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EC number: 240-004-1 | CAS number: 15875-13-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was considered to be weakly mutagenic in the OECD Test Guideline 471 "Bacterial Reverse Mutation Test".
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).
The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 ug/plate. As Experiment 1 was considered to be weakly positive the plate incorporation method was again employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix) to confirm initial findings.
The experiment was repeated using fresh cultures of the bacterial strains and fresh test item formulations with an amended dose range employed of 50, 150, 500, 750 1000, 1500, 2000, 3000 and 5000 µg/plate. Nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both reproducibility and a better dose-related response.
A third (confirmatory) experiment was performed in TA100 and TA98 (with and without metabolic activation) using pre-incubation methodology following the results from Experiments 1 and 2. The dose ranges were 500, 700, 800, 900, 1000, 1250 and 1500 µg/plate for TA100 and 700, 800, 900, 1000, 1250, 1500 and 2000 µg/plate for TA98. As for Experiment 2, intermediate dose levels were selected in the confirmatory experiment in order to confirm and potentially enhance the mutagenic responses observed in both Experiments 1 and 2.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in the absence of metabolic activation from 1500 µg/plate (TA1537) and at 5000 µg/plate (remaining Salmonella strains).
In the presence of metabolic activation, weakened lawns were noted from 1500 µg/plate to TA100 and TA1537 and at 5000 µg/plate to TA98 and TA1535.
Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (plate incorporation method). The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in both the presence and absence of metabolic activation, initially from 2000 µg/plate (TA100 and TA1537). Toxicity was also observed in the confirmatory experiment from 1250 µg/plate in the absence of metabolic activation and 1500 µg/plate in the presence of metabolic activation.
No weakened bacterial background lawns were observed in the E.coli strain, WP2uvrA in any of the experiments performed.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in any of the experiments performed.
In Experiment 1 (plate incorporation method), the test item induced a greater than two-fold increase in the frequency of TA98 revertant colonies at 1500 µg/plate in the presence of metabolic activation only. Smaller, statistically significant increases in revertant colony frequency were observed in TA100 at 500 µg/plate in the presence of metabolic activation, TA1535 (1500 µg/plate in the presence of metabolic activation), TA98 (1500 µg/plate in the absence of metabolic activation) and WP2uvrA (5000 µg/plate in the absence of metabolic activation).
In Experiment 2, no increase greater than two or three times the concurrent solvent control (depending on bacterial strain type) was observed in any of the strains tested (either in the presence or absence of S9-mix). However, several strains exhibited statistically significant increases in the frequency of revertant colonies in the absence and presence of metabolic activation, particularly TA100 which exhibited increases in colony frequency at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria. There was also a small dose-response relationship noted between 500 and 1000 µg/plate in both the absence and presence of metabolic activation.
In the confirmatory experiment, in the absence of metabolic activation, increases greater than two times the concurrent solvent control were observed in TA100 between 500 and 1000 µg/plate and TA98 between 900 and 1250 µg/plate. In the presence of metabolic activation, increases of greater than two times the concurrent solvent control were observed in TA100 only between 500 and 1250 µg/plate. Smaller but statistically significant increases were noted in TA98 at 900 and 1500 µg/plate.
The substance was also assayed in a Mouse Lymphoma Forward mutation assay (according to OECD TG 476) in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.
Under the conditions of this test, the substance was tested up to a cytotoxic concentration of 250 µg/mL, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, the test substance also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that the test substance, tested up to a cytotoxic concentration of 250 µg/mL in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.
The test substance was also evaluated in an in Vitro Mammalian Cell Micronucleus Test assayed performed according to OECD Test Guideline No. 487. Human peripheral blood lymphocytes from healthy non-smoking donors were used for testing.
Two experiments were performed:
To determine cytotoxicity, in the first experiment without metabolic activation the concentrations 100, 500, 250 and 1000 μg/ml were used for analysis of genotoxic effect. In the first experiment with metabolic activation the concentrations 500, 250 and 100 μg/ml were used for analysis of genotoxic effect.
A concentration of 1000 μg/ml was found to highly cytotoxic in experiment with metabolic activation.
In the second experiment (prolonged exposition without activation) concentrations of 1000 and 500 μg/mL were highly cytotoxic. Therefore only the concentrations 250, 125 and 100 ug/mL were used for analysis of potential genotoxic effects.
The results from the first experiment without metabolic activation with short exposure did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc <2).
The results from the first experiment with metabolic activation with short exposure showed increases in the number of binucleated cells with micronuclei, however the ratio of number of binucleated cells with micronuclei at tested dose to number of binucleated cells with micronuclei in negative control (Mt/Mc) was lower than 2.
As the first experiments gave negative results, the second experiment without metabolic activation was performed with extended exposure (23 hours) in the presence of cytochalasin B.
The results of the second experiment did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc <2), and no experiment gave evidence of rising trend in the number of binucleated cells with micronuclei with increasing dose.
Comparison of current control values to historical ranges showed that test system responds adequately and obtained results are biologically relevant.
Under the experimental design , the test substance was non mutagenic for the human peripheral blood lymphocytes in experiments both without and with metabolic activation.
A ToxTracker assay was also performed with Polycat-41. ToxTracker is a mammalian stem cell-based assay that monitors activation of specific cellular signalling pathways for detection of the biological reactivity of compounds.
ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response.
In ToxTracker ACE (Aneugen Clastigen Evaluation), a DNA staining after 4 and 24 hours of treatment is included to assess clastogenic and/or aneugenic properties of compounds.
The results of the ToxTracker assay revealed significant cytotoxicity (>50%) at a concentration of 440.8 μM in the absence of a metabolising system. No increased cytotoxicity was observed in the presence of a metabolising system. No autofluorescence was observed for this test substance.
Activation of the Rtkn-GFP genotoxicity reporter was observed in absence or presence of a metabolising system. Bscl2-GFP was weakly (>1.5-fold) activated, but the induction levels did not reach the 2 -fold threshold for a positive ToxTracker response in absence or presence of S9.
Activation of the p53 response in absence and presence of a metabolising system was observed.
Activation of the Srxn1-GFP oxidative stress reporters was observed in the ToxTracker assay in the absence and presence of a metabolising system. For Blvrb-GFP a weak induction (>1.5 fold) was observed, but the induction levels did not reach the 2-fold threshold for a positive ToxTracker response in absence or presence of S9.
No activation of the unfolded protein response in absence and presence of S9 was observed.
No accumulation in G2/M phase or an increase in aneuploid cells above the threshold was observed.
Polycat-41 activated the Rtkn-GFP genotoxicity reporter more than 2-fold and was therefore classified as genotoxic in this assay. Activation of the Rtkn-GFP ToxTracker reporter is associated with induction of DNA double strand breaks and indicates the induction of chromosome damage. Activation of Rtkn-GFP but not Bscl2-GFP is often associated with an aneugenic mode of action. However, no accumulation of cells in G2/M or aneuploidy was observed after exposure to Poycat-41. Activation of the oxidative stress reporters Srxn1 as well as the Btg2-GFP reporter for p53 activation was observed, both in absence and presence of S9.
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:
- October 2018 - March 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- See details below
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- See details below
- Principles of method if other than guideline:
- A discrepancy has been noted between the General Study Plan and original data regarding the incubation period of the test plates (currently stated in the General Study Plan as ‘approximately 48 hours’). It is acknowledged that this time period is not sufficiently reflected in the original data. In reality, the plates are incubated for significantly longer
(48 to 72 hours) prior to scoring. Initially Bruce Ames, who developed the method in 1975, recommended 48 hour incubation of plates. However, by the 1990’s, with many Ames test being performed, it was considered that the plates could be incubated for 48 to 72 hours without any detriment to the study result. This is confirmed in the current OECD 471 test guideline which states the following “All plates in a given assay should be incubated at
37° C for 48 – 72 hours.” Therefore, the discrepancy detailed above is considered due to an over familiarity to the wording and long term acceptance of the phrase ‘approximately 48 hours’.
This deviation is considered to have no impact on either the result or integrity of the study. - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Identification: N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine
Batch Number: 2305905
Purity: 98.62%
Expiry Date: 15 July 2020
Appearance: Clear pale yellow liquid
Storage Conditions: Room temperature, in the dark
No correction for purity was required. - Target gene:
- Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor
Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized
in-house procedures (outside the confines of this study). Lot No.’s PB/NF S9 25 May 2018 (Experiments 1 and 2) and 28 October 2018 (Experiment 3 Confirmatory) were used in this study.
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test. - Test concentrations with justification for top dose:
- The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 ug/plate.
Experiment 1 was considered to be weakly positive the plate incorporation method was again employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix) to confirm initial findings. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations with an amended dose range employed of 50, 150, 500, 750 1000, 1500, 2000, 3000 and 5000 µg/plate. Nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both reproducibility and a better dose-related response.
A third (confirmatory) experiment was performed in TA100 and TA98 (with and without metabolic activation) using pre-incubation methodology following the results from Experiments 1 and 2. The dose ranges were 500, 700, 800, 900, 1000, 1250 and 1500 µg/plate for TA100 and 700, 800, 900, 1000, 1250, 1500 and 2000 µg/plate for TA98. As for Experiment 2, intermediate dose levels were selected in the confirmatory experiment in order to confirm and potentially enhance the mutagenic responses observed in both Experiments 1 and 2. - Vehicle / solvent:
- Sterile distilled water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 2104309 04/2022 (Experiments 1 and 2) and 2216012 11/2022 (Experiment 3 only)) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
All of the plates were incubated at 37 ± 3 C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). - Rationale for test conditions:
- The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These Salmonella typhimurium strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium, revertants may be produced after exposure to a chemical mutagen which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.
Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre treated with a mixture known to induce an elevated level of these enzymes. - Evaluation criteria:
- Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- Experiment 1 at 1500 µg/plate in the presence and absence of metabolic activation only
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Experiment 1 at 500 µg/plate in the presence of metabolic activation
- 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
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Experiment 1 @ 1500 µg/plate in the presence of metabolic activation
- 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
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- Experiment 2
- 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
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- Experiment 2 1500 µg/plate in the absence of metabolic activation and at 1500 and 2000 µg/plate in the presence of metabolic activation
- 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
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- Experiment 3 @ between 500 and 1000 µg/plate
- 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
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- Experiment 3 @ between 900 and 1250 µg/plate
- 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
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Experiment 3 @ between 500 and 1250 µg/plate
- 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
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In Experiment 1 (plate incorporation), the test item induced a greater than two-fold increase in the frequency of TA98 revertant colonies at 1500 µg/plate in the presence of metabolic activation only.
Smaller increases in revertant colony frequency were observed in TA100 at 500 µg/plate in the presence of metabolic activation, TA1535 at 1500 µg/plate in the presence of metabolic activation, TA98 at 1500 µg/plate in the absence of metabolic activation and WP2uvrA at 5000 µg/plate in the absence of metabolic activation and were all considered statistically significant using Dunnets Regression analysis.
In Experiment 2 (plate incorporation), no increase greater than two or three times the concurrent solvent control (depending on bacterial strain type) was observed in any of the strains tested (either in the presence or absence of S9-mix). However, several strains exhibited statistically significant increases in the frequency of revertant colonies in the absence and presence of metabolic activation, particularly TA100 which exhibited increases at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria.
There was also a small dose-response relationship noted between 500 and 1000 µg/plate in both the absence and presence of metabolic activation. The responses were much more clear following the inclusion of intermediate dose levels. Increases in TA98 revertant colony frequency were noted at 1500 µg/plate in the absence of metabolic activation and at 1500 and 2000 µg/plate in the presence of metabolic activation. Smaller responses were also noted for TA1535 (1500 and 2000 µg/plate in the presence of metabolic activation) and TA1537 (1500 µg/plate in the presence of metabolic activation).
In experiment 3, in the absence of metabolic activation, increases greater than two times the concurrent solvent control were observed in TA100 between 500 and 1000 µg/plate and TA98 between 900 and 1250 µg/plate. In the presence of metabolic activation, increases of greater than two times the concurrent solvent control were observed in TA100 only between 500 and 1250 µg/plate. Smaller but statistically significant increases were noted in TA98 at 900 and 1500 µg/plate. The response was much clearer following the inclusion of intermediate dose levels and employing the pre-incubation modification, particularly in TA100 which exhibited increases at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria.
In all three experiments, no test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix). - Conclusions:
- N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was considered to be weakly mutagenic under the conditions of this test.
- Executive summary:
N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was tested according to OECD Test Guideline 471 "Bacterial Reverse Mutation Test".
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).
The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 ug/plate. As Experiment 1 was considered to be weakly positive the plate incorporation method was again employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix) to confirm initial findings.
The experiment was repeated using fresh cultures of the bacterial strains and fresh test item formulations with an amended dose range employed of 50, 150, 500, 750 1000, 1500, 2000, 3000 and 5000 µg/plate. Nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both reproducibility and a better dose-related response.
A third (confirmatory) experiment was performed in TA100 and TA98 (with and without metabolic activation) using pre-incubation methodology following the results from Experiments 1 and 2. The dose ranges were 500, 700, 800, 900, 1000, 1250 and 1500 µg/plate for TA100 and 700, 800, 900, 1000, 1250, 1500 and 2000 µg/plate for TA98. As for Experiment 2, intermediate dose levels were selected in the confirmatory experiment in order to confirm and potentially enhance the mutagenic responses observed in both Experiments 1 and 2.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in the absence of metabolic activation from 1500 µg/plate (TA1537) and at 5000 µg/plate (remaining Salmonella strains).
In the presence of metabolic activation, weakened lawns were noted from 1500 µg/plate to TA100 and TA1537 and at 5000 µg/plate to TA98 and TA1535.
Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (plate incorporation method). The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in both the presence and absence of metabolic activation, initially from 2000 µg/plate (TA100 and TA1537). Toxicity was also observed in the confirmatory experiment from 1250 µg/plate in the absence of metabolic activation and 1500 µg/plate in the presence of metabolic activation.
No weakened bacterial background lawns were observed in the E.coli strain, WP2uvrA in any of the experiments performed.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in any of the experiments performed.
In Experiment 1 (plate incorporation method), the test item induced a greater than two-fold increase in the frequency of TA98 revertant colonies at 1500 µg/plate in the presence of metabolic activation only. Smaller, statistically significant increases in revertant colony frequency were observed in TA100 at 500 µg/plate in the presence of metabolic activation, TA1535 (1500 µg/plate in the presence of metabolic activation), TA98 (1500 µg/plate in the absence of metabolic activation) and WP2uvrA (5000 µg/plate in the absence of metabolic activation).
In Experiment 2, no increase greater than two or three times the concurrent solvent control (depending on bacterial strain type) was observed in any of the strains tested (either in the presence or absence of S9-mix). However, several strains exhibited statistically significant increases in the frequency of revertant colonies in the absence and presence of metabolic activation, particularly TA100 which exhibited increases in colony frequency at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria outlined in Section 3.4. There was also a small dose-response relationship noted between 500 and 1000 µg/plate in both the absence and presence of metabolic activation.
In the Confirmatory Experiment, in the absence of metabolic activation, increases greater than two times the concurrent solvent control were observed in TA100 between 500 and 1000 µg/plate and TA98 between 900 and 1250 µg/plate. In the presence of metabolic activation, increases of greater than two times the concurrent solvent control were observed in TA100 only between 500 and 1250 µg/plate. Smaller but statistically significant increases were noted in TA98 at 900 and 1500 µg/plate.
The observed increases in revertant colony frequency which were two times greater than the concurrent solvent control were inconsistent between Experiment 1 and 2. In Experiment 1, only TA98 at 1500 µg/plate in the presence of S9-mix achieved a twofold increase and no strain achieved twofold increases over the solvent control in Experiment 2. However, both experiments exhibited statistically significant increases that approached two times the concurrent solvent control, particularly in TA98 and TA100, both in the presence and absence of metabolic activation. The number of revertant colonies observed in these strains also approached or exceeded the upper limit of the expected range for these strains.
A third, confirmatory experiment using the pre-incubation method was performed in TA100 and TA98, both in the presence and absence of metabolic activation and a narrowed dose range. The results of this experiment showed that the test item exhibited increases in revertant colony frequency of greater than two times the concurrent solvent control over all of the sub-toxic dose levels in TA100 both with and without metabolic activation. In TA98, increases in revertant colony frequency of greater than two times the concurrent solvent control were observed at 900 and 1250 µg/plate only and none in the presence of S9 mix (although fold increases were noted from 1.6 to 1.9 times the concurrent vehicle control).
As it is, there is no concordance between the experiments other than there are small but reproducible increases in revertant colony frequency in TA100 and TA98 in particular. However, these statistically significant increases were observed consistently over all three experiments and, where the change in methodology to the pre-incubation method was implemented, the increased sensitivity of the assay exhibited a much clearer response, particularly in the frequency of TA100 revertant colonies. Consequently, the overall conclusion of the study must be that the test substance should be considered weakly mutagenic.
The weakly mutagenic results of the Ames test were evaluated against historical control data for the period 2017 -2018 (attached ). It was observed that for TA98 only one value was out of the historical control range (Experiment 1 – with metabolic activation, 1500 µg). The mean values of the negative control data from the Ames test were within the range of 2017, but near to the maximum values and some single values are also out of the range. For TA 100, with and without metabolization and increase of 32% in the number of mutations within the negative control data from 2017 to 2018, which was not observed for the other strains. Historical data for 2019 were not yet available, so a trend could not yet be confirmed.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- July - August 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- yes
- Remarks:
- See details below
- Principles of method if other than guideline:
- Some deviations from study plan occurred:
- The experimental part of study lasted longer than was planned.
- The first experiments with and without metabolic activation must have been repeated because of low CBPI index in all cultures. Data from both experiments are given in this report and they are part of primary data.
- The second experiment with prolonged treatment period was repeated because of equivocal results. Data from experiment with equivocal results are not given in this report, however they are part of primary data. Records and specimens are archived as a part of study documentation.
These deviations had no impact on the outcome of the study. - GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- CAS No.: 15875-13-5
Molecular formula: C18H42N6
Molecular weight: 342.57
Batch No.: 1166046
Purity: N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
(CAS No. 15875-13-5) 98.0% (w/w)
Impurities: Water (CAS No. 7732-18-5) 0.14 % (w/w) - Species / strain / cell type:
- lymphocytes: Human Peripheral lymphocytes
- Details on mammalian cell type (if applicable):
- Human peripheral blood lymphocytes from healthy donors were used for testing.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs).
- Test concentrations with justification for top dose:
- In the first experiment without metabolic activation the concentrations 1000, 500, 250 and 1000 μg/ml were used for analysis of genotoxic effect. In the first experiment with metabolic activation the concentrations 500, 250 and 100 μg/ml were used for analysis of genotoxic effect. A concentration of 1000 μg/ml was highly cytotoxic in experiment with metabolic activation.
In second experiment (prolonged exposition without activation) the concentrations 1000 and 500 μg/mL revealed themselves as highly cytotoxic. Therefore only concentrations 250, 125 and 100 ug/mL were used for analysis of genotoxic effect. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- colchicine
- cyclophosphamide
- Details on test system and experimental conditions:
- Principle of test is the detection of binucleated cells with micronuclei, which are induced by the test substance in human peripheral blood lymphocytes. Lymphocytes are cultured in growth medium and test substance is added to them. The cell cycle is then stopped by cytochalasin B, cultures are sampled and microscopic preparations are prepared. Preparations are then analysed by microscope.
Genotoxicity is indicated by increased incidence of binucleated cells with micronuclei.
Experiments with and without metabolic activation with short treatment (3 hours) are done at first.
If both experiments of the short treatments are negative or equivocal, subsequently, extended exposure treatment without metabolic activation is performed. - Rationale for test conditions:
- The test was performed according to the methods published in OECD TG 487.
- Evaluation criteria:
- The genotoxic potential is indicated by doubling of the number of binucleated cells with micronuclei in comparison to the negative control (two-fold increase rule) and/or by dependence of increasing number of binucleated cells with micronuclei on dose (dose-response relationship).
- Statistics:
- According to OECD TG 487, the biological relevance of the results is the criterion for the interpretation of results; a statistical evaluation of the results is assessed with respect to dose response relationship.
- Key result
- Species / strain:
- lymphocytes: Human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 - Cytotoxicity evident at 1000 μg/mL and above
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: Human peripheral lymphocytes
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Experiment 2 - without metablic activation
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- As the first experiments gave negative results, the second experiment without metabolic activation had to be done with extended exposure.
- Conclusions:
- Under the test condition used, the test substance does not induce chromosome breaks and/or gain or loss of chromosomes in cultured human peripheral blood lymphocytes in experiments both without and with metabolic activation.
- Executive summary:
N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was tested in an in Vitro Mammalian Cell Micronucleus Test assayed performed according to OECD Test Guideline No. 487.
Human peripheral blood lymphocytes from healthy non-smoking donors were used for testing.
The test substance was suspended in culture medium and assayed in concentrations of 100 5000 μg/mL, which were applied to cultures in volume of 20 μl.
Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.
Two experiments were performed:
To determine cytotoxicity, in the first experiment without metabolic activation the concentrations 100, 500, 250 and 1000 μg/ml were used for analysis of genotoxic effect. In the first experiment with metabolic activation the concentrations 500, 250 and 100 μg/ml were used for analysis of genotoxic effect.
A concentration of 1000 μg/ml was found to highly cytotoxic in experiment with metabolic activation.
In the second experiment (prolonged exposition without activation) concentrations of 1000 and 500 μg/mL were highly cytotoxic. Therefore only the concentrations 250, 125 and 100 ug/mL were used for analysis of potential genotoxic effects.
The results from the first experiment without metabolic activation with short exposure did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc <2).
The results from the first experiment with metabolic activation with short exposure showed increases in the number of binucleated cells with micronuclei, however the ratio of number of binucleated cells with micronuclei at tested dose to number of binucleated cells with micronuclei in negative control (Mt/Mc) was lower than 2.
As the first experiments gave negative results, the second experiment without metabolic activation was performed with extended exposure (23 hours) in the presence of cytochalasin B.
The results of the second experiment did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc <2), and no experiment gave evidence of rising trend in the number of binucleated cells with micronuclei with increasing dose.
Comparison of current control values to historical ranges showed that test system responds adequately and obtained results are biologically relevant.
Under the experimental design described above, the test substance was non mutagenic to human peripheral blood lymphocytes in experiments performed both without and with metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Nov 2012 - May 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- See details below
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- See details below
- Principles of method if other than guideline:
- There were no major deviations from this Study Plan. However, the following minor deviations were noted:
- The following additional parameters were evaluated using the existing data following the recommendations of OECD guideline for testing of chemicals (No. 476, July 21, 1997 and the Mouse Lymphoma Workgroup, Aberdeen, 2003): suspension growth (SG), cloning efficiency (CE), relative suspension growth (RSG) and relative total growth (RTG).
- In the presence of TFT the plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted and not 11 to 12 days as stated in the Study Plan caused by a typing error.
- A 4th Sponsor was added (title page and page 8, section 2.2 “Sonsor / Test Facility / Responsible personnel) on request of Air Products and Chemicals, Inc.: Momentive Performance Materials GmbH Chempark, Building T39 51368 Leverkusen Germany
- In the preliminary experiment, after exposure to the test item, the cells were incubated in growth medium for 7 days and not for 3 days as stated in the Study Plan caused by a typing error.
These minor deviations did not affect the validity of the scientific results obtained in this study - GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Specific details on test material used for the study:
- CAS name N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-
1,3,5(2H,4H,6H)-tripropanamine
Other names N-Dimethylaminopropylhexahydrotriazine
2,4,6-Tris[(3-dimethylamino)propyl]hexahydrotriazine
CAS No. 15875-13-5
EC No. 240-004-1
Molecular formula C18H42N6
Molecular weight 342.57 g/mol
Batch no. 1166046
Receipt no. 51894
Date of receipt November 05, 2012
Characteristics Colorless to pale yellow liquid
Stability/Expiry date June 9, 2014
Storage conditions At room temperature, not to store near acids, to
store in steel containers, containers to be kept
tightly closed in a dry, cool and well-ventilated
place.
Purity 98.0% (w/w) - Target gene:
- The objective of this study was to evaluate the ability of the test item to induce forward mutations at the thymidine kinase (TK) locus in L5178Y TK +/- mouse lymphoma cells as assayed by colony growth in the presence of 5-trifluorothymidine (TFT) and to determine the quality of the mutation (base pair substitutions and deletions or larger genetic changes frequently visible as chromosome aberrations).
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Supplier: ATCC (American Type Culture Collection), 0801
University Blvd., Manassas, VA 20110-2209, USA - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
The in vitro metabolic activation system was comprised of rat liver enzymes (S9 fraction) and an energy producing system comprised of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate. The enzymes
were contained in a 9000 x g supernatant from liver homogenate prepared from male rats treated with 500 mg/kg of Aroclor 1254 five days prior to sacrifice. - Test concentrations with justification for top dose:
- A preliminary cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiment. This study was performed without and with S9 metabolic activation.
A wide range of test item concentrations of 25, 100, 250, 1000, 2500, and 5000 µg were tested for cytotoxicity, and this information was then used to select concentration levels for the mutation assay.
Concentrations were selected using the following criteria:
At least four analysable concentrations were used. Where there is cytotoxicity, these concentrations cover a range from the maximum to little or no toxicity and are separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival (relative cloning efficiency) or relative total growth.
Based on the results of the preliminary study five concentrations of 15.63, 31.3, 62.5, 125 and 250 µgt/mL medium were employed in the main mutagenicity tests.
The lowest separation factor of 2 as recommended by the guidelines was used. No increase in the mutant frequency was observed. Hence, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information. - Vehicle / solvent:
- The test item was completely dissolved in water for injection (aqua ad iniectabilia)
A correction factor of 1.02 was used as the purity the test item was 98.0%.
As recommended by the guidelines at least 106 cells were suspended in treatment medium and diluted to 5 x 105 cells/mL - Untreated negative controls:
- yes
- Remarks:
- aqua ad iniectablia
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- The cells for the first and second experiments were obtained from logarithmically growing laboratory stock cultures of 8.45 x 106 cells/mL and were seeded into a series of tubes, diluted to 5 x 105 cells/mL per tube.
Cell densities were adjusted to 2 x 105/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates
(^ 192 wells, averaging 1.6 cells/well) and incubated for 1 week (plating efficiency step 1) whereas the rest of the cells was incubated for 2 days for the expression period.
The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells incubated for the expression period were maintained below 106 cells per mL and a minimum of 4 concentration levels plus positive and negative control was selected for 5-trifluoro-thymidine (TFT) resistance.
At the end of the second expression period the selected cultures were diluted to 1 x 104 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency for TFT resistance). The plating for survival was identical to the above described method (plating efficiency step 1 in 192 wells with average 1.6 cells/well). For the plating for TFT resistance 3 µg/mL TFT (final concentration) was added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (^ 384 wells, averaging 2 x 103 cells/well). The plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted - Evaluation criteria:
- Test items are evaluated in the Mouse Lymphoma Forward Mutation Assay on the basis of a combination of a minimum increase in mutant frequency and a series of assay evaluation criteria. These criteria take into account the variability that can occur in an assay because of variable mutant frequency at high toxicity. An assay may need to be repeated with different concentrations in order to properly evaluate a test item.
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is ≥ 2 times the concurrent background mutant frequency.
The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test item as a mutagen. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the main study, cytotoxicity (decreased survival) was noted in the absence and presence of metabolic activation at the top concentration of 250 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the preliminary experiment without and with metabolic activation (3-h exposure) cytotoxicity (decreased survival) was noted starting at a concentration of 250 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The values of mutation frequencies of the negative controls ranged from 56.84 to 64.68 per 10^8 clonable cells in the experiments without metabolic activation and from 53.97 to 96.09 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.
- Remarks on result:
- other: No mutagenicity observed.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this test, the substance was tested up to a cytotoxic concentration of 250 µg/mL, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, the test substance also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that the test substance, tested up to a cytotoxic concentration of 250 µg/mL in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential. - Executive summary:
The test substance was assayed in a Mouse Lymphoma Forward mutation assay (according to OECD TG 476) in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.
The substance was completely dissolved in aqua ad iniectabilia. A correction factor of 1.02 was used as the purity of the substance was 98.0% only. The vehicleaqua ad iniectabiliaserved as the negative control.
In the preliminary experiment without and with metabolic activation (24-hour or 3-h exposure) cytotoxicity (decreased survival) was noted starting at a concentration of 250 µg/mL.
Hence, in the main study the concentration-range of 15.63 to 250 µg/mL was used in the experiments without and with metabolic activation.
Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3 -Methylcholanthrene (at 2.5 or 4.0 µg/mL) in the presence of exogenous metabolic activation.
In the main study,cytotoxicity(decreased survival) was noted in the absence and presence of metabolic activation at the top concentration of 250 µg/mL.
The values of mutation frequencies of the negative controls ranged from 56.84 to 64.68 per 106 clonable cells in the experiments without metabolic activation and from 53.97 to 96.09 per 10^6 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.
The mutation frequencies of the cultures treated with the substance ranged from 72.29 to 102.61 per 106 clonable cells (3 hours exposure) and from 65.55 to 103.04 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 54.99 to 80.92 per 10^6 clonable cells (3 hours exposure, first assay) and from 57.80 to 118.46 per 10^6 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.
In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.44 to 1.20 for treated cells and from 0.52 to 1.40 for the negative controls.
The positive controls Methylmethanesulfonate (MMS) and 3 -Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 1803.22 to 2939.36 per 10^6 clonable cells in the case of MMS and ranging from 765.77 to 1088.19 per 10^6 clonable cells in the case of 3 -MC.
The colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.67 to 2.59 in the case of MMS. Therefore, the test substance did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that the test substance, tested up to a cytotoxic concentration of 250 µg/mL in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.
- Endpoint:
- genetic toxicity in vitro, other
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 12/06/2019 to 21/06/2019
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Justification for type of information:
- The ToxTracker assay is a panel of six validated GFP-based mouse embryonic stem (mES) reporter cell lines that can be used to identify the biological reactivity and potential carcinogenic properties of newly developed compounds in a single test.
ToxTracker is a mammalian stem cell-based assay that monitors activation of specific cellular signaling pathways for detection of the biological reactivity of compounds. In contrast to the cancer-derived cell lines that are currently used for in vitro genotoxicity testing, stem cells are genetically stable and proficient in all cellular pathways required for accurate detection of potentially carcinogenic properties of compounds. Extensive whole-genome transcription profiling has led to identification of a panel of biomarker genes that are preferentially activated upon exposure to different classes of carcinogens and toxicants. To allow easy assessment of the activation status of these biomarker genes, we have generated green fluorescent (GFP) mES reporter cell lines. These reporters were created using bacterial artificial chromosomes (BAC) that contain the complete biomarker gene including promoter and regulatory elements ensuring physiological regulation of the GFP reporters following transfection into stem cells.
ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response.
In ToxTracker ACE (Aneugen Clastigen Evaluation), a DNA staining after 4 and 24 hours of treatment is included to assess clastogenic and/or aneugenic properties of compounds.
Treatments with aneugenic compounds generally lead to accumulation of cells in the G2/M phase of the cells cycle after 4 hours of treatment.
After 24 hours of treatment, cells with an abnormal DNA content (aneuploid cells) appear due to missegregation and other problems during mitosis.
For clastogenic compounds, cell cycle distributions can shift, but generally there is no increase in the number of aneuploid cells after 24 hours of treatment.
The inclusion of a DNA stain allows the quantification of cell cycle distributions to detect changes in cell cycle distributions as well as the appearance of aneuploid cells. The specificity of the ToxTracker reporter cell lines was extensively validated using different libraries of reference compounds as demanded by various regulatory agencies4. Over 97% of all tested chemicals were classified correctly, confirming the outstanding performance of the assay. Importantly, compounds that are often erroneously identified as genotoxic compounds by the conventional in vitro tests were identified by the assay as causing predominantly oxidative stress, explaining their positive results. - Qualifier:
- no guideline available
- Principles of method if other than guideline:
- ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage.
The differential induction of the GFP reporters as well as cytotoxicity of the tested compound is determined by flow cytometry. The sample is analysed in the absence and presence of an S9 rat liver extract-based metabolising system. Additionally, cellular DNA content is analysed after 4 and 24 hours of treatment to investigate aneugenic properties of the tested sample by assessing cell cycle distributions. Exposures to control compounds are included in each test to determine technical performance and reproducibility of the ToxTracker ACE assay. Quantitive data analysis was done using ToxPlot software and FlowLogic. - GLP compliance:
- no
- Remarks:
- This experiment was conducted as a non-GLP study, however general principles to conduct proper scientifically correct in vitro experiments were adhered to. Toxys strictly follows the Good Cell Culture Practice guidelines from the OECD
- Type of assay:
- other: ToxTracker Assay (Toxys protocol)
- Specific details on test material used for the study:
- identification: Polycat-41
Toxys reference: Txs000585
Solvent: DMSO
Stock Conc: 0.5M
Storage: Room Tenmp, Dark - Target gene:
- Reporter genes as follows:
DNA damage
Bscl2
Rtkn
Cellular stress (p53)
Btg2
Oxidative stress
Srxn1
Blvrb
Protein damage
Ddit3 - Species / strain / cell type:
- mammalian cell line, other: Mammalian stem cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- Metabolic activation was included in the ToxTracker assay by addition of S9 liver extract from aroclor1254-induced rats (Moltox).
- Test concentrations with justification for top dose:
- For chemical testing, first a dose range finding was performed using wild-type mES cells (strain B4418). Wild type mES cells were exposed to 20 different concentrations of the test substance, with a maximum concentration of 5 mM.
Cytotoxicity was estimated by cell count after 24 h exposure using a flow cytometer and was expressed as the percentage of viable cells after 24 h exposure compared to vehicle control exposed cells. From this dose range finding, 5 concentrations were selected. - Vehicle / solvent:
- DMSO for all test compounds, except PBS for cisplatin.
- Positive controls:
- yes
- Positive control substance:
- other: Reference compounds: Cisplatin, 3.3 mM in PBS Diethyl maleate, 6.2 M in DMSO Tunicamycin, 1 mg/ml in DMSO Aflatoxin B1, 10 mM in DMSO Taxol, 10 mM in DMSO
- Evaluation criteria:
- The ToxTracker assay was considered to have a positive response when a compound induces at least a 2 fold increase in GFP expression in any of the reporters.
Activation of the Bscl2-GFP or Rtkn-GFP reporters indicate induction of DNA damage, Srxn1-GFP and Blvrb-GFP indicate induction of cellular oxidative stress and Ddit3-GFP activation is associated with the unfolded protein response.
The Btg2-GFP reporter is controlled by the p53 tumor suppressor and is activated by DNA damage but can also be induced by oxidative stress, hypoxia, metabolic stress and apoptosis. Only GFP inductions at compound concentrations that showed < 75% cytotoxicity are used for the ToxTracker analysis. Data from measurements > 75% cytotoxicity can not be interpreted in a meaningful way and are therefore discarded.
The No Observed Effect Level (NOEL) defines the highest concentrations where none of the ToxTracker reporters showed a >1.5-fold increase in fluorescence.
The Lowest Observed Effect Level (LOEL) indicates the lowest concentration were at least one of the ToxTracker reporters showed an induction of ≥2.0. The No Observed Genotoxicity Effect Level (NOGEL) is the highest concentration that did not activate the Bscl2-GFP and/or Rtkn-GFP reporters with a >1.5-fold increase in fluorescence.
The Lowest Observed Genotoxicity Effect Level (LOGEL) is the lowest concentration that activated the Bscl2-GFP and/or Rtkn-GFP genotoxicity reporters ≥2.0-fold.
In the aneuploidy analysis, a threshold of 4 % of cells showing more than 4n DNA content is used. This threshold is based on historical control data and the analysis of clastogenic, aneugenic and non-genotoxic reference compounds. - Statistics:
- In order to allow comparison of induction levels of the ToxTracker reporter cell lines for large number of compounds Toxys have developed Toxplot, a dedicated data analysis software package.
Toxplot imports raw GFP reporter data from the flow cytometer, calculates GFP induction levels and cytotoxicity, performs statistical analysis of the data and hierarchical clustering of the tested compounds, and visualises the data in a heatmap allowing convenient interpretation of obtained test results. ToxPlot software uses agglomerative hierarchical clustering to visualize the ToxTracker data. Agglomerative clustering uses the ‘bottom-up’ approach, which puts each observation in its own cluster and pairs of clusters are merged as one moves up the hierarchy. To compare the induction of the six GFP
reporters for a collection of compounds, each with different biological reactivities, dose-response relationships and kinetics, Toxplot calculates for each compound the level of GFP induction for every individual reporter at a specified level of cytotoxicity (typically 10%, 25%, 50% and 75%). GFP induction
levels are calculated by linear regression between two data points around the specified cytotoxicity level. In case the specified level of cytotoxicity can not be reached at the highest tested compound concentration, Toxplot displays the GFP induction level at this top concentration. In the heatmap, Toxplot clearly marks the compounds that do not induce the selected level of cytotoxicity. Because the cytotoxicity for a compound can vary between the ToxTracker cell lines, calculations of the GFP induction levels of the individual reporters by Toxplot can slightly deviate from the GFP induction and cytotoxicity figures. - Key result
- Species / strain:
- mammalian cell line, other: mouse embryonic stem (mES) reporter cell line
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- Polycat-41 activated the Rtkn-GFP genotoxicity reporter more than 2-fold and was thus classified as genotoxic. Activation of the Rtkn-GFP reporter is associated with induction of DNA double strand breaks and indicates the induction of chromosome damage.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At the maximum tested concentrations in the absence of a metabolising system cytotoxicity was observed for Polycat-41. In the presence of a metabolising system, no significant increase in cytotoxicity was observed.
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- Polycat-41 activated the Rtkn-GFP genotoxicity reporter more than 2-fold and was therefore classified as genotoxic in the ToxTracker assay. Activation of the Rtkn-GFP ToxTracker reporter is associated with induction of DNA double strand breaks and indicates the induction of chromosome damage. Activation of Rtkn-GFP but not Bscl2-GFP is often associated with an aneugenic mode of action. However, no accumulation of cells in G2/M or aneuploidy was observed after exposure to Poycat-41.
Activation of the oxidative stress reporters Srxn1 as well as the Btg2-GFP reporter for p53 activation was observed, both in absence and presence of S9.
The validity of the ToxTracker assay results was confirmed by exposure to various reference compounds and assessing the specificity of the different reporter cell lines. - Executive summary:
The ToxTracker assay is a panel of six validated GFP-based mouse embryonic stem (mES) reporter cell lines that can be used to identify the biological reactivity and potential carcinogenic properties of newly developed compounds in a single test.
ToxTracker is a mammalian stem cell-based assay that monitors activation of specific cellular signaling pathways for detection of the biological reactivity of compounds. In contrast to the cancer-derived cell lines that are currently used for in vitro genotoxicity testing, stem cells are genetically stable and proficient in all cellular pathways required for accurate detection of potentially carcinogenic properties of compounds. Extensive whole-genome transcription profiling has led to identification of a panel of biomarker genes that are preferentially activated upon exposure to different classes of carcinogens and toxicants. To allow easy assessment of the activation status of these biomarker genes, we have generated green fluorescent (GFP) mES reporter cell lines. These reporters were created using bacterial artificial chromosomes (BAC) that contain the complete biomarker gene including promoter and regulatory elements ensuring physiological regulation of the GFP reporters following transfection into stem cells.
ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response.
In ToxTracker ACE (Aneugen Clastigen Evaluation), a DNA staining after 4 and 24 hours of treatment is included to assess clastogenic and/or aneugenic properties of compounds.
Treatments with aneugenic compounds generally lead to accumulation of cells in the G2/M phase of the cells cycle after 4 hours of treatment.
After 24 hours of treatment, cells with an abnormal DNA content (aneuploid cells) appear due to missegregation and other problems during mitosis.
For clastogenic compounds, cell cycle distributions can shift, but generally there is no increase in the number of aneuploid cells after 24 hours of treatment.
The inclusion of a DNA stain allows the quantification of cell cycle distributions to detect changes in cell cycle distributions as well as the appearance of aneuploid cells. The specificity of the ToxTracker reporter cell lines was extensively validated using different libraries of reference compounds as demanded by various regulatory agencies4. Over 97% of all tested chemicals were classified correctly, confirming the outstanding performance of the assay. Importantly, compounds that are often erroneously identified as genotoxic compounds by the conventional in vitro tests were identified by the assay as causing predominantly oxidative stress, explaining their positive results.
The validity of the ToxTracker assay was confirmed using exposure to the reference compounds specific for the pathways evaluated. The genotoxic compound cisplatin showed induction of the DNA damage response (Bscl2, Rtkn) and p53-mediated cellular stress (Btg2). Diethyl maleate (DEM) induced primarily the oxidative stress related reporters Srxn1 and Blvrb, tunicamycin induced the unfolded/misfolded protein stress response (Ddit3). The positive control compound aflatoxin B1, which requires metabolic activation to become genotoxic, selectively induced the Bscl2 and Rtkn reporters when tested in the presence of S9 liver extract. Generally, the controls showed GFP induction levels compliant with historical data and demonstrated the functionality of the mES reporter cell lines.
Cytotoxicity:
At the maximum tested concentrations in the absence of a metabolising system cytotoxicity was observed for Polycat-41. In the presence of a metabolising system, no significant increase in cytotoxicity was observed. The six ToxTracker reporter cell lines showed a comparable cytotoxic response to the test sample. For this reason, the cell survival graphs in the GFP induction figures show the average cytotoxicity of the six different cell lines.
Genotoxicity:
Exposure to Polycat-41 activated the Rtkn-GFP reporter in absence and presence of S9. Bscl2-GFP was weakly activated (>1.5 fold) in absence and presence of S9, but the induction levels did not reach the 2-fold threshold for a positive ToxTracker response.
Btg2-GFP, the reporter for p53 activation was activated in response to exposure to Polycat-41 in absence and presence of S9. The Btg2-GFP reporter is associated with activation of the p53 tumor suppressor, which can be activated by DNA damage as well as oxidative stress, heat shock, hypoxia or apoptosis.
Oxidative stress:
Activation of Srxn1-GFP, one of the oxidative stress markers, was observed for Polycat-41 in absence and presence of S9. Blvrb-GFP, the second marker for oxidative damage, was only weakly induced (>1,5-fold), but induction levels did not reach the 2-fold threshold for a positive response in ToxTracker. Induction of the Srxn1-GFP reporter is associated with activation of the Nrf2 antioxidant response and activation of the BlvrbGFP reporter is associated with the Hmox1 antioxidant response.
Protein damage:
The Ddit3-GFP reporter, associated with protein damage and the unfolded protein response, was not activated by Polycat-41.
Aneugenicity:
Analysis of the cell cycle distributions showed no arrest of cells in G2/M phase for Polycat-41. An increase in aneuploid cells above the 4% threshold for aneuploidy was not observed for Polycat-41 either, although the percentage of aneuploid cells increased with increasing concentrations of Polycat-41.
In summary, the ToxTracker assay performed with Polycat-41 revealed:
• Significant cytotoxicity (>50%) was observed at a concentration of 440.8 μM in the absence of a metabolising system. No increased cytotoxicity was observed in the presence of a metabolising system.
• No autofluorescence was observed for this test substance.
• Activation of the Rtkn-GFP genotoxicity reporter was observed in absence or presence of a metabolising system. Blvrb-GFP was weakly (>1.5-fold) activated, but the induction levels did not reach the 2 -fold threshold for a positive ToxTracker response in absence or presence of S9.
• Activation of the p53 response in absence and presence of a metabolising system was observed.
• Activation of the Srxn1-GFP oxidative stress reporters was observed in the ToxTracker assay in the absence and presence of a metabolising system. For Blvrb-GFP a weak induction (>1.5 fold) was observed, but the induction levels did not reach the 2-fold threshold for a positive ToxTracker response in absence or presence of S9.
• No activation of the unfolded protein response in absence and presence of S9 was observed.
• No accumulation in G2/M phase or an increase in aneuploid cells above the threshold was observed.
Conclusion:
Polycat-41 activated the Rtkn-GFP genotoxicity reporter more than 2-fold and was therefore classified as genotoxic. Activation of the Rtkn-GFP ToxTracker reporter is associated with induction of DNA double strand breaks and indicates the induction of chromosome damage. Activation of Rtkn-GFP but not Bscl2-GFP is often associated with an aneugenic mode of action. However, no accumulation of cells in G2/M or aneuploidy was observed after exposure to Poycat-41. Activation of the oxidative stress reporters Srxn1 as well as the Btg2-GFP reporter for p53 activation was observed, both in absence and presence of S9.
Referenceopen allclose all
Spontaneous Mutation Rates (Concurrent Negative Controls)
Experiment 1
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
147 |
|
15 |
|
27 |
|
28 |
|
10 |
|
150 |
(143) |
22 |
(16) |
27 |
(29) |
24 |
(29) |
17 |
(14) |
133 |
|
12 |
|
32 |
|
34 |
|
16 |
|
Experiment 2
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
123 |
|
22 |
|
25 |
|
21 |
|
15 |
|
132 |
(137) |
18 |
(20) |
30 |
(31) |
28 |
(25) |
22 |
(16) |
155 |
|
21 |
|
37 |
|
25 |
|
11 |
|
Experiment 3
Number of revertants (mean number of colonies per plate) |
|||
Base-pair substitution type |
Frameshift type |
||
TA100 |
TA98 |
||
118 |
|
29 |
|
97 |
(107) |
24 |
(24) |
107 |
|
20 |
|
Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 04 September 2018 |
To: 07 September 2018 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
133 138 147 |
(139) 7.1# |
10 19 25 |
(18) 7.5 |
26 25 29 |
(27) 2.1 |
21 29 25 |
(25) 4.0 |
9 11 12 |
(11) 1.5 |
||
1.5 µg |
138 132 124 |
(131) 7.0 |
16 16 22 |
(18) 3.5 |
23 19 33 |
(25) 7.2 |
21 22 25 |
(23) 2.1 |
14 4 9 |
(9) 5.0 |
||
5 µg |
131 124 135 |
(130) 5.6 |
29 12 23 |
(21) 8.6 |
36 28 34 |
(33) 4.2 |
22 23 21 |
(22) 1.0 |
13 14 14 |
(14) 0.6 |
||
15 µg |
117 138 139 |
(131) 12.4 |
11 26 14 |
(17) 7.9 |
27 22 25 |
(25) 2.5 |
11 21 27 |
(20) 8.1 |
20 13 7 |
(13) 6.5 |
||
50 µg |
126 137 117 |
(127) 10.0 |
21 15 15 |
(17) 3.5 |
36 27 30 |
(31) 4.6 |
21 19 26 |
(22) 3.6 |
11 14 11 |
(12) 1.7 |
||
150 µg |
118 118 123 |
(120) 2.9 |
21 17 14 |
(17) 3.5 |
28 21 34 |
(28) 6.5 |
32 24 27 |
(28) 4.0 |
20 20 5 |
(15) 8.7 |
||
500 µg |
147 150 148 |
(148) 1.5 |
19 20 17 |
(19) 1.5 |
29 28 19 |
(25) 5.5 |
32 34 26 |
(31) 4.2 |
9 18 15 |
(14) 4.6 |
||
1500 µg |
109 107 92 |
(103) 9.3 |
21 25 28 |
(25) 3.5 |
27 29 29 |
(28) 1.2 |
40 43 47 |
*** (43) 3.5 |
21 S 16 S 13 S |
(17) 4.0 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
37 40 43 |
* (40) 3.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
628 694 757 |
(693) 64.5 |
982 1717 1880 |
(1526) 478.4 |
1088 1010 969 |
(1022) 60.5 |
245 201 194 |
(213) 27.6 |
210 611 559 |
(460) 218.1 |
|||
Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 04 September 2018 |
To: 07 September 2018 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
133 130 132 |
(132) 1.5# |
18 15 14 |
(16) 2.1 |
49 45 42 |
(45) 3.5 |
22 21 34 |
(26) 7.2 |
14 10 12 |
(12) 2.0 |
||
1.5 µg |
147 137 138 |
(141) 5.5 |
26 14 12 |
(17) 7.6 |
31 46 46 |
(41) 8.7 |
25 39 31 |
(32) 7.0 |
8 11 12 |
(10) 2.1 |
||
5 µg |
146 138 112 |
(132) 17.8 |
18 11 11 |
(13) 4.0 |
25 23 42 |
(30) 10.4 |
38 27 24 |
(30) 7.4 |
24 15 10 |
(16) 7.1 |
||
15 µg |
129 143 125 |
(132) 9.5 |
9 11 14 |
(11) 2.5 |
32 25 28 |
(28) 3.5 |
26 35 35 |
(32) 5.2 |
12 8 15 |
(12) 3.5 |
||
50 µg |
143 144 149 |
(145) 3.2 |
14 11 17 |
(14) 3.0 |
41 38 47 |
(42) 4.6 |
26 27 22 |
(25) 2.6 |
14 14 17 |
(15) 1.7 |
||
150 µg |
141 121 154 |
(139) 16.6 |
11 15 12 |
(13) 2.1 |
48 34 36 |
(39) 7.6 |
29 32 35 |
(32) 3.0 |
17 20 11 |
(16) 4.6 |
||
500 µg |
183 194 166 |
*** (181) 14.1 |
14 12 19 |
(15) 3.6 |
44 40 36 |
(40) 4.0 |
34 32 28 |
(31) 3.1 |
14 9 15 |
(13) 3.2 |
||
1500 µg |
174 S 207 S 169 S |
*** (183) 20.6 |
36 20 37 |
** (31) 9.5 |
41 40 41 |
(41) 0.6 |
61 65 72 |
*** (66) 5.6 |
20 S 14 S 14 S |
(16) 3.5 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
42 40 48 |
(43) 4.2 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1987 1843 1912 |
(1914) 72.0 |
396 383 342 |
(374) 28.2 |
227 270 304 |
(267) 38.6 |
262 241 259 |
(254) 11.4 |
337 335 331 |
(334) 3.1 |
|||
Test Results: Experiment 2 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 11 September 2018 |
To: 14 September 2018 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
137 131 131 |
(133) 3.5# |
23 29 17 |
(23) 6.0 |
40 34 24 |
(33) 8.1 |
30 21 23 |
(25) 4.7 |
13 17 12 |
(14) 2.6 |
||
50 µg |
144 136 147 |
(142) 5.7 |
22 20 17 |
(20) 2.5 |
32 37 46 |
(38) 7.1 |
30 28 19 |
(26) 5.9 |
10 12 16 |
(13) 3.1 |
||
150 µg |
133 150 141 |
(141) 8.5 |
31 35 22 |
(29) 6.7 |
41 40 33 |
(38) 4.4 |
15 39 19 |
(24) 12.9 |
16 12 20 |
(16) 4.0 |
||
500 µg |
184 159 151 |
** (165) 17.2 |
31 22 36 |
(30) 7.1 |
22 44 30 |
(32) 11.1 |
27 31 36 |
(31) 4.5 |
20 23 24 |
(22) 2.1 |
||
750 µg |
200 200 204 |
*** (201) 2.3 |
20 32 35 |
(29) 7.9 |
25 30 52 |
(36) 14.4 |
36 32 26 |
(31) 5.0 |
18 13 15 |
(15) 2.5 |
||
1000 µg |
239 219 242 |
*** (233) 12.5 |
29 21 38 |
(29) 8.5 |
41 33 30 |
(35) 5.7 |
35 33 39 |
(36) 3.1 |
19 11 19 |
(16) 4.6 |
||
1500 µg |
163 158 185 |
** (169) 14.4 |
20 39 36 |
(32) 10.2 |
41 43 43 |
(42) 1.2 |
46 38 46 |
* (43) 4.6 |
14 12 21 |
(16) 4.7 |
||
2000 µg |
47 S 78 S 54 S |
(60) 16.3 |
25 36 22 |
(28) 7.4 |
43 35 40 |
(39) 4.0 |
23 43 37 |
(34) 10.3 |
4 S 8 S 16 S |
(9) 6.1 |
||
3000 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
25 39 37 |
(34) 7.6 |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
18 43 31 |
(31) 12.5 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
562 544 616 |
(574) 37.5 |
606 623 702 |
(644) 51.2 |
424 441 501 |
(455) 40.5 |
126 131 125 |
(127) 3.2 |
289 319 485 |
(364) 105.6 |
|||
Test Results: Experiment 2 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 11 September 2018 |
To: 14 September 2018 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
122 132 141 |
(132) 9.5# |
10 20 14 |
(15) 5.0 |
32 36 32 |
(33) 2.3 |
26 39 31 |
(32) 6.6 |
13 12 22 |
(16) 5.5 |
||
50 µg |
141 128 120 |
(130) 10.6 |
22 15 13 |
(17) 4.7 |
35 40 56 |
(44) 11.0 |
27 24 30 |
(27) 3.0 |
22 24 13 |
(20) 5.9 |
||
150 µg |
141 149 177 |
(156) 18.9 |
16 32 17 |
(22) 9.0 |
34 50 32 |
(39) 9.9 |
32 34 21 |
(29) 7.0 |
11 16 13 |
(13) 2.5 |
||
500 µg |
166 183 172 |
** (174) 8.6 |
16 20 18 |
(18) 2.0 |
43 32 33 |
(36) 6.1 |
40 29 35 |
(35) 5.5 |
16 17 19 |
(17) 1.5 |
||
750 µg |
226 223 207 |
*** (219) 10.2 |
31 26 21 |
(26) 5.0 |
34 29 42 |
(35) 6.6 |
43 46 43 |
(44) 1.7 |
19 18 13 |
(17) 3.2 |
||
1000 µg |
223 280 261 |
*** (255) 29.0 |
32 19 21 |
(24) 7.0 |
42 40 39 |
(40) 1.5 |
37 42 57 |
(45) 10.4 |
17 14 19 |
(17) 2.5 |
||
1500 µg |
260 248 255 |
*** (254) 6.0 |
36 37 21 |
** (31) 9.0 |
35 42 43 |
(40) 4.4 |
41 70 47 |
** (53) 15.3 |
22 27 21 |
* (23) 3.2 |
||
2000 µg |
71 S 89 S 87 S |
(82) 9.9 |
30 33 25 |
* (29) 4.0 |
37 38 48 |
(41) 6.1 |
59 51 60 |
** (57) 4.9 |
25 S 26 S 20 S |
* (24) 3.2 |
||
3000 µg |
0 T 0 T 0 T |
(0) 0.0 |
8 S 6 S 2 S |
(5) 3.1 |
45 42 47 |
(45) 2.5 |
10 10 8 |
(9) 1.2 |
8 S 6 S 9 S |
(8) 1.5 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
45 50 40 |
(45) 5.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1891 1990 1837 |
(1906) 77.6 |
363 362 295 |
(340) 39.0 |
238 211 240 |
(230) 16.2 |
169 157 168 |
(165) 6.7 |
392 370 380 |
(381) 11.0 |
|||
Experiment 3 – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 29 January 2019 |
To: 01 February 2019 |
|||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||
Base-pair substitution strain |
Frameshift strain |
||||
TA100 |
TA98 |
||||
Solvent Control (Water) |
98 103 77 |
(93) 13.8# |
16 17 14 |
(16) 1.5 |
|
500 µg |
220 219 188 |
*** (209) 18.2 |
N/T |
||
700 µg |
211 254 242 |
*** (236) 22.2 |
21 29 26 |
* (25) 4.0 |
|
800 µg |
232 201 230 |
*** (221) 17.3 |
27 29 34 |
** (30) 3.6 |
|
900 µg |
181 231 210 |
*** (207) 25.1 |
33 28 41 |
*** (34) 6.6 |
|
1000 µg |
233 248 250 |
*** (244) 9.3 |
27 21 35 |
** (28) 7.0 |
|
1250 µg |
110 S 76 S 76 S |
(87) 19.6 |
39 32 26 |
*** (32) 6.5 |
|
1500 µg |
67 S 71 S 53 S |
(64) 9.5 |
23 S 28 S 28 S |
* (26) 2.9 |
|
2000 µg |
N/T |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
4NQO |
||
3 µg |
0.2 µg |
||||
589 538 591 |
(573) 30.0 |
211 171 211 |
(198) 23.1 |
||
Experiment 3 – With Metabolic Activation (Pre-Incubation)
Test Period |
From: 29 January 2019 |
To: 01 February 2019 |
|||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||
Base-pair substitution strain |
Frameshift strain |
||||
TA100 |
TA98 |
||||
Solvent Control (Water) |
101 125 93 |
(106) 16.7# |
26 29 13 |
(23) 8.5 |
|
500 µg |
211 222 211 |
** (215) 6.4 |
N/T |
||
700 µg |
229 247 244 |
*** (240) 9.6 |
32 43 29 |
(35) 7.4 |
|
800 µg |
257 291 254 |
*** (267) 20.6 |
36 31 38 |
(35) 3.6 |
|
900 µg |
259 277 276 |
*** (271) 10.1 |
54 36 39 |
* (43) 9.6 |
|
1000 µg |
281 255 241 |
*** (259) 20.3 |
28 43 49 |
(40) 10.8 |
|
1250 µg |
188 216 236 |
** (213) 24.1 |
36 36 38 |
(37) 1.2 |
|
1500 µg |
140 S 108 S 263 S |
* (170) 81.8 |
35 35 56 |
* (42) 12.1 |
|
2000 µg |
N/T |
8 S 23 S 6 S |
(12) 9.3 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
BP |
||
1 µg |
5 µg |
||||
571 601 574 |
(582) 16.5 |
89 90 118 |
(99) 16.5 |
||
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
N/T Not tested at this dose level
S Sparse bacterial background lawn
* p<=0.05
** p<=0.01
*** p<=0.00
# Standard deviation
Table 1: Evaluation of cytotoxic effect without metabolic activation from the first and repeated experiment
- MA I |
||||||
Culture No. |
Treatment/Test substance concentration |
Number of MNC |
Number of BNC |
Number of MTNC |
CBPI |
Cytotoxicity (%) |
1 |
UTC |
589 |
393 |
18 |
1.43 |
100.0 |
2 |
5000 μg/mL |
977 |
22 |
1 |
1.02 |
94.4 |
3 |
2500 μg/mL |
965 |
33 |
2 |
1.04 |
91.4 |
4 |
1000 μg/mL |
923 |
76 |
1 |
1.08 |
81.8 |
5 |
500 μg/mL |
643 |
346 |
11 |
1.37 |
14.2 |
6 |
250 μg/mL |
650 |
336 |
14 |
1.36 |
15.2 |
7 |
Colchicine |
832 |
162 |
6 |
1.17 |
82.2 |
- MA I (repeated) |
||||||
Culture No. |
Treatment/Test substance concentration |
Number of MNC |
Number of BNC |
Number of MTNC |
CBPI |
Cytotoxicity (%) |
1 |
UTC |
383 |
598 |
19 |
1.64 |
100.0 |
2 |
5000 μg/mL |
974 |
25 |
1 |
1.03 |
95.8 |
3 |
2500 μg/mL |
904 |
92 |
4 |
1.10 |
84.3 |
4 |
1000 μg/mL |
669 |
319 |
12 |
1.34 |
46.1 |
5 |
500 μg/mL |
478 |
522 |
10 |
1.54 |
15.6 |
6 |
250 μg/mL |
442 |
526 |
32 |
1.59 |
7.2 |
7 |
100 μg/mL |
461 |
664 |
10 |
1.56 |
11.3 |
8 |
Colchicine |
765 |
226 |
9 |
1.24 |
61.6 |
Table 2: Evaluation of cytotoxic effect with metabolic activation from the first and repeated experiment
+MA I |
||||||
Culture No. |
Treatment/Test substance concentration |
Number of MNC |
Number of BNC |
Number of MTNC |
CBPI |
% cytotoxicity |
1 |
S9 |
452 |
534 |
14 |
1.56 |
100.0 |
7 |
5000mg/mL + S9 |
987 |
13 |
0 |
1.01 |
97.7 |
8 |
2500mg/mL + S9 |
946 |
54 |
0 |
1.05 |
90.4 |
9 |
1000mg/mL + S9 |
833 |
167 |
0 |
1.17 |
70.3 |
10 |
500mg/mL + S9 |
633 |
362 |
5 |
1.37 |
33.8 |
11 |
250mg/mL + S9 |
653 |
344 |
3 |
1.35 |
37.7 |
12 |
100 mg/mL + S9 |
600 |
387 |
13 |
1.45 |
20.1 |
13 |
Cyclophosphamide + S9 |
517 |
467 |
16 |
1.50 |
11.2 |
+MA I (repeated) |
||||||
Culture No. |
Treatment/Test substance concentration |
Number of MNC |
Number of BNC |
Number of MTNC |
CBPI |
% cytotoxicity |
9 |
S9 |
355 |
628 |
17 |
1.66 |
100.0 |
10 |
500mg/mL + S9 |
530 |
460 |
10 |
1.48 |
27.5 |
11 |
250mg/mL + S9 |
365 |
619 |
16 |
1.65 |
1.7 |
12 |
100 mg/mL + S9 |
334 |
654 |
12 |
1.68 |
-2.4 |
13 |
Cyclophosphamide + S9 |
546 |
454 |
10 |
1.47 |
29.1 |
Table 3: Evaluation of genotoxicity without metabolic activation (first experiment)
Culture No. |
Treatment/Test substance concentration |
Number of binucleated cells with MN |
Number of MN |
Average number of BN cells with MN per 1000 binucleated cells |
Average number of MN per 1000 binucleated cells |
Mt/Mc |
1 |
UTC |
22 |
24 |
11 |
12 |
- |
4 |
1000mg/mL |
40 |
42 |
20 |
21 |
1.82 |
5 |
500mg/mL |
34 |
37 |
17 |
18.5 |
1.55 |
6 |
250mg/mL |
35 |
37 |
17.5 |
18.5 |
1.59 |
7 |
100mg/mL |
22 |
23 |
11 |
11.5 |
1.00 |
8 |
Colchicine |
93 |
122 |
46.5 |
61 |
4.23 |
Table 4: Evaluation of genotoxicity with metabolic activation (first experiment)
Culture No. |
Treatment/Test substance concentration |
Number of binucleated cells with MN |
Number of MN |
Average number of BN cells with MN per 1000 binucleated cells |
Average number of MN per 1000 binucleated cells |
Mt/Mc |
9 |
S9 |
18 |
18 |
9 |
9 |
- |
10 |
500mg/mL + S9 |
34 |
37 |
17 |
18.5 |
1.89 |
11 |
250mg/mL + S9 |
21 |
26 |
10.5 |
13 |
1.17 |
12 |
100mg/mL + S9 |
17 |
19 |
8.5 |
9.5 |
0.94 |
13 |
Cyclophosphamide + S9 |
38 |
39 |
19 |
19.5 |
2.11 |
1 |
UTC |
22 |
24 |
11 |
12 |
1.22 |
The numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls (provided in the attachment) at the performing laboratory. Values of negative and positive controls in this study were within the ranges of historical data, so that test system responds adequately and the experiment is acceptable.
The results from the first experiment without metabolic activation with short exposure did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc <2). The results from the first experiment with metabolic activation with short exposure showed increase in the number of binucleated cells with micronuclei, however the ratio of number of binucleated cells with micronuclei at tested dose to number of binucleated cells with micronuclei in negative control (Mt/Mc) was lower than 2.
Table 5: Evaluation of genotoxicity without metabolic activation (second experiment)
Culture No. |
Treatment/Test substance concentration |
Number of binucleated cells with MN |
Number of MN |
Average number of binucleated cells with MN per 1000 binucleated cells |
Average number of MN per 1000 binucleated cells |
Mt/Mc |
7 |
UTC |
26 |
30 |
13 |
15 |
- |
10 |
250mg/mL |
22 |
22 |
11 |
11 |
0.85 |
9 |
125mg/mL |
42 |
51 |
21 |
25.5 |
1.62 |
11 |
100mg/mL |
31 |
33 |
15 |
16 |
1.15 |
The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure (23 hours) in the presence of cytochalasin B. The results did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc <2), and no experiment gave evidence of rising trend in the number of binucleated cells with micronuclei with increasing dose. Comparison of current control values to historical ranges showed that test system responds adequately and obtained results are biologically relevant.
Mouse Lymphoma Forward Mutation Assay
Additional tables (individual results, colony ratios) are presented in the attachment.
Table 1:Preliminary Cytotoxicity Experiment - plating efficiency
Culture Number |
S9 -without + with |
Concentration of Polycat 41 catalyst (µg/mL) |
Total Wells |
Wells Containing Cells |
Empty Wells |
Probable number of clones/well (P) |
Plating Efficiency (PE) |
% Relative Survival (RS) |
1 |
- |
0 (control) |
32 |
21 |
11 |
1.0678 |
0.6674 |
100 |
7 |
- |
25 |
32 |
19 |
13 |
0.9008 |
0.5630 |
84 |
6 |
- |
100 |
32 |
19 |
13 |
0.9008 |
0.5630 |
84 |
5 |
- |
250 |
32 |
3 |
29 |
0.0984 |
0.0615 |
9 |
4 |
- |
1000 |
32 |
0 |
32 |
0.0000 |
0.0000 |
0 |
3 |
- |
2500 |
32 |
0 |
32 |
0.0000 |
0.0000 |
0 |
2 |
- |
5000 |
32 |
0 |
32 |
0.0000 |
0.0000 |
0 |
|
||||||||
8 |
+ |
0 (control) |
32 |
15 |
17 |
0.6325 |
0.3953 |
100 |
14 |
+ |
25 |
32 |
15 |
17 |
0.6325 |
0.3953 |
100 |
13 |
+ |
100 |
32 |
12 |
20 |
0.4700 |
0.2938 |
74 |
12 |
+ |
250 |
32 |
2 |
30 |
0.0645 |
0.0403 |
10 |
11 |
+ |
1000 |
32 |
0 |
32 |
0.0000 |
0.0000 |
0 |
10 |
+ |
2500 |
32 |
0 |
32 |
0.0000 |
0.0000 |
0 |
9 |
+ |
5000 |
32 |
0 |
32 |
0.0000 |
0.0000 |
0 |
Table 2a:Plating Efficiency 1 for Survival -withoutS9 activation
Culture Number |
S9 -without
|
Concentration of Polycat 41 catalyst (µg/mL) |
Total Wells |
Wells Containing Cells |
Empty Wells |
Probable number of clones/well (P) |
Plating Efficiency (PE) |
% Relative Survival (RS) |
1st experiment (3-hour exposure)
|
||||||||
1, 2 |
- |
0 (control) |
192 |
152 |
40 |
1.5699 |
0.9812 |
100 |
7 |
- |
15.63 |
192 |
128 |
64 |
1.0986 |
0.6866 |
70 |
6 |
- |
31.3 |
192 |
156 |
36 |
1.6740 |
1.0463 |
107 |
5 |
- |
62.5 |
192 |
122 |
70 |
1.0090 |
0.6306 |
64 |
4 |
- |
125 |
192 |
128 |
64 |
1.0986 |
0.6866 |
70 |
3 |
- |
250 |
192 |
32 |
160 |
0.1823 |
0.1139 |
12 |
9 |
- |
MMS 10 |
192 |
96 |
96 |
0.6931 |
0.4332 |
44 |
8 |
- |
MMS 15 |
192 |
92 |
100 |
0.6523 |
0.4077 |
42 |
2nd experiment (24-hour exposure)
|
||||||||
10,11 |
- |
0 (control) |
192 |
154 |
38 |
1.6213 |
1.0133 |
100 |
16 |
- |
15.63 |
192 |
160 |
32 |
1.7918 |
1.1199 |
111 |
15 |
- |
31.3 |
192 |
128 |
64 |
1.0986 |
0.6866 |
68 |
14 |
- |
62.5 |
192 |
106 |
86 |
0.8031 |
0.5019 |
50 |
13 |
- |
125 |
192 |
146 |
46 |
1.4289 |
0.8931 |
88 |
12 |
- |
250 |
192 |
8 |
184 |
0.0426 |
0.0266 |
3 |
18 |
- |
MMS 10 |
192 |
50 |
142 |
0.3017 |
0.1886 |
19 |
17 |
- |
MMS 15 |
192 |
24 |
168 |
0.1335 |
0.0834 |
8 |
# = mean value of two parallel cultures; individual results are presented in Table 5a (attachment)
P
= probable number of clones/well
PE
= plating efficiency
%
RS = percent relative survival
MMS
= Methylmethanesulfonate [µL/mL]
Table 2b:Plating Efficiency 2 for Mutant Frequency -withoutS9 activation
Culture Number |
S9 -without
|
Concentration of Polycat 41 catalyst (µg/mL) |
Total Wells |
Wells Containing Cells |
Empty Wells |
Probable number of clones/well (P) |
Plating Efficiency (PE) |
% Relative Survival (RS) |
1st experiment (3-hour exposure)
|
||||||||
1, 2 |
- |
0 (control) |
192 |
136.5 |
55.5 |
1.2461 |
0.7788 |
100 |
7 |
- |
15.63 |
192 |
121 |
71 |
0.9948 |
0.6218 |
80 |
6 |
- |
31.3 |
192 |
129 |
63 |
1.1144 |
0.6965 |
89 |
5 |
- |
62.5 |
192 |
127 |
65 |
1.0831 |
0.6769 |
87 |
4 |
- |
125 |
192 |
151 |
41 |
1.5439 |
0.9649 |
124 |
3 |
- |
250 |
192 |
51 |
141 |
0.3087 |
0.1929 |
25 |
9 |
- |
MMS 10 |
192 |
33 |
159 |
0.1886 |
0.1179 |
15 |
8 |
- |
MMS 15 |
192 |
29 |
163 |
0.1637 |
0.1023 |
13 |
2nd experiment (24-hour exposure)
|
||||||||
10,11 |
- |
0 (control) |
192 |
137.5 |
54.5 |
1.2597 |
0.7873 |
100 |
16 |
- |
15.63 |
192 |
121 |
71 |
0.9948 |
0.6218 |
79 |
15 |
- |
31.3 |
192 |
133 |
59 |
1.1800 |
0.7375 |
94 |
14 |
- |
62.5 |
192 |
127 |
65 |
1.0831 |
0.6769 |
86 |
13 |
- |
125 |
192 |
153 |
39 |
1.5939 |
0.9962 |
127 |
12 |
- |
250 |
192 |
55 |
137 |
0.3375 |
0.2109 |
27 |
18 |
- |
MMS 10 |
192 |
33 |
159 |
0.1886 |
0.1179 |
15 |
17 |
- |
MMS 15 |
192 |
42 |
150 |
0.2469 |
0.1543 |
20 |
# = mean value of two parallel cultures; individual results are presented in Table 5b (attachment)
P
= probable number of clones/well
PE
= plating efficiency
%
RS = percent relative survival
MMS
= Methylmethanesulfonate [µL/mL]
Table 2c:Plating Efficiency 1 for Survival -withS9 activation
Culture Number |
S9 +with
|
Concentration of Polycat 41 catalyst (µg/mL) |
Total Wells |
Wells Containing Cells |
Empty Wells |
Probable number of clones/well (P) |
Plating Efficiency (PE) 1 |
% Relative Survival (RS) |
1st experiment (3-hour exposure)
|
||||||||
1, 2# |
+ |
0 (control) |
192 |
148 |
44 |
1.4744 |
0.9215 |
100 |
7 |
+ |
15.63 |
192 |
124 |
68 |
1.0380 |
0.6488 |
70 |
6 |
+ |
31.3 |
192 |
128 |
64 |
1.0986 |
0.6866 |
75 |
5 |
+ |
62.5 |
192 |
152 |
40 |
1.5686 |
0.9804 |
106 |
4 |
+ |
125 |
192 |
160 |
32 |
1.7918 |
1.1199 |
122 |
3 |
+ |
250 |
192 |
48 |
144 |
0.2877 |
0.1798 |
20 |
9 |
+ |
3-MC 2.5 |
192 |
110 |
82 |
0.8508 |
0.5318 |
58 |
8 |
+ |
3-MC 4.0 |
192 |
98 |
94 |
0.7142 |
0.4464 |
48 |
2nd experiment (3-hour exposure)
|
||||||||
10,11# |
+ |
0 (control) |
192 |
157 |
35 |
1.7026 |
1.0641 |
100 |
16 |
+ |
15.63 |
192 |
118 |
74 |
0.9534 |
0.5959 |
56 |
15 |
+ |
31.3 |
192 |
162 |
30 |
1.8563 |
1.1602 |
109 |
14 |
+ |
62.5 |
192 |
112 |
80 |
0.8755 |
0.5472 |
51 |
13 |
+ |
125 |
192 |
150 |
42 |
1.5198 |
0.9499 |
89 |
12 |
+ |
250 |
192 |
50 |
142 |
0.3017 |
0.1886 |
18 |
18 |
+ |
3-MC 2.5 |
192 |
88 |
104 |
0.6131 |
0.3832 |
36 |
17 |
+ |
3-MC 4.0 |
192 |
118 |
74 |
0.9534 |
0.5959 |
56 |
# = mean value of two parallel cultures; individual results are presented in Table 5c (attachment)
P
= probable number of clones/well
PE
= plating efficiency
% RS = percent relative survival
3-MC= 3-Methylcholanthrene
Table 2d:Plating Efficiency 2 for Mutant Frequency - with S9 activation
Culture Number |
S9 +with
|
Concentration of Polycat 41 catalyst (µg/mL) |
Total Wells |
Wells Containing Cells |
Empty Wells |
Probable number of clones/well (P) |
Plating Efficiency (PE) 2 |
% Relative Survival (RS) |
1st experiment (3-hour exposure)
|
||||||||
1, 2# |
+ |
0 (control) |
192 |
158.5 |
33.5 |
1.7461 |
1.0913 |
100 |
7 |
+ |
15.63 |
192 |
121 |
71 |
0.9948 |
0.6218 |
57 |
6 |
+ |
31.3 |
192 |
161 |
31 |
1.8235 |
1.1397 |
104 |
5 |
+ |
62.5 |
192 |
162 |
30 |
1.8563 |
1.1602 |
106 |
4 |
+ |
125 |
192 |
135 |
57 |
1.2144 |
0.7590 |
70 |
3 |
+ |
250 |
192 |
48 |
144 |
0.2877 |
0.1798 |
16 |
9 |
+ |
3-MC 2.5 |
192 |
42 |
150 |
0.2469 |
0.1543 |
14 |
8 |
+ |
3-MC 4.0 |
192 |
55 |
137 |
0.3375 |
0.2109 |
19 |
2nd experiment (3-hour exposure)
|
||||||||
10,11# |
+ |
0 (control) |
192 |
138 |
54 |
1.3505 |
0.8441 |
100 |
16 |
+ |
15.63 |
192 |
122 |
70 |
1.0090 |
0.6306 |
75 |
15 |
+ |
31.3 |
192 |
127 |
65 |
1.0831 |
0.6769 |
80 |
14 |
+ |
62.5 |
192 |
163 |
29 |
1.8902 |
1.1814 |
140 |
13 |
+ |
125 |
192 |
139 |
53 |
1.2872 |
0.8045 |
95 |
12 |
+ |
250 |
192 |
48 |
144 |
0.2877 |
0.1798 |
21 |
18 |
+ |
3-MC 2.5 |
192 |
37 |
155 |
0.2141 |
0.1338 |
16 |
17 |
+ |
3-MC 4.0 |
192 |
49 |
143 |
0.2947 |
0.1842 |
22 |
# = mean value of two parallel cultures; individual results are presented in Table 5d (attachment)
P
= probable number of clones/well
PE
= plating efficiency
% RS = percent relative survival
3-MC= 3-Methylcholanthrene
Table 3a:Mutant Frequency/106 cells -withoutS9 activation
Culture Number |
S9 -without
|
Concentration of Polycat 41 catalyst (µg/mL) |
Total Wells |
Empty Wells |
Probable number of clones/well (P) |
PE (Mutant cells) |
Plating Efficiency (PE) 2 |
MF/106 |
1st experiment (3-hour exposure)
|
||||||||
1, 2# |
- |
0 (control) |
384 |
349.5 |
0.0942 |
4.7100E -05 |
0.7788 |
60.76 |
7 |
- |
15.63 |
384 |
338 |
0.1276 |
6.3800E -05 |
0.6218 |
102.61 |
6 |
- |
31.3 |
384 |
343 |
0.1129 |
5.6450E -05 |
0.6965 |
81.05 |
5 |
- |
62.5 |
384 |
335 |
0.1365 |
6.8250E -05 |
0.6769 |
100.83 |
4 |
- |
125 |
384 |
334 |
0.1395 |
6.9750E -05 |
0.9649 |
72.29 |
3 |
- |
250 |
384 |
371 |
0.0344 |
1.7200E -05 |
0.1929 |
89.17 |
9 |
- |
MMS 10 |
384 |
251 |
0.4252 |
2.1260E -04 |
0.1179 |
1803.22 |
8 |
- |
MMS 15 |
384 |
240 |
0.4700 |
2.3500E -04 |
0.1023 |
2297.17 |
2nd experiment (24-hour exposure)
|
||||||||
10,11# |
- |
0 (control) |
384 |
349.5 |
0.0941 |
4.7050E -05 |
0.7873 |
59.81 |
16 |
- |
15.63 |
384 |
338 |
0.1276 |
6.3800E -05 |
0.6218 |
102.61 |
15 |
- |
31.3 |
384 |
341 |
0.1188 |
5.9400E -05 |
0.7375 |
80.54 |
14 |
- |
62.5 |
384 |
334 |
0.1395 |
6.9750E -05 |
0.6769 |
103.04 |
13 |
- |
125 |
384 |
337 |
0.1306 |
6.5300E -05 |
0.9962 |
65.55 |
12 |
- |
250 |
384 |
369 |
0.0398 |
1.9900E -05 |
0.2109 |
94.36 |
18 |
- |
MMS 10 |
384 |
192 |
0.6931 |
3.4655E -04 |
0.1179 |
2939.36 |
17 |
- |
MMS 15 |
384 |
204 |
0.6325 |
3.1625E -04 |
0.1543 |
2049.58 |
MF/106 = Mutant frequency/106 cells
MMS = Methylmethanesulfonate [µL/mL]
Table 3b:Mutant Frequency/106cells -withS9 activation
Culture Number |
S9 +with
|
Concentration of Polycat 41 catalyst (µg/mL) |
Total Wells |
Empty Wells |
probable number of clones/well (P) |
PE (Mutant cells) |
Plating Efficiency (PE) 2 |
MF/106 |
1st experiment (3-hour exposure)
|
||||||||
1, 2# |
+ |
0 (control) |
384 |
338.5 |
0.1261 |
6.3050E -05 |
1.0913 |
57.84 |
7 |
+ |
15.63 |
384 |
358 |
0.0701 |
3.5050E -05 |
0.6218 |
56.37 |
6 |
+ |
31.3 |
384 |
333 |
0.1425 |
7.1250E -05 |
1.1397 |
62.52 |
5 |
+ |
62.5 |
384 |
338 |
0.1276 |
6.3800E -05 |
1.1602 |
54.99 |
4 |
+ |
125 |
384 |
342 |
0.1158 |
5.7900E -05 |
0.7590 |
76.28 |
3 |
+ |
250 |
384 |
373 |
0.0291 |
1.4550E -05 |
0.1798 |
80.92 |
9 |
+ |
3-MC 2.5 |
384 |
292 |
0.2739 |
1.3695E -04 |
0.1543 |
887.56 |
8 |
+ |
3-MC 4.0 |
384 |
278 |
0.3230 |
1.6150E -04 |
0.2109 |
765.77 |
2nd experiment (3-hour exposure)
|
||||||||
10,11# |
+ |
0 (control) |
384 |
339 |
0.1247 |
6.2350E -05 |
0.8441 |
79.05 |
16 |
+ |
15.63 |
384 |
357 |
0.0729 |
3.6450E -05 |
0.6306 |
57.80 |
15 |
+ |
31.3 |
384 |
341 |
0.1188 |
5.9400E -05 |
0.6769 |
87.75 |
14 |
+ |
62.5 |
384 |
334 |
0.1395 |
6.9750E -05 |
1.1814 |
59.04 |
13 |
+ |
125 |
384 |
333 |
0.1425 |
7.1250E -05 |
0.8045 |
88.56 |
12 |
+ |
250 |
384 |
368 |
0.0426 |
2.1300E -05 |
0.1798 |
118.46 |
18 |
+ |
3-MC 2.5 |
384 |
287 |
0.2912 |
1.4560E -04 |
0.1338 |
1088.19 |
17 |
+ |
3-MC 4.0 |
384 |
261 |
0.3861 |
1.9305E -04 |
0.1842 |
1048.05 |
MF/106= Mutant frequency/106cells
3-MC = 3-Methylcholanthrene
Table 8a: Relative Total Growth (RTG) -withoutS9 activation
Culture Number |
S9 -without
|
Concentration of Polycat 41 catalyst (µg/mL) |
SG |
% RSG |
CE |
P(0) |
% RTG |
MF/106 |
1st experiment (3-hour exposure)
|
||||||||
1, 2# |
- |
0 (control) |
26.7 |
100 |
77.56 |
0.2891 |
100 |
60.66 |
7 |
- |
15.63 |
30.1 |
113 |
62.17 |
0.3698 |
91 |
102.63 |
6 |
- |
31.3 |
22.6 |
85 |
69.65 |
0.3281 |
76 |
81.08 |
5 |
- |
62.5 |
19.4 |
72 |
67.70 |
0.3385 |
63 |
100.82 |
4 |
- |
125 |
23.4 |
88 |
96.51 |
0.2135 |
110 |
72.27 |
3 |
- |
250 |
29.2 |
109 |
19.29 |
0.7344 |
27 |
89.39 |
9 |
- |
MMS 10 |
17.5 |
66 |
11.79 |
0.8281 |
10 |
1803.48 |
8 |
- |
MMS 15 |
20.7 |
77 |
10.23 |
0.8490 |
10 |
2297.18 |
2nd experiment (24-hour exposure)
|
||||||||
10,11# |
- |
0 (control) |
47.6 |
100 |
78.70 |
0.2839 |
100 |
59.78 |
16 |
- |
15.63 |
62.4 |
131 |
62.17 |
0.3698 |
103 |
102.63 |
15 |
- |
31.3 |
61.1 |
128 |
73.75 |
0.3037 |
120 |
80.53 |
14 |
- |
62.5 |
36.9 |
77 |
67.70 |
0.3385 |
66 |
103.02 |
13 |
- |
125 |
62.4 |
131 |
99.63 |
0.2031 |
166 |
65.52 |
12 |
- |
250 |
9.7 |
20 |
21.10 |
0.7135 |
5 |
94.51 |
18 |
- |
MMS 10 |
35.3 |
74 |
11.79 |
0.8281 |
11 |
2939.56 |
17 |
- |
MMS 15 |
38.3 |
80 |
15.42 |
0.7813 |
16 |
2052.68 |
# = mean value of two parallel cultures
RTG
= Relative total growth
MMS
= Methylmethanesulfonate [µL/mL]
CE
= Cloning efficiency
P(0)
= number of well with no colony/total number of wells
Table 8b: Relative Total Growth (RTG) -withS9 activation
Culture Number |
S9 -with
|
Concentration of Polycat 41 catalyst (µg/mL) |
SG |
% RSG |
CE |
P(0) |
% RTG |
MF/106 |
1st experiment (3-hour exposure)
|
||||||||
1, 2# |
+ |
0 (control) |
24.4 |
100 |
109.11 |
0.1745 |
100 |
57.80 |
7 |
+ |
15.63 |
21.2 |
87 |
62.17 |
0.3698 |
50 |
56.38 |
6 |
+ |
31.3 |
28.1 |
115 |
113.95 |
0.1615 |
120 |
62.52 |
5 |
+ |
62.5 |
27.6 |
113 |
116.00 |
0.1563 |
120 |
55.00 |
4 |
+ |
125 |
20.2 |
83 |
75.90 |
0.2969 |
58 |
76.32 |
3 |
+ |
250 |
7.1 |
29 |
17.98 |
0.7500 |
5 |
80.69 |
9 |
+ |
3-MC 2.5 |
24.4 |
100 |
15.42 |
0.7813 |
14 |
888.17 |
8 |
+ |
3-MC 4.0 |
25.7 |
106 |
21.10 |
0.7135 |
20 |
765.32 |
2nd experiment (3-hour exposure)
|
||||||||
10,11# |
+ |
0 (control) |
17.5 |
100 |
79.27 |
0.2813 |
100 |
78.63 |
16 |
+ |
15.63 |
20.8 |
119 |
63.06 |
0.3646 |
95 |
57.80 |
15 |
+ |
31.3 |
30.5 |
175 |
67.70 |
0.3385 |
149 |
87.73 |
14 |
+ |
62.5 |
19.6 |
112 |
118.15 |
0.1510 |
167 |
59.03 |
13 |
+ |
125 |
21.7 |
124 |
80.46 |
0.2760 |
126 |
88.54 |
12 |
+ |
250 |
14.3 |
82 |
17.98 |
0.7500 |
19 |
118.45 |
18 |
+ |
3-MC 2.5 |
19.0 |
109 |
13.38 |
0.8073 |
18 |
1088.02 |
17 |
+ |
3-MC 4.0 |
21.7 |
124 |
18.41 |
0.7448 |
29 |
1048.63 |
# = mean value of two parallel cultures
RTG
= Relative total growth
3-MC
= 3-Methylcholanthrene
CE
= Cloning efficiency
P(0)
= number of well with no colony/total number of wells
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In an OECD Guideline 471 (Bacterial reverse mutation assay), in a first preliminary experiment, the test item induced a greater than two-fold increase in the frequency of TA98 revertant colonies at 1500 µg/plate in the presence of metabolic activation only. Smaller, statistically significant increases in revertant colony frequency were observed in TA100 at 500 µg/plate in the presence of metabolic activation, TA1535 (1500 µg/plate in the presence of metabolic activation), TA98 (1500 µg/plate in the absence of metabolic activation) and WP2uvrA (5000 µg/plate in the absence of metabolic activation).
The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in both the presence and absence of metabolic activation, initially from 2000 µg/plate (TA100 and TA1537). Toxicity was also observed in the confirmatory experiment from 1250 µg/plate in the absence of metabolic activation and 1500 µg/plate in the presence of metabolic activation.
In a second experiment, no increase greater than two or three times the concurrent solvent control (depending on bacterial strain type) was observed in any of the strains tested (either in the presence or absence of S9-mix). However, several strains exhibited statistically significant increases in the frequency of revertant colonies in the absence and presence of metabolic activation, particularly TA100 which exhibited increases in colony frequency at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria. There was also a small dose-response relationship noted between 500 and 1000 µg/plate in both the absence and presence of metabolic activation.
In a third confirmatory experiment in the absence of metabolic activation, increases greater than two times the concurrent solvent control were observed in TA100 between 500 and 1000 µg/plate and TA98 between 900 and 1250 µg/plate. In the presence of metabolic activation, increases of greater than two times the concurrent solvent control were observed in TA100 only between 500 and 1250 µg/plate. Smaller but statistically significant increases were noted in TA98 at 900 and 1500 µg/plate.
In an OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test) the material was tested up to a cytotoxic concentration of 250 µg/mL,in the absence and presence of metabolic activation in two independent experiments and was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cells. The test substance also did not exhibit clastogenic potential at the concentration-range investigated.
In an OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test) the material was tested up to a cytotoxic concentrationof 250 µg/mL in the absence metabolic activation (prolonged exposure) and 500 µg/mL in the presence of metabolic activation (short exposure) did not induce chromosome breaks and/or gain or loss of chromosomes in human lymphocytes.
The test substance was weakly mutagenic in the Bacterial reverse mutation assay, where there was evidence of cytotoxicity observed at highest dose levels. The test substance showed no mutagenic potential in the Mouse Lymphoma Forward mutation assay (OECD TG 476) and in the in Vitro Mammalian Cell Micronucleus Test (OECD TG) 487.
To further address the weak mutagenic response seen in the Bacerial Reverse Mutation Assay, a ToxTracker assay was conducted using Polycat 41 as test substance.
Significant cytotoxicity (>50%) was observed at a concentration of 440.8 μM in the absence of a metabolising system. No increased cytotoxicity was observed in the presence of a metabolising system. No autofluorescence was observed for this test substance.
Polycat-41 activated the Rtkn-GFP genotoxicity reporter more than 2-fold
and was therefore classified as genotoxic in this assay. Activation of
the Rtkn-GFP ToxTracker reporter is associated with induction of DNA
double strand breaks and indicates the induction of chromosome damage.
Activation of Rtkn-GFP but not Bscl2-GFP is often associated with an
aneugenic mode of action. However, no accumulation of cells in G2/M or
aneuploidy was observed after exposure to Poycat-41. Activation of the
oxidative stress reporters Srxn1 as well as the Btg2-GFP reporter for
p53 activation was observed, both in absence and presence of S9. Bscl2
-GFP and Btg2 -GFP activation suggest that repair mechanisms have been
triggered.
Justification for classification or non-classification
According to Guidance on the Application of the CLP Criteria (Version 5.0, July 2017), a weight of evidence approach using expert judgement has to be applied in the case of equivocal data on mutagenicity.
In the OECD Test Guideline 471 "Bacterial Reverse Mutation Test", observed increases in revertant colony frequency which were two times greater than the concurrent solvent control were inconsistent between Experiment 1 and 2. In Experiment 1, only TA98 at 1500 µg/plate in the presence of S9-mix achieved a twofold increase and no strain achieved twofold increases over the solvent control in Experiment 2. However, both experiments exhibited statistically significant increases that approached two times the concurrent solvent control, particularly in TA98 and TA100, both in the presence and absence of metabolic activation. The number of revertant colonies observed in these strains also approached or exceeded the upper limit of the expected range for these strains.
A third, confirmatory experiment using the pre-incubation method was performed in TA100 and TA98, both in the presence and absence of metabolic activation and a narrowed dose range. The results of this experiment showed that the test item exhibited increases in revertant colony frequency of greater than two times the concurrent solvent control over all of the sub-toxic dose levels in TA100 both with and without metabolic activation. In TA98, increases in revertant colony frequency of greater than two times the concurrent solvent control were observed at 900 and 1250 µg/plate only and none in the presence of S9 mix (although fold increases were noted from 1.6 to 1.9 times the concurrent vehicle control).
As it is, there is no concordance between the experiments other than that there are small but reproducible increases in revertant colony frequency in TA100 and TA98 in particular. However, these statistically significant increases were observed consistently over all three experiments and, where the change in methodology to the pre-incubation method was implemented, the increased sensitivity of the assay exhibited a much clearer response, particularly in the frequency of TA100 revertant colonies. Consequently, the overall conclusion of the study must be that the test substance should be considered weakly mutagenic.
Two further in vitro assays were performed using mammalian cells.
A Mouse Lymphoma Forward mutation assay (according to OECD TG 476) using the test substance, tested up to a cytotoxic concentration of 250 µg/mL in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.
In an in vitro Mammalian Cell Micronucleus Test (OECD Test Guideline No. 487) the test substance was non mutagenic to human peripheral blood lymphocytes in experiments both without and with metabolic activation.
A ToxTracker assay was also performed with Polycat-41. ToxTracker is a mammalian stem cell-based assay that monitors activation of specific cellular signaling pathways for detection of the biological reactivity of compounds. ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response.
In ToxTracker ACE (Aneugen Clastigen Evaluation), a DNA staining after 4 and 24 hours of treatment is included to assess clastogenic and/or aneugenic properties of compounds.
Polycat-41 activated the Rtkn-GFP genotoxicity reporter more than 2-fold and was therefore classified as genotoxic in this assay. Activation of the Rtkn-GFP ToxTracker reporter is associated with induction of DNA double strand breaks and indicates the induction of chromosome damage. Activation of Rtkn-GFP but not Bscl2 -GFP is often associated with an aneugenic mode of action. However, no accumulation of cells in G2/M or aneuploidy was observed after exposure to Poycat-41. Activation of the oxidative stress reporters Srxn1 as well as the Btg2-GFP reporter for p53 activation was observed, both in absence and presence of S9.
Interpretation and Conclusion:
Both in vitro tests with mammalian cells show a clear negative result, which should be interpreted to have a higher relevance.
The results of the ToxTracker investigation indicated an activiation of the marker for DNA double strand breaks, however the mammalian cell test on chromosome aberration (OECD 487) was negative, which has a higher relevance. Bscl2 -GFP and Btg2 -GFP activation suggest that repair mechanisms have been triggered.
The weakly mutagenic results of the Ames test were evaluated against historical control data for the period 2017 -2018 (attached to RSS). It was observed that for TA98 only one value was out of the historical control range (Experiment 1 – with metabolic activation, 1500 µg).
The mean values of the negative control data from the Ames test were within the range of 2017, but near to the maximum values and some single values are also out of the range. For TA 100, with and without metabolization and increase of 32% in the number of mutations within the negative control data from 2017 to 2018, which was not observed for the other strains. Historical data for 2019 were not yet available, so a trend could not be confirmed.
Based on weight of evidence, N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine is not classified for mutagenicity as it shows weak mutagenic activity in the bacterial mutagenicity assay, and is negative for mutagenicity in mammalian (mouse and human) cells.
The results of the ToxTracker assay indicates an activiation of the marker for the DNA double-strand breaks, however the mammalian cell test on chromosome aberration (OECD 487) was negative, and this was deemed to have a higher relevance. ToxTracker did also reveal Bscl2-GFP and Btg2-GFP activation, which suggests the activation of repair mechanisms.
Based on a toxicokinetic assessment of information from existing animal studies is it observed that toxic effects are primarily manifested on local level (irritation of skin and mucous membranes). The results of sensitisation studies (no sensitisation, potential skin irritation) support this conclusion.
After repeated oral exposure, subtle changes in some biochemical and haematological parameters were detected but those were not attributable to the test substance effect. Only pathological changes in forestomach could be considered as attributable to the test substance action. These findings lead to the conclusion that also in this case the substance did not enter the animal body and its effect is manifested only locally in the site of first contact with organism, in the forestomach.
Therefore, in the absence of significant systemic bioavialability, specific target tissue would likely not be adequately exposed to the test substance and/or its metabolites. Assays that use site of contact tissues, for example the in vivo comet assay (OECD 489) would be compromised by the local irritation effect.
Human exposure to the substance is anticipated to be low or insignificant. Only Industrial and Professional uses are supported. The primary hazard from a risk management perspective is the avoidance of skin/eye/respiratory contact with the substance due to the risk of severe irritation/corrosion. Therefore precautions are taken against exposures by all routes. The potential for inhaltion of vapours is ameliorated by the low vapour pressure of the substance. Once the substance is used for its purpose (catalyst) it is bound in a matrix, preventing the possibility of further exposure.
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