RD 14156

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:

The study was conducted between 14 April 2016 and 16 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 September 2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

31 March 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponros Lot/Batch number: 5399561P10
- Expiration date of the lot/batch: 25 January 2020
- Purity test date: 25 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: A&B

Details on mammalian cell type (if applicable):

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. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered 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.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 33 years
Main Experiment: male, aged 30 years

Cell Culture
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. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fractions

Test concentrations with justification for top dose:

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 100% and was not accounted for in the test item formulations.

The test item was insoluble in aqueous media at 50 mg/mL, and was insoluble in dimethyl sulphoxide and acetone at 125, 250, and 500 mg/mL. However, the test
item could be formulated into a best doseable suspension in acetone at 125 mg/mL in solubility checks performed in-house.
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. Consequently, the maximum practical concentration was 625 μg/mL.
Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Solubility

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

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. 1mL 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 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.

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.

Evaluation criteria:

Data Evaluation
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.

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

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. (Richardson et al. 1989).
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: A&B

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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 analyzed.
The test item 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 item did not induce any statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Any other information on results incl. tables

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 2.44 to 625 μg/mL. The maximum dose was initially set at the maximum recommended dose level of 5000 μg/mL. Due to solubility issues, the test item could only be formulated into a best doseable suspension at 125 mg/mL in solubility checks performed in-house. Additionally, 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. Consequently, the maximum practical concentration was 625 μg/mL.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 4.88 μg/mL in both the 4(20)-hour and 24-hours exposures in the absence of metabolic activation (S9) and at and above 19.53 μg/mL in the presence of S9. No hemolysis was observed following exposure to the test item in any of the exposure groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to maximum dose level of 625 μg/mL in the 4(20)-hour exposures in the presence and absence of S9. The maximum dose with metaphases present in the 24-hour continuous exposure was 312.5 μg/mL. The test item induced some 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 in all three exposure groups.

Chromosome Aberration Test – Main Experiment

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

Group                                                 Final concentration of RD 14156 (μg/mL)

4(20)-hour without S9               0*, 0.625*, 1.25*, 2.5*, 5*, 10, 20, 30, MMC 0.2*

4(20)-hour with S9 (2%)              0*, 2.5*, 5*, 10*, 20*, 30, 40, 50, CP 1*

24-hour without S9                      0*, 0.625, 1.25, 2.5*, 5*, 10*, 20*, 30, MMC 0.1*

*dose levels selected for metaphase analysis

The qualitative assessment of the slides determined that precipitate was evident at much higher concentrations and the toxicity was greater to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present in all three exposure groups at the maximum dose level of test item.

Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 10 μg/mL in the 4(20)-hour exposure group in the absence of S9 and at and above 20 μg/mL in the 4(20)-hour exposure in the presence of S9 and 24-hour continuous exposure group.

The mitotic index data confirm the qualitative observations in that no explicit dose-related inhibition of mitotic index was observed, however, there were exposure groups where a moderate plateau of toxicity was observed.

In the 4(20)-hour exposure group in the absence of S9, 69%, 73%, 52% and 88% mitotic inhibition was achieved at 1.25, 2.5, 5 and 10 μg/mL, respectively. Therefore, the maximum dose level selected for metaphase analysis was 5 μg/mL as this dose level exhibited optimum toxicity as specified in the OECD 473 test guideline (55±5%).

In the 4(20) hour exposure group in the presence of S9, a slight dose-related inhibition of mitotic index was observed at where 83%, 75% and 45% mitotic inhibition was observed at 5, 10 and 20 μg/mL, respectively. As the lowest precipitating dose level and approximately optimum toxicity was observed at 20 μg/mL, this dose level was chosen as the maximum concentration for metaphase analysis.

In the 24-hour continuous exposure group, 81%, 70% and 65% mitotic inhibition was observed at 5, 10 and 20 μg/mL, respectively. Therefore, the maximum dose level selected for metaphase analysis was 20 μg/mL. Although the inhibition of mitotic index approached optimum toxicity in the 24-hour continuous exposure group, the lowest precipitating dose level was selected as the maximum dose level for metaphase analysis (20 μg/mL).

In the 24-hour continuous exposure group, 81%, 70% and 65% mitotic inhibition was observed at 5, 10 and 20 μg/mL, respectively. Therefore, the maximum dose level selected for metaphase analysis was 20 μg/mL. Although the inhibition of mitotic index approached optimum toxicity in the 24-hour continuous exposure group, the lowest precipitating dose level was selected as the maximum dose level for metaphase analysis (20 μg/mL).

It should be noted that the some of the Mitotic Indices for the duplicate cultures of a single concentration varied but tended to be consistent with the quantitative slide assessment for the presence of metaphases and/or precipitate. It should also be noted that the variability in MI values between duplicates, which was also reflected by the qualitative assessment, was considered not to affect the integrity of the study. This was particularly true for the short term exposure groups. The unequal MI values in the duplicates depressed the overall MI value but not enough to alter the required number of metaphases available for analysis.

Applicant's summary and conclusion

Conclusions:

RD 14156 did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1990).

Methods

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 on toxicity for the 4-hour exposure group, in the absence of S9-mix and precipitate for the other two exposure groups.

Results

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items 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 item was toxic 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 or optimum toxicity.

Conclusion

The test item, RD 14156 was considered to be non-clastogenic to human lymphocytes in vitro.