4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance showed no evidence of mutagenic potential in a battery of three different in vitro assays (as required by Annexes VII and VIII of REACH): a reverse mutation test in Salmonella typhimurium and E. coli; a chromosome aberration assay using human lymphocytes, and a mouse lymphoma gene mutation assay.

Short description of key information:
The substance showed no evidence of mutagenic potential in three different in vitro assays.

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

TK gene (coding for thymidine kinase)

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

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.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th March 2011 - 18th July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Description/Appearance: Brown Solid
Batch/ Lot/ Notebook Ref: N.B. E00031-691 (Batch 30038-158)
Sample Expiration Date: 01/2014
Purity: 100%

Target gene:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were from Dr. Bruce Ames’ Master cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. The following tester strains were distributed by Moltox (Boone, NC), using cultures derived from the above sources: TA100 and TA1535 in the initial toxicity-mutation assay, TA1537 in the retest of the initial toxicity-mutation assay and TA1535 in the confirmatory mutagenicity assay.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Limit test at 5000 µg per plate

In the initial toxicity-mutation assay: dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. The test article formed soluble but cloudy solutions from 30 to 100 mg/mL and soluble and clear solutions from 0.030 to 10 mg/mL. Precipitate was observed beginning at 1500 or at 5000 µg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 µg per plate.

In the retest of the initial toxicity-mutation assay, dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate. No appreciable toxicity was observed.

In the confirmatory mutagenicity assay, dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strains TA100 and TA1535 in the absence of S9 activation and 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 1500 µg per plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were from Dr. Bruce Ames’ Master cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. The following tester strains were distributed by Moltox (Boone, NC), using cultures derived from the above sources: TA100 and TA1535 in the initial toxicity-mutation assay, TA1537 in the retest of the initial toxicity-mutation assay and TA1535 in the confirmatory mutagenicity assay.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Overnight cultures were prepared by inoculating from the appropriate master plate, appropriate frozen permanent stock or with a lyophilized pellet into a vessel, containing ~30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm at 37±2°C approximately 12 to 14 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Evaluation criteria:

Evaluation of Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Following completion of the study, a recording error was discovered for the incubation records of the confirmatory mutagenicity assay. The assay plates were not recorded as having been logged into the incubator after dosing and, consequently, there is no record to support proper incubation (i.e. 48-72 hours) of the assay plates. Therefore, the complete assay was retested in Experiment B4. The data from the confirmatory assay are included in the report, but were not used in the evaluation of the study results.

In Experiment B4 (Retest of the Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate with tester
strains TA100 and TA1535 in the absence of S9 activation and 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 1500 µg per plate. No appreciable toxicity was observed.

See attached background document

Conclusions:

Under the conditions of this study, the Substance was concluded to be negative in the Bacterial Reverse Mutation Assay.

Executive summary:

The Substance was tested in the Bacterial Reverse Mutation Assay usingSalmonella typhimuriumtester strains TA98, TA100, TA1535 and TA1537 andEscherichia colitester strain WP2uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. After sonication at 33°C for 15 minutes, the test article formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. The test article formed soluble but cloudy solutions from 30 to 100 mg/mL and soluble and clear solutions from 0.030 to 10 mg/mL. No positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation and with tester strains TA98, TA100, TA1535 and WP2uvrA in the absence of S9 activation. Precipitate was observed beginning at 1500 or at 5000 µg per plate. No appreciable toxicity was observed. Due to contamination on both replicate plates at the 5000 µg per plate dose level, tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profile observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 µg per plate.

In the retest of the initial toxicity-mutation assay, no positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate. No appreciable toxicity was observed.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strains TA100 and TA1535 in the absence of S9 activation and 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 1500 µg per plate. No appreciable toxicity was observed.

Following completion of the study, a recording error was discovered for the incubation records of the confirmatory mutagenicity assay. The assay plates were not recorded as having been logged into the incubator after dosing and, consequently, there is no record to

support proper incubation (i.e. 48-72 hours) of the assay plates. Therefore, the complete assay was retested. The data from the confirmatory assay are included in the report, but were not used in the evaluation of the study results.

In the retest of the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strains TA100 and TA1535 in the absence of S9 activation and 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 1500 µg per plate. No appreciable toxicity was observed.

 

Under the conditions of this study, the Substance was concluded to be negative in the Bacterial Reverse Mutation Assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10th December 2012 - 10th July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Description/Appearance: Brown Solid
Batch/ Lot/ Notebook Ref: N.B. E00031-691 (Batch 30038-158)
Sample Expiration Date: 01/2014
Purity: 100%

Target gene:

chromosomal aberrations in cultured mammalian cells

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking 25-year-old adult
male on 15 Jan 2013 for the preliminary toxicity assay and from the same donor on 29 Jan
2013 for the definitive assay. The donor had no recent history of radiotherapy, viral
infection or the administration of drugs. This system has been demonstrated to be sensitive
to the clastogenic activity of a variety of chemicals (Preston et al., 1981).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 metabolic activation system

Test concentrations with justification for top dose:

Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test. HPBL cells were first exposed to nine dose levels of the Substance,ranging from 0.5 µg/mL to 5000 µg/mL, as well as vehicle controls.

Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was observed at dose levels = 1500 µg/mL in the non-activated 4 and 20-hour exposure groups, and at dose levels = 500 µg/mL in the S9-activated 4-hour exposure group. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows: 2.5, 5, 10, 25, 50, 100, 150 µg/mL

Vehicle / solvent:

DMSO was used as the vehicle based on the solubility of the test article and compatibility with the target cells. In a solubility test conducted at BioReliance, the test article formed clear and soluble solution in DMSO at a concentration of approximately 500 mg/mL, the
maximum concentration tested for solubility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Test System
Peripheral blood lymphocytes were obtained from a healthy non-smoking 25-year-old adult male on 15 Jan 2013 for the preliminary toxicity assay and from the same donor on 29 Jan 2013 for the definitive assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston et al., 1981).
 
Preparation of Target Cells
In the preliminary toxicity assay, peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin, and 1% phytohemagglutinin) by adding 0.6 mL heparinized blood to a centrifuge tube containing 9.4 mL of complete medium. In the definitive assay, peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin, and 2% phytohemagglutinin) by adding 0.6 mL heparinized blood to a centrifuge tube containing 9.4 mL of complete medium. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2in air) for 44-48 hours.
 
Identification of Test System
Prior to treatment, each tube was identified by the BioReliance study number, dose level, test phase, treatment condition, activation system and/or replicate design.
 
Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 (Lot No. 3019) was obtained from Molecular Toxicology Inc. (Boone, NC). Each bulk preparation of S9 was assayed for sterility and its ability to metabolize at least two promutagens to forms mutagenic toSalmonella typhimuriumTA100 by the supplier.
 
Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per milliliter medium (RPMI 1640 serum-free medium supplemented with 100 units penicillin/mL and 100 µg streptomycin/mL and 2 mM L-glutamine).
 
Solubility Test
In order to determine the highest soluble or workable concentration, a solubility test was conducted up to 500 mg/mL in DMSO.
 
Experimental Design
Thein vitromammalian chromosome aberration assay was conducted using standard procedures (Evans and O'Riordan, 1975; Galloway et al, 1994; Preston et al, 1981; Swierenga et al, 1991) by exposing human peripheral blood lymphocytes (HPBL) to appropriate concentrations of the test article as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.
 
Preliminary Toxicity Test for Selection of Dose Levels
HPBL were exposed to vehicle alone and to nine concentrations of test article with half-log dose spacing using single cultures. Precipitation of test article dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the solvent, the highest dose level, lowest precipitating dose level and the highest soluble dose level was measured. The pH of the highest dose level of dosing solution in the treatment medium was measured using test tape. The number of cells in mitosis per 500 cells scored was determined in order to evaluate a possible test article effect on mitotic index. Dose levels for the definitive assay were based upon visible precipitation of the test article in treatment medium at the conclusion of the treatment period.
 
Chromosome Aberration Assay
Seven dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test article. Precipitation of test article dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The pH of the highest dose level of dosing solution in the treatment medium was measured using test tape. The highest dose level evaluated for the chromosome aberrations was selected based on visible precipitation of the test article in treatment medium at the conclusion of the treatment period. Two additional dose levels were included in the evaluation.
 
Treatment of Target Cells (Preliminary Toxicity Test and Definitive Assay)
Test article dosing solutions were prepared immediately prior to use. Treatment was carried out by refeeding the cultures with approximately 10 mL complete medium for the non-activated exposure or 10 mL S9 mix (8 mL culture medium + 2 mL of S9 cofactor pool) for the S9-activated exposure to which was added 0.1 µL of test article dosing solution, or vehicle alone. In the definitive assay, positive control cultures were resuspended in either 10 mL of complete medium for the non-activated studies, or 10 mL of the S9 reaction mixture (8 mL serum free medium + 2 mL of S9 cofactor pool), to which was added 0.1 mL of positive control in solvent.
 
After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium was aspirated, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium and returned to the incubator under standard conditions.
 
Collection of Metaphase Cells (Preliminary Toxicity Test and Definitive Assay)
For the preliminary toxicity and the definitive assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analysed in the first division metaphase. Two hours prior to harvest, Colcemid®was added to the cultures at a final concentration of 0.1 µg/mL.
 
Cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2-8°C. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted, and identified by the BioReliance study number and a code system to designate at least the treatment condition, dose level, and test phase.
 
Scoring for Metaphase Chromosome Aberrations (Definitive Assay)
The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted.
Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture may be reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated per 100 cells analyzed for each culture (a total of 200 per dose level).

Evaluation criteria:

Toxicity induced by treatment is based upon inhibition of mitosis and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure doseresponsiveness. A test article was considered positive if it induced a statistically significant and dose-dependent increase in the frequency of aberrant metaphases (p = 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Key result

Species / strain:

lymphocytes: Human peripheral Blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Prelim: Cytotoxicity was observed at dose levels>=500 ug/mL

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: statistically significant or dose-dependent increase

See attached background material

Conclusions:

The Substance is considered to be non-clastogenic to human lymphocytes, in vitro.

Executive summary:

The test substance was evaluated for clastogenic potential in thein vitrochromosome aberration assay using human lymphocytes. This GLP study was conducted in accordance with OECD Test Guideline 473. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was observed at dose levels = 1500 µg/mL in the non-activated 4 and 20-hour exposure groups, and at dose levels = 500 µg/mL in the S9-activated 4-hour exposure group. Based on the results of the preliminary toxicity test test material concentrations, in DMSO vehicle, of 0, 2.5, 5, 10, 25, 50, 100 and 150 micrograms/ml were selectyed for testing. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels = 100 µg/mL, while dose levels = 50 µg/mL were soluble in treatment medium. The pH of the highest dose level of test article in treatment medium was 7.5. There was no dose-related inhibition of mitotic index either in the presence or absence of metabolic activation (S9). The test material did not induce any statistically significant increases in the frequency of cells with aberrations or the frequency of polyploid cells at any dose level either in the presence or absence of metabolic activation. The test material was therefore considered to be non-clastogenic to human lymphocytesin vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Bacterial Reverse Mutation:

The Substance was tested in the Bacterial Reverse Mutation Assay usingSalmonella typhimuriumtester strains TA98, TA100, TA1535 and TA1537 andEscherichia colitester strain WP2uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. After sonication at 33°C for 15 minutes, the test article formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. The test article formed soluble but cloudy solutions from 30 to 100 mg/mL and soluble and clear solutions from 0.030 to 10 mg/mL. No positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation and with tester strains TA98, TA100, TA1535 and WP2uvrA in the absence of S9 activation. Precipitate was observed beginning at 1500 or at 5000 µg per plate. No appreciable toxicity was observed. Due to contamination on both replicate plates at the 5000 µg per plate dose level, tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profile observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 µg per plate.

In the retest of the initial toxicity-mutation assay, no positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate. No appreciable toxicity was observed.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strains TA100 and TA1535 in the absence of S9 activation and 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 1500 µg per plate. No appreciable toxicity was observed.

Following completion of the study, a recording error was discovered for the incubation records of the confirmatory mutagenicity assay. The assay plates were not recorded as having been logged into the incubator after dosing and, consequently, there is no record to

support proper incubation (i.e. 48-72 hours) of the assay plates. Therefore, the complete assay was retested. The data from the confirmatory assay are included in the report, but were not used in the evaluation of the study results.

In the retest of the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strains TA100 and TA1535 in the absence of S9 activation and 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 1500 µg per plate. No appreciable toxicity was observed.

 

Under the conditions of this study, the Substance was concluded to be negative in the Bacterial Reverse Mutation Assay.

In vitro Chromosome Aberration:

The test substance was evaluated for clastogenic potential in thein vitrochromosome aberration assay using human lymphocytes. This GLP study was conducted in accordance with OECD Test Guideline 473. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was observed at dose levels = 1500 µg/mL in the non-activated 4 and 20-hour exposure groups, and at dose levels = 500 µg/mL in the S9-activated 4-hour exposure group. Based on the results of the preliminary toxicity test test material concentrations, in DMSO vehicle, of 0, 2.5, 5, 10, 25, 50, 100 and 150 micrograms/ml were selectyed for testing. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels = 100 µg/mL, while dose levels = 50 µg/mL were soluble in treatment medium. The pH of the highest dose level of test article in treatment medium was 7.5. There was no dose-related inhibition of mitotic index either in the presence or absence of metabolic activation (S9). The test material did not induce any statistically significant increases in the frequency of cells with aberrations or the frequency of polyploid cells at any dose level either in the presence or absence of metabolic activation. The test material was therefore considered to be non-clastogenic to human lymphocytesin vitro.

In vitro: Mouse Lymphoma Assay:

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.

Justification for classification or non-classification

The substance showed no evidence of mutagenic potential in the standard battery of three in vitro assays (bacterial mutation, mammalian gene mutation, and mammalian cytogenetic). Consequently, no classification for gene mutagenicity is required under CLP.