4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

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:

12th October2015 - 14th December 2015

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation

Test material

Test material information

1

Reference substance name:

4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

EC Number:

299-434-3

EC Name:

4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

Cas Number:

93882-40-7

Molecular formula:

C48H86O8S

IUPAC Name:

2-octadecenylsuccinic anhydride, thiodiethanol esterification products

Test material form:

solid

Remarks:

Waxy Solid

Method

Target gene:

TK gene (coding for thymidine kinase)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine kinase (TK) locus. L5178Y/TK+/- cells, clone 3.7.2C, were received from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC. Each batch of frozen cells was tested and found to be free of mycoplasma contamination. This test system has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Assay: 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The test substance formed workable suspensions in DMSO at concentrations =3.13 mg/mL, while concentrations =1.56 mg/mL were solutions. Visible precipitate was observed >= 62.5 µg/mL at the beginning and end of the treatment period under the conditions of -S9, 24 hour treatment and +S9, 4 hour treatment .Visible precipitate was observed >= 62.5 µg/mL at the beginning of treatment and >= 125 µg/mL at the end of treatment under the condition of -S9, 4 hour treatment.

Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were:

-S9, 4 hours:7.81, 15.6, 31.3, 62.5, 125, 250, 275, 300, 350, 400 and 500 µg/mL
-S9, 24 hours:7.81, 15.6, 31.3, 62.5, 75, 100, 110, 120, 125, 135 and 250 µg/mL
+S9, 4 hours:7.81, 15.6, 31.3, 62.5, 75, 100, 110, 120, 125, 135 and 250 µg/mL

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, Water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Test System
L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine kinase (TK) locus. L5178Y/TK+/- cells, clone 3.7.2C, were received from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC. Each batch of frozen cells was tested and found to be free of mycoplasma contamination. This test system has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

Solubility Determination
DMSO was the vehicle of choice based on information provided by the Sponsor and compatibility with the target cells. Based on the information provided by the Sponsor, the test material is workable in DMSO at a concentration of approximately 200 mg/mL.

Preparation of Target Cells
Prior to use in the assay, L5178Y/TK+/- cells were cleansed to reduce the frequency of spontaneously occurring TK-/- cells. Using the procedure described by Clive and Spector (1975), L5178Y cells were cultured for 24 hours in the presence of thymidine, hypoxanthine, methotrexate and glycine to poison the TK-/- cells. L5178Y/TK+/- cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F 68 (F0P). All media contained antibiotics.

Identification of Test System
The cultures were identified by the BioReliance study number and a code system to designate the treatment condition, dose level and test phase.

Exogenous Metabolic Activation
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3437, Expiration Date: 25 March 2017) was purchased commercially from Moltox (Boone, NC). Upon arrival at Laboratory, the S9 was stored at 60°C or colder until used. Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.The S9 mix was prepared on the day of use.

Frequency and Route of Administration
Target cells were treated for 4 hours in the presence and absence of S9, and for 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture in the treatment medium.

Preliminary Toxicity Test for Selection of Dose Levels
L5178Y/TK+/- cells were exposed to the vehicle alone in duplicate cultures and ten concentrations of test substance using single cultures. The maximum concentration evaluated was the limit dose for this assay. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Osmolality of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured at the beginning of treatment. Precipitation was determined with the unaided eye at the beginning and end of treatment. Dose levels for the definitive assay were based upon post-treatment cytotoxicity (growth inhibition relative to the vehicle control).

Mouse Lymphoma Assays
Eleven (without S9 activation), ten (with S9 activation in the definitive mutagenicity assay) or Nine (with S9 activation in the repeat of definitive mutagenicity assay) concentrations were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Precipitation was determined with the unaided eye at the beginning and end of treatment.

Treatment of Target Cells
The preparation and addition of the test substance dose formulations was carried out under yellow or filtered lighting during the exposure period. Treatment was carried out by combining 100 µL of test substance dose formulation, vehicle or positive control dose formulation and F0P medium or S9 mix (as appropriate) with 6 x 106 L5178Y/TK+/- cells in a total volume of 10 mL. All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9 activated 10-mL culture contained 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1¿C for 4 or 24 hours.
For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post treatment, with cell concentration adjustment on the first day. After a 24 hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 105 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.
For the definitive assays only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1¿C for two days following treatment. Cell population adjustments to 3 x 105 cells/mL were made as follows:

• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal, and 2 and 3 days after treatment.



Selection of Mutant Phenotype
Cells from selected dose levels were cultured in triplicate with 2-4 ¿g TFT/mL at a density of 1 x 106 cells/100 mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1¿C in a humidified atmosphere of 5 ± 1% CO2 in air) for 11 or 12 days.
The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ¿10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

Extended Treatment and/or Confirmatory Assay
Verification of a clear positive response was not required (OECD Guideline 490). For negative results without activation, an extended treatment assay was performed in which cultures were continuously exposed to the test substance for 24 hours without S9 activation. The extended treatment assay was performed concurrently with the initial assay. For negative results with S9 activation, a confirmatory assay was not required unless the test substance was known to have specific requirements of metabolism.

Evaluation criteria:

In evaluation of the data, increases in induced mutant frequency which occurred only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data (Moore et al., 2006).
• A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of ¿90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/106 clonable cells, a doubling of mutant frequency over the vehicle would also be required (Mitchell et al., 1997).
• A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
There are some situations in which a chemical would be considered negative when there was no culture showing between 10 to 20% survival (Office of Food Additive Safety, 2001).
• There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/106) in a series of data points within 100 to 20% survival and there was at least one negative data point between 20 and 25% survival.
• There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/106) in a series of data points between 100 to 25% survival and there was also a negative data point between 10 and 1% survival. In this case, it would be acceptable to count the TFT colonies of cultures exhibiting <10% total growth.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

For results tables see attached background documents.

No increases in induced mutant frequency =90 mutants/10⁶clonable cells were observed were observed following 4-hour treatments with S9. However the average mutant frequency value for the vehicle controls was outside of the acceptable range (35-140 X 10^-6), and therefore this treatment condition was retested. The concentrations chosen for the repeat of the definitive mutagenicity assay were 4.22, 8.44, 16.9, 33.8, 67.5, 135, 150, 175 and 200 µg/mL.

In the repeat of the definitive mutagenicity assay following a 4-hour treatment with S9, visible precipitate was observed at concentrations =67.5 µg/mL at the beginning of treatment and at concentrations =33.8 µg/mL by the end of treatment. All criteria for a valid assay were met. Following a 4-hour treatment with S9, no increases in induced mutant frequency =90 mutants/10⁶clonable cells were observed.

Applicant's summary and conclusion

Conclusions:

The Substance did not induce any significant increases in the mutant frequency at the TK +/- locus in L5178Y cells when tested up to the maximum practical dose level. Therefore the Substance is considered non-mutagenic under the conditions of the test.

Executive summary:

The Substance was evaluated for mutagenic potential in mouse lymphoma L5178Y cells in vitro. The method followed that described by OECD Test Guideline 490 and the study was conducted to GLP.

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated was the limit dose for this assay. Visible precipitate was observed at concentrations from=62.5 µg/mL or=125 µg/mL in all treatments. Relative suspension growth (RSG) was 34, 34 and 57% at concentrations of 125 µg/mL (4-hour treatment with S9), 250 µg/mL (4-hour treatment without S9) and 62.5 µg/mL (24-hour treatment without S9), respectively. RSG was, or approximated, 0% at all higher concentrations tested. 

Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were:

-S9, 4 hours:7.81, 15.6, 31.3, 62.5, 125, 250, 275, 300, 350, 400 and 500 µg/mL

-S9, 24 hours:7.81, 15.6, 31.3, 62.5, 75, 100, 110, 120, 125, 135 and 250 µg/mL

+S9, 4 hours:7.81, 15.6, 31.3, 62.5, 75, 100, 110, 120, 125, 135 and 250 µg/mL

Cultures treated at other concentrations were discarded prior to cloning because a sufficient number of higher concentrations were available, and/or were excluded from evaluation of mutagenicity due to excessive toxicity. Relative total growth of the cloned cultures ranged from 18 to 135% (4-hour treatment without S9) and 33 to 96% (24-hour treatment without S9). 

No increases in induced mutant frequency =90 mutants/106 clonable cells were observed following 4- or 24-hour treatments without S9 and 4- hour treatment with S9. Therefore the Substance was considered to be non-mutagenic in this test.