hydrogenation products of (esterification products of 2-ethylhexan-1-ol with (Estolide formation products of oleic acid and Fatty acids, C8-18 and C18-unsatd. (branched or linear)).

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in the ames and mammalian in-vitro studies, no adverse effects were observed:

1. Under test conditions, test article CAS# 1365345-64-7 (SE7B Batch 2137-0), did not have mutagenicity potential in the Bacterial Reverse Mutation Test.
2. The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be nonclastogenic to human lymphocytes in vitro. Therefore, SE7B also did not exhibit clastogenic potential at the concentration-range investigated.
3. According to the evaluation criteria for this assay, these findings indicate that SE7B, tested up to the concentration of 5.0 μl per ml medium, that led to slight test item precipitation, did neither induce mutations nor have any chromosomal aberration potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 4th March 2013 Experimental completion date: 28th March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Tester strain Gene affected
E. coli WP2 uvrA T/PE
S. typh. TA-97a his D 6610
S. typh. TA-1535 his G 46
S. typh. TA-98 his D 3052
S. typh. TA-100 his G 46

Species / strain / cell type:

S. typhimurium, other: TA1535, TA-97a, TA-98 and TA-100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

A cytotoxicity screen was conducted in the Salmonella typhimurium TA-1 00 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µl/plate) of the test article, two plates per dose.

Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5 µl/plate) of the test article were tested in each of five bacterial tester strains (E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on test article solubility information provided by Sponsor, the Study Director chose Acetone as the vehicle for the assay.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA),

Remarks:

All bacterial strains with exogenous metabolic activation (S9).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

no

Positive control substance:

other: Acridine, 6-chloro-9-(3-((2-chloroethyl)amino) propyl)amino-2-methoxy, dihydrochloride

Remarks:

S. typh. TA-97a without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typh. TA-100 and TA-1535, without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin (DM)

Remarks:

S. typh. TA-98 without S9

Details on test system and experimental conditions:

Tester strains were dosed in triplicate with five concentrations of the test article. Six plates each were dosed with the Vehicle Control and the Positive Control specific for each tester strain.

Preparation of the Tester Strains:
Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. #26-555). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37°C ±2°C with agitation. The cultures were used after they reached the late exponential growth phase as determined by absorbance readings at 600 nm.

Treatment of the Test System:
Top agar supplemented with appropriate amino acids were prepared, as 2 ml aliquots, and maintained at 45-50°C in sterile culture tubes. Dulbecco's Phosphate Buffered Saline (DPBS) was added to the tubes not undergoing S9 activation (i.e. without S9, or -S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, Vehicle Control or Positive Control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were gently vortexed and overlaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37°C ±2°C for 48-72 hours. Plates that were not scored immediately following the incubation period were stored at 2-8°C until scoring.

Main Assay:
Five concentrations (0.05, 0.01, 0.5, 1 and 5 µl/plate) of the test article were tested in each of five bacterial tester strains (Escherichia coli WP2 uvrA, and Salmonella typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Two sets of culture plates were dosed per concentration (+S9 and No S9). A Vehicle Control and Positive Controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ±2°C for 48-72 hours.

Revertant Colony Count:
Counting of the revertants per plate was performed using an Alphalmagerm 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article. The plates were also examined visually for test article precipitate.

Independent Repeat Assay:
No positive response or dose-related increased response of the test article in any strain was found in the main assay, so an independent repeat assay was conducted with the same test conditions used in the main study.

Evaluation criteria:

Analysis of Data:
Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the Vehicle Control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account.

In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose related increases approaching a 2-fold increase are deemed equivocal.

A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.

Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA-97a, TA-98 and TA-100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no diminution and clearing of the background lawn observed at any dose level, indicating that the test article was not cytotoxic to TA-100 at 0.001 to 5 µl/plate. The test article precipitated out in the plates at concentrations of 0.5 to 5 µl/plate. The Study Director chose 5 µl/plate as the top test article concentration for the main test.

Main Assay:
The assay was run in all five strains on triplicate plates. Positive and Vehicle Controls were run concurrently for all five strains, on six plates per strain. All plating was with and without exogenous metabolic activation, S9. No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. The test article precipitated out in the plates at 0.5 to 5 µl/plate. However, the precipitation did not interfere with the automatic counting of the plates. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test article in the presence or absence of S9. All Positive and Negative Control values were within acceptable ranges, and all criteria for a valid study were met.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Sterility Test: No contaminating microorganisms were detected in any of the reagents used in the assay. No contaminating microorganisms were detected in any of the reagents used in the assay. The sterility test passed the quality checks.

Vehicle Controls The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain of bacteria was measured and compared to in-house historical ranges. All Vehicle Controls passed the quality check.

Positive Controls: All Vehicle Controls passed the quality check.

Conclusions:

Interpretation of results (migrated information):
negative

Under test conditions, test article CAS# 1365345-64-7 (SE7B Batch 2137-0), did not have mutagenicity potential in the Bacterial Reverse Mutation Test.

Executive summary:

Objective:

The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 - Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 - Bacterial Reverse Mutation Test.

Method Synopsis:

Based on test article solubility information provided by Sponsor, the Study Director chose Acetone as the vehicle for the assay. A cytotoxicity screen was conducted in the Salmonella typhimurium TA-1 00 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µl/plate) of the test article, two plates per dose. The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation mixture (S9) and overlaid onto minimal glucose agar plates. An Acetone Vehicle Control was run concurrently, with and without S9. Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5 µl/plate) of the test article were tested in each of five bacterial tester strains (E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Vehicle Controls and Positive Controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ±2°C for 48-72 hours. Revertant colony growth was determined by counting the colonies per plate using an Alphalmagerm imaging system. The number of revertants of the test article treatment plates and Positive Control plates was divided by the number of revertants of the vehicle plates. In general, a positive result is determined by a 2-fold increase above the Vehicle Control. Due to a negative result in the main assay, an independent repeat assay (confirmatory test) was conducted in all five tester strains using test article concentrations of 0.05, 0.1, 0.5, 1 and 5 µl/plate.

Summary:

Test article CAS# 1365345-64-7 (SE7B Batch 2137-0), in the vehicle, Acetone, was tested in a Bacterial Reverse Mutation Assay. The test article did not show obvious cytotoxicity to tester strain TA-100 at a dose range of 0.001 to 5µl/plate, with or without S9. The test article at 0.05 to 5µl/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay. As in the main test, in the independent repeat assay the test article at 0.05 to 5 µl/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, confirming that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

Conclusion: Under test conditions, test article CAS# 1365345-64-7 (SE7B Batch 2137-0), did not have mutagenicity potential in the Bacterial Reverse Mutation Test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Identification of the test item
After receipt at LPT the test item is inspected. Batch number(s), amount and any characteristics are compared with the information (if any) provided by the Sponsor/Supplier. An identification sheet will be filed with the raw data.

Description
Name: SE7B
Description: Biodegradable biobased ester
Chemical identity: Estolide (biobased ester)
Lot no.: 0000140602
CAS no: 1365345-64-7
Characteristics: Liquid; light yellow with little to no odor
Storage conditions: Keep container dry. Store in a cool place. Keep away from oxidizing agents, excessive heat, and ignitions sources.
Retention sample of the test item: Stored in LPT's archives.

Target gene:

The indicator cell used for this study is the L517 BY mouse lymphoma cell line that is heterozygous at the TK locus ( + /-).
The objective of this study is to evaluate the ability of the test item to induce forward mutations at the thymidine kinase (TK) locus in L5178Y TK+/mouse lymphoma cells as assayed by colony growth in the presence of 5-triflurothymidine (TFT) and to determine the quality of the mutation (base pair substitutions and deletions or larger genetic changes frequently visible as chromosome aberrations).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Indicator cells
The indicator cell used for this study is the L517 BY mouse lymphoma cell line that is heterozygous at the TK locus ( + /-). The particular clone (3. 7 .2C) used in this assay was isolated by ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA. Stock cultures were obtained from ATCC, and master stocks are maintained in liquid nitrogen. Laboratory cultures are periodically checked for the absence of mycoplasma contamination by culturing methods (in accordance to SOP 170 505). To reduce the background mutant frequency (spontaneous frequency) of TK-/- mutants to a level as low as possible, cell cultures are exposed to conditions that select against the TK-/- phenotype (exposure to aminopterin or methotrexate). Cell cultures are maintained in cleansing medium for three days, placed in recovery medium for three days and then returned to normal growth medium for three to eight days before use.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

A preliminary cytotoxicity experiment is performed to establish an appropriate concentration range for the mutation experiment. This study is performed both with and without S9 metabolic activation since substantial shifts in toxicity often occur for the two test conditions. A wide range of test article concentrations is tested for cytotoxicity. After an exposure time of 3 hours at approx. 37 °C on a roller drum at 10-15 rpm, the cells are washed and resuspended in growth medium. The cells are then adjusted to 8 cells/ml and for each dose 0.2 ml is plated into 32 microtiter wells. The plates are incubated at 37°C in a humidified incubator gassed with 5% C02 in air for 7 days. Wells containing viable clones are identified under a microscope and counted.
The preliminary results are then used to select dose levels for the mutation assay using the following criteria:
At least 4 analysable concentrations will be used. Where there is cytotoxicity, the concentrations will cover a range from the maximum to little or no toxicity; so that the concentrations will be separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10-20% relative survival or relative total growth. For relatively noncytotoxic compounds the maximum concentration will be 5 mg/ml, 5 μL/ml, or 0 .01M, whichever is the lowest. Also, among the criteria to be considered when determining the highest concentration are cytotoxicity and solubility in the test system and changes in pH or osmolality.
Although 5 doses may be used to initiate an experiment, the objective is to carry at least 4 doses through the entire experiment, to compensates for normal variations in cellular toxicity and help ensure the choice of at least 4 analysable doses appropriately spaced in the relative growth range of ~10-100%. Duplicate cultures are used at each test item concentration tested. Duplicate negative control cultures will be used.

Vehicle / solvent:

The test item will be diluted, suspended or dissolved in a suitable vehicle (e.g. water, culture media, DMSO) and serial dilutions will be made. A suitable solvent or vehicle will be determined in the course of the preliminary experiment. Details will be stated in the raw data and the report. The Monitor will be informed of the solvent to be used before the start of the main study. Treatment conditions consist of at least 5 x 106 cells suspended in 20.0 ml of treatment medium. Fresh preparations of test item in the vehicle are prepared on each day for biological testing.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

Nonactivation assay
The assay procedure used is based on that reported by COLE et al. (1990). Single cultures are used for each test item concentration for each independent experiment. The cells for the experiment are obtained from logarithmically growing laboratory stock cultures and are seeded into a series of tubes at at least 5 x 106 cells per tube. The cells are pelleted by centrifugation, the culture medium is removed, and the cells are resuspended in a final volume of 20.0 ml of treatment medium that contains 5% heat inactivated fetal bovine serum. The dosed tubes are closed, vortexed and placed on a roller drum at approximately 37°C at 10-15 rpm for an exposure period of 3 hours.
The cells are washed and resuspended in growth medium.
Cell densities are adjusted to 2 x 105/ml and the cells are incubated for the expression period and plated for survival in parallel, i.e. an aliquot of the cells is diluted to 8 cells/ml and 0.2 ml of each culture are placed in two 96 well microtiter plates ( = 192 wells, averaging 1 .6 cells/well) and incubated for 1 week whereas the rest of the cells is incubated for 2 to 3 days for the expression period.
The cells for the plating of survival are counted after 1 week and the number of viable clones is recorded. The cells in the expression period are maintained below 106 cells per ml and a minimum of 4 dose levels plus positive and vehicle reference items are selected for 5- Trifluorothymidine (TFT) resistance.
At the end of the expression period the selected cultures are diluted to 1 x 104 cells/ml and plated for survival and TFT resistance in parallel. The plating for survival is similar to the above described method. For the plating for TFT resistance 3 μg/ml TFT (final concentration) are added to the cultures and 0.2 ml of each suspensions placed into four 96-well microtiter plates r· 384 wells, averaging 2 x 103 cells/well). The plates are incubated for 11 to 14 days and wells containing clones are identified microscopically and counted.
In addition the number of large and small colonies is recorded with an automated colony counter that can detect colony diameters equal to or greater than 0.2 to 0.3 mm. If the test item is positive, colony sizing is performed on at least one of the test cultures (the highest positive concentration) and on the negative and positive reference items. If the test item is negative, colony sizing is performed on the negative and positive reference items only.
The results from the initial mutation assay are confirmed by performing an independent repeat mutation assay, whereby the exposure time will be 24 hours.
Activation assay
The activation assay is often run concurrently with the nonactivation assay; however, it is an independent assay performed with its own set of solvent and positive reference items. In this assay the above described activation system is added to the cells together with test item. The exposure time will be 3 hours for the activation experiment.
The activation assay will be repeated in an independent experiment. The exposure time will again be 3 hours.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A preliminary cytotoxicity study was conducted to establish the top concentration for the main study. Concentrations of 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 μL SE7B/mL medium were employed in an experiment without and with metabolic activation. No signs of cytotoxicity (decreased survival) were noted in the absence and in the presence of metabolic activation up to the top concentration of 5.0 μL SE7B/mL. Slight test item precipitation was noted in both experiments at the top concentration of 5.0 μL SE7B/mL.
Hence, in the main study the concentration-range of 0.313 to 5.0 μL SE7B/mL was used in the experiments without and with metabolic activation.
Methylmethanesulfonate (at 1.56 μg/mL and 3.13 μg/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 μg/mL) in the presence of exogenous metabolic activation.
In the main study, no signs of cytotoxicity were noted up to the top concentration of 5.0 μL SE7B/mL in the absence and presence of metabolic activation. Slight test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 5.0 μL SE7B/mL.
The values of mutation frequencies of the negative controls ranged from 75.87 to 147.48 per 106 cloneable cells in the experiments without metabolic activation and from 130.65 to 159.62 per 106 cloneable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.
The mutation frequencies of the cultures treated with SE7B ranged from 67.78 to 119.73 per 106 cloneable cells (3 hours exposure) and from 86.68 to 135.39 per 106 cloneable cells (24 hours exposure) in the experiments without metabolic activation and from 94.86 to 156.75 per 106 cloneable cells (3 hours exposure, first assay) and from 101.63 to 159.66 per 106 cloneable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.
In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.55 to 1.22 for SE7B-treated cells and from 0.53 to 1.23 for thenegative controls.
The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 436.09 to 1042.68 per 106 cloneable cells in the case of MMS and ranging from 1264.47 to 2738.93 per 106 cloneable cells in the case of 3-MC.
In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.48 to 2.17 in the case of MMS.

Conclusions:

Under the present test conditions, SE7B, tested up to the concentration of 5.0 μl per ml medium, that led to slight test item precipitation, in two independent experiments was negative with respect to the mutant frequency in the l5178Y TK +/-mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies.
Therefore, SE7B also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that SE7B, tested up to the concentration of 5.0 μl per ml medium, that led to slight test item precipitation, did neither induce mutations nor have any chromosomal aberration potential.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

Test System
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The test item was considered to be a mixture and therefore the maximum recommended dose was initially set at 5000 μg/mL. No purity correction was required as the test item was 100% pure.

The test item was insoluble in culture media at 50 mg/mL and dimethyl sulphoxide at 250 μg/mL. The test item was soluble in acetone at 500 mg/mL in solubility checks performed in-house. The test item was accurately weighed, dissolved in acetone and serial dilutions prepared. Due to the use of acetone as the solvent and the intolerance of the lymphocytes to it, the test item formulations were dosed at 0.5% and to achieve the maximum recommended dose of 5000 μg/mL the test item was dosed neat.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle / solvent:

Vehicle and positive controls were used in parallel with the test item.
The vehicle control used was acetone.
The positive control items were as follows:
In the absence of S9, mitomycin C (MMC) (Sigma, Batch No. SLBD1982V) was used at 0.4 and 0.2 μg/mL for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.
In the presence of S9, cyclophosphamide (CP) (Acros, Batch No. A0302605) was used at 5 μg/mL in both experiments. It was dissolved in dimethyl sulphoxide.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 °C, 5% C02 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture.
For the positive control, 0.1 mL of the appropriate solution was added to the cultures. lmL of 20% S9---mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 mL of 10% S9-mix (i.e. 1 % fmal concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 °C in 5% C02 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at approximately 37 °C with 5% C02 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C, 5% C02 in humidified air for 24 hours.
The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4 hours exposure to the test item with S9-mix (2% ), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 19.53 to 5000 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
Two exposure groups were used for Experiment 1:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 160 and 320 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2% ), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 160 and 320 μg/mL.

Experiment 2
Two exposure groups were used for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 160 and 320 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (1 %) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 160 and 320 μg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCL After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation prior to slide preparation.

Preparation of Meta phase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

The following criteria were used to determine a valid assay:

Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control
cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce clear positive responses (p:S0.01). Acceptable
positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Criteria for determining the Study Conclusion
A test item can be classified as non-genotoxic if:
1.The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis

A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations 1s not m the range of laboratory historical control data
And
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis

Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Statistics:

Statistical Analysis
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.(Richardson et al. 1989).

Key result

Species / strain:

mammalian cell line, other: Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL. The maximum dose was the maximum recommended dose level.

A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 39.06 μg/mL and 78.13 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9 respectively. Greasy/oily precipitate was seen in both the 4(20)-hour exposure groups at and above 312.5 μg/mL. In the continuous exposure group cloudy precipitate and greasy/oily precipitate was observed at the end of the exposure period at and above 625 μg/mL.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 μg/mL in all three exposure groups. The mitotic index data are presented in Table 1. Although there were some reductions in the mitotic index of the 24-hour exposure group, these were considered to be due to a high vehicle control value and overall the test item was considered to induce no evidence of toxicity in any of the exposure groups.

The selection of the maximum dose level was based on the absence of marked toxicity and the presence of greasy/oily precipitate and was 320 μg/mL for the 4(20)-hour exposure groups and for the continuous exposure group used in Experiment 2. It was considered that maximum exposure was achieved by the test item at this dose level.

 

Chromosome Aberration Test - Experiment 1

The qualitative assessment of the slides determined that the toxicity and precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 320 μg/mL in both exposure groups.

Precipitate observations were made at the end of exposure and greasy/oily precipitate was noted at and above 80 μg/mL in the absence of S9 and at and above 40 μg/mL in the presence of S9.

The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in either exposure group.

The maximum dose level selected for metaphase analysis was 320 μg/mL and the dose range selected for scoring included both precipitating and non-precipitating dose levels.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 

Chromosome Aberration Test - Experiment 2

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 320 μg/mL in the absence and presence ofS9.

A greasy/oily precipitate of the test item was observed at the end of exposure, at and above 40 μg/mL, in the 4(20)-hour exposure group in the presence of S9. In the 24-hour continuous exposure group, greasy/oily precipitate was seen at and above 80 μg/mL and cloudy precipitate was seen at and above 160 μg/mL.

The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in either exposure group.

The maximum dose level selected for metaphase analysis was 320 μg/mL, the same as Experiment 1. This maximum dose level was selected based on the precipitate observations made in the Preliminary Toxicity Test and was the lowest dose level at which greasy/oily precipitate was seen to occur in the parallel media only cultures of all three exposure groups.Both precipitating and non-precipitating dose levels were selected for metaphase analysis in Experiment 2.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

The test item was considered to be non-toxic and the dose range for the main experiments was limited to a maximum dose of 320 μg/mL. This maximum dose level was based on the precipitate observations made in the Preliminary Toxicity Test and was the lowest dose level at which greasy/oily precipitate was seen to occur in the parallel media only cultures of all three exposure groups. Dose levels scored for chromosome aberrations in Experiments 1 and 2 were selected to include both precipitating and non-precipitating dose levels.

Conclusion
The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be nonclastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

From the in-vitro studies performed, no adverse effects were observed, as such the substance is not considered classified for Genetic Toxicity enpoints