N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental condition chosen. Read-across from in vitro micronucleus and mouse lymphoma assays in structurally similar tertiary amines indicates no mutagenic activity or alerts.

In an OECD Guideline 487 study (In vitro Mammalian Cell Micronucleus Test), PU-2018-788 (Polycat 77) tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

In a OECD Guideline 490 study (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene) the substance PU-2018-788 (Polycat 77) tested up to cytotoxic concentrations in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed in accordance with OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay). A GLP certificate is provided.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The stability of the test substance in the vehicle water has not been determined analytically .

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Target gene:

TA. 1535, TA 100, TA 1537, TA 98 and E . coli WP2 uvrA

Test concentrations with justification for top dose:

20 - 5,000 ug/plate

Details on test system and experimental conditions:

Standard plate test and preincubation test both with and without metabolic activation (Aroclor-induced rat liver S-9 mix)

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precitation of the test sub stance was found.

Occasionally, a slight decrease in the number of revertants was observed in the standard plate test with S-9 mix at 5,000 ug/ plate . In the preincubation assay bacteriotoxicity was found depending on the strain and test conditions_at doses > 2,500 ug / plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

According to the result's of the present study, the substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental condition chosen.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2018 - January 2019

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)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 487: In Vitro Mammalian Cell Micronucleus Test (MNvit), adopted July 29, 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Designation: PU-2018-788 (Polycat 77)
Chemical name: N-[3-(Dimethylamino)propyl]-N,N’,N’-trimethylpropan-1,3-diamin
CAS no.: 3855-32-1
Batch no.: 2302776
Receipt no.: 66072
Date of receipt: 25 June 2018
Characteristics: Colourless, liquid
Storage conditions: Stored at +10°C to +25°C, in a tightly closed container and stored at a dry, cool and well-ventilated place
Production date: 13 April 2017
Stability / Expiry date: 13 April 2020
Purity: 96.3%

Species / strain / cell type:

lymphocytes: cultured human peripheral lymphocytes

Details on mammalian cell type (if applicable):

cultured human peripheral lymphocytes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

CytoB (Cytochalasin B)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 (Aroclor 1254-induced)

Test concentrations with justification for top dose:

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg PU-2018-788 (Polycat 77)/mL medium were employed. Complete cytotoxicity was noted at concentrations of 1000 and 2000 µg/mL medium in the experiments without and with metabolic activation (24- or 4-hour exposure, respectively). No changes in osmolality of the test item formulations compared to the negative control were noted at concentrations up to 2000 µg/mL medium. The pH value increased starting at a concentration of 100 µg/mL medium (up to pH = 9.74 at 2000 µg/mL compared to the control with pH = 7.72). The increase at 100 and 316 µg/mL medium (up to pH = 8.8) was considered to be of no biological relevance.
Hence, 800 µg/mL medium were employed as the top concentration for the genotoxicity tests.

In the main study complete cytotoxicity was noted at 800 µg PU-2018-788 (Polycat 77)/mL medium in all experiments. In addition, cytotoxicity was noted at 600 µg/mL medium in the absence and presence of metabolic activation (24- or 4-hour exposure, respectively).

Vehicle / solvent:

Highly purified water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cell cultures (human peripheral lymphocytes) were exposed to the test item both with and without an exogenous source of metabolic activation. Concurrently the vehicle highly purified water and positive controls were included in all tests. During or after exposure to the test item, the cells were grown for a period sufficient to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. Harvested and stained interphase cells were analysed for the presence of micronuclei.

0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. After initiation appropriate concentrations of the test item in the vehicle were added to the cell cultures for each target concentration of the test item in the test medium and each experiment. Precipitation of the test item was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.

Rationale for test conditions:

Test cnducted in accordance with OECD Guideline 487

Evaluation criteria:

Acceptance of a test is based on the following criteria:
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control limits). Where concurrent negative control data fall outside the 95% control limits, they may be acceptable for inclusion in the historical control data as long these data are not extreme outliers.
- Concurrent positive controls induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Adequate number of cells, cell proliferation criteria and concentrations are analysable
- Vehicle control and untreated cultures give reproducibly low and consistent micronucleus frequencies.

Statistics:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).

Key result

Species / strain:

lymphocytes: Human Cultured Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the main study complete cytotoxicity was noted at 800 µg PU-2018-788 (Polycat 77)/mL medium in all experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the main study complete cytotoxicity was noted at 800 µg PU-2018-788 (Polycat 77)/mL medium in all experiments. In addition, cytotoxicity was noted at 600 µg/mL medium in the absence and presence of metabolic activation (24- or 4-hour exposure, respectively).

Tests without metabolic activation (4- and 24-hour exposure):
The mean micronucleus frequencies of cultures treated with the concentrations of 100, 200, 400 and 600 µg PU-2018-788 (Polycat 77)/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 1.5 to 6.5 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 600 µg/mL medium (4- and 24-hour exposure, respectively). The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.
Vehicle controls showed mean frequencies of 6.0 or 7.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges of 1 to 16 or 2 to 13 micronucleate cells per 1000 binucleate cells in the first and second experiment, respectively, and acceptable for inclusion in the historical control distribution.
In the positive control cultures the mean micronucleus frequencies were increased to 24.5 or 28.0 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure):
The mean micronucleus frequencies of cultures treated with the concentrations of 100, 200, 400 and 600 µg PU-2018-788 (Polycat 77)/mL medium in the presence of metabolic activation (4-hour exposure) ranged from 3.0 to 5.0 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 600 µg/mL medium. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.
Vehicle controls showed a mean frequency of 3.5 micronucleate cells per 1000 binucleate cells was observed. The vehicle result was within the historical control range of 1 to 9 micronucleate cells per 1000 binucleate cells and acceptable for inclusion in the historical control distribution.
In the positive control culture the mean micronucleus frequency was increased to 16.0 micronucleate cells per 1000 binucleate cells for the 4-hour exposure with metabolic activation. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

In an OECD Guideline 487 study (In vitro Mammalian Cell Micronucleus Test), PU-2018-788 (Polycat 77) tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within the historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Executive summary:

Test samples of PU-2018-788 (Polycat 77) were assayed in an in vitro micronucleus test (OECD TG 487) using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

PU-2018-788 (Polycat 77) was completely dissolved in highly purified water.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg PU-2018-788 (Polycat 77)/mL medium were employed. Complete cytotoxicity was noted at concentrations of 1000 and 2000 µg/mL medium in the experiments without and with metabolic activation (24- or 4-hour exposure, respectively). No changes in osmolality of the test item formulations compared to the negative control were noted at concentrations up to 2000 µg/mL medium. The pH value increased starting at a concentration of 100 µg/mL medium (up to pH = 9.74 at 2000 µg/mL compared to the control with pH = 7.72). The increase at 100 and 316 µg/mL medium (up to pH = 8.8) was considered to be of no biological relevance.

Hence, 800 µg/mL medium were employed as the top concentration for the genotoxicity tests.

In the main study complete cytotoxicity was noted at 800 µg PU-2018-788 (Polycat 77)/mL medium in all experiments. In addition, cytotoxicity was noted at 600 µg/mL medium in the absence and presence of metabolic activation (24- or 4-hour exposure, respectively).

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure):

The mean micronucleus frequencies of cultures treated with the concentrations of 100, 200, 400 and 600 µg PU-2018-788 (Polycat 77)/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 1.5 to 6.5 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 600 µg/mL medium (4- and 24-hour exposure, respectively). The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls showed mean frequencies of 6.0 or 7.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges of 1 to 16 or 2 to 13 micronucleate cells per 1000 binucleate cells in the first and second experiment, respectively, and acceptable for inclusion in the historical control distribution.

In the positive control cultures the mean micronucleus frequencies were increased to 24.5 or 28.0 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure):

The mean micronucleus frequencies of cultures treated with the concentrations of 100, 200, 400 and 600 µg PU-2018-788 (Polycat 77)/mL medium in the presence of metabolic activation (4-hour exposure) ranged from 3.0 to 5.0 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 600 µg/mL medium. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls showed a mean frequency of 3.5 micronucleate cells per 1000 binucleate cells was observed. The vehicle result was within the historical control range of 1 to 9 micronucleate cells per 1000 binucleate cells and acceptable for inclusion in the historical control distribution.

In the positive control culture the mean micronucleus frequency was increased to 16.0 micronucleate cells per 1000 binucleate cells for the 4-hour exposure with metabolic activation. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Under the present test conditions, PU-2018-788 (Polycat 77) tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within the historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2018 - February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Designation: PU-2018-788 (Polycat 77)
Chemical name: N-[3-(Dimethylamino)propyl]-N,N’,N’-trimethylpropan-1,3-diamin
CAS no.: 3855-32-1
Batch no:. 2302776
Receipt no: 66072
Date of receipt: 25 June 2018
Characteristics: Colourless, liquid
Storage conditions: Stored at +10°C to +25°C, in a tightly closed container and stored at a dry, cool and well- ventilated place
Production date: 13 April 2017
Stability / Expiry date: 13 April 2020
Purity 96.3%

Target gene:

The objective of this study was to evaluate the ability of PU-2018-788 (Polycat 77) 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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The in vitro metabolic activation system comprised rat liver enzymes (S9 fraction) and an energy producing system comprising nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate. The treatment of rats with Aroclor 1254 was used to induce mixed function oxidase enzymes capable of transforming chemicals to more active forms.
The post-mitochondrial fraction (S9 fraction) prepared according to MARON and AMES (1983) was obtained from Trinova Biochem7. S9 was collected from male rats.

Test concentrations with justification for top dose:

A preliminary cytotoxicity study 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 10.0, 31.6, 100, 316, 1000 and 2000 μg PU-2018-788 (Polycat 77)/mL medium were tested for cytotoxicity. After an exposure time of 24 hours (-S9) or 3 hours (+S9) at approx. 37°C on a roller drum at approximately 40 rpm, the cells were washed and resuspended in growth medium. The cells were then adjusted to 8 cells/mL and for each concentration 0.2 mL suspension per well were plated into 32 wells of a 96-well microtiter plate (average 1.6 cells/well). The plates were incubated at 37°C in a humidified incubator gassed with 5% CO2 in air for 7 days. Wells containing viable clones were identified under a microscope and counted.

Based on the results of the preliminary study five concentrations of 62.5, 125, 250, 500 and 1000 μg/mL medium for the 3-hour exposure experiments without and with metabolic activation and 37.5, 75, 150, 300 and 400 μg/mL medium in the second experiment without metabolic activation (24-hour exposure) were employed

Vehicle / solvent:

Highly purified water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

The assay procedure used is based on that reported by COLE et al. (1990). Single cultures were used for each test item concentration level and reference item. The results from the initial mutation assay were confirmed by performing an independent repeat mutation assay.

The cells for the first and second experiments were obtained from logarithmically growing laboratory stock cultures. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were re-suspended in treatment medium that contained 5% heat inactivated horse serum, and the corresponding concentration of test substance was added. The final cell density at start of treatment was 0.5 x 106 cells/mL. The dosed tubes were closed, mixed, and placed on a roller drum at approx. 37°C at approx. 40 rpm for an exposure period of 3 hours.
Thereafter the cells were washed and re-suspended in growth medium.
Cell densities were adjusted to 2 x 105 cells/mL and incubated for 2 days for the expression period. Within the expression period cell densities were determined after incubation for 24 hours and, after adjustment to 2 x 105 cells/mL, the cells were incubated for another 24 hours.
At the end of the expression period, a minimum of 4 concentration levels plus positive and negative controls were selected for the determination of survival (plating efficiency) and 5-trifluoro-thymidine (TFT) resistance. The selected cultures were diluted to 1 x 10^4 cells/mL in plating medium. For the plating for survival, one aliquot of each cell suspension was diluted to 8 cells/mL in plating medium and 0.2 mL of each culture were placed in each well of two 96 well microtiter plates (^ 192 wells, average 1.6 cells/well).

After 1-week incubation, the number of viable clones was recorded. For the TFT resistance TFT was added to the cultures (final concentration 3 μg/mL) and 0.2 mL of each cell suspension were placed into each well of four 96-well microtiter plates (^ 384 wells, average 2 x 10^3 cells/well). The plates were incubated for 10 to 12 days and wells containing clones were identified microscopically and counted. In addition, the number of large and small colonies was recorded. Large colonies are defined as ≥ 1/4 and small colonies < 1/4 of the well diameter of 6 mm.

The activation assay is often run concurrently with the non-activation assay; however, it is an independent assay performed with its own set of solvent and positive controls. In this assay, the above-described activation system was added to the treatment medium together with the test item. An exposure time of 3 hours was used.

The non-activation and activation experiment were repeated in an independent experiment. An exposure time of 3 hours was used for the repeat experiment with metabolic activation and an exposure time of 24 hours instead of 3 hours was used for the repeat experiment without metabolic activation covering long term effects.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system. In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data is evaluated by expert judgement and/or further investigations.

Statistics:

To define positive and negative results and to assure that the increased MF is biologically relevant instead of a statistical analysis (generally used for other tests), the interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10^-6) is based on the analysis of the distribution of the negative control MF data from participating laboratories (M.M. Moore et al. (2006)).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

In the main study, slight to pronounced cytotoxicity (decreased relative total growth) was noted starting at a concentration of 125 μg/mL medium in the absence (3-hour exposure) and at 500 or 250 μg/mL medium in the presence of metabolic activation in the first and second experiment, respectively. The top concentration of 1000 μg/mL medium led to complete cytotoxicity (RTG = 0) in all 3-hour exposure experiments. In the 24-hour exposure experiment without metabolic activation pronounced to complete cytotoxicity was noted at concentrations of 300 and 400 μg/mL medium (decreased relative total growth RTG = 11 or RTG = 0, respectively). Hence, the top concentrations of 1000 and 400 μg/mL medium could not be evaluated.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

In a OECD Guideline 490 study (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene) the substance PU-2018-788 (Polycat 77) tested up to cytotoxic concentrations 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.
No change was noted in the ratio of small to large mutant colonies. Therefore, PU-2018-788 (Polycat 77) also did not exhibit clastogenic potential at the concentration-range investigated.

According to the evaluation criteria for this assay, these findings indicate that PU-2018-788 (Polycat77), tested up to cytotoxic concentrations neither induced mutations nor had any chromosomal aberration potential.

Executive summary:

In order to investigate the mutagenic potential on mammalian cells, the substance PU-2018-788 (Polycat 77) was assayed in a gene mutation assay 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.

PU-2018-788 (Polycat 77) was completely dissolved in highly purified water. The vehicle highly purified water was employed as the negative control.

A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation. A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000 and 2000 μg PU-2018-788 (Polycat 77)/mL medium were tested for cytotoxicity. Pronounced to complete cytotoxicity (decreased survival and a relative plating efficiency (RPE) of 23% at a concentration of 316 μg/mL and 0% at 1000 and 2000 μg/mL) was noted in the absence of metabolic activation (24-hour exposure). In the presence of metabolic activation (3-hour exposure) decreased viability (decreased survival and a relative plating efficiency (RPE) of 16% or 0%) was noted at concentrations of 1000 and 2000 μg/mL. No changes in osmolality of the test item formulations compared to the negative control were noted at concentrations up to 2000 μg/mL medium. The pH value markedly increased at the cytotoxic concentrations of 1000 and 2000 μg/mL medium (up to pH = 9.32 at 2000 μg/mL compared to the control with pH = 7.50).

Hence, in the main study the highest concentrations employed were 1000 μg PU-2018- 788 (Polycat 77)/mL medium in the 3-hour exposure experiments without and with metabolic activation and 400 μg/mL medium in the second experiment without metabolic activation (24-hour exposure).

Methylmethanesulfonate (13 or 12 μg/mL for a 3- and 24-hour exposure, respectively) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (1.0 μg/mL) in the presence of exogenous metabolic activation.

In the main study, slight to pronounced cytotoxicity (decreased relative total growth) was noted starting at a concentration of 125 μg/mL medium in the absence (3-hour exposure) and at 500 or 250 μg/mL medium in the presence of metabolic activation in the first and second experiment, respectively. The top concentration of 1000 μg/mL medium led to complete cytotoxicity (RTG = 0) in all 3-hour exposure experiments. In the 24-hour exposure experiment without metabolic activation pronounced to complete cytotoxicity was noted at concentrations of 300 and 400 μg/mL medium (decreased relative total growth RTG = 11 or RTG = 0, respectively). Hence, the top concentrations of 1000 and 400 μg/mL medium could not be evaluated.

The negative controls had mutation frequencies of 57.54 or 77.39 mutant colonies per 10^6 cells in the experiments without metabolic activation (3- or 24-hour exposure, respectively) and 51.27 or 70.64 mutant colonies per 10^6 cells in the experiments with metabolic activation and, hence, were all well within the historical data-range.

The mutation frequencies of the cultures treated with PU-2018-788 (Polycat 77) ranged from 51.77 to 66.59 mutant colonies per 10^6 cells (3 hours exposure) and from 50.74 to 84.33 mutant colonies per 10^6 cells (24 hours exposure) in the experiments without metabolic activation. In the experiments with metabolic activation, mutation frequencies ranged from 50.42 to 59.22 mutant colonies per 10^6 cells (3 hours exposure, first assay) and from 57.28 to 98.44 mutant colonies per 10^6 cells (3 hours exposure, second assay). These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10^6 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 0.19 to 0.63 for PU-2018-788 (Polycat 77)-treated cells and ratios of 0.58 to 0.68 for the negative controls.

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency of 667.47 and 447.51 mutant colonies per 10^6 cells in the case of MMS and of 368.63 and 513.96 mutant colonies per 10^6 cells in the case of 3-MC. All positive controls showed an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control and an absolute increase in total mutation frequency of at least 300 x 10^-6. Furthermore, the mean relative total growth (RTG) for the positive controls was greater than or equal to 10%. Hence, the acceptance criteria were met.

According to the evaluation criteria for this assay, these findings indicate that PU-2018-788 (Polycat77), tested up to cytotoxic concentrations neither induced mutations nor had any chromosomal aberration potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental condition chosen. Read-across from in vitro micronucleus and mouse lymphoma assays in structurally similar tertiary amines indicates no mutagenic activity or alerts.

In an OECD Guideline 487 study (In vitro Mammalian Cell Micronucleus Test), PU-2018-788 (Polycat 77) tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

In a OECD Guideline 490 study (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene) the substance PU-2018-788 (Polycat 77) tested up to cytotoxic concentrations in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test.

Additional information from genetic toxicity in vitro:

The substance does not need to be classified for genotoxicity according to classification criteria of Directive 67/548/EEC and Regulation (EC) No 1272/2008.