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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

EC Number:

951-985-7

Molecular formula:

C30H38N6O3

IUPAC Name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

Test material form:

solid: crystalline

Method

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

Controls

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.