Reaction products of Dihydro-3-(tetrapropenyl)furan-2,5 dione with propane-1,2,diol

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:

15 February 2016 to 23 March 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:

2-(tetrapropenyl)succinic acid, monoester with propane-1,2-diol

EC Number:

257-836-6

EC Name:

2-(tetrapropenyl)succinic acid, monoester with propane-1,2-diol

Cas Number:

52305-09-6

Molecular formula:

C17H30O5 - C21H38O5

IUPAC Name:

2-[2-(2-hydroxy-1-methylethoxy)-2-oxoethyl]tetradecanoic acid

Test material form:

solid

Details on test material:

- Apperance: Yellow solid block
- Storage: Room temperature in the dark

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
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. This is an exception with regard to GLP and has been reflected in the GLP compliance statement

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.
- Cell cycle length, doubling time or proliferation index: 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 under typical experimental exposure conditions. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- Sex, age and number of blood donors if applicable:
Preliminary Toxicity Test: Female, aged 21 years
Main Experiment: Male, aged 32 years
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood cultures
- 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).


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.

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 STUDY
0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1 250 and 2 500 μg/mL for 4 hour exposure with and without S9 and 24-hour without S9

MAIN STUDY
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 120, 160 and 240 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 10, 20, 40, 60, 80, 120 and 160 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 10, 20, 40, 60, 80, 120 and 160 μg/mL.

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 toxicity.

Vehicle / solvent:

- Vehicle/solvent used: Acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in culture medium at 50 mg/mL and dimethyl sulfoxide at 500 mg/mL but was soluble in acetone at 500 mg/mL in solubility checks performed in-house.

The test material was considered to be a UVCB and therefore the maximum recommended dose was 5 000 μg/mL. However, due to the necessity of using acetone as the solvent the maximum dose level that could be achieved was 2 500 μg/mL. Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed at 0.5 % in 50 μL aliquots. Consequently, the maximum achievable concentration was 2 500 μg/mL.

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

Remarks:

Absence of S9-mix: Mitomycin C (Minimal Essential Medium) 0.2 μg/mL for 4(20)-h exposure and 0.1 μg/mL for 24-hour continuous exposure. Presence of S9-mix: Cyclophosphamide (DMSO) at 2 mg/mL for 4(20)-h exposure.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION:
- Exposure duration: 4 or 24 hours
- Expression time:
4 h exposure: 20 hours
24 h exposure: None

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: 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: Duplicate lymphocyte cultures (A and B) were established for each dose level.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- 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.
- 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.
- Coding: The slides were coded using a computerized random number generator. Supplementary slides were coded manually.

NUMBER OF CELLS EVALUATED: A total of 2 000 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.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE
Where possible, 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 and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence endoreduplicated cells) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index


- OTHER: CULTURE CONDITIONS
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

- 4-Hour Exposure with Metabolic Activation (S9):
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.

- 4-Hour Exposure without Metabolic Activation (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 re-suspended 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.

- 24-Hour Exposure without Metabolic Activation (S9)
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 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.

- Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2 %), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix. The dose range of test item used was 9.77 to 2500 μg/mL.
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.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with 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

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: There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation: In the preliminary toxicity test, a precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in the 4(20)-hour exposure groups, at and above 312.5 μg/mL in the absence of S9 and at and above 625 μg/mL in the presence of S9. In the continuous exposure group a precipitate of the test item was observed at and above 312.5 μg/mL.
In the main test, no precipitate was observed at the end of the exposure period in any of the three exposure groups.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1 250 and 2 500 μg/mL. The maximum dose was the maximum achievable dose level due to formulation difficulties.
Haemolysis was observed following exposure to the test item at and above 39.06 μg/mL in the 4(20)-hour exposure group in the absence of S9 and at and above 78.13 μg/mL in the presence of S9. In the 24-hour continuous exposure group haemolysis was seen from 9.77 μg/mL to 625 μg/mL at the end of the exposure period. Haemolysis is an indication of a toxic response of the erythrocytes and not indicative of any genotoxic response of the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 156.25 μg/mL in the 4(20)-hour exposure in the absence of metabolic activation (S9) and in the 24-hour continuous exposure group. The maximum dose with metaphases present in the 4(20)-hour exposure group in the presence of S9 was 78.13 μg/mL.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 240 μg/mL for the 4(20)-hour exposure group in the absence of S9, and 160 μg/mL for both the 4(20)-hour exposure group in the presence of S9 and the 24-hour continuous exposure group.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Preliminary Toxicity Test: The test item induced marked evidence of toxicity in all three exposure groups.
- Main Test: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 160 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9. In the 24-hour exposure group the maximum test item dose level with metaphases suitable for scoring was 80 μg/mL.

ADDITIONAL INFORMATION ON GENOTOXICITY
The mitotic index data for the Main Experiment confirms the qualitative observations in that a dose-related inhibition of mitotic index was observed in all three exposure groups. In the 4(20)-hour exposure group in the absence of S9, marginally greater than acceptable toxicity was achieved at 160 μg/mL with 67 % mitotic index inhibition. In the presence of S9, the toxicity was seen to plateau at 120 μg/mL and 160 μg/mL with 44 % and 43 % mitotic index inhibition, respectively. Although the 160 μg/mL dose level did not demonstrate optimum toxicity, there was a reduction in the number of cells on the slides demonstrating overall toxicity to the cell population. In the ‘B’ replicate of the 160 μg/mL dose level only 93 metaphases were available for scoring as a result of toxicity and if taken in isolation this replicate did achieve optimum toxicity of 50 %. In the 24-hour continuous exposure group an inhibition of mitotic index of 43 % and 68 % was noted at 60 μg/mL and 80 μg/mL, respectively. Extreme toxicity was noted in the ‘B’ replicate of the 80 μg/mL dose level as only 103 metaphases were available for scoring; this replicate exceeded acceptable toxicity, although the dose level achieved the upper limit of acceptable toxicity. The deficiency in metaphases in the 4(20)-hour exposure group in the presence of S9 at 160 μg/mL and in the 24-hour exposure group at 80 μg/mL was due to toxicity and it is considered that sufficient metaphases were evaluated to demonstrate the effects of the test item on the frequency of cells with chromosome aberrations.
The maximum dose level selected for metaphase analysis was based on toxicity and was 160 μg/mL for the 4(20)-hour exposure groups and 80 μg/mL for the 24-hour exposure group.
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 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 item did not induce any statistically significant increases in the frequency of cells with aberrations either in the 4(20)-hour exposure group in the presence of metabolic activation or in the 24-hour continuous exposure group. In the 4(20)-hour exposure group in the absence of S9 there was a small but statistically significant increase in the frequency of cells with aberrations at 160 μg/mL. Since this dose level marginally exceeded acceptable toxicity the response was considered to be of no biological relevance and due to cytotoxicity rather than any clastogenic effect.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure conditions.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material 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 item, 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 toxicity. The dose levels selected for the Main Experiment were 10, 20, 40, 80, 120 and 160 μg/mL for 4 hour exposure without S9, 4 hour exposure with S9 and 24 hour exposure without S9. An additional dose of 240 μg/mL was tested for 4 hour exposure 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 demonstrated marked toxicity in all three exposure groups. The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour exposure group in the presence of S9 or in the 24-hour exposure group, using a dose range that included a dose level that achieved near optimum toxicity. In the 4-hour exposure group in the absence of S9 there was a small but statistically significant increase in the frequency of aberrations at 160 μg/mL, but this was considered to be of no biological relevance since it exceeded acceptable toxicity and it was therefore considered that the response was as a result of cytotoxicity.

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.