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EC number: 700-903-6 | CAS number: 255830-15-0
- 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
In vitro bacterial mutagencity: WoE read-across from ATMP-H, negative with and without metabolic activation in S. typhimurium TA 98; 100; 1535 and 1537 (similar to OECD Test Guideline 471) (Monsanto, 1981)
In vitro bacterial mutagencity: WoE read-across from ATMP-H, negative with and without metabolic activation in E. coli WP2 uvr A (OECD Test Guideline 471) (Envigo, 2018)
In vitro cytogenicity in mammalian cells: ATMP-N-oxide-xK was negative with and without metabolic activation in peripheral human lymphocytes (OECD Test Guideline 473) (Safepharm Laboratories, 1994)
In vitro mutagenicity in mammalian cells: read-across from ATMP-xNa, negative with metabolic activation in mouse lymphoma L5178Y cells (similar to OECD Test Guideline 476) (SRI, 1988)
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1994-02-22 to 1994-05-12
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 1981
- Deviations:
- yes
- Remarks:
- experiment was not repeated to confirm negative result, exposure period without metabolic activation 20 hours
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: peripheral human
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 - induced rat liver S9
- Test concentrations with justification for top dose:
- 393.75-1575 µg/ml (without metabolic activation), 787.5-3150 µg/ml (with metabolic activation). This is equivalent to an active acid content of approximately 250-1000 500-2000 µg/ml and 500-2000 µg/ml.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water. Hepes buffer was added to maintain pH balance.
- Justification for choice of solvent/vehicle: none given in report. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation: 500 µg/ml
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- without metabolic activation: 25 µg/ml
- Details on test system and experimental conditions:
- ACTIVATION: Aroclor induced rat liver S9 mix included NADP, G6P, KCl and MgCl₂ in Phosphate buffer, final concentration S9 mix in cultures was 1%
METHOD OF APPLICATION: in medium;
DURATION
- Preincubation period:
- Exposure duration: 4 hours (with metabolic activation),
- Expression time (cells in growth medium): 16 hours (with metabolic activation),
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
SPINDLE INHIBITOR (cytogenetic assays): demecolcine (2 h before harvest)
STAIN (for cytogenetic assays): Giesma
NUMBER OF REPLICATIONS: duplicate cultures for each dose level
NUMBER OF CELLS EVALUATED: 100 well-spread metaphases from each culture were scored for gaps, breaks or rearrangements. 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as mitotic index and percentage of vehicle control values.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: percentage of vehicle control values
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no - Evaluation criteria:
- A positive response was recorded if the percentage of cells with aberrations markedly exceeded the concurrent vehicle control. For modest increases a dose-response relationship is generally required.
- Statistics:
- Fisher's exact test.
- Species / strain:
- lymphocytes: peripheral human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1575 µg/ml (without metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was controlled with buffer
- Effects of osmolality: not recorded
COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures gave values of aberrations within the range of historical controls. - Conclusions:
- In an in vitro cytogenicity / chromosome aberration study in mammalian cells, ATMP-N-oxide-xK was tested according to OECD TG 473 and in compliance with GLP. No increase in the number of cells with aberrations was observed when peripheral human lymphocytes were tested up to cytotoxic concentrations. Exposure lasted for 4 hours in the presence of metabolic activation and for 20 hours in the absence of metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.
- 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:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- : not tested in the absence of metabolic activation
- Principles of method if other than guideline:
- Method: Clive et al
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat liver S9
- Test concentrations with justification for top dose:
- 0-1.2 µl Dequest 2000/ml (calculated by previous reviewer as 0-0.78 µl active acid/ml). These concentrations were positive in the previous experiment.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no information - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Remarks:
- with activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days
SELECTION AGENT (mutation assays): 5ug/ml trifluorothymidine
NUMBER OF REPLICATIONS: duplicate cultures
NUMBER OF CELLS EVALUATED: 6x10E06 (1st experiment); 9x10E06 (2nd experiment)
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) - Evaluation criteria:
- Positive result: Dose-related increase in number of mutant colonies and mutant frequency at one or more concentrations of greater than or equal to twice control levels with an RTG of greater than 10%.
- Statistics:
- No statistical analysis of results was carried out.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Remarks:
- Concentrations based on data from previous experiment
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: test solution was neutralised to eliminate pH effects. Slight drop in pH seen at highest concentration but approximate fall in 0.08pH units only
- Precipitation: white precipitate reported at concentrations greater than 0.61µl/ml
RANGE-FINDING/SCREENING STUDIES: not conducted as this is a follow-up experiment
COMPARISON WITH HISTORICAL CONTROL DATA: no data
- Conclusions:
- In an in vitro gene mutation study in mammalian cells, conducted in a similar manner to OECD TG 476 and in compliance with GLP, a neutralized solution of ATMP acid did not increase the number of revertants in the presence of metabolic activation when tested up to the solubility limit. The test results indicates that the positive findings in the previous experiment with un-neutralised ATMP acid were an artefact of pH. It is concluded that ATMP acid is not mutagenic to mammalian cells under the conditions of this test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 07 November 2017 to 23 November 2017
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Only one test strain of E. coli was used to detect mutations via cross-linking.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- yes
- Remarks:
- Only one test strain of E. coli was used to detect mutations via cross-linking.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: On the day of the experiment, the test item ATMP-H was dissolved in deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.
- Preliminary purification step: The dose selection was adjusted to the purity of 33%.
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable - Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- To evaluate the toxicity of the test item, a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed, 5000 µg/plate were chosen as the maximal concentration.
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Remarks:
- no treatment
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionised water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
ACTIVATION: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C in the dark
SELECTION AGENT (mutation assays): selective agar
NUMBER OF REPLICATIONS: triplicate cultures
DETERMINATION OF CYTOTOXICITY
- Method: number of revertants
- Any supplementary information relevant to cytotoxicity: not specified - Rationale for test conditions:
- To evaluate the toxicity of the test item, a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: not observed
RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed, 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
HISTORICAL CONTROL DATA
- Positive historical control data: within the range of positive historical control data
- Negative historical control data: within the range of negative historical control data
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertants - Conclusions:
- In a valid bacterial reverse mutation assay, conducted according to OECD TG 471 and in compliance with GLP, ATMP-H has been tested using E. coli WP2 uvr A. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to the limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- The restrictions were that only four strains of bacteria were used.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- insufficient range of strains to meet requirements of current guideline
- GLP compliance:
- yes
- 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:
- Aroclor induced mouse and rat liver S9
- Test concentrations with justification for top dose:
- 0.01, 0.04, 0.2, 1, 3,10 μl Dequest 2000/plate and 25 μl Dequest 2000 Dequest/plate for spot test. Expressed as ATMP: 0.0065, 0.026, 0.13, 0.65, 1.95, 6.5 μl/plate for plate incorporation; up to 9 μl for spot test.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- TA 98 and TA 100 without activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium nitrite
- Remarks:
- TA 1535 without activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA 1537 without activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- TA 98 and TA 100 with activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- TA 1535 and TA 1537 with activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation); as impregnation on paper disk
DURATION
- Preincubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours
NUMBER OF REPLICATIONS: single applications (spot test), triplicate plates (plate incorporation)
DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in microbial lawn - Evaluation criteria:
- A positive response is indicated if three of more treatments are significantly greater than the solvent control with a significant dose-response.
- Statistics:
- Analysis of log10 revertants per plate used Bartlett's test for homogeneity of variance and comparison of treatments with controls used within-levels pooled variance and a one-sided t-test. Where values were found to be significant, a Grubb's test was applied and significance of dose-response was evaluated by a t-test.
- 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
- Remarks:
- 1-3ul/plate for plate incorporation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main study toxicity was seen as follows: +S9; 10 μl, 3 μl (TA98, TA1535); 10 μl, 3 μl, 1 μl (TA100, TA1537) -S9: 10 μl, 3 μl (all strains)
- Remarks on result:
- other: No mutagenic potential
- Conclusions:
- In an in vitro gene mutation study in bacteria with the test substance ATMP acid (ATMP-H), conducted according to OECD TG 471 and in compliance with GLP, a lack of mutagenic potential occurred. Four Salmonella strains (S. typhimurium TA 98; 100; 1535; 1537) were tested in the absence and presence of rat and mouse liver S9-mix up to the toxicity limit (1-3μl/plate). It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Referenceopen allclose all
Results of Chromosome Aberration Assay
Test material: BRIQUEST 3010-25K
Harvest time: 20 hours
Treatment Group |
Replicate Identification |
No. of Cells Scored |
Total Gaps |
Chromatid |
Chromosome |
Others |
Total Aberrations |
Aberrant Cells |
Mean Mitotic Index |
||||
Breaks |
Exchanges |
Breaks |
Exchanges |
X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
|||||
Without Metabolic Activation |
|||||||||||||
Solvent Control |
Total |
200 |
3 |
1 |
0 |
0 |
0 |
0 |
4 |
7 |
4 |
1 |
8.05 |
393.75 (µg/ml) |
Total |
192 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
6.15 |
787.5 (µg/ml) |
Total |
200 |
3 |
0 |
0 |
0 |
1 |
0 |
4 |
1 |
4 |
1 |
4.73 |
1575 (µg/ml) |
Total |
200 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
2 |
1 |
3.25 |
Positive Control EMS 500 (µg/ml) |
Total |
200 |
30 |
41 |
0 |
14 |
0 |
0 |
85 |
55 |
57*** |
38*** |
4.58 |
With Metabolic Activation |
|||||||||||||
Solvent Control |
Total |
200 |
3 |
2 |
0 |
0 |
0 |
0 |
5 |
2 |
5 |
2 |
5.05 |
787.5 (µg/ml) |
Total |
200 |
4 |
0 |
0 |
1 |
0 |
0 |
5 |
1 |
5 |
1 |
4.15 |
1575 (µg/ml) |
Total |
200 |
4 |
2 |
0 |
0 |
0 |
0 |
6 |
2 |
6 |
2 |
4.15 |
3150 (µg/ml) |
Total |
200 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
1 |
1 |
4.03 |
Positive Control CP 25 (µg/ml) |
Total |
200 |
24 |
13 |
1 |
4 |
0 |
1 |
42 |
18 |
30*** |
17*** |
1.70 |
X = > 10 aberrations per cell (not included in total aberrations)
*** = p < 0.001
In the first experiment all treated cultures had higher RTGs than the control culture and a dose-related increase in mutant frequency was seen (see Table 1). The experiment was discounted because of the low control growth.
Table 1: Results of Mammalian Mutagenicity assay 1 with tester strain L5178Y in presence of metabolic activation
Concentration µl/ml |
Mutant frequency x10E-06 |
RTG % |
0* |
23 |
100 |
0.49 |
22 |
195 |
0.51 |
33 |
134 |
0.77 |
48 |
149 |
0.96 |
59 |
133 |
1.2 |
58 |
121 |
In the second experiment no increases in mutant frequency and no cytotoxicity were seen. It had been proposed that a selective advantage of mutants in the presence of test substance was the reason for the increase in revertants observed in the previous experiment. No effects on levels of spontaneous mutants were seen when cells were plated immediately after treatment. This finding does not support the proposed reason for the previously observed positive result.
Table 2: Results of Mammalian Mutagenicity assay 2 with tester strain L5178Y in presence of metabolic activation
Concentration µl/ml |
Mutant frequency x10E-06 |
RTG % |
0* |
101 |
100 |
0.61 |
91 |
100 |
0.77 |
112 |
74 |
0.96 |
98 |
92 |
1.2 |
100 |
88 |
1.5 |
112 |
103 |
Positive control |
659 |
46 |
Table 1: Summary of experiment 1
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
|
|
|
Without Activation |
|
WP2 uvrA |
|
|
|
Deionised water |
|
48 ± 8 |
Untreated |
|
48 ± 4 |
ATMP-H |
3 µg |
43 ± 5 |
|
10 µg |
48 ± 8 |
|
33 µg |
44 ± 8 |
|
100 µg |
48 ± 10 |
|
333 µg |
43 ± 9 |
|
1000 µg |
47 ± 11 |
|
2500 µg |
40 ± 7 |
|
5000 µg |
36 ± 5 |
MMS |
2.0 µL |
953 ± 87 |
|
|
|
With Activation |
|
|
Deionised water |
|
55 ± 3 |
Untreated |
|
56 ± 11 |
ATMP-H |
3 µg |
51 ± 8 |
|
10 µg |
57 ± 8 |
|
33 µg |
51 ± 13 |
|
100 µg |
49 ± 15 |
|
333 µg |
51 ± 7 |
|
1000 µg |
55 ± 7 |
|
2500 µg |
39 ± 9 |
|
5000 µg |
47 ± 13 |
2-AA |
10.0 µg |
534 ± 112 |
|
|
|
Table 2: Summary of experiment 2
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ± SD) |
Without Activation |
|
|
WP2 uvrA |
Deionised water |
|
|
32 ± 5 |
Untreated |
|
|
34 ± 4 |
ATMP-H |
33 µg |
|
36 ± 5 |
|
100 µg |
|
35 ± 5 |
|
333 µg |
|
32 ± 6 |
|
1000 µg |
|
31 ± 2 |
|
2500 µg |
|
33 ± 4 |
|
5000 µg |
|
19 ± 2 |
MMS |
2.0 µL |
|
945 ± 40 |
|
|
|
|
With Activation |
|
|
|
Deionised water |
|
|
42 ± 2 |
Untreated |
|
|
49 ± 12 |
ATMP-H |
33 µg |
|
34 ± 3 |
|
100 µg |
|
43 ± 2 |
|
333 µg |
|
37 ± 4 |
|
1000 µg |
|
36 ± 4 |
|
2500 µg |
|
35 ± 5 |
|
5000 µg |
|
28 ± 3 |
2-AA |
10.0 µg |
|
495 ± 16 |
|
|
|
|
MMS: methyl methane sulfonate
2-AA: 2-aminoanthracene
No increase in revertants in any strain/metabolic activation condition. Full details of results presented in report. Positive and negative values acceptable.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
n vivo micronucleus study: read-across from ATMP-xNa, negative (OECD Test Guideline 474) (Covance, 1998)
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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 12/96 final draft
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: Crl: CD-1 (ICR) BR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Labs Raleigh NC
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 30.6 - 33.9 grams (males); 23.6-28.4 grams (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 5 animals per polycarbonate cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 °C
- Humidity (%): 30-70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 10 ml/kg - Duration of treatment / exposure:
- 2 days
- Frequency of treatment:
- 24, 48h
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Range finding experiments: 3 male and 3 female per dose; 6 males per dose in main experiment.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - cyclophosphamide;
- Justification for choice of positive control(s): none given
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg - Tissues and cell types examined:
- Bone marrow erythrocytes
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Based on two range-finding studies.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 500 mg/kg bw, 1000 mg/kg bw and positive control harvested at 24 hours; 2000 mg/kg bw and vehicle control harvested at 24 and 48 hours.
DETAILS OF SLIDE PREPARATION: Giesma stain
METHOD OF ANALYSIS: scored for micronuclei and PCE NCE ratio - Evaluation criteria:
- Criteria for positive: Statistically significant increase in micronucleated PCEs or at least one dose level and a statistically significant dose-related response.
- Statistics:
- ANOVA followed by Dunnett's t-test when ANOVA is positive
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Remarks:
- up to limit concentration
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- In an in vivo mammalian erythrocyte micronucleus study, conducted according to OECD 174 and in compliance with GLP, ATMP sodium salt did not increase the frequency of micronucleated erythrocytes. It is concluded that ATMP-xNa is negative for the induction of micronuclei under the conditions of this test.
Reference
No clinical toxicity and no cytotoxicity to the bone marrow was observed in any treated animals. No increase in the frequency of micronucleated erythrocytes occurred in any test substance dose.
Table 1: Mean Results of in vivo micronucleus test in mouse bone marrow
- |
Solvent Control (water) |
Positive Control (Cyclophosphamide) |
Low dose 500 mg/kg |
Mid dose 1000 mg/kg |
High dose 2000 mg/kg |
||
Sampling time (h) |
24 |
48 |
24 |
24 |
48 |
24 |
48 |
Number of cells analysed |
2000 |
2000 |
2000 |
2000 |
2000 |
2000 |
2000 |
Micronucleated cells per animal % |
0.07 |
0.07 |
2.73 |
0.04 |
0.09 |
0.02 |
0.06 |
Ratio PCE/NCE |
0.52 |
0.47 |
0.50 |
0.59 |
0.44 |
0.41 |
0.60 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro studies:
Reliable information is available on the cytogenicity (in vitro) of ATMP-N-oxide-5K. For endpoints where reliable studies were not available for ATMP-N-oxide-5K, key studies for ATMP-H or ATMP-xNa were read across. Read-across between acids and salts is considered to be scientifically justified as the counterions are not significant in genotoxicity and have been assessed in depth in the public literature. The results of all the studies on ATMP-N-oxide-5K were negative.
An in vitro gene mutation study in bacteria with the test substance ATMP-H, conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP, showed a lack of mutagenic potential. Four Salmonella strains (S. typhimurium TA 98; 100; 1535 and 1537) were tested in the absence and presence of rat and mouse liver S9-mix up to the toxicity limit (1-3μl/plate). Appropriate vehicle and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Monsanto, 1981).
In a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 and in compliance with GLP, ATMP-H was tested using E. coli WP2 uvr A. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to the limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Envigo, 2018).
In a low-reliability supporting in vitro bacterial gene mutation study, conducted according to OECD Test Guideline 471 and in compliance with GLP, ATMP-N-oxide-5K was tested in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. No evidence of induction of revertants was observed. Cytotoxicity was not recorded, and the substance was tested up to a concentration equivalent to approximately 1350 µg/plate. It is concluded that the substance is negative for mutagenicity to bacteria under the conditions of the test (Instituto di Ricerche Biomediche, 1992).
In an additional low-reliability supporting in vitro bacterial mutagenicity study, conducted in a similar manner to OECD Test Guideline 471 and in compliance with GLP, ATMP-N-oxide-5K was tested in Salmonella typhimurium strains TA98 and TA100. No test substance related increase in the number of revertants was observed in the presence and absence of metabolic activation and no toxicity to bacteria was observed. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Safepharm Laboratories, 1994).
In an in vitro cytogenicity / chromosome aberration study in mammalian cells, conducted according to OECD Test Guideline 473 and in compliance with GLP, ATMP-N-oxide-5K did not cause any increase in the number of cells with aberrations when peripheral human lymphocytes were tested up to cytotoxic concentrations. Exposure lasted for 4 hours in the presence of metabolic activation and for 20 hours in the absence of metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test (Safepharm Laboratories, 1994).
In a supporting in vitro chromosome aberration test, conducted according to OECD Test Guideline 473 and in compliance with GLP, ATMP-xNa did not induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence and absence of metabolic activation. In absence of metabolic activation, no cytotoxicity was observed with 3-hour treatment with <500 µg/ml. With continuous treatment, cytotoxicity was induced. The top dose of 250 µg/ml had a relative mitotic index of 60%. The higher dose of 500 µg/ml had a relative mitotic index of <10%. It is concluded that ATMP-xNa does not cause chromosomal aberrations under the conditions of the test (Covance, 1998).
In an in vitro gene mutation study in mammalian cells, conducted according to a protocol similar to OECD Test Guideline 476 and in compliance with GLP, no increase in the number of revertants was observed in the presence of metabolic activation when ATMP-xNa was tested up to the solubility limit. It is concluded that ATMP-xNa is not mutagenic to mammalian cells under the conditions of this test (SRI, 1988).
In vivo study:
In an in vivo mammalian erythrocyte micronucleus study, conducted according to OECD Test Guideline 474 and in compliance with GLP, ATMP-xNa did not increase the frequency of micronucleated erythrocytes. It is concluded that ATMP-xNa is negative for the induction of micronuclei under the conditions of this test (Covance, 1998).
Justification for classification or non-classification
The available data indicate that when tested in vitro, ATMP-N-oxide-5K did not cause chromosomal aberrations when tested in vitro in the presence or absence of metabolic activation. The information on mutagenicity comes from tests on ATMP acid, which did not cause an increase in revertants in bacterial cells. This is supported by the reliability 4 data for ATMP-N-oxide-5K. A neutralised solution of ATMP acid did not cause mutations in mammalian cells. On the basis of this data, it is considered that classification for mutagenicity is not required according to Regulation (EC) No 1272/2008.
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