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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 2017 - 10 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Name of substance : Greenbiol ((2R,3R)-butanediol rich)
Cas No. : 24347-58-8
Batch No.: GSC160928-110
Serial No.: GSC170403B
Purity : 91.11 %
Date of manufacture: 04.03.2017
Physical description : Transparent liquid
Storage condition : Room temperature, (1 – 30) °C

Method

Species / strain
Species / strain / cell type:
other: Chinese hamster lung cell line / CHL/IU
Details on mammalian cell type (if applicable):
Cell line : CHL/IU (derived from the lungs of female Chinese hamster)
Supplier : American Type Culture Collection (ATCC CRL-1935™)

The properties of the frozen cells were confirmed to have following properties from May 08, 2017 until May 15, 2017:
Modal chromosome number (2n): 25
Doubling time: 15.5 hours
Mycoplasma: negative

Culture flask: 25 cm2 Cell culture flask, Canted neck (Corning)
Temperature: 37 °C
CO2 concentration: 5 %
Humidity: Under moist atmosphere
Incubator: CO2 incubator (Sanyo, MCO-19AIC)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from male rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Range finding study: 62.5, 125, 250, 500, 1000, 2000 µg/mL (max. concentration as required by the guideline)
Main test I (short-term S9+, short-term S9-): 125, 250, 500, 1000, 2000 µg/mL
Main test II (long-term S9-, short-term S9+): long-term: 31.3, 62.5, 125, 250 and 500 μg/mL; shot-term: 500, 1000 and 2000 μg/mL
Vehicle / solvent:
- Vehicle: sterilised distilled water (SDW) (DAIHAN PHARM CO. LTD.)
- Justification for choice of solvent/vehicle: In the preliminary test for the selection of the vehicle, the test substance at 200 mg/mL was soluble in SDW. Heat, discoloration or foaming were not observed in the preparation using SDW. SDW was selected as the vehicle for the test substance and used as the negative control substance in this study.

Vehicle for positive controle substances:
Mitomycin C (MMC): SDW
Cyclophosphamide monohydrate (CPA): SDW
Controls
Untreated negative controls:
yes
Remarks:
sterilised distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other:
Details on test system and experimental conditions:
Chromosome preparation
(1) Two hours before the treatment terminated, colcemid was applied to each culture flask at a final concentration of 0.2 μg/mL to accumulate the metaphase cells.
(2) After treatment terminated, cells were washed by PBS (-).
(3) 0.25 w/v % Trypsin-EDTA was treated (37 °C, 5 minutes), MEM medium was added and the cells were detached by pipetting.
(4) The cell suspension was collected into the centrifugal tubes, and then the cells were collected by centrifugation (1000 rpm, for 5 minutes; less than or equal to).
(5) After removing the supernatant, 0.075 mol/L potassium chloride 4 mL was added to each centrifugal tube for the hypotonic treatment of cells (37 °C, 15 minutes).
(6) 0.5 mL of cooled fixing fluid (methanol, glacial acetic acid [3:1, v/v]) was added, mixed, centrifuged, and then the supernatant was removed.
(7) The cells and 4 mL of fixing fluid were mixed. The mixture was centrifuged, and the supernatant was removed.
(8) The procedure in step (7) conducted again.
(9) The cells were floated in the proper amount of cooled fixing fluid.
(10) On a slide glass, placed in a slide tray, each of one drop fell in 2 ~3 sites and dried. Two slides were prepared from each plate.
(11) After staining for 5 minutes with 5 % (v/v) Giemsa’s solution, specimens were washed and dried.

Condition of treatment
The result (short-term treatment) was negative, so the chromosomal aberration test (continuous treatment test & second short-term treatment) was continuously conducted.

Measurement of cell growth index
(1) After a portion of cell suspension was obtained , the cells were counted using the cell count analyzer. The cell growth index of positive control group was not measured.
(2) Measured value of negative control (average) was set as 100 %, and the rate of cell proliferation was calculated.

Observation
Code of specimen:
(1) The slides of test and control group are randomly method, and slide number recording paper is written except by observer.
(2) At the time of specimen preparation, the number of each specimen is written in the slide.
(3) The sample observer conducts observation with blind test following slide number recording paper.
(4) After the end of observation, the data is aggregated based on the slide number recording paper.

Selection of methaphase cells on specimen:
(1) Chromosomes are well widened.
(2) Structural aberration : Chromosome' number 25 ± 2
(3) Numerical aberration : Chromosome' number 25 ± 2 or more than 35.

The number of the observed cells:
150 cells/plate (300 cells/concentration)

Structural aberration:
(1) Chromatid-type breaks
(2) Chromatid-type exchanges
(3) Chromosome-type breaks
(4) Chromosome-type exchanges (dicentric, circular chromosome, etc.)
(5) Fragmentation

Gap:
Gap seen in the width of the non-strained in the chromatid had to be narrower than the width of the strained. Record it by distinguishing from other abnormality; gaps were not included in the structural aberrations.

Numerical aberration:
(1) Polyploid cells with the number of 35 or more of centromeres
(2) Endoreduplicated cells

Evaluation of the results
Chromosome aberration cell:
Structural aberration cell: cells with one or more structural aberration of chromosome
Numerical aberration cell: cells with numerical aberration in chromosome number
Rationale for test conditions:
Top concentration is the highest concentration to be tested according to guideline, if neither cytotoxicity nor solubility is the limiting factor. Thereof at least three lower doses were evaluated.
Evaluation criteria:
According to the test results, the frequency of the aberration cells were less than 5%. Therefore statistical analysis was not performed and it was judged to the following criteria.
• Negative: For any test substance treatment group, the frequency of the structural aberration cells and the numerical aberration cells were less than 5 %.
• Inconclusive: In some test substance treatment group, the frequency of the,structural aberration cells and the numerical aberration cells were more than 5 % and less than 10 %.
• Positive: In some test substance treatment group, the frequency of the structural aberration cells and the numerical aberration cells were more than 10 %, and there was a tendency to concentration-dependent increase.
Statistics:
Because frequency of appearance of aberration cells were less than 5%, statistical analysis was not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: Chinese hamster lung cell line / CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: Chinese hamster lung cell line / CHL/IU
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Chromosomal aberration test (short-term S9+ and short-term S9-)
At the start of the treatment, precipitation or suspension by test substance were not observed. According to the result of the observation, the frequency of the structural anomaly of chromosome in 0, 125, 250, 500, 1000 and 2000 μg/mL of –S9 mix was 0.7 %, 0.0 %, 0.3 %, 0.0 %, 0.7 % and 0.3 %, respectively; the frequency of the numerical abnormality was 0.0 %, 0.0 %, 0.0 %, 0.0 %, 0.0 % and 0.3 %, respectively. Also, the frequency of the structural anomaly of chromosome in 0, 125, 250, 500, 1000 and 2000 μg/mL of +S9 mix was 0.0 %, 0.0 %, 0.3 %, 0.3 %, 0.3 % and 0.3 % and the frequency of the numerical abnormality was 0.0 %, 0.0 %, 0.0 %, 0.0 %, 0.0 % and 0.0 %, respectively. For the negative control group, the frequency of the structural anomaly of chromosome and the numerical abnormality were less than 5 %. For the positive control group, the frequency of the structural anomaly of chromosome was more than 10 %.

Chromosomal aberration test (long-term S9- and short-term S9+)
At the start of the treatment, precipitation or suspension by test substance were not observed. According to the result of the observation, the frequency of the structural anomaly of chromosome in 0, 31.3, 62.5, 125, 250 and 500 μg/mL of 24 hour exposure was 0.3 %, 0.3 %, 0.3 %, 0.0 %, 0.0 % and 0.3 %, respectively; the frequency of the numerical abnormality was 0.0 %, 0.0 %, 0.0 %, 0.0 %, 0.0 % and 0.0 % respectively. Also, the frequency of the structural anomaly of chromosome in 0, 500, 1000 and 2000 μg/mL of +S9 mix was 0.0 %, 0.3 %, 0.3 % and 0.0 % and the frequency of the numerical abnormality was 0.0 %, 0.0 %, 0.0 % and 0.0 %, respectively. For the negative control group, the frequency of the structural anomaly of chromosome and the numerical abnormality were less than 5 %. For the positive control group, the frequency of the structural anomaly of chromosome was more than 10 %.
Remarks on result:
other: short-term test: 6 hours exposure

Applicant's summary and conclusion

Conclusions:
The test item was considered to have not the ability to induce the chromosomal aberrations in CHL/IU cells under the present experimental test conditions.
Executive summary:

The test substance (2R,3R)-butane-2,3 -diol was evaluated for its potential to induce chromosome aberration by performing the in vitro mammalian chromosomal aberration test with cultured Chinese hamster lung cell line (CHL) in the absence (S9 -) and presence (S9 +) of metabolic activation system. The study was performed according to OECD 473 (adopted 2016) and in compliance with GLP. Concentration range-finding test was performed on cell cultures using a short-term treatment assay in the absence of S9 mix (referred to as –S9 mix) and in the presence of S9 mix (referred to as +S9 mix) and continuous treatment test (referred to as 24 hour exposure, S9 -). The concentration range used was 62.5, 125, 250, 500, 1000 and 2000 μg/mL. After 24 hours exposure, relative increase in cell counts (RICC) was observed by more than 60% at –S9 mix and +S9 mix. The RICC (55 ± 5) % was 443.72 μg/mL (24 hours exposure).

Based on the result of concentration range-finding test, concentrations of 125, 250, 500, 1000 and 2000 μg/mL were chosen for the main test. First, the chromosomal aberration test (short-term treatment method) was conducted with and without S9-mix. Results showed that the frequencies of aberration cells with structural aberration and numerical aberrations of chromosome were less than 5% for both S9- and S9+. Since all results were negative under both conditions of short-term treatments, chromosomal aberration test continuous treatment for 24 hour exposure without S9-mix and a second short-term treatment (S9+) followed. Continuous treatment (24 hour exposure) was conducted at 31.3, 62.5, 125, 250 and 500 μg/mL, and the second short-term treatment (+S9 mix) was conducted at 500, 1000 and 2000 μg/mL. Observation of specimens were conducted at all treatment groups in the 24 hour exposure and the second short-term test. Results showed that frequencies of aberration cells with structural aberration and numerical aberrations of chromosome were less than 5% in the long-term (S9-) and short-term (S9+) test. Therefore, the test substance was considered to be non-clastogenic (Negative) to CHL/IU cells under the present experimental condition.

The study was considered reliable and adequate for risk assessment.