1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 November 2015 to 22 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Test material

Test material information

1

Reference substance name:

1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]

EC Number:

264-637-8

EC Name:

1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]

Cas Number:

64051-50-9

Molecular formula:

C52H95N5O4

IUPAC Name:

3-octadecyl-1-[2-({2-[(2-{[2-(3-octadecyl-2,5-dioxopyrrolidin-1-yl)ethyl]amino}ethyl)amino]ethyl}amino)ethyl]pyrrolidine-2,5-dione

Test material form:

liquid: viscous

Details on test material:

Appearance: Brown viscous liquid
Storage: Room temperature in the dark under nitrogen

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 µg/mL. The purity of the test item was 83 % and was accounted for in the test item formulations due to the amount of base oil present.
Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 µL aliquots. The test item could not be formulated at 1000 mg/mL, therefore, the maximum practical concentration was limited to 2500 µg/mL.
Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared.
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines.

OTHER SPECIFICS
Formulated concentrations were adjusted to allow for the stated oil content (17 %) of the test material.

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Remarks:

Human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 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.
- Suitability of cells: Human peripheral blood lymphocytes are recognized in the OECD 473 guideline as being a suitable cell line for the Mammalian Chromosome Aberration Test.
- Sex, age and number of blood donors if applicable:
Preliminary Toxicity test: One male donor aged 30 years.
Main Experiment: One female donor aged 31 years.
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Methods for maintenance in cell culture if applicable: The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Normal (negative control) cell cycle time: The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours. Therefore using this average, the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.
- Properly maintained: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL for 4 hour exposure with and without S9 mix, and 24-hour without S9 mix.

Main Experiment: 0, 2.5, 5, 10, 20, 40 and 80 µg/mL for 4 hour exposure with and without S9 mix, and 24-hour without S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was considered incompatible with aqueous media and immiscible in dimethyl sulphoxide at 200 mg/mL. However, the test material was fully miscible in acetone at 500 mg/mL in solubility checks performed in-house.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10 % (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

- With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20 % S9-mix (i.e. 2 % final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.

- Without Metabolic Activation (S9) Treatment
For the 4(20)-hour exposure in the absence of S9, after approximately 48 hours incubation at approximately 37 ºC with 5 % CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
For the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5 % CO2 in humidified air for 24 hours.
The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

DURATION
- Exposure duration: 4 hours for Experiment 1 with and without S9 and 24 hours for Experiment 2 without S9.
- Expression time (cells in growth medium): 20 hours for Experiment 1 with and without S9 and 24-hour continuous exposure to the test material without S9-mix prior for Experiment 2.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours for all experiments.

SPINDLE INHIBITOR: Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

STAIN: 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. Each slide was permanently labelled with the appropriate identification data.
When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS: Performed in duplicate.

NUMBER OF CELLS EVALUATED: 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate); where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index. A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.
-Qualitative slide assessment: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

PRELIMINARY TOXICITY TEST
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made.
Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Rationale for test conditions:

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p=0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitate was the dose limiting factor rather than the toxicity of the test item.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test material was considered to have no significant effect on change in pH when the test material was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation: The qualitative assessment of the slides determined that precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test item (80 µg/mL) in all three exposure groups.
Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 40 µg/mL in all three exposure groups.
Results confirmed qualitative obervations that no dose-related inhibition of mitotic index was observed becasue precipitate was the dose-limiting factor rather than the toxicity of the test item. The maximum dose level selected for metaphase analysis was therefore the lowest precipitating dose level (40 µg/mL).

RANGE-FINDING/SCREENING STUDIES
The dose range for the Preliminary Toxicity Test was 9.77 to 2500 µg/mL. The maximum dose was the maximum practical dose level (2500 µg/mL).
A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 39.06 µg/mL, in all three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 78.13 µg/mL in the 4(20)-hour exposures in the absence of metabolic activation (S9). The maximum dose with metaphases present in the the 4(20)-hour exposures in the presence of S9 and the 24-hour continuous exposure was 156.25 µg/mL. The test material induced marked evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for all three exposure groups.

CHROMOSOME ABERRATION TEST
The qualitative assessment of the slides determined that precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test material (80 µg/mL) in all three exposure groups.

VALIDITY
The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p=0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix. The positive control chemical concentrations assessed for metaphase analysis were those that were closest to the 55 ± 5 % mitotic index value given as optimum toxicity in the test guideline.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analysed.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test material only induced one polyploid cell in all three exposure groups and was, therefore, within the in-house historical range for polyploidy.

HISTORICAL CONTROL DATA
- Positive historical control data: All the positive control chemicals induced a demonstrable positive response (p=0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix. The positive control chemical concentrations assessed for metaphase analysis were those that were closest to the 55 ± 5 % mitotic index value given as optimum toxicity in the test guideline.
- Negative (solvent/vehicle) historical control data: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

Executive summary:

The potential of the test material to induce structural chromosomal aberrations was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 473 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

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 were 0, 2.5, 5, 10, 20, 40 and 80 µg/mL for 4 hour exposure with and without S9 and 24-hour without S9.

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material was toxic at the recommended dose level but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.