N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity Data Summary

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 14 July 2020 to 03 September 2020

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:

The retest date stated in the certificate of analysis provided by the sponsor did not match the expiry date in the study plan. It was advised by the sponsor that the retest date in the study plan is the correct date.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal Enzyme Fraction was purchased from Moltox and Lot no 4222 with the expiry date of 12 March 2022 was used in this study. The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control and Production Certificate” which is presented in Annex 2. The protein content was adjusted to approximately 20 mg/ml prior to use.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

4-hour without S9: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/ml

4-hour with S9 (2%): 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/ml

24-hour without S9 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/ml

The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level observed in a preliminary toxicity test which was 125µg/mL for all three of the exposure groups. The top dose was subsequently not used for analysis due precipitation of the test substance.

Vehicle / solvent:

Dimethyl Sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

CELLS

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.


The details of the donors used are:
Preliminary Toxicity Test: female, aged 28 years
Main Experiment: female, aged 24 years


CULTURE CONDITIONS
Lymphocyte cultures were established for each dose level, by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.20-9.28 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.52-0.6 mL heparinised whole blood

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: (Main Test) duplicate for each dose level, quadruplicate for the solvent

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium


CELL HARVEST
At the end of the treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.


PREPARATION OF MICROSCOPE SLIDES
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labelled with the appropriate identification data.

STAINING
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.



CYTOKINESIS BLOCK PROLIFERATION INDEX (CBPI)
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the solvent controls.


SCORING OF MICRONUCLEI
The micronucleus frequency in 1000 binucleated cells was analysed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the solvent controls). Cells with 1, 2 or more micronuclei were recorded and included in the total. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.


The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent solvent control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.

The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Key result

Species / strain:

other: Human whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: All tests were conducted up to precipitating concentrations. No cytotoxicity was seen in the 4-hour exposures, but cytotoxic was observed at 62.5 µg/mL in the 24-hour exposure.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Micronucleus Test – Main Experiment

 

 The dose levels of the controls and the test item are given in the table below:

 

Exposure Group	Final concentration of test item (µg/mL)
4-hour without S9	0*, 3.91, 7.81, 15.63*, 31.25*, 62.5*, 125, MMC 0.2*
4-hour with S9 (2%)	0*, 3.91, 7.81, 15.63*, 31.25*, 62.5*, 125, CP 6*
24-hour without S9	0*, 3.91, 7.81, 15.63*, 31.25*, 62.5*, 125, DC 0.075*

* = Dose levels selected for analysis of micronucleus frequency in binucleate cells

 

MMC = Mitomycin C
DC = Demecolcine
CP = Cyclophosphamide

 

The maximum dose level selected for analysis of binucleate cells was the lowest precipitating dose level and was 62.5 µg/mL for all three exposure groups.  

 

 In the 24-hour continuous exposure group there was modest dose related toxicity where 22% cytostasis was observed at 62.5 µg/mL.

 

The solvent control cultures had frequencies of cells with micronuclei within the expected range and were considered acceptable for addition to the laboratory historical negative control data range.

 

 The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that were compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation, and the results were within the distribution of the historical solvent data (within 95% control limits). There was no statistically significant concentration related increase in any of the three exposure groups when evaluated with a trend test.

Conclusions:

The test item, , N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-
aminobenzamide) did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

Introduction

 This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. The test method used was designed to meet the requirements of the following guideline:

• OECD Guidelines for Testing of Chemicals No. 487 "In Vitro Mammalian Cell
  Micronucleus Test", adopted 29 July 2016.

 

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with solvent (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolising system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation.

 

At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Experiment were as follows:

 

Exposure Group	Final concentration of test item N,N'-(2-(4-(2- aminobenzamido)butyl)pentane-1,5-diyl)bis(2- aminobenzamide) (µg/mL)
4-hour without S9	0, 3.91, 7.81, 15.63, 31.25, 62.5, 125
4-hour with S9 (2%)	0, 3.91, 7.81, 15.63, 31.25, 62.5, 125
24-hour without	0, 3.91, 7.81, 15.63, 31.25, 62.5, 125

 

Results

Precipitation occurred in the 125 µg/mL dose concentration in all experiments and was not used for analysis.

 

All solvent (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical solvent control data range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level in all three of the exposure groups.

 

Conclusion

The test item, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 08 September 2020 to 02 November 2020

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

GLP compliance:

yes

Type of assay:

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

Target gene:

Thymidine kinase, TK +/-, locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.


CELL CLEANSING
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

CELL CULTURE
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were
routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier. Master stocks of cells were tested and found to be free of mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal Enzyme Fraction was purchased from Moltox, Lot no 4222, with the expiry date of 12 March 2022, was used in this study. A copy of the Quality Control and Production Certificate provided by the supplier confirmed its capability to activate known mutagens. The protein content was adjusted to 20 mg/ml prior to use.

Test concentrations with justification for top dose:

Main Test Doses Applied (corrected for purity)

4-hour without S9: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250* µg/mL

4-hour with S9: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250* µg/mL

* The onset of test item precipitate was observed at 125 µg/mL in both the absence and presence of metabolic activation; therefore, following the recommendations of the OECD 490 guideline, the 250 µg/mL dose was not plated for cloning efficiency or 5-TFT resistance.

The doses for the main test were based on the results of a preliminary range-finding toxicity test.

Vehicle / solvent:

Dimethyl Sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (suspension growth)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10E^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universal bottles.


TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No
- Exposure duration/duration of treatment: The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.



MEASUREMENT OF SURVIVAL, CLONING EFFICIENCY AND MUTANT FREQUENCY
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL.


On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for cloning efficiency (%V) in non-selective medium.


The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the solvent control, and when combined with the cloning efficiency (%V) data, a Relative Total Growth (RTG) value.


PLATE SCORING
96 well plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black coloUr, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

There was no evidence of any toxicity, in either the absence or presence of metabolic activation, following exposure to the test item, as indicated by the %RSG and RTG values.

 

 There was also no evidence of any marked reductions in cloning efficiency (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. The onset of test item precipitate was observed at 125 µg/mL in both the absence and presence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the subsequent 250 µg/mL dose level was not plated for cloning efficiency or 5-TFT resistance due to the presence of test item precipitate. Acceptable levels of toxicity were seen with both positive control substances).

 

The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

 

 The test item did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

Conclusions:

The test item, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the following guidelines:

 

• Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016.
  
  


• Method B67 of Commission Regulation (EC) No. 440/2008 of 26 September 2019, and the US EPA OPPTS 870.5300 Guideline of August 1998.

 

Method

 One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with solvent (dimethyl sulfoxide (DMSO)), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose levels in the Mutagenicity Test were limited by the onset of test item precipitate in both the absence and presence of metabolic activation.

The dose levels plated for cloning efficiency and expression of mutant colonies were as follows:

Group	Concentration of Test Substance N,N’-(2-(4-(2-aminobenzamido)butyl)pentane1,5-diyl)bis(2-aminobenzamide) - (µg/mL) plated for cloning efficiency and mutant frequency
4-hour without S9	0, 3.91, 7.81, 15.63, 31.25, 62.5, 125
4-hour with S9 (2%)	0, 3.91, 7.81, 15.63, 31.25, 62.5, 125

 

Results

The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

 

The test item did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

 

 Conclusion

The test item, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2- aminobenzamide), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 17 JUne 2020 to 02 July 2020.

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:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella
Trptophan for E.coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4146 and the protein level was adjusted to 20 mg/mL.
- concentration or volume of S9 mix and S9 in the final culture medium: A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Experiment one: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate
Experiment two: 15, 50, 150, 1500 and 5000 µg/plate

Vehicle / solvent:

The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Negative solvent / vehicle controls:

yes

Remarks:

Spontaneous mutation rate

Positive controls:

yes

Remarks:

For TA100, TA1535 and WP2uvrA

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation

Positive controls:

yes

Remarks:

For TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Positive controls:

yes

Remarks:

For TA98

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Positive controls:

yes

Remarks:

For TA100, TA1535, TA1537 and WP2uvrA

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With metabolic activation

Positive controls:

yes

Remarks:

For TA98

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): tripilcate
- Number of independent experiments: Two - experiment 1 plate incorporation, Experiment 2 pre-incubation


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 48-72 hours


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Plates were assessed for numbers of revertant colonies and examined for effects on the growth of bacterial background lawn.

Evaluation criteria:

Any, one, or all of the following criteria could 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).

Statistics:

Statistical significance was confirmed by using Dunnett’s 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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A test item precipitate (white/cloudy in appearance) was noted at 5000 µg/plate in both the presence and absence of metabolic activation (S9-mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A test item precipitate (white/cloudy in appearance) was noted at 5000 µg/plate in both the presence and absence of metabolic activation (S9-mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A test item precipitate (white/cloudy in appearance) was noted at 5000 µg/plate in both the presence and absence of metabolic activation (S9-mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A test item precipitate (white/cloudy in appearance) was noted at 5000 µg/plate in both the presence and absence of metabolic activation (S9-mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A test item precipitate (white/cloudy in appearance) was noted at 5000 µg/plate in both the presence and absence of metabolic activation (S9-mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Experiment 1 (plate incorporation)

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. 

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

A test item precipitate (white/cloudy in appearance) was noted at 5000 µg/plate in both the presence and absence of metabolic activation (S9-mix).  This observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). 

Experiment 2 (pre-incubation)

The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate). 

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

A test item precipitate (white/cloudy in appearance) was noted at 5000 mg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Conclusions:

Under the conditions of the test, the test substance N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) was considered to be non mutagenic.

Executive summary:

Introduction

The bacterial mutagenic potential of the test substance was assessed using a test method designed to be compatible with the following guidelines:

• OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test"
• Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008,
• ICH S2(R1)guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
• USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.
  

Method

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 eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising 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 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. 

Results

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). 

Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Conclusion

 Under the conditions of the test, the test substance N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)was considered to be non‑mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo studies conducted.

Justification for classification or non-classification

The test substance was negative in the Ames, mouse lymphoma and in vitro micronucleus assays and it is therefore concluded that it is negative for genotoxicity and no classification is required.