Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Well conducted studies using OECD guidelines provided negative results for three (3) of the four (4) studies with the exception of the chromosome aberration study conducted in HPBL cells which gave an equivocal result. Because of the equivocal result the study was repeated with CHO cells which gave a clearly negative result.

These results provide evidence that the registration substance does not produce in vitro genotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark

- Analytical purity: 100%
- Lot/batch No.: E00275-350
- Expiration date of the lot/batch: 31 Dec 2013
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 derived from Dr. Bruce
Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine
Bacteria, Aberdeen, Scotland.

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 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Vehicle / solvent:
EXP1200078 - tetrahydrofuran (CAS 109-99-9)
Positive controls - dimethyl sulfoxide (CAS 67-68-5)
Water (Sodium Azide control)
Untreated negative controls:
yes
Remarks:
Water, dimethyl sulfoxide, ethanol, acetone
Negative solvent / vehicle controls:
yes
Remarks:
Water, dimethyl sulfoxide, ethanol, acetone
True negative controls:
no
Positive controls:
yes
Remarks:
Table of Positive Controls for Study AD63KG.503.BTL included below.
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (CAS 613-13-8)
Details on test system and experimental conditions:
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock
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 125 to 175 rpm and
incubating at 37±2°C for approximately 12 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.

Metabolic Activation System
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. 3017, Exp. Date: 17 October 2014) was purchased commercially from Moltox (Boone,
NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk
preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and
2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Metabolic Activation System
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. 3017, Exp. Date: 17 October 2014) was purchased commercially from Moltox (Boone,
NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk
preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and
2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
Evaluation criteria:
Initial Toxicity-Mutation Assay
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory
mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control,
positive controls and eight dose levels of the test article were plated, two plates per dose, with
overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal
agar in the presence and absence of Aroclor-induced rat liver S9.

Confirmatory Mutagenicity Assay
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential
of the test article. Five dose levels of test article along with appropriate vehicle control and
positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and
WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver
S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.
Statistics:
means and standard deviations
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the conditions of this study, test article EXP1200078 was concluded to be negative in the
Bacterial Reverse Mutation Assay. The increase observed with tester strain TA1537 in the
presence of S9 activation in the initial toxicity-mutation assay was not considered indicative of
mutagenic activity because (1) the increase was non-dose responsive and not reproducible and
(2) all of the plate counts for each treated dose level were within the historical vehicle control
range. Therefore, the test article was concluded to be negative in this assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012 November 29 to 2013 April 22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 21 July 1997
GLP compliance:
yes
Type of assay:
other: The purpose of this study was to evaluate the genotoxic potential of the test article based on quantitation of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells and sizing of the resulting colonies.
Specific details on test material used for the study:
Test Article I.D.: EXP1200078
Test Article Chemical Name: Capped overbased calcium phenate
Test Article Batch/Lot No.: E00275-350
Test Article Purity: 100%
Description/Appearance: Very dark brown (almost black) viscous liquid
Target gene:
L5178Y/TK+/- Mouse Lymphoma Assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y cells, clone 3.7.2C, were obtained from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC on 14 August 1995. Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination. Prior to use in the assay, L5178Y cells were cleansed of spontaneous TK-/- cells by culturing in a restrictive medium (Clive and Spector, 1975).
Additional strain / cell type characteristics:
other: Thymidine Kinase (TK)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
Based on the viscosity of the test article during test article dilution, the maximum concentration of EXP1200078 in treatment medium was 2000 µg/mL in the preliminary toxicity assay. Visible precipitate was present in the treatment medium at concentrations = 150 µg/mL at the beginning and end of treatment. Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control. Suspension growth relative to the solvent control was 59% and 18% at a concentration of 2000 µg/mL for the non activated and S9 activated cultures with a 4-hour exposure, respectively, and 0% at concentrations = 500 µg/mL for the non-activated cultures with a 24-hour exposure.
Vehicle / solvent:
Solubility tests were conducted to determine the vehicle. The tests were conducted using dimethyl sulfoxide (DMSO), ethanol (EtOH), acetone and tetrahydrofuran (THF) to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 500 mg/mL.

Tetrahydrofuran (THF) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in THF at approximately 500 mg/mL, the maximum concentration tested in the solubility test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
Test System
L5178Y cells, clone 3.7.2C, were obtained from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC on 14 August 1995. Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination. Prior to use in the assay, L5178Y cells were cleansed of spontaneous TK-/- cells by culturing in a restrictive medium.

Metabolic Activation System
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. 2878, Expiration Date: 07 March 2014) was purchased commercially from Moltox (Boone, NC). Upon arrival at BioReliance, 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 Record of Analysis is on file with the testing facility.

The S9 mix was prepared on the day of use. The final concentrations of the components in the test system were as indicated below.
Component Final Concentration in Cultures
Component Final Concentration in Cultures
DL-isocitric acid 17.4 mM
NADP (sodium salt) 3.0 mM
S9 homogenate 10 µL


Rationale for test conditions:
Test conditions based on solubilty limits of test material and cytotoxicy of the test material to the cell cultures.
Evaluation criteria:
The rationale for this procedure is as follows: Mutant L5178Y TK-/- colonies exhibit a characteristic frequency distribution of colony sizes. The precise distribution of large and small TFT-resistant mutant colonies appears to be the characteristic mutagenic "finger-print" of carcinogens in the L5178Y TK+/- system.

(Clive et al., 1979; DeMarini et al., 1989). Clive et al. (1979) and Hozier et al. (1981) have presented evidence to substantiate the hypothesis that the small colony variants carry chromosome aberrations associated with chromosome 11, the chromosome on which the TK locus is located in the mouse. They suggested that large colony mutants received localized damage, possibly in the form of a point mutation or small deletion within the TK locus, while small colony mutants received damage to collateral loci concordant with the loss of TK activity.
Statistics:
None.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
other: Negative control is the same as vehicle control within this study.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Positive and vehicle controls were clear and unambiguous.
Remarks on result:
other: Non-mutagenic within an in vitro mammalian system based on experimental results from this study.

Data tables in attached file ( AD63KG 704 BTL_Tables-ONLY.PDF_.

Table Content
01 PRELIMINARY TOXICITY ASSAY USING EXP1200078, 4-Hour Exposure
01 (continued) PRELIMINARY TOXICITY ASSAY USING EXP1200078, 24-Hour Exposure
02 DATA SUMMARY FOR L5178Y/TK+/- MOUSE LYMPHOMA CELLS TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; Initial Assay (4-hour exposure)
03 DATA SUMMARY FOR L5178Y/TK+/- MOUSE LYMPHOMA CELLS TREATED WITH EXP1200078 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION; Initial Assay (4-hour exposure)
04 DATA SUMMARY FOR L5178Y/TK+/- MOUSE LYMPHOMA CELLS TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; Extended Treatment Assay (24-hour exposure)
Conclusions:
Under the conditions of this study, test article EXP1200078 was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Assay.
Executive summary:

A well conducted, mammalian in vitro gene mutation assay was conducted according to OECD guideline 476 of 21 July 1997. All validation criteria were met, and positive and vehicle controls were comparable to historical records at this testing facility. Therefore, the negative result obtained with the conditions of this assay were met, and EXP1200078 is considered to be negative in the mammalian in vitro gene mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013 Oct 04 to 2013 Dec 30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
26 September 2014
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome abberation test
Specific details on test material used for the study:
Test Substance I.D.: EXP1200078
Test Substance Lot Number: E00275-350
Test Substance Purity: 100%
Test Substance Description: Blackish orange clear viscous liquid
Target gene:
None
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary (CHO K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
For the definitive assay only, two hours prior to cell harvest, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts, and then Colcemid® was added to all cultures at a final concentration of 0.1 µg/mL.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was the metabolic activation system. The S9 (Lot No. 3095) was obtained from Molecular Toxicology Inc. Each lot of S9 was assayed by the supplier for sterility and its ability to transform Salmonella typhimurium TA100.
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the dose levels tested ranged from 5 to 50 µg/mL. Dose levels for the preliminary toxicity assay were selected based on the precipitate profile of the test article in treatment medium. Substantial toxicity (at least 50% cell growth inhibition, relative to the vehicle control) was not observed at any dose level in all three exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 5 to 30 µg/mL for all three exposure groups.
Vehicle / solvent:
Tetrahydrofuran (THF) was used as the vehicle based on solubility data obtained from BioReliance Study, In Vitro Mammalian Chromosome Aberration Assay in Human Peripheral Blood Lymphocytes (HPBL) (AD63KG.341.BTL).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Mitomycin C was used as the positive control article in the non-activated test system. Cyclophosphamide was used as the positive control article in the S9 activated test system.
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Both were dissolved in and diluted in sterile distilled water (Gibco; CAS No. 7732-18-5, Lot No. 1247860, Exp. Date November 2014).
Details on test system and experimental conditions:
Preparation of Target Cells
Exponentially growing CHO K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 2 mM L glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1¿C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16 24 hours.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 (Lot No. 3095) was obtained from Molecular Toxicology Inc. (Boone, NC). Each bulk preparation of S9 was assayed by the supplier for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.

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 (McCoy's 5A serum-free medium supplemented with
100 units penicillin/mL, 100 µg streptomycin/mL, 2 mM L-glutamine and 2.5 µg/mL amphotericin B).

Experimental Design
The in vitro mammalian chromosome aberration assay was conducted using standard procedures (Galloway et al, 1994; Preston et al, 1981; Swierenga et al, 1991) by exposing Chinese hamster ovary (CHO) cells to appropriate concentrations of the test substance 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
Test article precipitate in the treatment medium without S9-activation was evaluated by light microscopy by adding appropriate volume of stock dosing formulations (1% v/v for organic
vehicle) in 5 mL treatment medium in culture flasks at 5000, 1500, 500, 150, 50, 40, 30, 20, 10, 5, 3.5, 2.0, 1.0, 0.5, and 0.1 µg/mL. Flasks were incubated for 4 and 20 hours under standard conditions (37 ± 1C in a humidified atmosphere of 5 ± 1% CO2 in air). Following incubation, the test article precipitate in the treatment medium was again evaluated by light microscopy.

Based on the precipitate profile of the test article in the treatment medium, CHO cells were exposed to vehicle alone and at dose levels of 50, 45, 40, 35, 30, 25, 20, 10, and 5 µg/mL of test article using single culture. Precipitation of test substance 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. Dose levels for the definitive assay were based upon visible precipitation of the test substance in treatment medium.


Rationale for test conditions:
standard test conditions that have good historical record in the testing facility
Evaluation criteria:
The results for the positive and negative controls indicate that all criteria for valid assay were met. Based on these criteria, the negative result is justified and does not require a repeat of any portions of the study.

Criteria for Determination of a Valid Test
The frequency of cells with structural chromosome aberrations in the vehicle control must be within the historical control range. The percentage of cells with aberrations must be statistically increased (p ¿ 0.05, Fisher's exact test) in the positive control condition relative to the vehicle control. The Historical Control Data are included in Appendix III.
Statistics:
Fisher's exact test; Cochran-Armitage test
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
other: No recordable chromosome aberations relative to positive, vehicle and historical controls.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All controls performed as expected when compared to historical information at this testing facility.
Remarks on result:
other: EXP1200078 was negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems.

Content of PDF attachment.

Table Content
1 PRELIMINARY TOXICITY TEST USING EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD
2 PRELIMINARY TOXICITY TEST USING EXP1200078 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD
3 PRELIMINARY TOXICITY TEST USING EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 20-HOUR CONTINUOUS TREATMENT
4 CONCURRENT TOXICITY TEST USING EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD
5 CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD
6 CONCURRENT TOXICITY TEST USING EXP1200078 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD
7 CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH EXP1200078 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 16-HOUR RECOVERY PERIOD
8 CONCURRENT TOXICITY TEST USING EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 20-HOUR CONTINUOUS TREATMENT
9 CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 20-HOUR CONTINUOUS TREATMENT
10 SUMMARY
Conclusions:
The positive and vehicle controls fulfilled the requirements for a valid test.

Under the conditions of the assay described in this report, EXP1200078 was determined to be negative for the induction of structural and numerical chromosome aberrations in CHO cells using both the non-activated and the S9-activated test systems.
Executive summary:

EXP1200078 was negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Remarks:
The experimental result was equivocal rather than negative like other genotoxicity studies on this material which were all negative. Our experience with this material has been that it has proved to have low reactivity in all biological systems.
Adequacy of study:
supporting study
Study period:
29 Novenber 2012 to 22 April 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
26 September 2014
Qualifier:
equivalent or similar to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome abberation test
Specific details on test material used for the study:
Test Substance I.D.: EXP1200078
Test Substance Lot Number: E00275-350
Test Substance Purity: 100%
Test Substance Description: Blackish orange clear viscous liquid
Target gene:
None
Species / strain / cell type:
lymphocytes: Human Peripheral Blood Lymphocytes (HPBL)
Details on mammalian cell type (if applicable):
Peripheral blood lymphocytes were obtained from healthy adults, 18-35 years of age, non-smokers, without a recent history of radiotherapy, viral infections 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).
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
For the definitive assay only, two hours prior to cell harvest, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts, and then Colcemid® was added to all cultures at a final concentration of 0.1 µg/mL.
Metabolic activation:
with and without
Metabolic activation system:
Activation using aroclor 1254-induced rat liver S9, (Lot No. 3019, Molecular Toxicology Inc., Boone, NC). Bulk preparations assayed for sterility and ability to metabolize at least two pro-mutagens. S9 thawed prior to use and mixed with a cofactor pool.
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the doses tested ranged from 0.25 to 2500 µg/mL. The top dose was based on limited solubility of the test article in the treatment medium. Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Substantial toxicity was observed at 2500 µg/mL in the S9 activated 4-hour and the non activated 20-hour exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 2.5 to 100 µg/mL for all three exposure groups.
Vehicle / solvent:
Solubility tests were conducted to determine the vehicle using DMSO, ethanol, acetone and THF, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 500 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Mitomycin C was used as the positive control article in the non-activated test system. Cyclophosphamide was used as the positive control article in the S9 activated test system.
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Experimental Design
The in vitro mammalian 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 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.

Chromosome Aberration Assay
Seven dose levels were tested using duplicate cultures at appropriate dose intervals based on the precipitate 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 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. Whenever possible, the highest dose level evaluated for the chromosome aberrations was selected based on visible precipitation of the test article in treatment medium. At least 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 0.05 mL 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.
Rationale for test conditions:
standard test conditions that have good historical record in the testing facility
Evaluation criteria:
The results for the positive and negative controls indicate that all criteria for valid assay were met. Based
on these criteria, the negative result is justified and does not require a repeat of any portions of the study.

Criteria for Determination of a Valid Test
The frequency of cells with structural chromosome aberrations in the vehicle control must be within the historical control range. The percentage of cells with aberrations must be statistically increased (p <= 0.05, Fisher's exact test) in the positive control condition relative to the vehicle control.
Statistics:
Fisher's exact test; Cochran-Armitage test
Key result
Species / strain:
lymphocytes: Human Primary Blood Lymphocytes
Metabolic activation:
with and without
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Attached data tables:

Table Content
1 PRELIMINARY TOXICITY TEST USING EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 20-HOUR HARVEST
2 PRELIMINARY TOXICITY TEST USING EXP1200078 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 20-HOUR HARVEST
3 PRELIMINARY TOXICITY TEST USING EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 20-HOUR TREATMENT, 20-HOUR HARVEST
4 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; DEFINITIVE ASSAY: 4-HOUR TREATMENT, 20-HOUR HARVEST
5 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH EXP1200078 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION; DEFINITIVE ASSAY: 4-HOUR TREATMENT, 20-HOUR HARVEST
6 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; DEFINITIVE ASSAY: 20-HOUR TREATMENT, 20-HOUR HARVEST
7 Summary
8 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; DEFINITIVE ASSAY: 4-HOUR TREATMENT, 20-HOUR HARVEST (COMBINED DATA)
9 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH EXP1200078 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION; DEFINITIVE ASSAY: 4-HOUR TREATMENT, 20-HOUR HARVEST (COMBINED DATA)
10 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH EXP1200078 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; DEFINITIVE ASSAY: 20-HOUR TREATMENT, 20-HOUR HARVEST (COMBINED DATA)
10

SUMMARY (COMBINED DATA)

Attached appendices:

Appendix Content
III Historical Control Data
IV Common Technical Document (CTD) Summary Tables
Conclusions:
All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ¿ 0.01). Thus, the study fulfilled all criteria required for a valid study.

Under the conditions of the assay described in this report, EXP1200078 was concluded to be equivocal for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in HPBL in both non-activated and S9 activated test systems.
Executive summary:

It can not be determined if EXP1200078 is capable of causing structural alterations to structural chromosomes using HPBL as the target in an in vitro experimental system.

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

Genetic toxicity in vivo

Description of key information

A well conducted study (OECD 474) in rats examining the production of bone marrow micronuclei resulted in negative findings at a dose of 2000 mg/kg. These results demonstrate that the registration substance does not produce genotoxicity using an in vivo protocol.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2013 to 07 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
other: The mammalian in vivo micronucleus test detects damage induced by the test chemical to the chromosomes or the mitotic apparatus of erythroblasts. It evaluates micronucleus formation in erythrocytes sampled from bone marrow or peripheral blood cells.
Specific details on test material used for the study:
Test Substance I.D.: EXP1200078
Test Substance Lot Number: E00275-350
Test Substance Purity: 100%
Test Substance Description: Blackish orange clear viscous liquid
Species:
rat
Strain:
other: Sprague Dawley Hsd:SD rats
Details on species / strain selection:
6 weeks old at study initiation
Sex:
male
Details on test animals and environmental conditions:
Housing
Animals were housed in a controlled environment at 72 ± 3¿F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Bedding, Food and Water
Heat treated hardwood chips were used for bedding to absorb liquids. A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Housing
Animals were housed in a controlled environment at 72 ± 3¿F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Bedding, Food and Water
Heat treated hardwood chips were used for bedding to absorb liquids. A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.
Route of administration:
oral: gavage
Vehicle:
The vehicle control was Mineral Oil (CAS No.: 8042-47-5, Lot No.: MKBP0802V, Expiration Date: 31 March 2018 obtained from Sigma Aldrich. The dose formulation was prepared prior to dose administration.
Details on exposure:
A single oral dose at 2000 mg/kg was given via gavage. The dose was based on results of the 28d and 90d repeat dose studies which demonstrated that 2000 mg/kg was the MTD and well tolerated.
Duration of treatment / exposure:
The were two groups of control/vehicle and two groups at the MTD (2000 mg/kg). Group 1 was kept for 24 hours, and group 2 was kept for 48 hours.
Frequency of treatment:
One single dose.

Dose Administration and Observation
All dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the mammalian bone marrow erythrocyte micronucleus assay. Body weights were recorded prior to the first dose for the purpose of dose volume calculations. Animals were observed prior to dose, approximately one and two hours after dose administration and daily thereafter for clinical signs of toxicity.
Post exposure period:
Group 1 - 24 hours
Group 2 - 48 hours
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
MTD based on OECD 407 and OECD 409
No. of animals per sex per dose:
Group 1 - 5 animals
Group 2 - 5 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide; 40 mg/kg; duration = 24 hours.
Tissues and cell types examined:
Femoral bone marrow was collected at approximately 24 or 48 hours after the single dose.
Details of tissue and slide preparation:
Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of slides was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.
Evaluation criteria:
Bone marrow was evaluated by fluorescent microscopy. The staining procedure permitted the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost like, dark green NCEs, respectively). The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus were counted as one micronucleated PCE (mnPCE), not two (or more) micronuclei.

At least 1000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE/EC proportions <20% of vehicle control value were considered excessively cytotoxic and the animal data was excluded from evaluation. The frequency of mnPCEs and the proportion of PCEs to total erythrocytes was determined for each animal and treatment group.
Statistics:
Statistical significance (p = 0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Criteria for a Valid Test
The vehicle control group should be consistent with the historical vehicle control range, and must be = 0.4% mnPCEs (Aikihiro et al., 1998), and the positive control must induce significant increase (p = 0.05) in mnPCE frequency as compared to the concurrent vehicle control.

Five animals/group were available for analysis

Evaluation of Test Results

Once the criteria for a valid assay were met, the results were evaluated. Test substance was considered to be positive if it induced a significant increase in mnPCE frequency (p£0.05) at any dose level or sampling time compared to the concurrent vehicle control. The test substance was considered to be negative if no significant increase in mnPCE frequency was observed (p > 0.05) compared to the concurrent vehicle control. Other criteria may have been used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Definitive Micronucleus Assay

No mortality occurred during the course of the definitive assay. All rats appeared normal throughout the observation period. Clinical signs are presented inTable 1.

Bone Marrow Analysis

No appreciable reductions in the PCEs/EC ratio in the test substance group compared to the vehicle control group were observed indicating the test did not induce cytotoxicity.

No statistically significant increase in the incidence of mnPCEs in the test-substance treated group was observed relative to the negative control group (p > 0.05, Kastenbaum-Bowman tables). The positive control induced a statistically significant increase in the incidence of mnPCEs (p < 0.05, Kastenbaum-Bowman tables). The number of mnPCEs in the vehicle control groups did not exceed the historical control range.

The incidence of mnPCEs per 10,000 PCEs scored (2000 PCEs/animal) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time inTable 2. Individual animal data is presented inTable 3. 

Based upon this, all criteria for a valid test were met as specified in the protocol. The Common Technical Document (CTD) Summary Table is included inAppendix IV.

Conclusions:
Under the conditions of this study, the administration of EXP1200078 at a dose of 2000 mg/kg was concluded to be negative in the Micronucleus assay.
Executive summary:

A well conducted guideline study to examine in vivo mutagenesis (chromosome aberration) of EXP1200078 at the MTD (2000 mg/kg) did not result in any mutagenic response. The test substance, EXP1200078,was evaluated for its clastogenic activity and/or disruption
of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone marrow.

No adverse effects were found at the dose of 2000 mg/kg in this in vivo genetic toxicity assay.

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

Additional information

Justification for classification or non-classification

The registration substance did not produce adverse effects in well conducted in vitro and in vivo genetic toxicity studies.

Therefore, in accordance to Regulation (EC) No 1272/2008 it is concluded that the registration substance does not merit any classification related to the genotoxicity endpoint.