Reaction mass of oxybispropanediol and tetraglycerol and triglycerol, esterification products with lactic acid and lauric acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 23rd 2021 to April 1st 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of oxybispropanediol and tetraglycerol and triglycerol, esterification products with lactic acid and lauric acid

Molecular formula:

not applicable - UVCB substance

IUPAC Name:

Reaction mass of oxybispropanediol and tetraglycerol and triglycerol, esterification products with lactic acid and lauric acid

Specific details on test material used for the study:

Test item name: Finestar 2009
CAS Number: 2225876-48-0
Water solubility: soluble in water
Molecular formula: C12 H22 O8
Molecular weight: 294.30
Batch/Lot number: 3253082031
analyzed purity: 100% active
Saponification value: 45.87
Date of manufacture: August 27, 2020
Date of expiry: August 26, 2021
Appearance: Pale yellow viscous liquid
Storage temperature: Room temperature (15 to 30°C)
Storage condition: Keep away from light, moisture, and oxidizing - reducing chemicals
Storage container: Keep in original container

Method

Target gene:

hprt locus of CHO-K1 cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cell line (free from mycoplasma contamination), a subclone of Chinese hamster ovary cell line obtained from the Japanese Collection of Research Bioresources (JCRB) Lot No # 072620055, maintained in the Mutagenicity Section at Jai Research Foundation was used for this study. The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study. CHO-K1 cell line passage number 34 was used in cytotoxicity test and passage number 26 was used in the main study.

Metabolic activation:

with and without

Metabolic activation system:

In order to test indirectly acting mutagens, a metabolically active extract of rat liver (treated
with Aroclor 1254) called S9 fraction was used.

The S9 fraction procured from Meshram Genotox service was used in this assay. The S9 fraction is buffered and supplemented with the
essential co-factors β-NADP, KCL and Glucose-6- phosphate to form the “S9 mix”. The S9 fraction
was used at a concentration of 2% v/v in the final culture medium for main study.

Preparation of S9 Mix
For treatment in presence of metabolic activation, S9 fraction was used at a final concentration of 2% v/v.

Constituent of Co-factor Mix and S9 Mix
a. D-Glucose-6-phosphate : 180 mg/mL
b. β-Nicotinamide adenine dinucleotide phosphate (β-NADP) : 25 mg/mL
c. Potassium chloride : 11.18 mg/mL

Ratio of the individual constituents in Co-factor Mix a: b: c: S9 mix: 1:1:1:2

The medium for treatment in presence of metabolic activation was prepared by addition of required volume of co-factor mix to the required volume of α-MEM with nucleosides (without serum) media. α-MEM with nucleosides without serum media was used for testing in the absence of metabolic
activation system.

Test concentrations with justification for top dose:

In mutagenicity experiment, the treatment was performed both in the absence and presence of
metabolic activation (2% v/v S9 mix). Two flasks were maintained for each test concentration,
negative and positive controls for presence of metabolic activation system, while four flask were
maintain for each test concentration, negative and positive controls for absence of metabolic
activation system.
Cultures were exposed at the concentrations of 31.25, 62.5, 125, 250, 500, 1000 μg/mL in the
absence and 62.5, 125, 250, 500, 1000, and 2000 μg/mL in the presence of metabolic activation (2% v/v S9 mix) for a period of 4 hours.

Vehicle / solvent:

The test item was soluble in distilled water at 200000 μg/ml. Hence, distilled
water was selected as vehicle for study. Test item was further diluted with distilled water to check
the precipitation, changes in the pH and osmolality at the different concentrations.

Controls

Untreated negative controls:

yes

Remarks:

Distilled Water

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

Test System
CHO-K1 cell line (free from mycoplasma contamination), a subclone of Chinese hamster ovary cell line obtained from the Japanese Collection of Research Bioresources (JCRB) Lot No # 072620055, maintained in the Mutagenicity Section at Jai Research Foundation was used for this study. The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study. CHO-K1 cell line passage number 34 was used in cytotoxicity test and passage number 26 was used in the main study.

Culture Medium
α-MEM (Minimum Essential Medium, Eagle α-Modification with nucleosides) with nucleosides
(Gupta R.S., 1984) with 10% heat inactivated, sterile, fetal bovine serum was used as the culture
medium to grow the CHO-K1 cell line. Culture medium was supplemented with antibiotic and
antimycotic solution (Penicillin: 50 IU/mL; Streptomycin: 50 μg/mL and Amphotericin B: 0.25-0.5
μg/mL). At the time of selection Minimum Essential Medium Eagle -modification without
nucleosides (-MEM w/o NS) with 10% dialyzed fetal bovine serum was used.The medium to eliminate the existing mutants in the culture for treatment was prepared by addition
of 2 mL of reconstituted HAT supplement to 98 mL of α-MEM w/o NS with 5% fetal bovine serum [50X vial of HAT media supplement was reconstituted using 10 mL of sterile α-MEM w/o NS. The reconstituted supplement contains 5 x 10-5M Hypoxanthine, 2 x 10-5 M Aminopterine and 8 x 10-4M Thymidine].

Selective Agent
2-amino-6-mercaptopurine (6-thioguanine) was used as selective agent at a concentration of
5 μg/mL -MEM without nucleosides.
Metabolic Activation System
The in vitro test systems lack the necessary oxidative enzyme systems for metabolising foreign
compounds to electrophilic metabolites capable of reacting with DNA. Sometimes these foreign
compounds, when reacting with a mammalian enzyme system, yield mutagenic metabolic products. In order to test these indirectly acting mutagens, a metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction was used.

Culture Vessels
Disposable tissue cultures flasks of 25 cm2 (Nunc during cytotoxicity test) and 75 cm2 culture (Nunc and corning during mutagenicity test) area with canted neck were used to culture the cell line and the treatment was given in the same flask. 60 mm disposable culture dishes (Corning) were used to determine the cloning efficiency. Tissue culture dishes (Corning) of 100 mm were used to select mutant colonies.

The S9 fraction procured from Meshram Genotox service was used in this assay. The S9 fraction is buffered and supplemented with the essential co-factors -NADP, KCL and Glucose-6- phosphate to form the “S9 mix”. The S9 fraction was used at a concentration of 2% v/v in the final culture medium for main study.

Preparation of S9 Mix
For treatment in presence of metabolic activation, S9 fraction was used at a final concentration of 2% v/v (Main Study).Ratio of the individual constituents in Co-factor Mix a: b: c: S9 mix: 1:1:1:2 The medium for treatment in presence of metabolic activation was prepared by addition of required volume of co-factor mix to the required volume of α-MEM with nucleosides (without serum) media. α-MEM with nucleosides without serum media was used for testing in the absence of metabolic activation system.

Cytotoxicity Test
Based on the results of solubility, precipitation and pH tests, 2000 μg/mL of culture medium was
selected as the highest dose concentration to be tested for cytotoxicity test. The cytotoxicity test was performed both in the absence and presence of metabolic activation (2% v/v S9 mix) at the concentrations of 62.5, 125, 250, 500, 1000 and 2000 μg/mL of culture medium. A concurrent negative control (DW) was also maintained. 400 mg of Finester 2009 was dissolved and made up to 2 mL with distilled water to attain the stock solution concentration of 200000 μg/mL (stock A). A volume of 1 mL of stock A was added to 1 mL distilled water to obtain 100000 μg/mL (stock B). Further stock solutions of 50000 μg/mL (stock C), 25000 μg/mL (stock D), 12500 μg/mL (stock E) and 6250 μg/mL (stock F) were prepared by serial dilution.

Volumes of 120 μL of relevant stock solutions were added into respective tubes containing
11.88 mL of medium to obtain the required test concentrations for the treatment in the absence and presence of metabolic activation system. To each 25 cm2 culture flask 5 mL of the treatment medium was added.

Based on the results of solubility, precipitation and cytotoxicity tests, 1000 and 2000 μg/mL of
culture medium has been proposed as a highest concentration in the absence and in the presence of metabolic activation, respectively.

Mutagenicity Experiment
In mutagenicity experiment, the treatment was performed both in the absence and presence of
metabolic activation (2% v/v S9 mix). Two flasks were maintained for each test concentration,
negative and positive controls for presence of metabolic activation system, while four flask were
maintain for each test concentration, negative and positive controls for absence of metabolic
activation system.

Cultures were exposed at the concentrations of 31.25, 62.5, 125, 250, 500, 1000 μg/mL in the
absence and 62.5, 125, 250, 500, 1000, and 2000 μg/mL in the presence of metabolic activation (2%v/v S9 mix) for a period of 4 hours.

For the treatment, the first stock solution (stock A) of the test item was prepared by dissolving 400mg of Finester 2009 in distilled water and volume made up to 2 mL 200000 μg/mL. Further stock solutions of 100000 μg/mL (stock B), 50000 μg/mL (stock C), 25000 μg/mL (stock D) and 12500μg/mL (stock E), 6250 μg/mL (stock F), 3125 μg/mL (stock G) were prepared by serial dilution.

Volumes of 250 μL of relevant stock solutions were added into 24.75 mL of culture medium. 10 mL of this treatment medium was added to each respective flask of replicate (2 Flasks) (75 cm2 culture flask) in presence of metabolic activation. Volumes of 500 μL of relevant stock solutions were added into 49.5 mL of culture medium. 10 mL of this treatment medium was added to each respective flask of replicate (4 Flasks) (75 cm2 culture flask) in absence of metabolic activation.

Serum free culture medium was used in the absence of metabolic activation while medium with S9 mix (2% v/v S9 mix) was used in the presence of metabolic activation. All the test concentrations were prepared just prior the treatment and used within 2 hours of preparation.

Rationale for test conditions:

Justification for Selection of Test System
The test system used for the in vitro mammalian cell gene mutation test was the CHO-K1 cell line.
The CHO-K1 cell line is easy to maintain, culture, and a recommended test system by the various
regulatory authorities, to detect the mutation at hgprt locus. It is suited for determining forward
mutations at the hgprt locus because it is a well characterised and validated system (Hsie et al.,
1981). These cells have a high cloning efficiency, rapid doubling time (12-14 hour) and a low
spontaneous mutation frequency (1-20 mutants per 106 cells). These cells also show a dose response with a variety of physical and chemical mutagens and a genetic basis for the presumptive mutational event has been well established.

Evaluation criteria:

Assay Acceptance and Evaluation Criteria

Assay Acceptance Criteria
A mutation assay was considered acceptable as it met the following criteria:
a. The concurrent negative control was considered acceptable for addition to the laboratory
historical negative control database.
b. Concurrent positive controls induce responses that were compatible with those generated in the historical positive control data base and produce a statistically significant increase compared
with the concurrent negative control.
c. Two experimental conditions (i.e., with and without metabolic activation) were tested.
d. Six concentrations (not including the solvent and positive controls) with adequate number of
cells and appropriate cytotoxicity were evaluated.

Assay Evaluation Criteria
Providing that all acceptability criteria are fulfilled, a test item was considered clearly negative as it met, in all experimental conditions examined:
a. None of the test concentrations exhibited a statistically significant increase compared with the
concurrent negative control.
b. There was no concentration-related increase when evaluated with an appropriate trend test.
c. All results were inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit).
The test item was considered unable to induce gene mutations in cultured mammalian cells in this test system. There was no requirement for verification of a clearly negative response.

Statistics:

Statistical Analysis
Weighted regression analysis was performed to evaluate the dose response relationship on treatment groups against the negative control group (excluding positive controls).

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility, Precipitation, pH and Osmolality Tests
Solubility of the test item (Finester 2009) was performed at 200000 μg/mL. The test item was
soluble in distilled water (DW) at 200000 μg/ml. A dilution series of 6250, 12500, 25000, 50000,
100000 and 200000 μg/mL was performed in distilled water for pH and osmolality test.
Any significant change in pH (± 1 unit) or osmolality (≥ 50 mOsm/kg H2O) was not observed at 0
and at 4 h in at tested concentration 62.5, 125, 250, 500, 1000 and 2000 μg/mL of culture medium.
Precipitation was not observed up to the tested concentration 2000 μg/mL Hence, as per the
guideline, limit test concentration of 2000 μg/mL of the culture media was selected as the highest
concentration for the cytotoxicity test.

Cytotoxicity Test
Cytotoxicity test was conducted at the tested concentration of 62.5, 125, 250, 500, 1000, 2000 μg of Finester 2009/mL. Cytotoxicity due to the test item was assessed by calculating the percent relative cloning efficiency, following the treatment. Precipitation was not observed up to the tested concentration 2000 μg/mL either in the absence or presence of the metabolic activation (2% v/v S9 mix).
The pH and osmolality at the beginning of the treatment at 2000 μg/mL were 7.28 and 310 mOsm/kg H2O, respectively (compared to 7.30 and 306 mOsm/kg H2O in the negative control) in the absence of the metabolic activation, while pH and osmolality at 2000 μg/mL were 7.40 and 306 mOsm/kg H2O, respectively (compared to 7.28 and 309 mOsm/kg H2O in the negative control), in the presence of the metabolic activation. Hence, any significant change in the pH or osmolality was not observed up to the concentration 2000 μg/mL either in the absence or
presence of the metabolic activation. pH and osmolality of other treatment concentration were concurrent to vehicle control group. Normal confluency (70-80%) was observed up to 500 μg/ mL in absence of metabolic activation system and up to 2000 μg/ mL in presence of metabolic activation system, while average confluency was observed at test concentration 1000 μg/ mL in absence of metabolic activation system. Dead cells was observed at test concentration 2000 μg/ mL in absence of metabolic activation system.
The percent relative cloning efficiency observed was 84.63, 82.79, 78.27, 65.98, 20.79 and 0.70 in
the absence of the metabolic activation and 90.61, 82.88, 79.07, 77.97, 74.95, and 71.41 in the
presence of the metabolic activation at tested concentration 62.5, 125, 250, 500, 1000, and 2000 μg/ mL of media, respectively.

Since required cytotoxicity, i.e., relative cell survival between 10-20%, was observed at tested
concentration of 1000 μg/mL in absence of metabolic activation, while significant toxicity was not
observed in the presence of metabolic activation (2% v/v S9 mix) up to the limit test concentration of 2000 μg/mL.
Based on the results of solubility, precipitation and cytotoxicity tests: 1000 μg/mL of culture media has been selected in absence of metabolic activation system, while 2000 μg/mL has been selected in presence metabolic activation system as highest concentration.

Mutagenicity Test
No relevant influence of the test item on osmolality and pH value was observed in the absence and presence of the metabolic activation during the main study. Normal confluency (70-80%) was
observed up to 500 μg/ mL in absence of metabolic activation system and up to 2000 μg/ mL in
presence of metabolic activation system, while average confluency was observed at test
concentration 1000 μg/ mL in absence of metabolic activation system.

Applicant's summary and conclusion

Conclusions:

From results of this study, it is concluded that Finester 2009 does not have any potential to induce
gene mutations at the hprt locus of CHO-K1 cells, in the absence and presence of the metabolic
activation (2% v/v S9 mix) under these experimental conditions, and it is considered to be negative for mutagenicity.

Executive summary:

Study Guidelines
The present study was conducted according to:
OECD 2016: The Organisation for Economic Co-operation and Development (OECD), Guidelines for Testing of Chemicals 476, “In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes” adopted by the Council on July 29, 2016.

Results-

Solubility, Precipitation, pH and Osmolality Tests
Solubility of the test item (Finester 2009) was performed at 200000 μg/mL. The test item was soluble in distilled water (DW) at 200000 μg/ml. A dilution series of 6250, 12500, 25000, 50000, 100000 and 200000 μg/mL was performed in distilled water for pH and osmolality test.
Any significant change in pH (± 1 unit) or osmolality (≥ 50 mOsm/kg H2O) was not observed at 0 and at 4 h in at tested concentration 62.5, 125, 250, 500, 1000 and 2000 μg/mL of culture medium.
Precipitation was not observed up to the tested concentration 2000 μg/mL Hence, as per the guideline, limit test concentration of 2000 μg/mL of the culture media was selected as the highest concentration for the cytotoxicity test.

Cytotoxicity Test
Cytotoxicity test was conducted at the tested concentration of 62.5, 125, 250, 500, 1000, 2000 μg of Finester 2009/mL. Cytotoxicity due to the test item was assessed by calculating the percent relative cloning efficiency, following the treatment. Precipitation was not observed up to the tested concentration 2000 μg/mL either in the absence or presence of the metabolic activation (2% v/v S9 mix).
The pH and osmolality at the beginning of the treatment at 2000 μg/mL were 7.28 and 310 mOsm/kg H2O, respectively (compared to 7.30 and 306 mOsm/kg H2O in the negative control) in the absence of the metabolic activation, while pH and osmolality at 2000 μg/mL were 7.40 and 306 mOsm/kg H2O, respectively (compared to 7.28 and 309 mOsm/kg H2O in the negative control), in the presence of the metabolic activation. Hence, any significant change in the pH or osmolality was not observed up to the concentration 2000 μg/mL either in the absence or
presence of the metabolic activation. pH and osmolality of other treatment concentration were concurrent to vehicle control group. Normal confluency (70-80%) was observed up to 500 μg/ mL in absence of metabolic activation system and up to 2000 μg/ mL in presence of metabolic activation system, while average confluency was observed at test concentration 1000 μg/ mL in absence of metabolic activation system. Dead cells was observed at test concentration 2000 μg/ mL in absence of metabolic activation system.
The percent relative cloning efficiency observed was 84.63, 82.79, 78.27, 65.98, 20.79 and 0.70 in the absence of the metabolic activation and 90.61, 82.88, 79.07, 77.97, 74.95, and 71.41 in the presence of the metabolic activation at tested concentration 62.5, 125, 250, 500, 1000, and 2000 μg/ mL of media, respectively.

Since required cytotoxicity, i.e., relative cell survival between 10-20%, was observed at tested concentration of 1000 μg/mL in absence of metabolic activation, while significant toxicity was not observed in the presence of metabolic activation (2% v/v S9 mix) up to the limit test concentration of 2000 μg/mL.
Based on the results of solubility, precipitation and cytotoxicity tests: 1000 μg/mL of culture media has been selected in absence of metabolic activation system, while 2000 μg/mL has been selected in presence metabolic activation system as highest concentration.

Mutagenicity Test
No relevant influence of the test item on osmolality and pH value was observed in the absence and presence of the metabolic activation during the main study. Normal confluency (70-80%) was observed up to 500 μg/ mL in absence of metabolic activation system and up to 2000 μg/ mL in presence of metabolic activation system, while average confluency was observed at test concentration 1000 μg/ mL in absence of metabolic activation system.