Registration Dossier
Registration Dossier
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EC number: 274-778-7 | CAS number: 70693-62-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro Bacteria Mutation Assay: Negative
In vitro Chromosome Aberration Test: Positive
In vitro Mouse Lymphoma Assay: Negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2001-03-15 - 2001-10-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- not indicated
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- other: histidine missense mutation (hisG46), deficient in a DNA repair system (uvrB), defective lipopolysaccharide coat on the wall (rfa)
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- other: histidine frameshift mutation (hisC3076), defective in a DNA repair system and lipopolysaccharide coat
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: histidine frameshift mutation (hisD3052), defective in a DNA repair system and lipopolysaccharide coat
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: histidine missense mutation (hisG46), deficient in a DNA repair system (uvrB), defective lipopolysaccharide coat on the wall (rfa)
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Additional strain / cell type characteristics:
- other: tryptophan dependent mutant
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Aroclor 1254 induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- First test (range finding):
5000, 1500, 500, 150, 50, 15 and 5 µg/plate (with and without metabolic activation). Based on the results of the range-finding (cytotoxicity) test, the following concentrations were selected for the main test:
Second (main) test:
5000, 1500, 500, 150 and 50 µg/plate with metabolic activation
500, 150, 50, 15 and 5 µg/plate without metabolic activation - Vehicle / solvent:
- not indicated
- Untreated negative controls:
- yes
- Remarks:
- water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: Without metabolic activation: Sodium azide, 9 Aminoacridine, 2 Nitrofluorene, 2 (2 Furyl) 3 (5 nitro 2 furyl)acrylamide With metabolic activation: 2-Aminoanthracene, Benzo[a]pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Aliquots of 0.1 mL of the test dilution (in water), positive or negative control, were placed in glass vessels.
0.5 mL S9 mix or 0.5 mL 0.1 M phosphate buffer (pH 7.4) was added, followed by 0.1 mL of a 10 hour bacterial culture and 2 mL of agar containing histidine (0.5 mM) and tryptophan (0.5 mM) at 45+/-2°C. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each concentration. All plates were incubated at 37°C for ca. 72 hours.
DURATION
- Preincubation period: In the second test:
Tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 min. with shaking before the addition of the agar overlay.
- Exposure duration: 72 hours
NUMBER OF CELLS EVALUATED: 10 E09 - Evaluation criteria:
- For a test to be considered valid the mean of the solvent/vehicle control revertant colony number for each strain should lie within the historical control range.
The positive control compounds must cause at least a doubling of mean revertant colony number over the negative control.
The mean number of revertant colonies for each treatment group was compared with those obtained for the solvent/vehicle control groups. - Statistics:
- not indicated
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate with S9 mix; 500 µg/plate without S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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:
- 5000 µg/plate with S9 mix; 500 µg/plate without S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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:
- 5000 µg/plate with S9 mix; 500 µg/plate without S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate with S9 mix; 500 µg/plate without S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate with S9 mix; 1500 µg/plate without S9 mix;
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXICITY:
Please refer to tables 1 and 2, which are presented under "Remarks on results including tables and figures"
- without metabolic activation: No increase in the number of revertants/plate observed
- with metabolic activation: No increase in the number of revertants/plate observed
CYTOTOXICITY:
Toxicity was seen in all strains following exposure to KMPS triple salt at 5000 µg/plate in the presence of S9 mix, and at 1500 µg/plate (E. coli) or 500 µg/plate (S. typhimurium) in the absence of S9 mix. - Conclusions:
- The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). Under the test conditions employed, KMPS triple salt showed no evidence of mutagenic activity in this bacterial system in the absence or presence of metabolic activation.
- Executive summary:
Materials and methods
The mutagenic potential of KMPS triple salt was investigated in the Ames test using the histidine auxotroph S. typhimurium strains TA98, TA100, TA1535, TA1537, and a tryptophan dependent mutant of E. coli, WP2uvrA/pKM101, all with and without metabolic activation by rat liver S9 mix from Aroclor 1254 induced male Sprague-Dawley rats. KMPS triple salt was tested at concentrations of 5000, 1500, 500, 150 and 50 µg/plate with metabolic activation and 500, 150, 50, 15 and 5 µg/plate without metabolic activity. The solvent used for the test substance was water.
Results and discussion
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to KMPS triple salt at any concentration in the presence or absence of S9 mix.
Toxicity was seen in all strains following exposure to KMPS triple salt at 5000 µg/plate in the presence of S9 mix, and at 1500 µg/plate (E. coli) or 500 µg/plate (S. typhimurium) in the absence of S9 mix.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2001-03-05 - 2001-10-31
- 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
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- mammalian cell line, other: human lymphocytes
- Details on mammalian cell type (if applicable):
- Deficiency:
- Limited capacity for metabolic activation of potential mutagens (exogenous activation system required)
- PHA (phythaemagglutinin) to be added to cultures to stimulate cell division in vitro of human lymphocytes
Proficiency:
- Chromosome constitution remains diploid in stimulated human peripheral blood lymphocytes
- Stable karyotype with a chromosome number of 46
- Population of lymphocytes is only partially synchronised and single treatment at or close to the time when metaphase stages first appear in the culture will include cells in all stages of the division cycle - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from rat livers
- Test concentrations with justification for top dose:
- First test: Treatment time 3 h, 17 h recovery time
39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg KMPS triple salt/mL (with and without S9 mix)
Second test: Treatment time 3 h, 17 h recovery time
0, 250, 500, 750, 1000, 1250 and 1500 µg KMPS triple salt/mL (with and without S9 mix) - Vehicle / solvent:
- water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: without metabolic activation: Mitomycin C at 0.1 µg/mL (test 1) and 0.2 µg/mL (test 2) dissolved in sterile water; with metabolic activation: Cyclophosphamide (CPA) at 6 µg/mL dissolved in sterile water
- Details on test system and experimental conditions:
- Metabolic activation system
- S9 mix from rat livers
- Source: male rats; strain: Sprague Dawley; age: 7 – 8 weeks; weight: < 300 g; supplier: Charles River UK;
- Induction of rats: single i.p. injection of 500 mg/kg bw Aroclor 1254 in corn oil as the vehicle
- Isolation of S9 mix 5 days after i.p. treatment by washing and homogenising isolated livers and centrifugation at 9000 g for 10 min. at 4°C
- S9 mix contained: S9 fraction (10 % v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose 6 phosphate (5 mM) and NADP (4 mM)
Way of application: Solvent for KMPS triple salt:
Water (on dosing at 10 % v/v into aqueous tissue culture medium, giving a final concentration of 5000 µg/mL, no precipitate was observed
Incubation times:
- after collection of blood, human lymphocytes were cultured for 48 h prior to treatment in the presence of phythaemagglutinin (PHA) to stimulate cell division
- treatment time with test article was 3 h (with and without S9 mix)
Further remarks:
2 h prior to harvest, cells were incubated with colcemid at 0.1 µg/mL to arrest mitotic activity
EXAMINATIONS
Number of cells evaluated
Cytotoxicity investigation (harvest time 20 hours after initiation of exposure):
Proportion of mitotic cells per 1000 cells in each culture was recorded
On the basis of these data, the following concentrations were selected for metaphase analysis:
First test: 312.5, 625 and 1250 µg/mL
Second test: 500, 1000 and 1250 µg/mL
Chromosomal aberrations (harvest time 20 hours after initiation of exposure):
100 metaphases per culture (each concentration in duplicate resulting in a total of 200 metaphases per concentration level)
The incidence of polyploid metaphase cells (numerical aberrations), out of 500 metaphase cells (1000 cells in total per concentration level), was determined quantitatively for negative control and cultures treated with the highest dose level of the test substance used in the analysis for chromosomal aberrations. - Evaluation criteria:
- Criteria for evaluation of a mutagenic effect:
- The number of chromosomal aberrations in the negative control and/or solvent control and positive control falls within the historical control range of the laboratory
The test substance is considered to cause a positive response if the following conditions are met:
- Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one test concentration.
- The increases exceed the negative control range of the laboratory, taken at the 99 % confidence limit.
- The increases are reproducible between replicate cultures.
- The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
- Evidence of a dose-relationship is considered to support conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level. - Statistics:
- One-tailed Fisher´s test was performed.
- Key result
- Species / strain:
- mammalian cell line, other: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- with and without metabolic activation
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The tables mentioned below are presented under: "Remarks on results including tables and figures"
GENOTOXICITY
- without metabolic activation:
First test (please refer to Table 1):
KMPS triple salt caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 1250 µg/mL (P<0.001; both excluding and including gaps), when compared with the solvent control value.
Second test (please refer to Table 2):
The test substance caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 1250 µg/mL (P<0.001 excluding gaps; P<0.01 including gaps), when compared with the solvent control value.
- with metabolic activation:
First test (please refer to Table 1):
KMPS triple salt caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 1250 µg/mL (P<0.01 excluding gaps; P<0.001 including gaps), when compared with the solvent control value.
Second test (please refer to Table 2):
The test substance caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 1250 µg/mL (P<0.001 both excluding and including gaps), when compared with the solvent control value.
CYTOTOXICITY
First test (please refer to Table 1):
In both in the absence and presence of S9 mix, KMPS triple salt caused a reduction in the mitotic index to 53 % of the solvent control value at 1250 µg/mL.
Second test (please refer to Table 2):
KMPS triple salt caused a mitotic index to 52 % of the solvent control in the absence of S9 mix and to 45 % in the presence of S9 mix at 1250 µg/mL. - Conclusions:
- The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). Under the experimental conditions described, the test substance KMPS triple salt has shown evidence of clastogenic activity in this in vitro cytogenetic test system in both the absence and presence of a metabolic activation system.
- Executive summary:
Materials and methods
The mutagenic potential of KMPS triple salt was investigated in human blood lymphocytes in vitro both with and without metabolic activation.
Human lymphocytes were obtained from healthy, non-smoking male donors. Cells were cultured for 48 h in the presence of a cell mitogen (PHA) to stimulate mitosis prior to treatment with the test substance.
The study was performed in two separate occasions. In the first test, a three hour treatment was used without and with S9 mix. In the second test, a three hour treatment was used with a narrower concentration range in both the absence and presence of S9 mix.
In both tests, cultured cells were treated for a period of 3 h with the test substance with and without metabolic activation. Thereafter, the test substance was removed, cells resuspended and cultured for another 17 h (20 h harvest time). Two hours before harvesting, cells were treated with Colcemide for a period of 2 h in order to arrest cells in the metaphase. Thereafter, cells were fixed with methanol : glacial acetic acid (3:1), slides were prepared and cells stained with Giemsa. Slides were evaluated for structural and numerical chromosomal aberrations. 100 well spread metaphases per culture (200 metaphases for duplicate cultures per concentration level) were scored for cytogenetic damage on coded slides. In addition, cytotoxicity (expressed as the mitotic index) and the number of polyploid cells was recorded as well.
Results and discussion
KMPS triple salt was tested for structural and numerical chromosomal aberrations in human blood lymphocytes in vitro. In the absence and presence of metabolic activation, the concentrations tested were 312.5, 625 and 1250 µg/mL in the first test and 500, 1000 and 1250 µg/mL in the second test for the 20 h harvest time point (3 hours incubation time in the presence of test material, 17 hours recovery time).
In the first test KMPS triple salt caused a reduction in the mitotic index to 53 % of the solvent control value at 1250 µg/mL (in both, the absence and presence of S9 mix). In the second test also a reduction in the mitotic index to 52 % (without S9 mix) and 45 % (with S9 mix) was recorded.
In both tests KMPS triple salt caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 1250 µg/mL (with and without S9 mix), when compared with the solvent control value. Increases exceeded the negative control range of the performing laboratory. Evidence of a dose-relationship is considered to support the conclusion. However, the increases in CA frequency were not dramatic and seen mainly at relatively high levels of cytotoxicity.
Both tests showed no statistically significant increases in the number of polyploid metaphase cells when compared with the solvent control, i.e. no increase in numerical chromosomal aberrations was noted.
No significant increases in osmolality and pH were seen at dose levels of 312.5, 500, 625, 1000 and 1250 µg/mL of test substance.
Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-04-25 to 2019-08-05
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- clone TK+/- -3.7.2C
- Details on mammalian cell type (if applicable):
- For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of the laboratory. This allows the repeated use of the same cell batch in experiments. Each cell batch is routinely checked for mycoplasma infection. Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Doubling time: 10 - 12 h; cloning efficiency: >50%
- Modal number of chromosomes: 40 +/- 2 chromosomes
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route
- method of preparation of S9 mix : according to Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.75 mg/mL
- quality controls of S9: The following quality control determinations were performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test - Test concentrations with justification for top dose:
- without and with metabolic activation:
50, 100, 125, 250, 500, 1000 and 2000 μg/mL
Concentrations are based on results of a pre-test. - Vehicle / solvent:
- - Vehicle/solvent used: deionized water
- Justification for choice of solvent/vehicle: standard vehicle according to the guideline - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionized water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- methylmethanesulfonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1 x 107 cells
- Test substance added: in suspension
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (incubation with a selective agent): 7 days
- Method used: microwell plates
- Selective agent: trifluorothymidine
- Criteria for small (slow growing) and large (fast growing) colonies:
- Small colonies approximately ≤ ¼ of well diameter
- Large colonies approximately > ¼ of well diameter
Size is the key factor and morphology should be secondary.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative cloning efficiency; relative total growth (RTG) - Rationale for test conditions:
- according to guidelines
- Evaluation criteria:
- The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a concentration-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
For statistical analysis the evaluation of the results by the Mann-Whitney test was performed using GraphPad prism and might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: acceptable
- Data on osmolality: acceptable
- Possibility of evaporation from medium: no
- Water solubility: well
- Precipitation and time of the determination: no - Conclusions:
- In the described mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells. Moreover, there are no indications of a clastogenic potential.
- Executive summary:
The test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations:
without and with metabolic activation:
50, 100, 125, 250, 500, 1000 and 2000 μg/mL
No precipitation of the test item was noted in the experiment. No growth inhibition was observed without and with metabolic activation. No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation) was not exceeded by the induced mutant frequency at any concentration.
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.
Referenceopen allclose all
Table 1: Results test 1 (plate-incorporation method)
Plate |
S9 mix |
Revertant colony mean counts |
||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvr/pKM101 |
||
S9mix, sterility check |
+ |
0 |
0 |
0 |
0 |
0 |
Buffer, sterility check |
- |
0 |
0 |
0 |
0 |
0 |
KMPS triple salt, sterility check |
- |
0 |
0 |
0 |
0 |
0 |
KMPS triple salt, 5000 µg/plate |
+ |
4±3* |
8±7* |
3±4* |
1±1* |
34±9 |
KMPS triple salt, 1500 µg/plate |
+ |
39±4 |
129±7 |
15±2 |
15±2 |
130±12 |
KMPS triple salt, 500 µg/plate |
+ |
40±5 |
133±9 |
17±6 |
20±7 |
124±9 |
KMPS triple salt, 150 µg/plate |
+ |
38±6 |
145±15 |
22±2 |
20±4 |
150±19 |
KMPS triple salt, 50 µg/plate |
+ |
39±5 |
135±14 |
17±5 |
20±6 |
163±22 |
KMPS triple salt, 15 µg/plate |
+ |
45±10 |
140±11 |
20±5 |
22±3 |
141±12 |
KMPS triple salt, 5 µg/plate |
+ |
48±5 |
143±10 |
19±2 |
18±2 |
143±12 |
Water, 0.1 mL/plate |
+ |
45±3 |
148±3 |
21±3 |
22±2 |
162±8 |
KMPS triple salt, 5000 µg/plate |
- |
0±0* |
0±0* |
0±0* |
0±0* |
0±0 |
KMPS triple salt, 1500 µg/plate |
- |
0±0* |
0±0* |
0±0* |
0±0* |
12±9 |
KMPS triple salt, 500 µg/plate |
- |
1±1* |
13±6* |
2±2* |
1±1* |
129±12 |
KMPS triple salt, 150 µg/plate |
- |
37±4 |
121±4 |
13±4 |
14±3 |
117±1 |
KMPS triple salt, 50 µg/plate |
- |
37±4 |
139±8 |
15±4 |
11±3 |
114±3 |
KMPS triple salt, 15 µg/plate |
- |
36±6 |
132±13 |
17±3 |
11±2 |
131±12 |
KMPS triple salt, 5 µg/plate |
- |
37±2 |
115±16 |
20±4 |
14±2 |
125±15 |
Water, (0.1 ml/plate) |
- |
39±5 |
135±8 |
18±3 |
14±2 |
131±10 |
Benzo[a]pyrene, (5 µg/plate) |
+ |
702±77 |
989±24 |
452±56 |
209±13 |
806±72 |
2-Nitrofluorene, (1 µg/plate) |
- |
319±25 |
622±58 |
291±46 |
382±20 |
527±21 |
10-6 dilution of overnight culture, plated on nutrient agar |
- |
104±6 |
106±1 |
130±8 |
110±11 |
169±16 |
* cytotoxicity
Table 2: Results test 2 (with pre‑incubation)
Plate |
S9 mix |
Revertant colony mean counts |
||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvr/pKM101 |
||
S9mix, sterility check |
+ |
0 |
0 |
0 |
0 |
0 |
Buffer, sterility check |
- |
0 |
0 |
0 |
0 |
0 |
KMPS triple salt, sterility check(5000 µg/plate) |
- |
0 |
0 |
0 |
0 |
0 |
KMPS triple salt, 5000 µg/plate |
+ |
13±4 |
41±5 |
6±2 |
2±1 |
21±2 |
KMPS triple salt, 1500 µg/plate |
+ |
41±6 |
138±6 |
14±1 |
14±3 |
115±25 |
KMPS triple salt, 500 µg/plate |
+ |
42±8 |
134±8 |
19±7 |
14±1 |
138±21 |
KMPS triple salt, 150 µg/plate |
+ |
49±8 |
139±11 |
18±4 |
18±4 |
136±8 |
KMPS triple salt, 50 µg/plate |
+ |
38±7 |
122±14 |
16±4 |
15±2 |
141±5 |
Water, 0.1 mL/plate |
+ |
49±4 |
151±5 |
22±3 |
18±3 |
146±4 |
KMPS triple salt, 500 µg/plate |
- |
12±3 |
73±11 |
4±1 |
2±2 |
8±5 |
KMPS triple salt, 150 µg/plate |
- |
31±4 |
122±11 |
17±5 |
8±1 |
108±6 |
KMPS triple salt, 50 µg/plate |
- |
31±9 |
114±13 |
19±2 |
12±3 |
117±13 |
KMPS triple salt, 15 µg/plate |
- |
33±6 |
131±10 |
18±7 |
9±2 |
121±12 |
KMPS triple salt, 5 µg/plate |
- |
32±5 |
122±15 |
16±5 |
10±1 |
107±10 |
Water, (0.1 ml/plate) |
- |
33±6 |
123±6 |
19±1 |
13±3 |
120±13 |
Benzo[a]pyrene, (5 µg/plate) |
+ |
778±71 |
1006±67 |
251±32 |
386±20 |
1461±396 |
2-Nitrofluorene, (1 µg/plate) |
- |
268±65 |
711±189 |
419±38 |
967±177 |
772±146 |
10-6 dilution of overnight culture, plated on nutrient agar |
- |
116±3 |
176±10 |
131±16 |
120±8 |
180±14 |
Table 1: Summary of results after treatment (3 h) of human lymphocytes with KMPS triple salt and harvest after 20 h including data for cytotoxicity (Test 1)
S9 mix | Concentration of test substance (µg/mL) | Cells with aberrations Excluding gaps | Cells with aberrations Including gaps | Relative Mitotic Index (%) | ||||
- | - | Individual values (%) | Mean (%) | Individual values (%) | Mean (%) | - | ||
- | 0 (water) | 0 | 2 | 1.0 | 3 | 3 | 3.0 | 100 |
- | 312.5 | 2 | 3 | 2.5 | 3 | 5 | 4.0 | 96 |
- | 625 | 3 | 2 | 2.5 | 3 | 5 | 4.0 | 79 |
- | 1250 | 8 | 9 | 8.5*** | 11 | 19 | 15.0*** | 53 |
- | 0.1 (Mitomycin C) | 12 | 12 | 12.0*** | 15 | 15 | 15.0*** | - |
- | - | - | - | - | - | - | - | - |
+ | 0 (water) | 2 | 0 | 1.0 | 2 | 1 | 1.5 | 100 |
- | 312.5 | 1 | 2 | 1.5 | 2 | 3 | 2.5 | 85 |
- | 625 | 2 | 2 | 2.0 | 4 | 5 | 4.5 | 76 |
- | 1250 | 4 | 10 | 7.0** | 5 | 12 | 8.5*** | 53 |
- | 6 (Cyclophosphamide) | 17 | 17 | 17.0*** | 19 | 19 | 19.0*** | - |
Statistical analysis – one-tailed Fisher’s test
*** P<0.001
** P<0.01
Otherwise P>=0.01
Table 2: Summary of results after treatment (3 h) of human lymphocytes with KMPS triple salt and harvest after 20 h including data for cytotoxicity (Test 2)
S9 mix | Concentration of test substance (µg/mL) | Cells with aberrations Excluding gaps | Cells with aberrations Including gaps | Relative Mitotic Index (%) | ||||
- | - | Individual values (%) | Mean (%) | Individual values (%) | Mean (%) | - | ||
- | 0 (water) | 1 | 1 | 1.0 | 3 | 4 | 3.5 | 100 |
- | 500 | 3 | 0 | 1.5 | 7 | 5 | 6.0 | 89 |
- | 1000 | 4 | 5 | 4.5 | 7 | 9 | 8.0 | 65 |
- | 1250 | 8 | 9 | 8.5*** | 10 | 12 | 11.0** | 52 |
- | 0.2 (Mitomycin C) | 23 | 16 | 19.5*** | 25 | 22 | 23.5*** | - |
- | - | - | - | - | - | - | - | - |
+ | 0 (water) | 0 | 1 | 0.5 | 3 | 1 | 2.0 | 100 |
- | 500 | 1 | 2 | 1.5 | 5 | 3 | 4.0 | 79 |
- | 1000 | 2 | 5 | 3.5 | 4 | 5 | 4.5 | 66 |
- | 1250 | 9 | 3 | 6.0*** | 13 | 8 | 10.5*** | 45 |
- | 6 (Cyclophosphamide) | 19 | 16 | 17.5*** | 23 | 21 | 22.0*** | - |
Statistical analysis – one-tailed Fisher’s test
*** P<0.001
** P<0.01
Otherwise P>=0.01
Table 1. Summary Table main experiment, without and with metabolic acitivation
| Test Group | Conc. [µg/mL] | RCEa[%] | RTGb[%] | MFc[mutants/ 106cells] | IMFd[mutants/ 106cells] | GEFeexceeded | Statistical Significant Increasef | Precipitate |
Exp without S9
| C1 | 0 | 125.7 | 111.1 | 134.2 | / | / | / | - |
C2 | 108.1 | 108.2 | / | / | / | - | |||
S1 | 0 | 100.0 | 100.0 | 165.7 | / | / | / | - | |
S2 | / | / | / | - | |||||
2 | 50 | 97.0 | 86.5 | 156.2 | -9.6 | - | - | - | |
3 | 100 | 123.5 | 112.0 | 111.8 | -53.9 | - | - | - | |
4 | 125 | 100.0 | 101.7 | 162.4 | -3.4 | - | - | - | |
5 | 250 | 94.1 | 82.8 | 172.2 | 6.5 | - | - | - | |
6 | 500 | 113.5 | 104.6 | 142.0 | -23.7 | - | - | - | |
7 | 1000 | 104.7 | 89.9 | 162.8 | -3.0 | - | - | - | |
8 | 2000 | 104.7 | 98.8 | 128.9 | -36.8 | - | - | - | |
EMS | 300 | 94.1 | 69.2 | 672.4 | 506.6 | + | + | - | |
MMS | 10 | 63.0 | 42.9 | 638.8 | 473.0 | + | + | - | |
| |||||||||
Exp with S9
| C1 | 0 | 101.4 | 106.5 | 149.4 | / | / | / | - |
C2 | 115.7 | 131.6 | / | / | / | - | |||
S1 | 0 | 100.0 | 100.0 | 142.4 | / | / | / | - | |
S2 | / | / | / | - | |||||
2 | 50 | 111.8 | 128.8 | 132.7 | -9.7 | - | - | - | |
3 | 100 | 99.8 | 103.3 | 146.0 | 3.6 | - | - | - | |
4 | 125 | 124.2 | 138.7 | 94.0 | -48.4 | - | - | - | |
5 | 250 | 87.1 | 96.3 | 167.1 | 24.7 | - | - | - | |
6 | 500 | 99.8 | 114.2 | 134.7 | -7.7 | - | - | - | |
7 | 1000 | 98.3 | 104.6 | 158.0 | 15.6 | - | - | - | |
8 | 2000 | 96.8 | 117.5 | 156.7 | 14.3 | - | - | - | |
B[a]P | 2.5 | 89.7 | 43.3 | 611.4 | 469.0 | + | + | - | |
C: Negative Controls
S: Solvent control (10%Aqua ad iniectabilia; v/v)
a: Relative Cloning Efficiency, RCE = [(CEtest group/ CEof solvent controls) x 100]
Cloning Efficiency, CE = ((-LN (((96 - (mean number of cultures from Plate and Plate 2)) / 96)) cells/well / 1.6 cells/well) x 100)
b: Relative Total Growth, RTG = (RSG x RCE)/100
c: Mutant Frequency, MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded
f: statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test, p<0.05).
+: significant;
-not significant
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
B[a]P: Benzo[a]pyrene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In vivo Mouse Bone Marrow Micronucleus Test: Negative
In vivo Mammalian Alkaline Comet Assay: Negative
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021-06-23 to 2021-12-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- Version / remarks:
- 2016-06-29
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian comet assay
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl: WI(Han) (Full Barrier)
- Details on species / strain selection:
- The Wistar rat is a commonly used strain for this type of assay. Furthermore, a wide historical control database is available for this strain at the testing laboratory.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 315-366 g
- Assigned to test groups randomly: Yes, randomization was performed by a validated software or by excel-file.
- Fasting period before study: No
- Housing: In groups of 2-3 animals / sex / group / cage in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet: Ad libitum (Altromin 1324 maintenance diet for rats and mice)
- Water: Ad libitum (Tap water)
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From day 0 to day 1 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle: Deionised water
- Justification for choice of solvent/vehicle: Solubility properties and non-toxicity in the test system
- Concentration of test material in vehicle: 15, 30, 60 and 75 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Batch No: 2005066 - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulations were kept under magnetic stirring until visual homogeneity was achieved. The test item formulations were prepared freshly on each administration day within one hour prior to administration. The prepared formulations were stored protected from light and at room temperature. - Duration of treatment / exposure:
- Approx. 28 hours (animals were sacrificed 4 hours after the last treatment) for treatment and negative control groups or 4 hours for the positive control group
- Frequency of treatment:
- 2 treatments at 24 (± 1) hour intervals for test substance treatment groups and negative control group; 1 treatment 4 hours before sacrifice for the positive control group
- Post exposure period:
- 4 hours
- Dose / conc.:
- 750 mg/kg bw/day (nominal)
- Dose / conc.:
- 600 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 male animals per group
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethyl methanesulfonate
- Justification for choice of positive control: According to the JaCVAM validation trial EMS was selected as positive control and was administered orally by gavage 4 h before animal sacrifice.
- Route of administration: Oral gavage
- Doses: 250 mg/kg bw - Tissues and cell types examined:
- Liver, forestomach, glandular stomach and duodenum
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Doses were selected based on the results of a dose-range finding study. The highest dose administered in the dose range-finding study was 2000 mg/kg bw/day. Further doses of 75, 125, 250, 500, 750 and 1000 mg/kg bw were administered as well. The animals received the test item twice at 0 h and 24 h after the first administration. The application was done in parallel with male and female rats. Toxic effects leading to humane killing (prone position, reduced spontaneous activity, ataxia, piloerection, half eyelid closure, and lacrimation) occured after the the first administration of 2000 mg/kg bw in one male and one female rat. Reduction of the dose to 1000 mg/kg bw/day in one male and one female animal lead to similar clinical signs and necropsy showed dark and reddened stomach mucosa which was dilated by gas. Administration of 750 mg/kg bw to three male and female rats led to similar clinical signs. No difference between sexes were found. The test item caused inflammatory and degenerative lesions in the stomach from 250 mg/kg bw onwards. From 750 mg/kg bw onwards, there were subacute submucosal inflammation associated with edema, erosion and/or ulceration in the stomach and clinical signs. The findings in the stomach at a dose of 750 mg/kg bw were considered adverse in nature. Based on these local effects, doses selected for the main study were 150, 300, 600 and 750 mg/kg bw/day to demonstrate a dose-dependency in histopathological findings and to induce clinical symptoms in the highest dose of 750 mg/kg bw.
TREATMENT AND SAMPLING TIMES: After euthanasia of the animals, the abdominal aorta was cut and the blood was released. The liver, the forestomach, the glandular stomach and the duodenum were removed, rinsed with cold mincing buffer to remove residual blood and kept in ice-cold mincing buffer on ice until further processing. The times to remove the tissues until the preparations of the slides were recorded in the raw data.
DETAILS OF SLIDE PREPARATION: A portion of the respective tissue was minced, further crushed (stomach tissue) and cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 μL of the supernatant was pipetted into a tube and mixed with 270 μL low-melting agarose (LMA) solution. The slides used were pre-coated with normal-melting agarose. A volume of 75 μL of cell suspension embedded in LMA was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue). Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts. Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order. After electrophoresis, the slides were neutralized by rinsing with neutralization buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards. The cells were stained by applying 75 μL gel red staining solution on top of the slides and covering with a cover slip.
METHOD OF ANALYSIS: Comet slides are analyzed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software “Comet Assay IV” (Perceptive Instruments, Software version 2.1.2). Each slide is screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. Calculations were done automatically by the software but might be corrected manually. All cells of the visual field were scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (heavily damaged cells). Therefore, cells will be classified into three categories: scorable, non-scorable and “hedgehog”. To avoid artefacts only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) were scored. The %-tail intensity was evaluated as the main parameter for interpretation of DNA damage. It was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus. - Evaluation criteria:
- Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value.
Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).
To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion. - Statistics:
- Statistical analysis was performed by testing for normality according to Kolmogorov-Smirnov-test. Statistical significances in comparison with the negative control were evaluated with one-way ANOVA and Dunnett’s post-hoc test at a 5 % level (p < 0.05). In addition, dose-dependency was tested with a linear trend test at the 5 % level (p < 0.05).
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 75, 125, 250, 500, 750, 1000 and 2000 mg/kg bw
- Solubility: Soluble in water at all concentrations
- Clinical signs of toxicity in test animals: Toxic effects leading to humane killing (including prone position, reduced spontaneous activity, ataxia, piloerection, half eyelid closure, and lacrimation) occured after the the first administration of 2000 mg/kg bw in one male and one female rat. Reduction of the dose to 1000 and 750 mg/kg bw/day lead to similar clinical signs. No difference between sexes were found.
- Evidence of cytotoxicity in tissue analysed: The test item caused inflammatory and degenerative lesion in the stomach from 250 mg/kg bw onwards. From 750 mg/kg bw onwards, there were subacute submucosal inflammation associated with edema, erosion and/or ulceration in the stomach. The findings in the stomach at a dose of 750 mg/kg bw were considered adverse in nature. Dose ranges for the main study were based on these local effects and were supported by histopathological examination of liver, forestomach, glandular stomach and duodenum.
- Harvest times: 4 hours after the last treatment
RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The dose levels were shown to be appropriate as the different doses induced graded signs of toxicity. The highest dose induced clinical signs like reduction of spontaneous activity, prone position and ataxia after the first and second application. Rats treated with the second highest dose only showed a reduction of spontaneous activity. No clinical signs were present in the two lowest dose groups. The test substance caused inflammatory and reactive lesions in the gastrointestinal tract at ≥300 mg/kg bw and in the duodenum at ≥600 mg/kg bw in a dose-dependent manner. Thus, it was shown that the test substance reached target tissues and the oral route of exposure was appropriate. The oral route is also the likely route of human exposure.
- Statistical evaluation: The statistical evaluation showed no significant differences in any of the parameters examined between vehicle control and dose groups. - Conclusions:
- In an in vivo Mammalian Alkaline Comet Assay according to OECD guideline 489, the test item KMPS Triple Salt did not induce biologically relevant DNA-strand breaks in liver, forestomach, glandular stomach and duodenum after oral administration to rats under the experimental conditions reported. Therefore, KMPS Triple Salt is considered to be non-DNA damaging under these experimental conditions in the in vivo mammalian Alkaline Comet Assay.
- Executive summary:
In an in vivo Mammalian Alkaline Comet Assay according to OECD guideline 489 and GLP, the genotoxic potential of KMPS Triple Salt was assessed by measuring its ability to induce DNA-strand breaks in the liver, the forestomach, the glandular stomach and the duodenum of rats. The organs were selected to cover three first-contact organs of chemicals upon peroral exposure and the liver as the primary organ for the metabolism of absorbed chemicals.
In addition, a formulation analysis for verification of concentration of KMPS Triple Salt in formulation samples was performed using an iodometric titration method. Nominal concentrations of KMPS Triple Salt Salt in formulation samples were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. A histopathological evaluation was performed to evaluate the possible toxicity in liver, stomach (fore and glandular stomach) and duodenum induced by repeated administration of KMPS Triple Salt. Under the conditions of this study, KMPS Triple Salt inflammatory and reactive lesions in the gastrointestinal tract at ≥300 mg/kg bw and in the duodenum at ≥600 mg/kg bw in a dose-dependent manner. The test item was suspended in deionized water and administered to 5 rats/sex and dose by daily gavage at 10 mL/kg bw (body weight) for two consecutive days. The organs were collected 4 h after the second administration of the test item. Based on local and systemic effects observed in the dose range finding study, a dose of 750 mg/kg bw was selected as the highest dose. In the main experiment, four dose levels (LD:150 mg/kg bw, MD: 300 mg/kg bw, HD1: 600 mg/kg bw/d and HD2: 750 mg/kg bw/d) were used covering a range from the little or no toxicity to the maximum tolerated dose. The animals treated with the LD and the MD showed no signs of systemic toxicity. The animals treated with the HD1 showed reduced spontaneous activity, while animals treated with the highest dose (HD2) showed moderate signs of systemic toxicity such as reduction of spontaneous activity, prone position, piloerection and ataxia. Cells from the liver, forestomach, glandular stomach and duodenum were isolated, embedded in agarose and lysed. DNA was allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity.
The validity criteria were met:
The tail intensities of the negative control group were within the historical control limits and therefore accepted for addition to the laboratory control data set. Ethyl methanesulfonate (250 mg/kg bw) administered orally was used as positive control and induced a statistically significant increase in DNA damage for all evaluated organs. The mean values noted for the dose groups, which were treated with the test item, were within the range of the concurrent negative control and within the historical control limits. No biologically relevant increase of tail intensity was found after treatment with the test item in any of the dose groups and organs evaluated compared to the negative controls. The test item did not induce DNA damage under the conditions tested.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item KMPS Triple Salt did not induce biologically relevant DNA-strand breaks in liver, forestomach, glandular stomach and duodenum after oral administration to rats. Therefore, KMPS Triple Salt is considered to be non-DNA damaging under these experimental conditions in the in vivo mammalian Alkaline Comet Assay.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2001-05-23 - 2001-11-13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK Ltd, Margate, Kent, UK
- Age at study initiation: not stated
- Weight at study initiation: 28 – 30 g (males) 22 – 24 g (females) - Route of administration:
- oral: gavage
- Vehicle:
- Purified water
- Details on exposure:
- Stability/homogeneity:
Concentration and stability of the dosing solutions was not assessed during the study. This is not considered to be required for this kind of tests for the following reason:
According to the description in the report on the in vivo MNT in mice, the dosing solutions were freshly prepared and immediately administered to the animals (“Solutions of the test substance were freshly prepared on the day of the test and were diluted to the concentrations…in purified water”. This dosing regime is usual practice and it is very unlikely that the test substance degraded in the short time period between preparation of dosing solutions and administration to the animals. Therefore, the lack of the determination of the stability and homogeneity of the test material is not considered to have compromised the study. In addition, KMPS triple salt is a readily water-soluble substance (25 – 30 % (w/v)) and, thus, homogeneity of the dose preparations was warranted. - Duration of treatment / exposure:
- Animals were killed and examined after 24 and 48 hours, respectively
- Frequency of treatment:
- One application only
- Post exposure period:
- 24 and 48 hours
- Dose / conc.:
- 1 500 mg/kg bw (total dose)
- Remarks:
- Preliminary toxicity test, corresponding to 75 mg/mL (nominal in water)
- Dose / conc.:
- 1 750 mg/kg bw (total dose)
- Remarks:
- Preliminary toxicitxy test, corresponding to 87.5 mg/mL (nominal in water)
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Remarks:
- Preliminary toxicity test, corresponding to 100 mg/mL (nominal in water)
- Dose / conc.:
- 0 mg/kg bw (total dose)
- Remarks:
- males
- Dose / conc.:
- 437.5 mg/kg bw (total dose)
- Remarks:
- males
- Dose / conc.:
- 1 750 mg/kg bw (total dose)
- Remarks:
- males
- Dose / conc.:
- 0 mg/kg bw (total dose)
- Remarks:
- females
- Dose / conc.:
- 500 mg/kg bw (total dose)
- Remarks:
- females
- Dose / conc.:
- 1 000 mg/kg bw (total dose)
- Remarks:
- females
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Remarks:
- females
- No. of animals per sex per dose:
- Preliminary toxicity test: 2 per sex and group
Micronucleus test:
vehicle control: 10 per sex
KMPS triple salt (437.5, 875,500 and 1000 mg/kg bw): 5 per sex and group
KMPS triple salt (1750 and 2000 mg/kg bw): 12 per sex and group (2 additional animals to replace any that died)
Positive control: 5 per sex - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Mitomycin C: 12 mg/kg bw
- Tissues and cell types examined:
- Tissue: Bone marrow
Type of cells: Erythrocytes
Further details on Examinations are given under "Any other information on materials and methods incl. tables". - Details of tissue and slide preparation:
- not indicated
- Evaluation criteria:
- not indicated
- Statistics:
- Statistical analysis was performed. For incidences of micronucleated immature (polychromatic) erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, USA). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected; for individual inter-group comparisons (i.e. the positive control group) this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, i.e. exact versions of Wilcoxon’s sum of ranks test and Jonckheere’s test for trend.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- only in females
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- CLINICAL SIGNS, MORTALITY
Preliminary toxicity test:
1500 mg/kg bw – males: fast respiration, hunched posture, underactivity, abnormal gait and piloerection
1500 mg/kg bw – females: fast respiration, underactivity and piloerection
2000 mg/kg bw – males: deep, fast slow or irregular respiration, unresponsive, pallor, hunched posture, underactivity, abnormal gait, partially closed eyelids or closed eyes, piloerection, prostrate, fasiculations and reduced body temperature.
One animal was dead app. 20 hours after dosing, the remaining animal was killed in extremis app. 44hours after dosing.
2000 mg/kg bw – females: fast respiration, hunched posture, underactivity, abnormal gait and piloerection
1750 mg/kg bw – males: irregular respiration, hunched posture, underactivity, abnormal gait, partially closed eyes and piloerection.
1750 mg/kg bw – females: fast and irregular respiration, hunched posture, underactivity, abnormal gait and piloerection
Main micronucleus test:
1750 mg/kg bw males: fast and irregular respiration, flat and hunched posture, underactivity, abnormal gait, partially closed eyes, piloerection/ungroomed and incidence of weight loss was recorded for eight of ten animals.
2000 mg/kg bw – females: fast respiration, flat and hunched posture, underactivity, abnormal gait, partially closed eyes and piloerection/ungroomed
No mortalities were observed at any dose level given.
No clinical signs were observed for the vehicle and positive control groups.
HAEMATOLOGY / TISSUE EXAMINATION
Not stated
GENOTOXICITY
The test substance did not cause any statistically significant increases in the number of micronucleated immature (polychromatic) erythrocytes at either sampling time in both male and female animals (P>0.01) when compared to vehicle control values.
It also did not cause any substantial increases in the incidence of micronucleated mature (normochromatic) erythrocytes at either sampling time in either sex when comparison to vehicle control values was made.
The test substance failed to cause any significant decreases in the proportion of immature erythrocytes at either sampling time in male animals (P>0.01).
There was a dose-related decrease in the proportion of immature erythrocytes at the 24 hour sampling time in female animals. The Jonckheere’s test for trend was significant at the 1 % level when all test substance treated groups were included in the analysis, compared to the vehicle control group values (P<0.001). When the high treatment group (2000 mg/kg bw) was excluded from the analysis, there was some evidence of a significant trend but this was not formally statistically significant (P>0.01).
At the 48 hour sampling interval, the difference in the proportion of immature erythrocytes between the 2000 mg/kg bw level and the control group of female animals was not significant. - Conclusions:
- Interpretation of results: negative
The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). KMPS triple salt did not show any evidence of causing chromosome damage in either male or female mice, but according to the interpretation in the study report, showed some evidence of bone marrow cell toxicity in female mice only, when administered orally by intragastric gavage in this in vivo test. The bioavailability was ensured by the clinical signs of toxicity observed in both sexes of mice.
The applicant further concludes that the depression in the PCE/NCE ratio noted in the in vivo MNT with KMPS triple salt at the high dose level of female mice at the 24 hour time point but not at the 48 hours sampling time point is due to the acute toxicity of KMPS triple salt. In view of the degradation of KMPS triple salt to hydrogen peroxide an influence of hydrogen peroxide on this particular effect cannot be excluded which has been demonstrated to depress the PCE/NCE ratio in an in vivo MNT in mice also but, most importantly, not to increase the incidence of micronucleated polychromatic erythrocytes. - Executive summary:
Materials and methods
Groups of CD 1 mice were orally administered a single dose of KMPS triple salt at concentrations of 1500, 1750, and 2000 mg/kg bw in a preliminary toxicity test. In the micronucleus test doses of 0, 437.5, 875,1750 mg/kg bw (males) and 0, 500, 1000 and 2000 mg/kg bw were administered. The animals were killed by cervical dislocation 24 and 48 hours, respectively, after the administration. Femoral marrow cells were flushed out and pooled in a total volume of 2 mL of pre-filtered foetal calf serum. The cells were sedimented by centrifugation and resuspended in a small volume of fresh serum. Three smears were made from each animal. Cells were fixed in methanol and stained with 10 % Giemsa. Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water and air-dried. The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The proportion of immature erythrocytes for each animal was assessed of at least 1000 erythrocytes.
Results and discussion
Please refer to Table 1; which is presented under "Remarks on results including tables and figures".
No statistically significant increases in the frequency of micronucleated immature erythrocytes were observed in mice treated with KMPS triple salt and killed 24 or 48 hours later, compared to vehicle control values (P>0.01 in each case).
No statistically significant decrease in the proportion of immature erythrocytes was recorded at either sampling time in male mice. In female mice, a significant decrease was observed at the high treatment level (2000 mg/kg bw) at the 24 hour sampling time only while no such an effect was evident at the 48 hour sampling time point.
The statistically significant reduction in the PCE/NCE ratio observable at the high dose level of female mice at the 24 hour sampling time point is considered to be a direct consequence of the acute toxicity of KMPS triple salt rather than related to systemic toxicity. The oral doses given to male and female mice were in the range of the LD50 observed in rats (please refer to Document IIIA, Section 6, A6.1.1/01) and although no mortalities occurred in mice there were clinical signs of toxicity evident at the top dose levels. These signs were noted during the first 24 hours of treatment which is consistent with the observations made in the acute oral study in rats. This supports the conclusion above that the decreased PCE/NCE ratio at the high dose level of female mice at the 24 hours sampling time point is related to the acute toxicity of KMPS triple salt.
Furthermore, in view of the chemical nature of KMPS triple salt and considering the mode of action, it will rapidly degrade to hydrogen peroxide which has also been demonstrated to depress the PCE/NCE ratio in an in vivo MNT in mice but, most importantly, no increases in the incidence of micronucleated polychromatic erythrocytes were noted. In an overview of the genotoxicity of hydrogen peroxide which has been compiled in the available EU Risk Assessment Report, hydrogen peroxide was demonstrated to be clearly negative in the in vivo tests performed amongst of which was an in vivo UDS. The overall conclusion was that the available studies are not in support of significant genotoxicity/mutagenicity of H2O2 under in vivo conditions. According to the principles followed in the EU, hydrogen peroxide is not classified as a mutagen.
Referenceopen allclose all
Dose Formulation Analysis
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.
Clinical signs
Animals treated with the highest dose (HD2) showed slight to moderate toxic effects such as reduction of spontaneous activity, prone position and ataxia after the first application. One animal additionally showed piloerection after 30 minutes. After 24 h before the second administration, no symptoms were left. After the second administration, reduction of spontaneous activity, prone position and ataxia were present after 4h. Rats treated with 600 mg/kg bw (HD1) only showed a reduction of spontaneous activity. Rats in the dose groups 300 mg/kg bw (MD) and 150 mg/kg bw (LD) showed no clinical signs.
Histopathology
For evaluation of possible toxicity in liver, stomach (fore and glandular stomach) and duodenum induced by repeated administration of the test item, a histopathological evaluation was performed. Under the conditions of this study, KMPS Triple Salt caused inflammatory and reactive lesions in the gastrointestinal tract at ≥300 mg/kg bw and in the duodenum at ≥600 mg/kg bw in a dose-dependent manner.
Table 1: KMPS triple salt – Mammalian Erythrocyte Mouse Micronucleus Test: Summary of results and statistical analysis |
||||||
MALES |
||||||
Sampling time |
Treatment |
Dose |
%ie/(ie+me)+ |
Incidence mie |
Incidence mme |
|
24 hours |
Purified water |
- |
43 |
0.2 |
0.7 |
|
- |
KMPS triple salt |
437.5 |
43 |
0.4 |
0.0 |
|
- |
KMPS triple salt |
875 |
42 |
0.4 |
1.3 |
|
- |
KMPS triple salt |
1750 |
38 |
0.2 |
0.0 |
|
- |
Mitomycin C |
12 |
44 |
31.2** |
0.7 |
|
48 hours |
Purified water |
- |
54 |
0.6 |
0.0 |
|
- |
KMPS triple salt |
1750 |
42 |
0.0 |
0.0 |
|
FEMALES |
|
|||||
Sampling time |
Treatment |
Dose |
%ie/(ie+me)+ |
Incidence mie |
Incidence mme |
|
24 hours |
Purified water |
- |
49 |
0.0 |
0.8 |
|
- |
KMPS triple salt |
500 |
49 |
0.6 |
0.8 |
|
- |
KMPS triple salt |
1000 |
37 |
0.0 |
0.0 |
|
- |
KMPS triple salt |
2000 |
36*** |
0.4 |
0.6 |
|
- |
Mitomycin C |
12 |
45 |
23.8** |
0.0 |
|
48 hours |
Purified water |
- |
51 |
0.4 |
0.0 |
|
- |
KMPS triple salt |
2000 |
37 |
0.2 |
0.6 |
|
%ie/(ie+me)+
Proportion of immature
(polychromatic) erythrocytes
mie Number
of micronucleated cells observed per 2000 immature erythrocytes examined
mme Number
of micronucleated cells calculated per 2000 mature (normochromatic)
erythrocytes
Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):
***
P<0.001 (highly significant)
** P<0.01
(significant)
otherwise P>0.01 (not significant)
+ Occasional apparent errors of ± 1 % may occur due to rounding of values for presentation in the table
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
KMPS triple salt was tested in vitro and in vivo for genotoxicity in the following tests:
Key, Ames test, RL1
The mutagenic potential of KMPS triple salt was investigated in the Ames test using the histidine auxotrophic S. typhimurium strains TA98, TA100, TA1535, TA1537, and a tryptophan dependent mutant of E. coli, WP2uvrA/pKM101, all with and without metabolic activation (S9 mix). KMPS triple salt was tested at concentrations of 5000, 1500, 500, 150 and 50 µg/plate with metabolic activation and 500, 150, 50, 15 and 5 µg/plate without metabolic activity. The solvent used for the test substance was water. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to KMPS triple salt at any concentration in the presence or absence of S9 mix. Toxicity was seen in all strains following exposure to KMPS triple salt at 5000 µg/plate in the presence of S9 mix, and at 1500 µg/plate (E. coli) or 500 µg/plate (S. typhimurium) in the absence of S9 mix. KMPS triple salt showed no mutagenic activity in this assay with and without metabolic activation.
Key, In vitro chromosomal aberration (CA) test, RL1
The mutagenic potential of KMPS triple salt was investigated in human blood lymphocytes in vitro both with and without metabolic activation. Human lymphocytes were obtained from healthy, non-smoking male donors. Cells were cultured for 48 h in the presence of a cell mitogen (PHA) to stimulate mitosis prior to treatment with the test substance. KMPS triple salt was tested for structural and numerical chromosomal aberrations in human blood lymphocytes in vitro in two separate occations. In the absence and presence of metabolic activation, the concentrations tested were 312.5, 625 and 1250 µg/mL in the first test and 500, 1000 and 1250 µg/mL in the second test for the 20 h harvest time point (3 hours incubation time in the presence of test material, 17 hours recovery time). The slides that were prepared from the cells and stained were evaluated for structural and numerical chromosomal aberrations. 100 well spread metaphases per culture (200 metaphases for duplicate cultures per concentration level) were scored for cytogenetic damage on coded slides. In addition, cytotoxicity (expressed as the mitotic index) and the number of polyploid cells was recorded as well. In the first test KMPS triple salt caused a reduction in the mitotic index to 53 % of the solvent control value at 1250 µg/mL (in both, the absence and presence of S9 mix). In the second test also a reduction in the mitotic index to 52 % (without S9 mix) and 45 % (with S9 mix) was recorded. In both tests KMPS triple salt caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 1250 µg/mL (with and without S9 mix), when compared with the solvent control value. Increases exceeded the negative control range of the performing laboratory. Evidence of a dose-relationship is considered to support the conclusion. However, the increases in CA frequency were not dramatic and seen mainly at relatively high levels of cytotoxicity.
Both tests showed no statistically significant increases in the number of polyploid metaphase cells when compared with the solvent control, i.e. no increase in numerical chromosomal aberrations was noted. No significant increases in osmolality and pH were seen at dose levels of 312.5, 500, 625, 1000 and 1250 µg/mL of test substance. Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells. KMPS triple salt caused a statistically significant increase in the proportion of cells with chromosomal aberrations with and without metabolic activation.
Key, Mouse lymphoma tk gene mutation assay (MLA), RL1
The test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations:
without and with metabolic activation:
50, 100, 125, 250, 500, 1000 and 2000 μg/mL
No precipitation of the test item was noted in the experiment. No growth inhibition was observed without and with metabolic activation. No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation was not exceeded by the induced mutant frequency at any concentration.
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.
Disregarded, Mouse lymphoma tk gene mutation assay (MLA), RL3
KMPS triple salt was investigated for its potential to induce forward gene mutations in vitro in mouse lymphoma L5178Y cells. Cells were exposed to different concentrations of the test substance in the presence and absence of S9 mix. Concentration used spanned a wide range with respect to cloning efficiency of the cells, i.e. from low to high cytotoxicity. At a defined time period after treatment cells were incubated with Trifluorethymidine (TFT) as the selective agent and monitored for the loss of the functional TK+/- enzyme. Cells deficient in the TK locus due to a forward mutation are resistant to the toxic effects of TFT and do proliferate in the presence of TFT whereas non-mutant cells are not able to proliferate. Mutant frequency was determined by seeding a known number of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival. At least four dose levels were tested for the establishment of a dose-response relationship. Dose levels were selected on the basis of the results of a pretest to yield concentration-related toxic effects. The highest concentration produced a low level of survival and survival at the lowest concentration was approx. in the range of the negative control. The assay was performed in two independent experiments using identical procedures. In the first test, the following concentrations were used: 0, 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1200 µg/mL. In the second a narrower concentration range was used: 0, 200, 300, 400, 500, 550, 600, 700, 800, 900, 1000 µg/mL. The sensitivity and reliability of the test system was confirmed by testing appropriate positive controls in the absence (Methyl methanesulphonate) and presence (3 Methylcholanthrene) of metabolic activation. As a result, in the first test without metabolic activation exposure to 400 or 500 µg/mL KMPS triple salt resulted in a statistically significant increase in the mean mutant frequencies when compared with the solvent control value. Without metabolic activation, exposure to 600 or 800 µg/mL KMPS triple salt, also resulted in statistically significantly increased mean mutant frequencies compared to the solvent control value. In the second test without metabolic activation exposure to 400 – 600 µg/mL KMPS triple salt, resulted in statistically significantly increased mean mutant frequencies when compared with the solvent control value. In the test with metabolic activation a statistically increased mutant frequency was also observed after exposure to 700 or 800 µg/mL KMPS triple salt.
However, an Expert Statement prepared by David Kirkland, who is an acknowledged expert in the field of regulatory genetic toxicology, is available concluding the following on the above mentioned study:
The top concentrations from which mutant frequency data were obtained in the main mutation experiments reduced relative survival to between 2 and 13% (i.e. 87-93% cytotoxicity). The lower relative survival values exceed the range recommended in OECD guideline 476 (OECD, 1997b; to aim for 10-20% but not to go below 10% relative survival) which was in place at the time the KMPS study was performed. In the study report the mutant frequency (MF) data were presented in an unusual way (as decimal values rather than mutants per 10^6 viable cells). By converting the numbers David Kirkland demonstrated that the MFs in solvent control cultures exceeded the currently recommended upper limit for the microwell method (170/10^6 viable cells; Moore et al., 2006; OECD 2016c). Thus, under current recommendations this study would not meet internationally agreed acceptance criteria, and would be rejected. The MF results in treated cultures were evaluated by statistical comparison to concurrent controls but also by comparison with the historical negative control range. However, only the mean and maximum values of the historical negative controls were given (177 and 288 mutants/10^6 viable cells respectively) – no minimum was given, and separate values in the absence and presence of S9 were also not given. Therefore, both the mean and maximum historical negative control MFs exceeded the recommended upper limit of 170/10^6 viable cells (Moore et al., 2006). Rather than comparison to historical control ranges it is now accepted practice to evaluate the increase in MF above concurrent control relative to a global evaluation factor (GEF), which is considered to be indicative of a biologically relevant response (Moore et al., 2006). For the microwell version of the MLA, the GEF is an additional 126 mutants per 10^6 viable cells. Even using these more stringent and current criteria, it can be seen that biologically relevant increases in MF were observed in KMPS-treated cultures, both in the absence and presence of S9, at levels of relative survival that were not considered excessive. It is notable that despite the increased MFs in treated cultures, the mutant colonies were not ”sized” into small and large categories. Large colony tk mutants tend to be associated with gene mutation events whereas small colony tk mutants tend to be associated with chromosome breakage (clastogenic) events. Thus, it is not known whether KMPS was primarily inducing gene mutation or clastogenic damage. Colony sizing is clearly recommended in the previous OECD guideline 476 (OECD, 1997b), which was in force at the time the KMPS study was performed. The study report states that colony sizing would be done in the event of a positive response, but no colony sizing data were presented, and it is not known why this was not done. It would have given valuable information on mode of action. It is concluded that KMPS did induce tk mutants in both the absence and presence of S9 at levels of cytotoxicity that were probably not excessive (although the recommended measure of cytotoxicity was not used). However, the solvent control mutant frequencies were outside the current recommended range and would (nowadays) have led to the study being rejected on acceptability grounds. Also, the mutant colonies were not sized, and so it is not known whether KMPS was acting via a mutagenic or clastogenic mode of action.
Please refer to IUCLID section 13 for the Expert Statement.
Key, In vivo bone marrow micronucleus (MN) test in mice, RL1
In an in vivo micronucleus test single doses of 0, 437.5, 875,1750 mg/kg bw (males) and 0, 500, 1000 and 2000 mg/kg bw of KMPS triple salt were administered to CD 1 mice. After the animals were killed the femoral marrow cells were flushed out and pooled together. Three smears were made from each animal. The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The proportion of immature erythrocytes for each animal was assessed of at least 1000 erythrocytes. No statistically significant increases in the frequency of micronucleated immature erythrocytes were observed in mice treated with KMPS triple salt and killed 24 or 48 hours later, compared to vehicle control values (P>0.01 in each case). No statistically significant decrease in the proportion of immature erythrocytes was recorded at either sampling time in male mice. In female mice, a significant decrease was observed at the high treatment level (2000 mg/kg bw) at the 24 hour sampling time only while no such an effect was evident at the 48 hour sampling time point. The statistically significant reduction in the PCE/NCE ratio observable at the high dose level of female mice at the 24 hour sampling time point was considered a direct consequence of the acute toxicity of KMPS triple salt rather than related to systemic toxicity. The oral doses given to male and female mice were in the range of the LD50 observed in rats and although no mortalities occurred in mice there were clinical signs of toxicity evident at the top dose levels. These signs were noted during the first 24 hours of treatment, consistent with the observations made in the acute oral study in rats. This supports the conclusion above that the decreased PCE/NCE ratio at the high dose level of female mice at the 24 hours sampling time point is related to the acute toxicity of KMPS triple salt. Furthermore, in view of the chemical nature of KMPS triple salt and considering the mode of action, it will rapidly degrade to hydrogen peroxide, demonstrated to depress the PCE/NCE ratio in an in vivo MNT in mice but, most importantly, without increasing the number of micronucleated polychromatic erythrocytes (EU RAR Hydrogen Peroxide, 2003). In an overview of the genotoxicity of hydrogen peroxide which has been compiled in the available EU Risk Assessment Report, hydrogen peroxide was demonstrated to be clearly negative in the in vivo tests performed amongst of which was an in vivo UDS. The overall conclusion was that the available studies are not in support of significant genotoxicity/mutagenicity of H2O2 under in vivo conditions.
In his Expert Statement David Kirkland concludes the following:
No blood samples were taken for analysis of KMPS in plasma, which is a common approach to demonstrating that systemic, and therefore bone marrow, exposure occurred. There were small reductions (12-23%) in %PCE in male bone marrow which were not significant. However, there was significant reduction (around 30%) in the %PCE in females, at both sampling times. Usually this would be taken as an indication of bone marrow toxicity, and therefore of bone marrow exposure. However, the lack of systemic bioavailability following oral dosing in other studies suggests that direct exposure of the bone marrow to KMPS is highly unlikely. The apparent bone marrow toxicity could be a “chance” observation, or could be a secondary symptom caused by localised corrosivity in the GI tract. Therefore, it is concluded that KMPS did not induce MN in mouse bone marrow at high acute doses, but the result may not be meaningful since bone marrow exposure would not be expected. It is important to know whether KMPS does induce gene mutations as well as chromosomal damage, or whether it only shows clastogenic activity. David Kirkland therefore recommends to test KMPS for induction of hprt mutations in mammalian cells in vitro in order to clarify mode of action, before considering any follow-up testing. For the whole Expert Statement, please refer to IUCLID section 13.
Key, In vivo Mammalian Alkaline Comet Assay, RL1
In an in vivo Mammalian Alkaline Comet Assay according to OECD guideline 489 and GLP, the genotoxic potential of KMPS Triple Salt was assessed by measuring its ability to induce DNA-strand breaks in the liver, the forestomach, the glandular stomach and the duodenum of rats. The organs were selected to cover three first-contact organs of chemicals upon peroral exposure and the liver as the primary organ for the metabolism of absorbed chemicals.
In addition, a formulation analysis for verification of concentration of KMPS Triple Salt in formulation samples was performed using an iodometric titration method. Nominal concentrations of KMPS Triple Salt in formulation samples were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. A histopathological evaluation was performed to evaluate the possible toxicity in liver, stomach (fore and glandular stomach) and duodenum induced by repeated administration of KMPS Triple Salt. Under the conditions of this study, KMPS Triple Salt inflammatory and reactive lesions in the gastrointestinal tract at ≥300 mg/kg bw and in the duodenum at ≥600 mg/kg bw in a dose-dependent manner. The test item was suspended in deionized water and administered to 5 rats/sex and dose by daily gavage at 10 mL/kg bw (body weight) for two consecutive days. The organs were collected 4 h after the second administration of the test item. Based on local and systemic effects observed in the dose range finding study, a dose of 750 mg/kg bw was selected as the highest dose. In the main experiment, four dose levels (LD:150 mg/kg bw, MD: 300 mg/kg bw, HD1: 600 mg/kg bw/d and HD2: 750 mg/kg bw/d) were used covering a range from the little or no toxicity to the maximum tolerated dose. The animals treated with the LD and the MD showed no signs of systemic toxicity. The animals treated with the HD1 showed reduced spontaneous activity, while animals treated with the highest dose (HD2) showed moderate signs of systemic toxicity such as reduction of spontaneous activity, prone position, piloerection and ataxia. Cells from the liver, forestomach, glandular stomach and duodenum were isolated, embedded in agarose and lysed. DNA was allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity.
The validity criteria were met:
The tail intensities of the negative control group were within the historical control limits and therefore accepted for addition to the laboratory control data set. Ethyl methanesulfonate (250 mg/kg bw) administered orally was used as positive control and induced a statistically significant increase in DNA damage for all evaluated organs. The mean values noted for the dose groups, which were treated with the test item, were within the range of the concurrent negative control and within the historical control limits. No biologically relevant increase of tail intensity was found after treatment with the test item in any of the dose groups and organs evaluated compared to the negative controls. The test item did not induce DNA damage under the conditions tested.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item KMPS Triple Salt did not induce biologically relevant DNA-strand breaks in liver, forestomach, glandular stomach and duodenum after oral administration to rats. Therefore, KMPS Triple Salt is considered to be non-DNA damaging under these experimental conditions in the in vivo mammalian Alkaline Comet Assay.
Overall Conclusion
Summarizing the above mentioned results, as a final conclusion, KMPS triple salt is not considered genotoxic based on the data available.
Table 1 summarizes the in vitro and in vivo genotoxicity studies available for KMPS.
Table 1. In vitro and in vivo genotoxicity studes available for KMPS
Study Type, Year of performance | Endpoint | Outcome | Remarks |
OECD 471 Ames Test in vitro, 2001 | Mutagenicity (procaryotes) | Negative |
|
OECD 476 MLA in vitro, 2001 | Mutagenicity (eucaryotes) & Clastogenicity | Equivocal | Validity questionable because: Validity criteria for solvent control were not met Validity criteria for cytotoxicity were not met Small and large colony differentiation missing |
OECD 473, Chromosome Aberration (CA) in vitro, 2001 | Clastogenicity | Positive | Stat. significance only at cytotoxic concentrations Test design not compliant with current OECD TG |
OECD 474, Micronucleus study in vivo, 2001 | Clastogenicity & Aneugenicity | Negative | Toxicokinetics: Target tissues may be missed (local MoA) |
OECD 490 MLA in vitro, 2019 | Mutagenicity (eucaryotes) & Clastogenicity | Negative | Valid study |
OECD 489, Comet Assay in vivo, 2021 | Clastogenicity & mutagenicity | Negative | Valid study |
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity in vitro and in vivo, the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.
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