the Sponsor and due to the equivocal results in experiment I, an independent repetition of experiment I without metabolic activation with different experimental conditions (closer spaced concentrations and double cultures) was conducted. The test item was investigated at the following concentrations: 5.0, 10, 25, 50 and 100 µg/mL (Experiment I, without and with metabolic activation) and 25, 50, 60, 70 and 80 µg/mL (Experiment I (repetition), without metabolic activation).
Precipitation of the test item was noted in the pre- and main experiment at concentration 100 µg/mL. In the repetition of experiment I precipitation occurred at concentration 80 µg/mL. No growth inhibition was observed in the experiment I without and with metabolic activation. A growth inhibition (relative survival < 70%) was observed in the repetition experiment without metabolic activation: the relative survival for all concentrations evaluated, with the exception of the highest concentration, was found slightly below the non-toxic section (within the range of 50-63 %). For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test substance is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.
Referenceopen allclose all
Table-1: Number of back-mutant colonies per plate (arithmetic mean) (without metabolic activation)
TA98 | TA100 | TA1535 | TA1537 | |
Control | 27 | 135 | 18 | 9 |
500 µg/0.1 ml | 29 | 109 | 14 | 7 |
1000 µg/0.1 ml | 26 | 129 | 13 | 5 |
2000 µg/0.1 ml | 26 | 132 | 12 | 6 |
4000 µg/0.1 ml | 24 | 118 | 22 | 7 |
8000 µg/0.1 ml | 22 | 105 | 16 | 9 |
Positive controls | ||||
Daunorubicin-HCl | ||||
Control | 26 | |||
5 µg/0.1 ml | 160 | |||
10 µg/0.1 ml | ---- | |||
4-nitroquinoline-N-oxide | ||||
control | 139 | |||
0.125 µg/0.1 ml | 683 | |||
0.25 µg/0.1 ml | 1184 | |||
Sodium azide | ||||
Control | 12 | |||
2.5 µg/0.1 ml | 461 | |||
5.0 µg/0.1 ml | 699 | |||
9(5)aminoacridine-hydrochloride | ||||
Control | 7 | |||
50 µg/0.1 ml | 1553 | |||
100 µg/0.1 ml | 2608 |
Table-2: Number of back-mutant colonies per plate (arithmetic mean) (with metabolic activation)
TA98 | TA100 | TA1535 | TA1537 | |
Control | 45 | 145 | 20 | 19 |
500 µg/0.1 ml | 42 | 127 | 16 | 17 |
1000 µg/0.1 ml | 40 | 113 | 20 | 20 |
2000 µg/0.1 ml | 34 | 123 | 18 | 19 |
4000 µg/0.1 ml | 41 | 117 | 19 | 20 |
8000 µg/0.1 ml | 54 | 96 | 25 | 20 |
Positive control of microsomal activation | ||||
cyclophosphamide | ||||
control | 16 | |||
250 µg/0.1 ml | 983 | |||
2-aminoanthracene | ||||
control | 30 | 147 | 18 | |
5 µg/0.1 ml | 1171 | 586 | 165 |
Experiment I – Toxicity, without metabolic activation |
|||||||||
Dose Group |
Concentration [µg/mL] |
Number of cells at |
|
Number of colonies per flask |
CE [%] |
Adjusted CE [%] |
Relative Survival (RS) [%] |
||
beginning of treatment |
endo of treatment |
I |
II |
mean |
|
||||
NC1 |
0 |
10000000 |
12665000 |
140 |
165 |
153 |
76 |
97 |
120 |
NC2 |
10000000 |
12818000 |
159 |
172 |
166 |
83 |
106 |
132 |
|
S1 |
0 |
10000000 |
10642000 |
141 |
120 |
131 |
65 |
69 |
100 |
S2 |
10000000 |
10846000 |
166 |
170 |
168 |
84 |
91 |
||
1 |
0.05 |
10000000 |
10625000 |
147 |
150 |
149 |
74 |
79 |
98 |
2 |
0.1 |
10000000 |
9537000 |
157 |
176 |
167 |
83 |
79 |
99 |
3 |
0.25 |
10000000 |
10013000 |
174 |
177 |
176 |
88 |
88 |
109 |
4 |
0.5 |
10000000 |
9622000 |
144 |
158 |
151 |
76 |
73 |
90 |
5 |
1 |
10000000 |
10217000 |
141 |
147 |
144 |
72 |
74 |
92 |
6 |
2.5 |
10000000 |
10591000 |
146 |
152 |
149 |
75 |
79 |
98 |
7 |
5 |
10000000 |
9418000 |
177 |
195 |
186 |
93 |
88 |
109 |
8 |
10 |
10000000 |
9265000 |
178 |
218 |
198 |
99 |
92 |
114 |
9 |
25 |
10000000 |
8993000 |
166 |
170 |
168 |
84 |
76 |
94 |
10 |
50 |
10000000 |
8041000 |
159 |
164 |
162 |
81 |
65 |
81 |
11 P |
100 |
10000000 |
8041000 |
191 |
180 |
186 |
93 |
75 |
93 |
12 P |
200 |
10000000 |
7854000 |
202 |
188 |
195 |
98 |
77 |
95 |
EMS |
300 |
10000000 |
13107000 |
181 |
168 |
175 |
87 |
114 |
142 |
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate |
Experiment I – Mutagenicity, without metabolic activation |
||||||||||||||
|
CE in non-selective medium |
CE in selective medium |
|
|||||||||||
Dose Group |
Concen-tration [µg/mL] |
Number of colonies per flask |
CE [%] |
Number of colonies per flask |
CE [%] |
Mutant Frequency per 106cells |
||||||||
I |
II |
mean |
I |
II |
III |
IV |
V |
mean |
SD |
|||||
NC1 |
0 |
185 |
183 |
184 |
92 |
18 |
11 |
17 |
13 |
14 |
14.6 |
2.6 |
0.0037 |
39.7 |
NC2 |
184 |
186 |
185 |
93 |
7 |
14 |
15 |
9 |
13 |
11.6 |
3.1 |
0.0029 |
31.4 |
|
S1 |
0 |
148 |
187 |
168 |
84 |
14 |
12 |
7 |
17 |
7 |
11.4 |
3.9 |
0.0029 |
34.0 |
S2 |
143 |
186 |
165 |
82 |
19 |
9 |
11 |
13 |
13 |
13.0 |
3.3 |
0.0033 |
39.5 |
|
7 |
5 |
182 |
164 |
173 |
87 |
16 |
13 |
14 |
15 |
11 |
13.8 |
1.7 |
0.0035 |
39.9 |
8 |
10 |
187 |
181 |
184 |
92 |
10 |
14 |
16 |
17 |
10 |
13.4 |
2.9 |
0.0034 |
36.4 |
9 |
25 |
185 |
149 |
167 |
84 |
11 |
15 |
7 |
12 |
16 |
12.2 |
3.2 |
0.0031 |
36.5 |
10 |
50 |
189 |
186 |
188 |
94 |
15 |
15 |
19 |
18 |
21 |
17.6 |
2.3 |
0.0044 |
46.9 |
11 P |
100 |
183 |
160 |
172 |
86 |
12 |
8 |
14 |
26 |
11 |
14.2 |
6.2 |
0.0036 |
41.4 |
EMS |
300 |
134 |
115 |
125 |
62 |
78 |
89 |
92 |
90 |
94 |
88.6 |
5.6 |
0.0222 |
355.8 |
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate |
Experiment I – Toxicity, with metabolic activation |
|||||||||
Dose Group |
Concen-tration |
Number of cells at the |
Number of colonies per flask |
CE [%] |
Adjusted CE [%] |
Relative Survival (RS) |
|||
[µg/mL] |
beginning of treatment |
end of treatment |
I |
II |
mean |
[%] |
|||
NC1 |
0 |
10000000 |
11288000 |
175 |
148 |
162 |
81 |
91 |
97 |
NC2 |
10000000 |
10863000 |
157 |
158 |
158 |
79 |
86 |
91 |
|
S1 |
0 |
10000000 |
11458000 |
143 |
179 |
161 |
81 |
92 |
100 |
S2 |
10000000 |
12257000 |
147 |
168 |
158 |
79 |
97 |
||
1 |
0.5 |
10000000 |
11237000 |
173 |
175 |
174 |
87 |
98 |
104 |
2 |
1 |
10000000 |
11424000 |
154 |
156 |
155 |
78 |
89 |
94 |
3 |
2.5 |
10000000 |
11900000 |
153 |
167 |
160 |
80 |
95 |
101 |
4 |
5 |
10000000 |
11730000 |
125 |
138 |
132 |
66 |
77 |
82 |
5 |
10 |
10000000 |
11305000 |
200 |
203 |
202 |
101 |
114 |
121 |
6 |
25 |
10000000 |
10744000 |
189 |
195 |
192 |
96 |
103 |
109 |
7 |
50 |
10000000 |
10591000 |
204 |
207 |
206 |
103 |
109 |
115 |
8:00 PM |
100 |
10000000 |
9979000 |
177 |
160 |
169 |
84 |
84 |
89 |
DMBA |
1 |
10000000 |
11441000 |
151 |
132 |
142 |
71 |
81 |
86 |
NC: negative control |
Experiment I – Mutagenicity, with metabolic activation |
||||||||||||||
|
CE in non-selective medium |
CE in selective medium |
|
|||||||||||
Dose Group |
Concen-tration [µg/mL] |
Number of colonies per flask |
CE [%] |
Number of colonies per flask |
CE [%] |
Mutant Frequency per 106cells |
||||||||
I |
II |
mean |
I |
II |
III |
IV |
V |
mean |
SD |
|||||
NC1 |
0 |
199 |
215 |
207 |
104 |
21 |
17 |
21 |
19 |
11 |
17.8 |
3.7 |
0.0045 |
43.0 |
NC2 |
166 |
179 |
173 |
86 |
12 |
16 |
14 |
14 |
10 |
13.2 |
2.0 |
0.0033 |
38.3 |
|
S1 |
0 |
174 |
197 |
186 |
93 |
5 |
16 |
8 |
5 |
7 |
8.2 |
4.1 |
0.0021 |
22.1 |
S2 |
189 |
194 |
192 |
96 |
16 |
15 |
15 |
12 |
17 |
15.0 |
1.7 |
0.0038 |
39.2 |
|
4 |
5 |
157 |
173 |
165 |
83 |
15 |
15 |
12 |
14 |
12 |
13.6 |
1.4 |
0.0034 |
41.2 |
5 |
10 |
174 |
167 |
171 |
85 |
9 |
9 |
10 |
10 |
15 |
10.6 |
2.2 |
0.0027 |
31.1 |
6 |
25 |
157 |
154 |
156 |
78 |
10 |
13 |
15 |
10 |
12 |
12.0 |
1.9 |
0.0030 |
38.6 |
7 |
50 |
168 |
162 |
165 |
83 |
8 |
15 |
12 |
11 |
7 |
10.6 |
2.9 |
0.0027 |
32.1 |
8 P |
100 |
157 |
173 |
165 |
83 |
8 |
14 |
8 |
8 |
10 |
9.6 |
2.3 |
0.0024 |
29.1 |
DMBA |
1.0 |
134 |
142 |
138 |
69 |
93 |
101 |
104 |
78 |
81 |
91.4 |
10.4 |
0.0229 |
331.2 |
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency DMBA: 7,12-dimethylbenz(a)anthracene |
Experiment I (repetition) – Toxicity, without metabolic activation |
|||||||||
Dose Group |
Concen-tration [µg/mL] |
Number of cells at the |
Number of colonies per flask |
CE [%] |
Adjusted CE [%] |
Relative Survival (RS) [%] |
|||
beginning of treatment |
end of treatment |
I |
II |
mean |
|||||
NC1 |
0 |
10000000 |
9010000 |
73 |
73 |
73 |
37 |
33 |
78 |
NC2 |
10000000 |
10081000 |
92 |
93 |
93 |
46 |
47 |
111 |
|
S1 |
0 |
10000000 |
7633000 |
125 |
128 |
127 |
63 |
48 |
100 |
S2 |
10000000 |
8058000 |
86 |
91 |
89 |
44 |
36 |
||
1 |
10 |
20000000 |
13090000 |
67 |
65 |
66 |
33 |
22 |
51 |
2 |
25 |
20000000 |
11424000 |
81 |
68 |
75 |
37 |
21 |
51 |
3 |
50 |
20000000 |
10914000 |
100 |
84 |
92 |
46 |
25 |
60 |
4 |
60 |
20000000 |
11322000 |
69 |
80 |
75 |
37 |
21 |
50 |
5 |
70 |
20000000 |
11356000 |
90 |
97 |
94 |
47 |
27 |
63 |
6 P |
80 |
20000000 |
12512000 |
104 |
126 |
115 |
58 |
36 |
86 |
7 P |
90 |
20000000 |
13430000 |
142 |
131 |
137 |
68 |
46 |
109 |
8 P |
100 |
20000000 |
12580000 |
120 |
141 |
131 |
65 |
41 |
98 |
EMS |
300 |
10000000 |
11475000 |
139 |
134 |
137 |
68 |
78 |
187 |
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate |
Experiment I (repetition) – Mutagenicity, without metabolic activation |
||||||||||||||
CE in non-selective medium |
CE in selective medium |
|||||||||||||
Dose Group |
Concen-tration [µg/mL] |
Number of colonies per flask |
CE [%] |
Number of colonies per flask |
CE [%] |
Mutant Frequency per 106cells |
||||||||
I |
II |
mean |
I |
II |
III |
IV |
V |
mean |
SD |
|||||
NC1 |
0 |
155 |
160 |
158 |
79 |
8 |
6 |
6 |
11 |
3 |
6.8 |
2.6 |
0.0017 |
21.6 |
NC2 |
147 |
144 |
146 |
73 |
13 |
15 |
13 |
12 |
10 |
12.6 |
1.6 |
0.0032 |
43.3 |
|
S1 |
0 |
133 |
130 |
132 |
66 |
7 |
5 |
8 |
9 |
8 |
7.4 |
1.4 |
0.0019 |
28.1 |
S2 |
152 |
151 |
152 |
76 |
7 |
11 |
11 |
14 |
9 |
10.4 |
2.3 |
0.0026 |
34.3 |
|
2 |
25 |
162 |
158 |
160 |
80 |
14 |
8 |
11 |
8 |
6 |
9.4 |
2.8 |
0.0024 |
29.4 |
3 |
50 |
179 |
165 |
172 |
86 |
6 |
12 |
12 |
5 |
12 |
9.4 |
3.2 |
0.0024 |
27.3 |
4 |
60 |
141 |
152 |
147 |
73 |
7 |
8 |
11 |
15 |
7 |
9.6 |
3.1 |
0.0024 |
32.8 |
5 |
70 |
150 |
140 |
145 |
73 |
12 |
12 |
15 |
5 |
6 |
10.0 |
3.8 |
0.0025 |
34.5 |
6 P |
80 |
169 |
151 |
160 |
80 |
6 |
7 |
7 |
3 |
6 |
5.8 |
1.5 |
0.0015 |
18.1 |
EMS |
300 |
138 |
152 |
145 |
73 |
91 |
80 |
96 |
80 |
86 |
86.6 |
6.2 |
0.0217 |
298.6 |
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Clastogenic effects (mammalian): Hamster micronucleus study with read-across substance. Equivalent to OECD 474; Negative. Reliability = 2.
Clastogenic effects (mammalian): Rat dominant lethal with read-across substance. Equivalent to OECD 478; Negative; Reliability = 2.
DNA damage and/or repair (mammalian): Sister chromatid exchange with read-across substance. Scientifically valid study; Negative; Reliability = 2.
Link to relevant study records
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Justification for type of information:
- Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The test was conducted to evaluate the cytotoxic or mutagenic effects on male germinal cells produced by test substance.
- GLP compliance:
- not specified
- Type of assay:
- rodent dominant lethal assay
- Species:
- mouse
- Strain:
- other: Tif: MAG f (SPF)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Animals were obtained from closed breeding colony
- Age at study initiation: Female: 2 months; Males: 2 1/2 to 6 months
- Diet: Standard diet (NAFAG No .890)
- Water: Tap water ad litium
ENVIRONMENTAL CONDITIONS
- Temperature : 21 ± 1°C
- Humidity: 60 ± 5%
- Photoperiod (hrs dark / hrs light): 14 hours dark and 10 hours light - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: CMC (carboxymethyl cellulose)
- Amount of vehicle: 0.2 mL/10 g of body weight - Duration of treatment / exposure:
- Single exposure
- Post exposure period:
- Each group consisted of 20 males, each of which was placed in a cage with 2 untreated females immediately after treatment. At the end of 1 week, the females were removed and replaced by another group of 2 females. The procedure was continued for six consecutive weeks. The females were daily examined for successful mating indicated by the occurrence of a vaginal plug. The day that the vaginal plug was observed was designated as "day 0" of gestation. The whole time of six "mating periods" comprises all the stages of the maturation of the germ cell from the A-spermatogonia to the mature spermatozoon.
- Males were observed first week after administration of test substance for general condition and symptomatology.
- Autopsy of females was done and progeny were examined. - Dose / conc.:
- 1 000 other: mg/kg
- Dose / conc.:
- 3 000 other: mg/kg
- No. of animals per sex per dose:
- 20 males/group
- Control animals:
- yes, concurrent vehicle
- Statistics:
- - Student's t test or Mann-Whitney u-test is used to compare the total no of implanatations indicating possible pre-implantation losses
- X²-test or fischer's exact test is used to compare total number of mated and pregnant dams or embryonic deaths.
- Experimental data, particularly on the number of implantations and early embryonic deaths were compared with spontaneous data of a cummulative of treated controls observed over a longer period of time (autopsy on day 18 of pregnancy). - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Remarks on result:
- other: No evidence of dominant lethal effect was observed in the progeny of male mice treated with test substance.
- Additional information on results:
- The females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions).
- Conclusions:
- No evidence of dominant lethal effects were observed in progeny of male mice tretaed with test substance.
- Executive summary:
The test was conducted to evaluate the cytotoxic or mutagenic effects on male germinal cells produced by test substance The test substance was administered orally by intubation in single doses to male albino mice which were then mated to untreated females from the same strain over a period of six weeks. At the end of each week the females were replaced by new ones. Doses of 1000 and 3000 mg/kg were given. The test was done to evaluate any cytotoxic or mutagenic effects on the male germinal cells as expressed by the loss of pre-implantation zygotes as well as by the rate of deaths of post-implantation stages of embryonic development.
The results showed that the females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of dominant lethal effect was observed in the progeny of male mice treated with test substance.
- Endpoint:
- genetic toxicity in vivo, other
- Remarks:
- Nucleus anomaly test
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Justification for type of information:
- Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo.
- GLP compliance:
- no
- Type of assay:
- other: Nuclues anomaly in bone marrow cells
- Species:
- hamster, Chinese
- Strain:
- other: Cricetulus griseus
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: Female: 20-26 g, Male: 22-30 g
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 51-59
- Photoperiod (hrs dark / hrs light): 12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: 0.7% aqueous solution of Sodium-Carboxymethylcellulose (CMC)
- Amount of vehicle: 20 mL/kg - Duration of treatment / exposure:
- 2 consecutive days
- Frequency of treatment:
- Once daily
- Dose / conc.:
- 1 500 mg/kg bw/day
- Dose / conc.:
- 3 000 mg/kg bw/day
- Dose / conc.:
- 6 000 mg/kg bw/day
- No. of animals per sex per dose:
- 3
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Route of administration: Oral
- Doses: 128 mg/kg - Tissues and cell types examined:
- Bone marrow cells
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION: Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approximately 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous, suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grunwald solution for 2 min then in May-Grunwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approximately 2 min. After rinsing with distilled water and air-drying, the slides were cleared in xylol and mounted in Eukitt.
SCORING: 1000 bone marrow cells each were scored per animal and the following anomalies were registered: Single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, polyploid cells. - Statistics:
- The significance of difference was assessed by Chi²-test
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. The mean percentage of anomalies was 8.52, whereas the negative control yielded a percentage of 0.12.
- Conclusions:
- Negative for nuclear anomalies in Chinese Hamester bone marrow cells
- Executive summary:
The mutagenic effects ot the test substance to the bone marrow of Chinese hamsters was evaluated. The test substance was administered by gavage at a daily dose of 1500, 3000 or 6000 mg/kg for two consecutive days. The animals were sacrificed 24 h after the second administration. From the bone marrow, smears were made. The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo. Mutagenic effects present themselves in interphase cells in form of nucleus anomalies of bone marrow cells.
The bone marrow smears from animals treated with various doses of the test substance showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a positive control experiment with cyclophosphamide (128 mg/kg) yielded 8.52% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (0.7 CMC) alone. It was concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Justification for type of information:
- Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The experiments were performed in order to detect a possible mutagenic property of the substance, manifested in somatic cells in vivo in the form of sister chromatid exchanges (SCE).
- GLP compliance:
- no
- Type of assay:
- sister chromatid exchange assay
- Species:
- hamster, Chinese
- Strain:
- other: Cricetulus griseus
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: Female: 20-28 g, Male: 25-30 g
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 22-24°C
- Humidity: 54-62%
- Photoperiod (hrs dark / hrs light): 12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: 0.7% aqueous solution of Sodium-Carboxymethylcellulose (CMC)
- Amount of vehicle: 20 mL/kg - Duration of treatment / exposure:
- Single dose
- Post exposure period:
- Animals were sacrificed 24 h after the application and 2 h after an intraperitoneal injection of colcemide (10 mg/kg).
- Dose / conc.:
- 1 500 mg/kg bw/day
- Dose / conc.:
- 3 000 mg/kg bw/day
- Dose / conc.:
- 6 000 mg/kg bw/day
- No. of animals per sex per dose:
- 2
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 7,12 Dimethylbenz(a)anthracene (DMBA)
- Route of administration: Oral
- Doses / concentrations: 100 mg/kg in 0.7% aqueous solution of sodium-carboxymethylcellulose (CMC) - Tissues and cell types examined:
- Bone marrow cells
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION: Animals were sacrificed 24 h after the application and 2 h after an intraperitoneal injection of colcemide (10 mg/kg). Bone marrow from the shafts of both femurs was suspended in balanced salt solution and diluted to hypotonicity with distilled water, kept in a water bath at 4 to 6°C for 23 min and then centrifuged for 10 min at 200 x g. The pellets were then fixed in methanolacetic acid 3:1 for a period of 30 min, resuspended, centrifuged for 5 min at 150 x g, and stored in renewed fixative overnight at 4°C. Finally, the pellets again were centrifuged for 5 min at 150 x g, and resuspended in some 0.5 ml fixative in order to obtain a more concentrated cell suspension.
These specimens were pipetted onto wet slides and air-dried. The air-dried slides then were treated with a solution of bisbenzimide for 15 min, rinsed in Mcilvaine-buffer pH 8.0 and irradiated in this buffer at 50°C with UV-light of 350 nm. Following the development of the fluorochrome-UV light reaction in 60°C 2 x SSC (standard sodium citrate) for 90 min, the slides were stained in 40% Giemsa for 20-40 min, well rinsed, cleared in Xylol and mounted in Eukitt.
Scoring of the slides: The slides of two females and two male animals each of the treatment groups and of the control groups were examined. Twenty-five differently stained metaphases of the second cell cycle with BUdR-substitution were analysed per animal for the number of SCE's. - Statistics:
- The significance of differences of the treatment groups compared to the negative control group was assessed by t-test on the level of one percent.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The positive control group showed a highly significant increase of SCE's per cell (12.8) in comparison with the negative control (5.28 SCE's/cell).
- Conclusions:
- The test substance does not provoke any effect interpretable as being suggestive of a mutagenic property.
- Executive summary:
The experiments were performed in order to detect a possible mutagenic property of the substance, manifested in somatic cells in vivo in the form of sister chromatid exchanges (SCE). The test substance was administered by gavage. Treatment consisted of a single dose of 1500, 3000 or 6000 mg/kg. The animals were sacrificed 24 h after the application and 2 h after an intraperitoneal injection of colcemide (10 mg/kg). From the bone marrow, drop-preparations were made and stained according to a modified fluorochrome plus Giemsa technique.
The number of SCE's in animals from the control group and in those treated with the various doses of the test substance was not significantly different. A "positive control" experiment with DMBA (100 mg/kg) yielded a mean value of 12.8 SCE's per cell. This is significantly different from the controls treated with the vehicle (0.7% CMC) alone.
The results obtained indicate that under the given experimental conditions, the test substance does not provoke any effect interpretable as being suggestive of a mutagenic property.
Referenceopen allclose all
Table-1: Animals sacrificed 24 h after the second application. Percent of cells with anomalies of nuclei
|
No. of animals |
Sex of animals |
Single jolly bodies |
Fragments of nuclei in erythrocytes |
Micronuclei in erythroblasts |
Micronuclei in leucopoietic cells |
Polyploid cells |
Total |
Control (CMC 0.7%) |
1 |
Female |
- |
- |
- |
- |
- |
0.0 |
2 |
Female |
- |
- |
- |
- |
- |
0.0 |
|
3 |
Female |
0.2 |
- |
- |
- |
- |
0.2 |
|
4 |
Male |
- |
- |
- |
0.1 |
|
0.1 |
|
5 |
Male |
0.3 |
- |
- |
- |
- |
0.3 |
|
6 |
Male |
0.1 |
- |
- |
- |
- |
0.1 |
|
Cyclophosphamide (128 mg/kg) |
1 |
Female |
8.9 |
0.9 |
0.8 |
0.2 |
0.2 |
11.0 |
2 |
Female |
8.4 |
1.7 |
0.5 |
0.5 |
|
11.1 |
|
3 |
Female |
10.0 |
0.7 |
2.1 |
0.9 |
0.1 |
13.8 |
|
4 |
Male |
3.2 |
0.9 |
0.3 |
0.4 |
0.1 |
4.9 |
|
5 |
Male |
3.1 |
1.5 |
0.7 |
0.2 |
- |
5.5 |
|
6 |
Male |
3.6 |
0.2 |
0.6 |
0.4 |
- |
4.8 |
|
1500 mg/kg |
1 |
Female |
- |
- |
- |
- |
- |
0.0 |
2 |
Female |
0.3 |
- |
- |
- |
- |
0.3 |
|
3 |
Female |
0.1 |
- |
- |
- |
- |
0.1 |
|
4 |
Male |
|
- |
- |
- |
- |
0.0 |
|
5 |
Male |
0.1 |
- |
- |
- |
- |
0.1 |
|
6 |
Male |
- |
- |
- |
- |
- |
0.0 |
|
3000 mg/kg |
1 |
Female |
- |
- |
- |
0.1 |
- |
0.1 |
2 |
Female |
- |
- |
- |
- |
- |
0.0 |
|
3 |
Female |
- |
- |
- |
- |
- |
0.0 |
|
4 |
Male |
- |
- |
- |
- |
- |
0.0 |
|
5 |
Male |
- |
- |
- |
- |
- |
0.0 |
|
6 |
Male |
0.1 |
- |
- |
- |
- |
0.1 |
|
6000 mg/kg |
1 |
Female |
0.1 |
- |
- |
- |
- |
0.1 |
2 |
Female |
0.1 |
- |
- |
- |
0.1 |
0.2 |
|
3 |
Female |
- |
- |
- |
- |
- |
0.0 |
|
4 |
Male |
- |
- |
- |
- |
- |
0.0 |
|
5 |
Male |
- |
- |
- |
- |
- |
0.0 |
|
6 |
Male |
0.1 |
- |
- |
- |
- |
0.1 |
Table 1: Effects of the test substance on Bone marrow cells of Chinese Hamster
Group |
No-of animals |
Sex |
Group SCE’s per cell (mean and SD) |
Observed t value |
Negative Control |
4 |
2M + 2F |
5.28 (2.95) |
- |
DMBA (positive control) |
4 |
2M + 2F |
12.79 (6.25) |
10.88* |
1500 mg/Kg |
4 |
2M + 2F |
6.02 (3.16) |
1.72 |
3000 mg/Kg |
4 |
2M + 2F |
4.81 (2.37) |
1.24 |
6000 mg/Kg |
4 |
2M + 2F |
4.88 (3.91) |
0.82 |
*statistically significant at 1% (critical t-value = 2.34, d.f. = 198)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In an Ames test according to OECD Guideline 471, the test substance was tested for gene mutations in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 with or without metabolic activation at concentrations up to 5000 or 8000 µg/mL in the first and repeat assays, respectively. None of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No evidence of the induction of point mutations by test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the test substance is considered not mutagenic in the Ames test.
The test substance was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster in accordance with OECD Guideline 476. The test item was investigated at the following concentrations: 5.0, 10, 25, 50 and 100 µg/mL (Experiment I, without and with metabolic activation) and 25, 50, 60, 70 and 80 µg/mL (Experiment I (repetition), without metabolic activation). For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed. The test substance is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.
The test substance was administered by gavage daily at doses of 1500, 3000 or 6000 mg/kg on two consecutive days to evaluate the mutagenic effect on somatic interphase cells in vivo. The animals were sacrificed 24 h after the second application and bone marrow smears were made. The bone marrow smears showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponded to the frequency observed in the control group. Therefore, the test substance is considered negative in this test.
The potential of the test substance was examined to evaluate the cytotoxic or mutagenic effects on male germinal cells as expressed by the loss of pre-implantation zygotes as well as by the rate of deaths of post-implantation stages of embryonic development. The test substance was administered orally by intubation in single doses to male albino mice which were then mated to untreated females from the same strain over a period of six weeks. At the end of each week the females were replaced by new ones. Doses of 1000 and 3000 mg/kg were given. The results showed that the females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of dominant lethal effect was observed in the progeny of male mice treated with test substance.
The ability of the test substance to manifest changes in somatic cells was studied in vivo in the form of sister chromatid exchanges (SCE). The test substance was administered by gavage at a single dose of 1500, 3000, or 6000 mg/kg. The results obtained indicate that under the given experimental conditions, the test substance does not provoke any effect interpretable as being suggestive of a mutagenic property and was negative in this test.
Justification for classification or non-classification
The test substance did not produce mutagenicity when evaluated in cell culture or laboratory animals. The substance does not need to be classified for mutagenicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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