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EC number: 229-146-5 | CAS number: 6419-19-8
- 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
The test substance ATMP-H (CAS 6419-19-8, EC No. 229-146-5) was negative with and without metabolic activation in four strains (S. typhimurium TA98, 100, 1535, 1537) tested for genetic mutation in the bacterial reverse mutation assay (Ames test). The test was conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP (Monsanto, 1981, Reliability 2).
There are no reliable studies for ATMP-H (CAS 6419-19-8, EC No. 229-146-5) for a fifth strain capable of detecting cross-linking agents, chromosomal aberrations or mammalian cell mutagenicity, therefore data were read-across from ATMP-xNa (CAS 20592-85-2, EC No., 243-900-0).
The results for ATMP-xNa (CAS 20592-85-2, EC No., 243-900-0) were negative for genetic mutation in the bacterial reverse mutation assay (Ames test) with and without metabolic activation in the E. coli WP2 uvr A strain tested according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP (Envigo, 2018, Reliability 2).
ATMP-xNa was negative in a cytogenicity test in CHO mammalian cells conducted according to OECD Test Guideline 473 and in compliance with GLP (Covance Laboratories, 1998a, Reliability 1).
ATMP-xNa was negative for mutagenicity with metabolic activation
in L5178Y mouse lymphoma cells. The test was conducted according to a
similar protocol to OECD Test Guideline 476 and in compliance with GLP
(SRI International, 1988, Reliability 1).
Key genetic toxicity studies on DTPMP acid and sodium salts are included in support of read-across of the reproductive toxicity study on DTPMP (5-7Na) only.
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:
- 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, ATMP-H was dissolved in deionised water to form the sodium salt. The solvent was chosen because of its solubility properties and its relative non-toxicity 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 each 3 plates. 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 selected 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: deionised water
- Justification for choice of solvent/vehicle: The solvent was selected 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 approximately 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 each 3 plates. 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 selected as the 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 (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Within the range of positive historical control data
- Negative (solvent/vehicle) 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 (reliability 2), conducted according to OECD Test Guideline 471 and in compliance with GLP, ATMP-xNa 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 limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that ATMP-xNa 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:
- key study
- Study period:
- Not specified
- 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 test material/plate and 25 μL test material/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 or 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
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1-3 μL/plate for plate incorporation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1-3 μL/plate for plate incorporation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1-3 μL/plate for plate incorporation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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
- Remarks:
- 1-3 μL/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 bacterial mutagenicity assay (reliability 2), conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP, ATMP-H was tested for mutagenicity in Salmonella typhimurium TA98, 100, 1535 and 1537. The test substance did not increase the number of revertants when tested up to cytotoxic concentration with or without metabolic activation. The vehicle and positive controls gave the expected results. ATMP-H was concluded to be negative for mutagenicity in bacteria 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
- Metabolic activation system:
- Aroclor induced rat liver S9
- Test concentrations with justification for top dose:
- 0-1.2 µL test material/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 0.08 pH units only
- Precipitation: White precipitate reported at concentrations greater than 0.61 µL/mL
RANGE-FINDING/SCREENING STUDIES: Not conducted as this is a follow-up experiment
COMPARISON WITH HISTORICAL CONTROL DATA: No data
- Conclusions:
- In a mammalian mutagenicity assay (reliability 1), conducted to a protocol similar to OECD Test Guideline 476 and in compliance with GLP, a neutralised solution of ATMP acid (ATMP-xNa) was tested in mammalian cells. No increase in the number of revertants was observed in the presence of metabolic activation when tested up to the solubility limit. The test results indicate that the positive findings in the previous experiment with un-neutralised ATMP acid were an artefact of pH. It is concluded that ATMP-xNa is not mutagenic to mammalian cells under the conditions of this test.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1998-06-25 to 1998-08-05
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat liver S9
- Test concentrations with justification for top dose:
- up to 4400 µg active salt/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: None given in report - Untreated negative controls:
- yes
- Remarks:
- growth medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without activation
- Untreated negative controls:
- yes
- Remarks:
- growth medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium
DURATION
- Exposure duration: 3 hours (initial assay) 17.8 hours (confirmatory assay)
- Expression time (cells in growth medium): 16.8 hours (initial assay 2 hours (confirmatory assay)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (present for last 2 hours of incubation)
STAIN (for cytogenetic assays): 5% Giesma solution
NUMBER OF REPLICATIONS: Duplicate cultures
NUMBER OF CELLS EVALUATED: 100 from each replicate culture where possible
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: Assessment of percent confluence of cell monolayer and presence of mitotic or dead cells floating in medium.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes - Evaluation criteria:
- The test substance is considered positive for inducing chromosomal aberrations if there is significant dose-dependent increase (p<=0.01) in the number of cells with aberrations at one or more concentrations.
- Statistics:
- Cochran-Armitage test for linear trend and Fisher's Exact test to compare percentage of cells with aberrations in treated cells with control results.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Non cytotoxic for 3h treatments. <500 µg/ml for continuous treatments without activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was measured at 9.0 (culture medium pH 8.5)
- Precipitation: No precipitation was observed
- Other confounding effects: Not specified
RANGE-FINDING/SCREENING STUDIES: No precipitate observed in the absence of cells at a concentration of 4400 μg/mL
COMPARISON WITH HISTORICAL CONTROL DATA: Control values were within historical control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: Dead monolayers and approximately 85% reduction in monolayer confluence reported at 4400 µg/mL. Unhealthy monolayers and approximately 70% reduction reported at 3080 µg/mL. Unhealthy monolayers and approximately 15% or 45% reduction in monolayer confluence at 2160 µg/mL. In absence of metabolic activation, no cytotoxicity was seen with 3 ou treatment. With continuous treatment cytotoxicity was induced. The top dose scored (250 µg/mL) had a relative mitotic index of 60%. The higher dose (500 µg/mL) had a relative mitotic index of <10%. - Conclusions:
- In an in vitro cytogenicity assay (Reliability 1), conducted according to OECD Test Guideline 473 and in compliance with GLP, ATMP-xNa has been tested for clastogenicity in Chinese hamster Ovary (CHO) cells. The test substance did not induce chromosome aberrations in vitro when tested up to cytotoxic concentration with or without metabolic activation. Positive, negative and solvent controls were included and gave the expected results. It is concluded that ATMP-xNa does not cause chromosomal aberrations under the conditions of the test.
Referenceopen allclose all
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.
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: Results of chromosome analysis Experiment 1 (3 h treatment, 20 incubation) without activation (total count from 2 cultures / 200 cells)
- |
Untreated |
Solvent* Control |
Positive Control |
Low dose 1510 µg/mL |
Mid dose 2160 µg/mL |
Mid dose 3080 µg/mL |
High dose 4400 µg/mL |
|
Cytotoxicity |
- |
- |
- |
no |
no |
no |
no |
|
|
Percentage from 200 cells |
|||||||
Percentage of cells with aberrations |
3.0 |
1 |
52 |
3.5 |
0 |
3.0 |
0 |
|
Mitotic index (%) |
14.8 |
20.8 |
NR |
23.1 |
23.5 |
24.0 |
17.8 |
|
Polyploidy (%) |
2.5 |
2.5 |
3.0 |
2.5 |
3.0 |
2.1 |
2.0 |
|
Endo reduplication (%) |
2 |
1.5 |
1.0 |
2.0 |
1.5 |
0.5 |
2.5 |
*Solvent control with water
NR not reported
Table 2: Results of chromosome analysis Experiment 2a (17.8 h treatment, 20 h incubation) without activation
- |
Untreated |
Solvent* Control |
Positive Control |
Low dose 31.3 µg/ml |
Mid dose 62.6µg/ml |
Mid dose 125 µg/ml |
High dose 250 µg/ml |
|
Cytotoxicity |
- |
- |
- |
no |
no |
no |
no |
|
|
Percentage from 200 cells |
|||||||
Percentage of cells with aberrations |
0 |
0 |
12.8 |
1.5 |
0 |
1.0 |
1.5 |
|
Mitotic index (%) |
6.5 |
7.0 |
NR |
4.0 |
6.9 |
4.0 |
4.2 |
|
Polyploidy (%) |
9.5 |
0.5 |
2.5 |
1.5 |
0 |
0.5 |
0.5 |
|
Endo reduplication (%) |
0 |
0 |
0 |
1.5 |
0 |
0 |
0 |
*Solvent control with water
NR not reported
Table 3: Results of chromosome analysis Experiment 1, (3 h treatment, 20 h incubation) with activation
- |
Untreated |
Solvent* Control |
Positive Control |
Low dose 1510 µg/mL |
Mid dose 2160 µg/mL |
Mid dose 3080 µg/mL |
High dose 4400 µg/mL |
|
Cytotoxicity |
- |
- |
- |
no |
no |
no |
no |
|
|
Percentage from 200 cells |
|||||||
Percentage of cells with aberrations |
1.5 |
1.0 |
60.0 |
1.0 |
8.0 |
1.5 |
1.0 |
|
Mitotic index (%) |
16.0 |
19.9 |
NR |
21.9 |
15.7 |
16.9 |
11.7 |
|
Polyploidy (%) |
3.0 |
2.5 |
2.5 |
2.0 |
2.0 |
2.0 |
1.0 |
|
Endo reduplication (%) |
1.5 |
0 |
1.5 |
1.0 |
6.0 |
0 |
0 |
*Solvent control with water
NR not reported
Table 4: Results of chromosome analysis Experiment 2, (3 h treatment, 20 h incubation) with activation
- |
Untreated |
Solvent* Control |
Positive Control |
Low dose 1510 µg/mL |
Mid dose 2160 µg/mL |
Mid dose 3080 µg/mL |
High dose 4400 µg/mL |
|
Cytotoxicity |
- |
- |
- |
no |
no |
no |
no |
|
|
Percentage from 200 cells |
|||||||
Percentage of cells with aberrations |
1.5 |
2.0 |
52 |
1.0 |
1.5 |
0.5 |
0.5 |
|
Mitotic index (%) |
13.1 |
14.1 |
NR |
12.8 |
13.8 |
13.3 |
13.3 |
|
Polyploidy (%) |
2.0 |
1.5 |
2.0 |
2.0 |
3.0 |
2.0 |
2.5 |
|
Endo reduplication (%) |
2.0 |
1.5 |
0.5 |
2.5 |
2.9 |
1.0 |
2.5 |
*Solvent control with water
NR not reported
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In an in vivo micronucleus assay, read-across from ATMP-xNa (CAS 20592-85-2, EC No., 243-900-0) was negative in mice when administered via oral gavage. Testing was conducted according to OECD Test Guideline 474 and in compliance with GLP (Covance Laboratories, 1998b, reliability 1).
Key genetic toxicity studies on DTPMP acid and sodium salts are included in support of read-across of the reproductive toxicity study on DTPMP (5-7Na) only.
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: approx 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: 10 mL/kg - Duration of treatment / exposure:
- 2 days
- Frequency of treatment:
- At 24 and 48 hours
- 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 the 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: 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 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 a reliable in vivo micronucleus study (reliability 1), conducted according to OECD Test Guideline 474 and in compliance with GLP, ATMP-xNa has been tested for clastogenicity. No increase in the frequency of micronucleated erythrocytes was observed. Vehicle and positive controls were included and gave expected results. 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
Reliable information is available on the mutagenicity of ATMP-H (CAS 6419-19-8, EC 229-146-5). For endpoints where reliable studies were not available for ATMP-H, key studies for ATMP-xNa (CAS 20592-85-2, EC 243-900-0) DTPMP (5-7Na) salt (EC 701-216-4) were read across. See attachment to IUCLID Section 13 for justification of read-across.
ATMP-H has been tested for mutagenicity in vitro in Salmonella typhimurium TA98, 100, 1535, 1537 in a study conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP (Monsanto, 1981, Reliability 2). ATMP-H did not increase the number of revertants when tested up to cytotoxic concentration with or without metabolic activation. The vehicle and positive controls gave the expected results. ATMP-H was concluded to be negative for mutagenicity in bacteria under the conditions of the test.
ATMP-xNa was tested in a valid bacterial reverse mutation assay, according to OECD Test Guideline 471 and in compliance with GLP, using E. coli WP2 uvr A (Envigo, 2018, Reliability 2). No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to 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.
ATMP-xNa has been tested for clastogenicity in vitro in Chinese hamster Ovary (CHO) cells in a study conducted according to OECD Test Guideline 473 and in compliance with GLP (Covance Laboratories, 1998a, Reliability 1). The test substance did not induce chromosome aberrations in vitro when tested up to cytotoxic concentration with or without metabolic activation. Positive, negative and solvent controls gave the expected results. It is concluded that ATMP-xNa does not cause chromosomal aberrations under the conditions of the test.
ATMP-H has been tested for mutagenicity to mammalian cells in mouse lymphoma L5178Y cells in a study conducted according to a protocol similar to OECD Test Guideline 476 and in compliance with GLP (SRI International, 1988, Reliability 1). No increase in the number of revertants was observed in the presence of metabolic activation when tested up to the solubility limit. The test results indicate that the positive findings in the previous experiment with unneutralised ATMP acid were an artefact of pH (SRI International, 1982, Reliability 1). It is concluded that ATMP-H is not mutagenic to mammalian cells under the conditions of this test.
ATMP-xNa has been tested for clastogenicity in vivo in a reliable study conducted according to OECD Test Guideline 474 and in compliance with GLP. No increase in the frequency of micronucleated erythrocytes was observed. It is concluded that ATMP-xNa is negative for the induction of micronuclei under the conditions of this test (Covance Laboratories, 1998b, Reliability 1).
DTPMP (5-7 Na) salt (EC 701-216-4) was tested for genotoxicity in the in vivo Alkaline Comet Assay in accordance with OECD Test Guideline 489 and in compliance with GLP (Charles River Laboratories, 2022, reliability 1). DTPMP (5 -7Na) salt was administered twice daily for two consecutive days via oral gavage to male Wistar rats at 500, 1000 and 2000 mg/kg bw/day in milli-Q water. No clinical signs of toxicity were observed. A positive control group was dosed twice by oral gavage with Ethyl Methane Sulfonate (EMS) at 200 mg /kg bw/day in physiological saline. Approximately 3-4 hours after the last dose the animals were sacrificed and single cell suspensions from liver, duodenum and stomach were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and stomach cells of test item treated animals compared to the vehicle (water) treated animals. In the stomach, mean Tail Intensities (%) were observed to be slightly higher than the 95% control limits of the distribution of the historical control data for the vehicle control (up to 12.63% versus 11.2%). However, the increase is minor and the results were within the maximum ranges observed in the historical data. Additionally, results were clearly distinct from the positive control (66.67%) and there was no statistically significant increase observed.
The mean Tail Intensity in liver, duodenum and stomach cells of vehicle-treated rats was 2.59 ± 0.52% (mean ± SD), 8.89 ± 0.68% (mean ± SD) and 11.34 ± 1.69% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control for liver and duodenum. The mean Tail Intensity in stomach cells of vehicle-treated rats was slightly above the 95% control limits (11.2%). However, this was considered to have no impact, as the increase is minor (0.14%), within the maximum range observed and it is clearly distinct from the positive control (66.67%). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 88.08 ± 2.64% (mean ± SD), 58.69 ± 2.75% (mean ± SD) and 66.67 ± 1.78% (mean ± SD) in male animals in liver, duodenum and stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed and the highest test dose was the maximum dose required by the guideline. Hence, all criteria for an acceptable assay were met. In conclusion, the test is valid and DTPMP (5-7 Na) salt is not genotoxic in the Comet assay in liver, duodenum and stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg (Charles River Laboratories, 2022, reliability 1).
Justification for classification or non-classification
Based on the available data, no classification for genetic toxicity is required according to Regulation (EC) No 1272/2008.
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