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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro

- Bacteria reverse mutation test: negative

- Mammalian chromosome aberration test: negative

- Mammalian cell gene mutation test: negative

Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 2017 - 04 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-, trp-, rfa, uvrB, uvrA
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: The test strains were checked for their genetic properties such as amino acid requirement, UV sensitivity, membrane mutation and drug resistance were verified in advance.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9 mix
Test concentrations with justification for top dose:
Range finding test: 50, 150, 500, 1500, 5000 µg/plate (top dose according to guideline)
Main test: 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle for test material: Sterile 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 was not observed in the preparation using SDW; therefore, SDW was selected as the vehicle for the test substance and used as the negative control substance in this study.

- Solvents for positive control substances:
Sodium azide: sterile distilled water
2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide: DMSO
2-aminoanthracene: DMSO
9-aminoacridine: DMSO
Untreated negative controls:
yes
Remarks:
sterile distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-aminoanthracene (2AA), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)
Details on test system and experimental conditions:
TEST METHOD
Selection of the test method
This test was carried out using the pre-incubation method under the presence and absence of S9 mix.

Pre-incubation Method
(1) For each treatment, 0.1 mL of the test substance solution, negative (vehicle) control or positive control solution was added into a sterilized test tube.
(2) For assays in the absence of S9 mix, 0.5 mL of 0.1 mol/L sodium phosphate buffer (pH 7.4) was mixed and 0.1 mL of a bacterial suspension was added to this mixture.
(3) For assays in the presence of S9 mix, 0.5 mL of S9 mix was mixed and 0.1 mL of a bacterial suspension was added to this mixture.
(4) The mixture was incubated with gentle shaking (Shaking frequency: 120 times/minute) for 20 minutes at 37 °C (pre-incubation).
(5) After pre-incubation, 2 mL of the molten top agar was added to this mixture and then poured onto a minimal glucose agar plate.
(6) After the overlaid agar had solidified, the plates were incubated for (48 ± 2) hours at 37 °C.

Observation
Precipitation : After incubation for (48 ±2) hours, presence of precipitation on the agar plates was examined with the unaided eye.
Microbial toxicity : After incubation for (48 ± 2) hours, microbial toxicity was examined with a stereoscopic microscope.

Colony count
The colonies in each plate were counted with the unaided eye.

Number of plate
Concentration range-finding test: 3 plate/concentration
Main tests: 3 plate/concentration

Sterility test
The sterility test was also performed, using one plate for each of the highest concentration of test substance solution and the S9 mix used in the test.
Rationale for test conditions:
In the concentration range-finding tests, microbial toxicity was not observed at any concentration in the presence and absence of S9 mix. Precipitation was not observed on the agar plates at the any concentration in the presence and absence of S9 mix.

Confirmation of the positive control value
The number of revertant colonies in the positive control solutions was confirmed to be within the acceptable ranges by the pre-incubation method.
Evaluation criteria:
A substance is considered to have mutagenic potential regardless of with or without metabolic activation system, if a reproducible increase of revertant colonies per plate can be observed exceeding an increase factor of 2 in at least one strain in one concentration. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
The mean and standard deviation of the measured number of revertant colonies was calculated for the negative (vehicle) control, the positive control and the test substance treatments. The mean was expressed by rounding to the first decimal place.
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
TA 1537 / TA 98 /TA 100 / E. coli WP2
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
Additional information on results:
Concentration range-finding test
Microbial toxicity was not observed at the any concentration in the presence and absence of S9 mix. Also, precipitation was not observed on the agar plates at the any concentration in the presence and absence of S9 mix. The number of revertant colonies in the test substance-treated groups was less than twice that in the corresponding negative (vehicle) control in any test strain regardless of the presence of absence of S9 mix.

Main test I, II
Microbial toxicity was not observed at the any concentration in the presence and absence of S9 mix. Also, precipitation was not observed on the agar plates at the any concentration in the presence and absence of S9 mix. The number of revertant colonies in the test substance-treated groups was less than twice that in the corresponding negative (vehicle) control in any test strain regardless of the presence of absence of S9 mix.

Sterility test
Microbial contamination was not observed in the highest concentration of test substance solution and S9 mix in the sterility test group in either the concentration range-finding tests or main tests.
Conclusions:
The test item did not exert mutagenic activity in the reverse bacterial mutation assay with and without metabolic activation.
Executive summary:

Mutagenicity of the test substance (2R,3R)-butane-2,3 -diol was assessed in a bacterial reverse mutation assay using histidine requirement strains of Salmonella typhimurium TA100, TA1535, TA98 and TA1537 and tryptophan requirement of strain Escherichia coli WP2uvr A. The study was performed according to OECD 471 (adopted 1997) and in compliance with GLP. The test was conducted by the pre-incubation method in the presence and absence of S9 mix.

The concentration range-finding test was conducted with test concentrations of 50, 150, 500, 1500 and 5000 μg/plate. Microbial toxicity was not observed at any concentration in the presence and absence of S9 mix. Also, precipitation was not observed on the agar plates at any concentration in the presence and absence of S9 mix. Based on the results of the preliminary test, the main test I and II were conducted at the following concentrations without and with S9 mix: 313, 625, 1250, 2500, 5000 μg/plate (for strains TA98, TA100, TA1535, TA1537, WP2uvrA).

Microbial toxicity was not observed at any concentration in the presence and absence of S9 mix in the main tests. Also, precipitation was not observed on the agar plates at any concentration in the presence and absence of S9 mix. The number of revertant colonies in the test substance-treated groups was less than twice that in the corresponding negative control (vehicle: sterilised distilled water) in any test strain regardless of the presence or absence of S9 mix. Reproducibility of the test result was confirmed in main tests I and II.

The number of revertant colonies in the negative (vehicle) control and positive control groups were within the acceptable ranges stipulated at the testing facility. The positive controls used in the assays with and without S9 mix showed clear positive responses by the respective test strains, as evidenced by the number of revertant colonies being greater than 2-fold of the respective negative (vehicle) control value. Base on this results, it was concluded that the test item was not mutagenic (negative) under the conditions employed in the present study.

The study was considered reliable and adequate for hazard assessment.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 December 2018 - 28 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: GSC180130-110
- Expiration date of the lot/batch: 28 January 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not applicable
Target gene:
hypoxanthine-guanine phosphoribosyl transferase gene (Hprt)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

For cell lines:
Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×106 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
56.3, 112.6, 225.3, 450.5, 901.0 μg/mL
The dose range of the main experiment was set according to data generated in the pre-experiment. The maximum test item concentration in the pre-experiment and in the main experiment (901.0 μg/mL) was equal to a molar concentration of about 10 mM. The individual concentrations were spaced by a factor of 2.0.
Vehicle / solvent:
deionized water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.7 to 1.2×10^7 Cells
- Test substance added in medium

TREATMENT:
- Preincubation period, if applicable: for 24 hours prior to treatment
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
Immediately after the end of treatment the cells were trypsinised and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.
The colonies used to determine the relative survival (RS) were fixed and stained approximately 6 to 8 days after treatment as described below.
Three or four days after the first sub-cultivation, at least 2.0×10^6 cells per experimental point were again, sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of approximately 7 days five 75 cm² cell culture flasks were seeded with about 4 - 5×10^5 cells each in medium containing 6-TG (11 μg/mL).
After 8 – 11 days the colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)
Evaluation criteria:
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mean mutant frequencies. The mean number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.35 for Solvent control, 7.30 for 2,3-butanediol ((2R,3R)-rich) (901.0 μg/mL)
- Data on osmolality: 308 mOsm for Solvent control, 313 mOsm for 2,3-butanediol ((2R,3R)-rich) (901.0 μg/mL)

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence (4 hours treatment) of metabolic activation. Test item concentrations between 7.0 μg/mL and 901.0 μg/mL (equal to a molar concentration of approximately 10 mM) were used.
In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below occurred up to the highest concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. No precipitation or phase separation occurred up to the highest concentration after 4 hours treatment with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The mean mutant frequency obtained in the solvent controls was 7.6 without S9 mix and 8.6 with S9 mix. The values were well within the 95% confidence interval of our laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a significant increase in induced mutant colonies.

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value
o with metabolic activation: 0.323
o without metabolic activation: 0.145

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: No cytotoxic effect indicated by a relative adjusted cloning efficiency I below 50% was noted up to the highest concentration in the presence and absence of metabolic activation.

- Genotoxicity results: The range of the mean mutant frequencies of the groups treated with the test item was from 5.5 up to 14.3 mutants per 10^6 cells.

 

conc.

μg/mL

S9 mix

relative cloning efficiency I

relative cell density

relative adjusted cloning efficiency I*

mutant colonies/10^6 cells**

95 confidence interval

Main Experiment / 4 h treatment

mean values of culture I and II

Solvent control with water

 

-

100.0

100.0

100.0

7.6

2.8-30.9

Positive control (EMS)

300

-

94.6

80.4

76.1

327.4

2.8-30.9

Test item

56.3

-

95.4

81.8

77.8

5.5

2.8-30.9

Test item

112.6

-

95.9

80.0

76.5

11.0

2.8-30.9

Test item

225.3

-

87.3

104.7

93.1

11.1

2.8-30.9

Test item

450.5

-

84.7

84.4

71.5

7.2

2.8-30.9

Test item

901.0

-

73.1

90.0

66.2

14.3

2.8-30.9

Solvent control with water

 

+

100.0

100.0

100.0

8.6

3.1-30.7

Positive control (DMBA)

2.3

+

81.5

111.4

91.3

152.8

3.1-30.7

Test item

56.3

+

81.8

113.9

93.4

7.1

3.1-30.7

Test item

112.6

+

94.4

101.0

95.1

10.2

3.1-30.7

Test item

225.3

+

77.5

98.8

76.6

6.8

3.1-30.7

Test item

450.5

+

83.1

89.7

73.7

5.8

3.1-30.7

Test item

901.0

+

91.9

103.0

94.0

6.9

3.1-30.7

*relative adjusted cloning efficiency I=relative cloning efficiency I x relative cell density at first subcultivation / 100

**mutant colonies / 10^6 cells=mean number of mutant colonies per flask found after plating in TG medium × 10^6 divided by the number of cells survived

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, 2,3-butanediol ((2R,3R)-rich) is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The test substance (2R,3R)-butane-2,3 -diol was investigated for its the potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study was conducted according to OECD 476 (adopted 2016) and in compliance with GLP. The experiment was performed with a treatment time of 4 hours with and without metabolic activation. Based on the results of a preliminary test, the main test were conducted at the following concentrations without and with S9 mix: 56.3, 112.6, 225.3, 450.5, 901.0 μg/mL.

No statistical significant increase of the mutation frequency at any test concentration or dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a significant increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. The study was considered reliable and adequate for risk assessment.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of information on species specific effects or metabolism the results are regarded as relevant for humans.

Additional information

Genotoxicity in vitro

Mutagenicity of the test substance (2R,3R)-butane-2,3 -diol was assessed in a bacterial reverse mutation assay using histidine requirement strains of Salmonella typhimurium TA100, TA1535, TA98 and TA1537, and tryptophan requirement of strain Escherichia coli WP2uvr A. The study was performed according to OECD 471 (adopted 1997) and in compliance with GLP. The test was conducted by the pre-incubation method in the presence and absence of S9 mix. Based on the results of a preliminary test, the main test I and II were conducted at the following concentrations without and with S9 mix: 313, 625, 1250, 2500, 5000 μg/plate (for strains TA98, TA100, TA1535, TA1537, WP2uvrA).

Microbial toxicity was not observed at the any concentration in the presence and absence of S9 mix in the main tests. Also, precipitation was not observed on the agar plates at any concentration in the presence and absence of S9 mix. The number of revertant colonies in the test substance-treated groups was less than twice that in the corresponding negative control (vehicle: sterilised distilled water) in any test strain regardless of the presence or absence of S9 mix. Reproducibility of the test result was confirmed in main tests I and II.

The number of revertant colonies in the negative (vehicle) control and positive control groups were within the acceptable ranges stipulated at the testing facility. The positive controls used in the assays with and without S9 mix showed clear positive responses by the respective test strains, as evidenced by the number of revertant colonies being greater than 2-fold of the respective negative (vehicle) control value. Based on the results, it was concluded that the test item was not mutagenic (negative) under the conditions employed in the present study. The study was considered reliable and adequate for hazard assessment.

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. 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.

The test substance (2R,3R)-butane-2,3 -diol was investigated for its the potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study was conducted according to OECD 476 (adopted 2016) and in compliance with GLP. The experiment was performed with a treatment time of 4 hours with and without metabolic activation. Based on the results of a preliminary test, the main test were conducted at the following concentrations without and with S9 mix: 56.3, 112.6, 225.3, 450.5, 901.0 μg/mL.

No statistical significant increase of the mutation frequency at any test concentration or dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a significant increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. The study was considered reliable and adequate for risk assessment.

Justification for classification or non-classification

Genetic toxicity

The submission substance does not need to be classified for germ cell mutagenicity according to the CLP regulation, because all results were negative obtained in experimental studies, i.e. in vitro studies according to OECD 471 (adopted 1997), OECD 473 (adopted 2016), and OECD 476 (adopted 2016).


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