Potassium sodium amino-hydroxy-[(3-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]-[(2-sulfonato-4-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

Study initiation date: 17 November 2014; Experimental starting date: 18 November 2014; Experimental completion date: 25 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See section "Deviation" in the field "Any other information on results incl. tables"

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

Induction of reverse mutations at selected loci of several strains of Salmonella typhimurium (histidine) and at the tryptophan locus of Escherichia coli strain WP2 uvrA without S9 activation, with Aroclor 1254-induced rat liver S9 activation (oxidative) and with uninduced hamster liver S9 activation (reductive).

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 activation (oxidative) and with uninduced hamster liver S9 activation (reductive).

Test concentrations with justification for top dose:

- Preliminary toxicity assay: 6,7 / 10 / 33 / 67 / 100 / 333 / 667 / 1000 / 3333 / 5000 microgr. per plate.
- Mutagenicity assay: 300 / 600 / 1000 / 3000 / 5000 microgr. per plate.
- Retest of the mutagenicity assay: 300 / 600 / 1000 / 3000 / 5000 microgr. per plate.

Vehicle / solvent:

- Vehicle: Sterile water
- CAS number: 7732-18-5
- Supplier: Mediatech, Inc.
- Lot: 25055624 and 25055615
- Purity grade: Water for injection quality
Expiration date: June 2017 and May 2017

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

steril water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA1535, TA100, TA1537 and WP2uvrA (with rat S9 metabolic activation).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

steril water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

methylmethanesulfonate

other: Sodium azide

Remarks:

without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

steril water

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

TA 98 with Hamster S9 metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: The test system was exposed to the test substance via the preincubation methodology described by Yahagi et al. (1977), and further modified for reductive activation conditions by Prival and Mitchell (1982).

DURATION
- Preincubation period: 60±2 minutes at 37±2°C (without S9 and with oxidative S9) or for 30±2 minutes at 30±2°C (reductive S9).
- Exposure duration: 48 to 72 hours at 37°C +/-2°C

NUMBER OF REPLICATIONS: All dose levels of test substance, vehicle control and positive controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
- The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in "Any other information on materials and methods inlc. tables" field. As appropriate, colonies were enumerated either by hand or by machine.

Evaluation criteria:

Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
- Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

The primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:

- LIMS Labware System: Test Substance Tracking
- Excel 2007 (Microsoft Corporation): Calculations
- Sorcerer Colony Counter and Ames Study Manager (Perceptive Instruments): Data Collection/Table Creation
- Kaye Lab Watch Monitoring system (Kaye GE): Environmental Monitoring
- BRIQS: Deviation and audit reporting

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

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

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Solubility Test

Water was selected as the solvent of choice based on information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water for injection-quality, cell culture grade water at a concentration of approximately 50 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Tester strain Titer results:

Experiment	Tester strain
	TA98	TA100	TA1535	TA1537	WP2 uvrA
	Titer value (X10E9 cells per ml)
B1	4.2	3.8	4.1	7.7	8.0
B2	5.3	1.8	3.1	3.7	8.2

Preliminary Toxicity Assay

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Increases in revertant counts (3.0- and 3.1-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

Mutagenicity Assay

In Experiment B1 (Mutagenicity Assay), positive mutagenic responses (2.0- and 2.6-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. No positive mutagenic responses were observed with the remaining test conditions. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed; however, reductions in revertant counts were observed beginning at 3000 or at 5000 μg per plate with a few test date provided by the supplier, it was more than six months beyond its preparation date as specified in the study protocol. Therefore, the reductive metabolic activation condition was retested in Experiment B2.

In Experiment B2 (Retest of the Mutagenicity Assay), a positive mutagenic response (2.5-fold maximum increase) was observed with tester strain WP2 uvrA in the presence of reductive S9 activation. No positive mutagenic responses were observed with the remaining test conditions in the presence of reductive S9 activation. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Deviation:

The lot of uninduced hamster liver (reductive) S9 homogenate used in the mutagenicity assay was not within six months of its preparation date at the time of use. The reductive metabolic activation condition was retested using uninduced hamster liver S9 homogenate that was within six months of its preparation date. Therefore, the Study Director has concluded that this deviation had no adverse impact on the integrity of the data or the validity of the study conclusion.

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, FAT 40868/A TE did cause positive mutagenic responses with tester strain WP2 uvrA in the presence of both oxidative and reductive S9 activation.

Executive summary:

The test substance, FAT 40868/A TE, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the absence of S9 activation and in the presence of both Aroclor-induced rat liver S9 activation (oxidative) and uninduced hamster liver S9 activation (reductive). The assay was performed in two phases, using the preincubation method. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test substance. Water was selected as the solvent of choice based on information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water for injection-quality, cell culture grade water at a concentration of approximately 50 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Increases in revertant counts (3.0- and 3.1-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, positive mutagenic responses (2.0- and 2.6-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. No positive mutagenic responses were observed with the remaining test conditions. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed; however, reductions in revertant counts were observed beginning at 3000 or at 5000 μg per plate with a few test conditions. Although the reductive S9 used in the mutagenicity assay was within the expiration date provided by the supplier, it was more than six months beyond its preparation date as required by the study protocol. Therefore, the reductive metabolic activation condition was retested.

In the retest of the mutagenicity assay, a positive mutagenic response (2.5-fold maximum increase) was observed with tester strain WP2 uvrA in the presence of reductive S9 activation. No positive mutagenic responses were observed with the remaining test conditions in the presence of reductive S9 activation. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Under the conditions of this study, FAT 40868/A TE was concluded to be positive with tester strain WP2 uvrA in the presence of both oxidative and reductive S9 activation in the Bacterial Reverse Mutation Assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date (first day test substance administered to test system): 09 December, 2014 and Experimental Completion Date:19 January, 2015

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

None

Target gene:

Not applicable

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. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).

Additional strain / cell type characteristics:

not applicable

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:

- Preliminary toxicity test without exogenous metabolic activation: 4 hours treatment, 16 hours recovery period:
0.5/1.5/5/15/50/150/500/1500 and 5000 microgr/mL.

- Preliminary toxicity test with exogenous metabolic activation: 4 hours treatment, 16 hours recovery period:
0.5/1.5/5/15/50/150/500/1500 and 5000 microgr/ml.

- Preliminary toxicity test without exogenous metabolic activation: 20 hours continuous treatment
0.5/1.5/5/15/50/150/500/1500 and 5000 microgr/ml.

- Initial chromosome aberration assay without exogenous metabolic activation: 4 hours treatment, 16 hours recovery period:
500/1500/3000/5000 microgr/ml

- Initial chromosome aberration assay with exogenous metabolic activation: 4 hours treatment, 16 hours recovery period:
500/1500/3000/5000 microgr/ml

- Initial chromosome aberration assay with exogenous metabolic activation: 20 hours continuous treatment:
100/250/500/700/800/900/1000/1250/1500 microgr/ml

- Repeat chromosome aberration assay without exogenous metabolic activation: 4 hours treatment, 16 hours recovery period:
100/200/400/800/1000/2000 microgr./ml

Vehicle / solvent:

The vehicle used was steril water.
- Supplier: Gibco
- CAS N°: 7732-18-5
- Lot: 1420169 and 1508375
- Exp. date: August 2015 and December 2015

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Scoring for Metaphase Chromosome Aberrations (Chromosome Aberration Assays):
The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture may be reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure was not be scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 100 cells per culture (200 cells per dose).

Evaluation criteria:

Toxicity induced by treatment is based upon inhibition of mitosis and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.
A test substance was considered positive if it induced a statistically significant and dose-dependent increase in the frequency of aberrant metaphases (p ≤ 0.05). If only Fisher's exact test was statistically significant without dose-dependent increase, the results may be considered equivocal. If neither criterion was met, the results were considered to be negative.

Statistics:

The primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:
- LIMS Labware System: Test Substance Tracking
- Excel (Microsoft Corporation): Calculations
- Kaye Lab Watch Monitoring system (Kaye GE): Environmental Monitoring
- BRIQS: Deviation and audit reporting

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Solubility Test

Water was used as the vehicle based on the information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a clear solution in sterile water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

Preliminary Toxicity Assay

A preliminary toxicity assay was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive chromosome aberration assay. CHO cells were first exposed to nine dose levels of FAT 40868/A TE, ranging from 0.5 to 5000 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test substance formed workable suspensions in water at concentrations ≥ 0.15 mg/mL, while concentrations ≤ 0.05 mg/mL were soluble in water. Visible precipitate was observed in treatment medium at the following dose levels:

		Visible precipitate
Treatment condition	Treatment time	At the beginning of Treatment period	At the conclusion of treatment period
Non-activated	4 hrs	≥ 500 μg/mL	≥ 1500 μg/mL
Non-activated	20 hrs	≥ 500 μg/mL	≥ 1500 μg/mL
S9 activated	4 hrs	≥ 500 μg/mL	≥ 1500 μg/mL

The osmolality in treatment medium was measured as follows:

Dose tested	Dose levels (μg/mL)	Osmolality (mmol/kg)
Vehicle	0	262
Highest	5000	287
Lowest precipitating	500	268
Highest soluble	150	265

The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest dose level of test substance in treatment medium was 7.5.

Substantial toxicity (at least 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. Substantial toxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 1500 μg/mL in the non-activated 20-hour exposure group. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

Treatment condition	Treatment time	Recovery time	Dode level (microgr/ml)
Non-activated	4 hrs	16 hrs	500/1500/3000/5000
Non-activated	20 hrs	0 hr	100/250/500/700/800/900/1000/1250/1500
S9 activated	4 hrs	16 hrs	500/1500/3000/5000

Initial Chromosome Aberration Assay

In the initial assay, the test substance formed workable suspensions in water at all concentrations tested. The pH of the highest dose level of test substance in treatment medium was 7.5. Visible precipitate was observed in treatment medium at the following dose levels:

		Visible precipitate
Treatment condition	Treatment time	At the beginning of Treatment period	At the conclusion of treatment period
Non-activated	4 hrs	≥ 500 μg/mL	≥ 1500 μg/mL
Non-activated	20 hrs	≥ 250 μg/mL	≥ 700 μg/mL
S9 activated	4 hrs	≥ 500 μg/mL	≥ 1500 μg/mL

Toxicity of FAT 40868/A TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was 55% at 700 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 700 μg/mL, was 13% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 100, 250, and 700 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (31.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Due to lack of requisite number of non-precipitating doses for scoring chromosome aberrations, the assay was repeated in the non-activated and S9-activated 4-hour exposure groups at dose levels of 100, 200, 400, 800, 1000, 2000 μg/mL.

Repeat Chromosome Aberration Assay

In the repeat assay, the test substance formed workable suspensions in water at all concentrations tested. The pH of the highest dose level of test substance in treatment medium was 7.0. Visible precipitate was observed in treatment medium at the following dose levels:

		Visible precipitate
Treatment condition	Treatment time	At the beginning of Treatment period	At the conclusion of treatment period
Non-activated	4 hrs	≥ 200 μg/mL	≥ 800 μg/mL
S9-activated	4 hrs	≥ 200 μg/mL	≥ 800 μg/mL

Toxicity of FAT 40868/A TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 15% at 800 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 800 μg/mL, was not reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 200, 400, and 800 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (27.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Toxicity of FAT 40868/A TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was not observed at 800 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 800 μg/mL, was 3% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 200, 400, and 800 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) treatment group (52.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

HISTORICAL CONTROL DATA:

IN VITRO MAMMALIAN CYTOGENETIC TEST USING

CHINESE HAMSTER OVARY (CHO) CELLS

HISTORICAL CONTROL VALUES

STRUCTURAL ABERRATIONS

2011-2013

NON-ACTIVATED TEST SYSTEM

Historical value	Solvent (%)	Positive control (%)
Mean	0.438	17.959
+/- SD1	0.529	4.220
Range	0.0 -2.5	9.0 -36.0

S9-ACTIVATED TEST SYSTEM

Historical value	Solvent (%)	Positive control (%)
Mean	0.656	24.220
+/- SD1	0.737	6.768
Range	0.0 -4.0	15.0 -44.0

1 SD = standard deviation.

2 Positive control for non-activated studies, Mitomycin C (MMC).

3 Positive control for S9-activated studies, cyclophosphamide (CP).

IN VITRO MAMMALIAN CYTOGENETIC TEST USING

CHINESE HAMSTER OVARY (CHO) CELLS

HISTORICAL CONTROL VALUES

COMBINED NUMERICAL ABERRATIONS

(POLYPLOID AND ENDOREDUPLICATED CELLS)

2011-2013

NON_ACTIVED TEST SYSTEM

Historical value	Solvent (%)	Positive control (%)
Mean	1.364	1.391
+/- SD1	1.183	1.152
Range	0.0 -5.5	0.0 -5.0

S9-ACTIVATED TEST SYSTEM

Historical value	Solvent (%)	Positive control (%)
Mean	1.978	1.482
+/- SD1	1.592	1.290
Range	0.0 -9.5	0.0 -4.5

1 SD = standard deviation.

2 Positive control for non-activated studies, Mitomycin C (MMC).

3 Positive control for S9-activated studies, cyclophosphamide (CP).

Conclusions:

FAT 40868/A TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

The test substance, FAT 40868/A TE, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, the CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Water was used as the vehicle based on the information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a clear solution in sterile water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 μg/mL. Substantial toxicity (at least 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. Substantial toxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 1500 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 500 to 5000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 100 to 1500 μg/mL for the non-activated 20-hour exposure group.

In the initial chromosome aberration assay, substantial toxicity was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. Substantial toxicity was observed at dose levels ≥ 700 μg/mL in the non-activated 20-hour exposure group. However, due to lack of requisite number of non-precipitating doses for scoring chromosome aberrations, the assay was repeated in the non-activated and S9-activated 4-hour exposure groups at dose levels ranging from 100 to 2000 μg/mL. In the repeat assay, substantial toxicity was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced an approximately 50% reduction in cell growth index which met the dose limit as recommended by testing guidelines for this assay. No significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). 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, all criteria for a valid study were met.

Under the conditions of the assay described in this report, FAT 40868/A TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date (First Day of Dosing): 30 March 2015, Experimental Completion Date: 15 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

None

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Sprague-Dawley (Hsd:SD) rats were received from Harlan, Frederick, MD on 25 March 2015.

The age at time of initiation and body weights of the rats assigned to the study groups at randomization were within the following ranges:

Sex: Male;

Age at Time of Initiation: 6 weeks;

Body Weight Range at Time of Randomization: 166.6-188.8 grams.

Animal Welfare Provisions, Receipt and Acclimation:
This study was not duplicative or unnecessary. The number of animals, procedures, and design used for this study, were reviewed and approved by the BioReliance Institutional Animal Care and Use Committee. All procedures performed at BioReliance which involved animals, followed the specifications recommended in the most current version of The Guide for the Care and Use of Laboratory Animals adopted by BioReliance. The animals were acclimated for 5 days. Animals were observed daily for signs of illness or poor health. All animals were judged to be healthy prior to use in 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.

Randomization and Identification:
Animals were assigned to groups using a randomization procedure within Microsoft Excel. At the time of randomization, the weight variation of animals did not exceed ±20% of the mean weight. Following randomization, animals were identified by sequentially numbered ear tags. The cage card contained, at least, the animal number(s), sex, study number, treatment group number, dose level, test substance ID and route of administration. Cage cards were color coded by treatment group. Raw data records and specimens were also identified by the unique animal number.

Route of administration:

oral: gavage

Vehicle:

Deionized water

Details on exposure:

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 oral 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, approximately one and two hours after dose administration and daily thereafter for clinical signs of toxicity.

Frequency of treatment:

Once

Post exposure period:

24 or 48 hour

Remarks:

Doses / Concentrations:
500, 1000, 2000
Basis:
nominal conc.

No. of animals per sex per dose:

Vehicle control: 10 males;
High dose group (2000 mg/kg): 10 males;
Other groups- low (500 mg/kg) and middle dose group (1000 mg/kg) and positve control (Cyclophosphamide 40 mg/kg): 5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide: 40 mg/kg

Details of tissue and slide preparation:

Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose, as indicated above. Animals were euthanized by carbon dioxide inhalation. 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 and will be archived at report finalization. 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:

Criteria for a Valid Test
The MnPCE frequency of the vehicle controls 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 to clearly report and describe any such considerations.

Statistics:

Kastenbaum-Bowman tables

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Definitive Micronucleus Assay
No mortality occurred at any dose level during the course of the definitive assay. All rats appeared normal throughout the observation period.
Clinical signs are presented in Table 1.

Bone Marrow Analysis
No appreciable reductions in the PCEs/EC ratio in the test substance groups compared to the vehicle control group was observed indicating the test article did not induce cytotoxicity.
No statistically significant increase in the incidence of MnPCEs in the test-substance treated groups 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 in Table 2. Individual animal data is presented in Table 3 and Table 4.
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 in Appendix III.

Table 1: Definitive Assay – Clinical Signs

Treatment	Observation	Number of Animals With Observed Signs/Number of Surviving Animals					Number of Animals Found Dead/Total Number of Animals Dosed
		Males					Males
		Day 0			Day 1	Day 2
		Pre-Dose	Post-Dose
			1 Hr	2 Hr
Deionized water	Normal	10/10	10/10	10/10	10/10	5/5	0/10
FAT 40868/A TE 500 mg/kg/day	Normal	5/5	5/5	5/5	5/5	NA	0/5
1000 mg/kg/day	Normal	5/5	5/5	5/5	5/5	NA	0/5
2000 mg/kg/day	Normal	10/10	10/10	10/10	10/10	5/5	0/10
CP 40 mg/kg/day	Normal	5/5	5/5	5/5	5/5	NA	0/5

N/A = No data due to 24 hour bone marrow collection.

Table 2: Summary of Bone Marrow Micronucleus Analysis

Treatment	Sex	Time (h)	Number of animals	PCE/Total Erythrocytes (Mean +/- SD)	Change from Control (%)	Number of MnPCE/1000 PCE (Mean +/- SD)	Number of MnPCE/PCE scored
Deionized water	M	24	5	0.531±0.00	---	0.1±0.22	1	/	1000
500 mg/kg	M	24	5	0.527±0.00	-1	0.3±0.27	3	/	10000
1000 mg/kg	M	24	5	0.528±0.01	-1	0.2±0.27	2	/	10000
2000 mg/kg	M	24	5	0.535±0.01	1	0.3±0.45	3	/	10000
40 mg/kg	M	24	5	0.548±0.00	3	23.6±1.71	*236	/	10000
Deionized water	M	48	5	0.526±0.01	---	0.2±0.27	2	/	1000
2000 mg/kg	M	48	5	0.527±0.01	0	0.3±0.27	3	/	10000

*Statistical significant increase compared to vehicle control p ≤ 0.05 (Kastenbaum-Bowman Tables)

PCE – polychromatic erythrocytes; MnPCE – micronucleated polychromatic erythrocytes

Table 3: Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Collected 24 Hours Post-Dose Administration

Treatment	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Deionized water	M	1	0.527	0/2000
		2	0.526	0/2000
		3	0.534	0/2000
		4	0.531	0/2000
		5	0.535	1/2000
FAT 40868/A TE 500 mg/kg	M	6	0.529	0/2000
		7	0.529	1/2000
		8	0.531	1/2000
		9	0.524	1/2000
		10	0.522	0/2000
FAT 40868/A TE 1000 mg/kg	M	11	0.531	0/2000
		12	0.527	1/2000
		13	0.524	1/2000
		14	0.536	0/2000
		15	0.521	0/2000
FAT 40868/A TE 2000 mg/kg	M	16	0.541	0/2000
		17	0.541	1/2000
		18	0.529	0/2000
		19	0.537	/22000
		20	0.526	0/2000
Cyclophosphamide 40 mg/kg	M	21	0.552	48/2000
		22	0.545	46/2000
		23	0.553	49/2000
		24	0.543	42/2000
		25	0.547	51/2000

 

Table 4: Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Collected 48 Hours Post-Dose Administration

Treatment	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Deionized water	M	26	0.528	0/2000
		27	0.535	1/2000
		28	0.521	1/2000
		29	0.517	0/2000
		30	0.530	0/2000
FAT 40868/A TE 2000 mg/kg	M	31	0.532	1/2000
		32	0.525	1/2000
		33	0.519	1/2000
		34	0.536	0/2000
		35	0.524	0/2000

PCE – polychromatic erythrocytes; MnPCE – micronucleated polychromatic erythrocytes

 

APPENDIX III: Common Technical Document (CTD) Table

Report Title:In VivoMicronucleus Assay in Rats			Test Substance:FAT 40868/A TE
Test for Induction of:	Bone marrow micronuclei	Treatment Schedule:	One dose	BioReliance Study No.:	AE04PT.125M012REACH.BTL
Species/Strain/Sex:	Hsd/SD rats/ males		Sampling Times:	24 & 48 hours post-dose
Age:	6 weeks old	Route of Administration:	Oral gavage	GLP Compliance:	Yes
Cells Evaluated:	Polychromatic erythrocytes (PCE)		Vehicle for the Test Substance Formulation: Vehicle/Positive control:	Deionized water Sterile water for injection/Cyclophosphamide (CP)
No. of Cells Analyzed/Animal:	2000 PCE/animal	Feeding Condition:	Ad libitum	Date of Dosing:	30 March 2015
Special Features:			N/A
Toxic/Cytotoxic Effects:			No mortality occurred at any dose level during the course of the definitive assay. All rats appeared normal throughout the observation period.
Genotoxic Effects:			No statistically significant increase in the incidence of MnPCEs in the test-substance treated groups 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.
Evidence of Exposure:			N/A

 

Sampling Time	Controls/Test Substances	Dose Level mg/kg	Sex/No. of Animals/Group	PCE/ Total Erythrocyte (Mean± SD)	Number MnPCE/PCE scored
24 hrs post-dose	Vehicle**	0	Males/5/group	0.531 ± 0.00	1/10000
	FAT 40868/A TE	500	Males/5/group	0.527 ± 0.00	3/10000
		1000	Males/5/group	0.528 ± 0.01	2/10000
		2000	Males/5/group	0.535 ± 0.01	3/10000
	Cyclophosphamide	40	Males/5/group	0.548 ± 0.00	*236/10000
48 hrs post-dose	Vehicle**	0	Males/5/group	0.526 ± 0.01	2/10000
	FAT 40868/A TE	2000	Males/5/group	0.527 ± 0.01	3/10000

*Statistically significant increase, p ≤ 0.05 (Kastenbaum-Bowman Tables, binomial distribution)

**Deionized water

PCE: Polychromatic Erythrocytes; MnPCE: Micronucleated Polychromatic Erythrocytes

Conclusions:

FAT 40868/A TE did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes. Therefore, FAT 40868/A was concluded to be negative.

Executive summary:

The test substance, FAT 40868/A TE, 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. Deionized water was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg by oral gavage. In the definitive assay, dose levels tested were 500, 1000 or 2000 mg/kg body weight. Groups 1 and 4 consisted of 10 animals each designated for either 24 or 48 hour bone marrow collections and Groups 2, 3 and 5 consisted of 5 animals each designated for 24 hour bone marrow collection. Following scheduled euthanasia times, femoral bone marrow was collected; bone marrow slides were prepared and stained with acridine orange. Bone marrow cells [polychromatic erythrocytes (2000 PCEs/animal)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MnPCEs) and statistical analysis of data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05). The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes (EC) in the test substance groups relative to the vehicle control groups was also evaluated to reflect the test substance’s cytotoxicity. Under the conditions of this study, the administration of FAT 40868/A TE at doses up to and including a dose of 2000 mg/kg was concluded to be negative in the Micronucleus assay.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date (First Day of Dosing): 02 March 2015, Experimental Completion Date: 09 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

None

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Sprague-Dawley male rats were received from Harlan, Frederick, MD on 25 February 2015.

Sex: Male; Age at Time of Initiation: 8 weeks; Body Weight Range at Time of Randomization: 258.2 – 283.1g.

Animal Receipt and Acclimation: Virus antibody-free (VAF) animals were acclimated for 5 days (12 to 16 hour exposure group) and 8 days (2 to 4 hour exposure group) in the assay. At BioReliance, they were observed each day for signs of illness or poor health. All animals were judged to be healthy prior to utilization in the study.

Housing: Animals were housed in a controlled environment at 72 ± 3°F (20.6 to 23.9°C) 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 four 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 (P.J. Murphy Forest Products) 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.

Randomization and Identification: Animals were assigned to groups using a randomization procedure. At the time of randomization, the weight variation of all animals assigned to the study did not exceed ±20% of the mean weight. A randomization function within Microsoft Excel™ was used to achieve random placement of animals throughout all groups. Following randomization, animals were identified by sequentially numbered ear tags. The cage card contained, at least, the animal number(s), sex, study number, treatment group number, dose level, test substance ID and route of administration. Cage cards were color coded by treatment group. Raw data records and specimens were also identified by the unique animal number.

Route of administration:

oral: gavage

Vehicle:

The vehicle used to deliver the FAT 40868/A TE to the test system was deionized water.

Details on exposure:

All dose formulations were administered at a volume of 10 mL/kg bw by oral gavage 2 to 4 hours and 12 to 16 hours prior to hepatocyte preparation using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles).

Frequency of treatment:

once

Post exposure period:

2 to 4 hours & 12 to 16 hours

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

Groups of 4 rats received a single oral dose of 35 mg/kg dimethylnitrosamine (DMN)

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

Preparation of Hepatocyte Cultures
The methods used for isolation and culturing of hepatocytes are modifications of the procedures used by Williams (1976 and 1979). For preparation of hepatocyte cultures, each animal was anesthetized by inhalation of isoflurane and a midventral incision was made to expose the liver. The liver was perfused with 0.5 mM ethylene glycol-bis(β-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) solution followed by collagenase solution (80-100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation, resuspended in complete Williams' Medium E (WME; buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 μg/mL gentamicin and 10% fetal bovine serum). Approximately 5 x 10^5 cells were seeded into each of six 35 mm tissue culture dishes containing 25 mm coverslips and preconditioned complete WME (i.e., complete WME medium in 35 mm tissue culture dishes incubated overnight in a humidified atmosphere of 5±1% CO2 and 37±1°C). A minimum of 6 cultures were set up for each animal. The hepatocyte cultures were maintained in a humidified atmosphere of 5±1% CO2 and 37±1°C.

Preparation of Slides
Ninety to 180 minutes after plating, the cells were washed once with complete WME and serum-free WME containing 10 μCi 3H-thymidine/mL. Four hours later, the radioactive medium was removed; the cultures were washed 3 times in serum-free WME containing 0.25 mM thymidine, and then refed with serum-free WME containing 0.25 mM non-titrated thymidine and incubated for 17-20 hours.
Seventeen to 20 hours after exposure to thymidine, the coverslips bearing cultures were washed once in serum-free WME. The nuclei were swelled in 1% sodium citrate solution and the cultures fixed in 3 changes of ethanol-glacial acetic acid fixative (3:1, v/v). The coverslips were allowed to air dry for at least 1.5 hours before mounting cell side up on glass slides. The slides were labeled with the study number, study phase and a code to identify the animal number.
At least 3 of the 6 slides for each animal were dipped in photographic emulsion at 43 to 45°C, allowed to drain and dry for at least 1.5 hours at room temperature and were stored for 7 days at 2-8°C in light tight boxes with desiccant. Slides were developed in D19 Developer (diluted 1:1 in deionized water), fixed in Fixer, and stained with hematoxylin-eosin stain.

Evaluation criteria:

Criteria for a Valid Test
The proportion of cells in repair in the negative (vehicle) control group must be less than 15% and the mean net nuclear grain count (MNNGC) must be less than one. The MNNGC of the positive control group must be at least 5 counts over that of the negative (vehicle) control group.

Evaluation of UDS Assay Results
All conclusions are based on sound scientific judgment; however, the following is offered as a guide to interpretation of the data.

Positive Results
• Any mean net nuclear grain count (MNNGC) that was increased by at least five counts over the negative (vehicle) control group is considered significant (Butterworth et al., 1987).
• The test substance would be judged positive if it induced a dose-related increase with no less than one dose significantly elevated above the negative (vehicle) control group.
• A significant increase in the MNNGC in at least two successive doses in the absence of a dose response could also be considered positive.

Equivocal Results
• A significant increase in net nuclear grain counts at the high dose group only with no evidence of a dose response would be considered suspect.
• A significant increase in net nuclear grain counts at one dose without a dose response would be judged to be equivocal.

Negative Results
• The test substance is considered negative if no significant increase in the net nuclear grain counts was observed.
The percentage of cells in repair (cells with ≥ 5 net nuclear grains) was also reported. These data may also be used by the Study Director in making a final evaluation of the activity of the test substance.

Statistics:

Scoring (Collection of UDS Data)
Slides were coded using a random number table performed and documented. All coded slides were evaluated without knowledge of treatment group. The slides were labeled with the study number, study phase and a code number with slide replicate identification. The slides were viewed microscopically under a 100X oil immersion lens. An automated colony counter was interfaced with the microscope so that silver grains within each nucleus and the surrounding cytoplasm can be counted. ProtoCOL (Version 3.07) system with accompanying support software was used for grain counts.
If possible, fifty nuclei were scored from each of three replicate cultures for a total of 150 randomly selected nuclei from each animal. A minimum of three animals per group were evaluated for UDS. Replicative DNA synthesis is evidenced by nuclei completely blackened with grains, and such cells were not counted. Cells exhibiting toxic effects of treatments, such as irregularly shaped or very darkly stained nuclei, were not counted.
Presentation of Data
A net nuclear grain count was calculated for each nucleus scored by subtracting the mean cytoplasmic area count from the nuclear area count. For each animal as well as for each treatment group, a mean net nuclear grain count and standard deviation (S.D.), as well as the proportion of cells in repair (percentage of nuclei showing ≥ 5 net nuclear grain counts) were determined.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs in the test substance treated groups for the 2 to 4 hour harvest included blue intestines, blue extremities and diarrhea (positive control group only), which were observed at the time of euthanasia.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Definitive Assay
No mortality occurred at any dose level or the control groups during the course of the definitive assay. Clinical signs in the test substance treated groups for the 2 to 4 hour harvest included blue intestines, blue extremities and diarrhea (positive control group only), which were observed at the time of euthanasia. Clinical signs for the test substance treated groups for the 12 to 16 hour harvest were also only observed at the time of euthanasia, which included blue tinted organs, piloerection and blue extremities. All other rats appeared normal throughout the observation period.
Clinical signs are presented in Table 1 and 2.

In Vivo UDS Assay
2 to 4 hour exposure
The results of the UDS assay using primary hepatocytes isolated 2 to 4 hours post-exposure are summarized in Table 3.
The mean net nuclear grain count for the vehicle control group was -2.3 with 3% of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment group was -2.2, -2.3 and -2.2 with 2%, 2% and 3% of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 14.5 with 97% of cells in repair. The mean net nuclear grain counts from the positive control and test substance-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. The test substance doses did not cause an increase in the mean net nuclear counts. The positive control, DMN, at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +16.8 over that of the vehicle control. According to the protocol criteria set for evaluating the test results, the induced increase was considered to be significant in the positive control (DMN)-treated animals.

12 to 16 hour exposure
The results of the UDS assay using primary hepatocytes isolated 12 to 16 hours post-exposure are summarized in Table 4.
The mean net nuclear grain count for the vehicle control group was -3.1 with 1% of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment group was -2.8, -2.7 and -2.1 with 2%, 3% and 2% of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 20.8 with 99% of cells in repair. The mean net nuclear grain counts from the positive control and test substance-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. The test substance doses did not cause an increase in the mean net nuclear counts. The positive control, DMN, at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +23.9 over that of the vehicle control. According to the protocol criteria set for evaluating the test results, the induced increase was considered to be significant in the positive control (DMN)-treated animals.

Table 1: Clinical Signs Following Dose Administrations: 2 to 4 Hour Exposure

Treatment	Observation	Number of Animals With Observed Signs/Number of Surviving Animals				Number of Animals Found Dead/Total Number of Animals Dosed
		Males				Males
		2 to 4 hour Harvest
		Pre- Dose	Post-Dose
			1 Hr	2 Hr	Sacrifice^A
Deionized water^B	Normal	4/4	4/4	4/4	4/4	0/4
FAT 40868/A TE 500 mg/kg/day	Normal Blue Intestines	4/4 0/4	4/4 0/4	4/4 0/4	3/3 3/3	0/4
1000 mg/kg/day	Normal Blue Extremities Blue Intestines	4/4 0/4 0/4	0/4 4/4 0/4	0/4 4/4 0/4	0/3 3/3 3/3	0/4
2000 mg/kg/day	Normal Blue Extremities Blue Intestines	4/4 0/4 0/4	0/4 4/4 0/4	0/4 4/4 0/4	0/3 3/3 3/3	0/4
Dimethyl nitrosamine (DMN, 35 mg/kg)	Normal Diarrhea	4/4 0/4	4/4 0/4	4/4 0/4	0/3 3/3	0/4

A = Observations and Mortality presented for the animals harvested in the study only.

B = Failed perfusion on one animal, replaced with extra animal.

 

Table 2: Clinical Signs Following Dose Administrations: 12 to 16 Hour Exposure

Treatment	Observation	Number of Animals With Observed Signs/Number of Surviving Animals				Number of Animals Found Dead/Total Number of Animals Dosed
		Males				Males
		12 to 16 hour Harvest
		Pre- Dose	Post-Dose
			1 Hr	2 Hr	Sacrifice^A
Deionized water	Normal	4/4	4/4	4/4	3/3	0/4
FAT 40868/A TE 500 mg/kg/day	Normal Blue Tinted Organs	4/4 0/4	4/4 0/4	4/4 0/4	3/3 3/3	0/4
1000 mg/kg/day	Normal Blue Tinted Organs	4/4 0/4	4/4 0/4	4/4 0/4	3/3 3/3	0/4
2000 mg/kg/day	Normal Piloerection Blue Extremities Blue Tinted Organs	4/4 0/4 0/4 0/4	4/4 0/4 0/4 0/4	4/4 0/4 0/4 0/4	0/3 3/3 3/3 3/3	0/4
Dimethyl nitrosamine (DMN, 35 mg/kg)^B	Normal	4/4	4/4	4/4	4/4	0/4

A = Observations and Mortality presented for the animals harvested in the study only.

B = Failed perfusion on one animal, replaced with extra animal.

 

Table 3: Summary of UDS Assay with FAT 40868/A TE: 2 to 4 Hour Exposure

Group	Ear Tag ID.	Slide Code	Cells Scored	per Animal				per Treatment Group
				Mean Grain Counts +/- SD^1			Cells in Repair	Mean Ne ± S.D.#^2	Cells in Repair
				Nuclear	Cytoplasmic	Net per Nucleus
Vehicle Deionized water -10 mL/kg	255	25	150	7.0±2.6	10.6±3.5	-3.6±3.2	1%	-2.3±1.3	3%
	256	37	150	8.0±3.7	9.2±3.4	-1.2±3.1	6%
	258	11	150	9.1±3.6	11.1±3.4	-2.1±3.2	3%
FAT 40868/A TE (mg/kg) -500 mg/kg	263	18	150	8.0±3.5	10.4±3.6	-2.4±3.2	1%	-2.2±0.1	2%
	264	21	150	7.8±3.8	9.8±3.5	-2.1±3.1	3%
	265	23	150	8.3±4.0	10.6±3.9	-2.2±3.3	2%
FAT 40868/A TE (mg/kg) -1000 mg/kg	271	26	150	8.6±3.5	10.9±3.5	-2.4±3.3	1%	-2.3±0.0	2%
	272	14	150	9.9±3.9	12.3±3.9	-2.4±3.3	1%
	273	13	150	7.6±3.2	9.9±3.4	-2.3±3.2	2%
FAT 40868/A TE (mg/kg) -2000 mg/kg	279	12	150	7.8±3.3	11.1±3.5	-3.3±3.2	1%	-2.2±1.1	3%
	280	24	150	7.8±4.0	8.9±3.6	-1.2±3.8	7%
	281	39	150	7.1±3.0	9.3±3.0	-2.2±2.6	1%
Positive Control: Dimethylnitrosamine -35 mg/kg	287	30	150	24.0±6.4	8.7±3.0	15.2±5.8	99%*	14.5±0.9	97%
	288	16	150	22.3±7.4	7.5±2.5	14.8±7.0	95%*
	289	32	150	21.0±6.4	7.4±3.3	13.5±5.5	97%*

1Standard deviation reflecting slide to slide variation

2S.D.#: Standard deviation reflecting variation between animals

* Significant (see protocol Section 10, Criteria for Determination of a Valid Test)

 

Table 4: Summary of UDS Assay with FAT 40868/A TE: 12 to 16 Hour Exposure

Group	Ear Tag ID.	Slide Code	Cells Scored	per Animal				per Treatment Group
				Mean Grain Counts +/- SD^1			Cells in Repair	Mean Ne ± S.D.#^2	Cells in Repair
				Nuclear	Cytoplasmic	Net per Nucleus
Vehicle Deionized water -10 mL/kg	251	27	150	9.1±3.6	11.9±3.6	-2.8±3.1	0%	-3.1±0.3	1%
	252	38	150	9.6±3.7	12.7±4.3	-3.1±3.6	2%
	253	28	150	9.4±4.4	12.7±5.0	-3.4±3.5	0%
FAT 40868/A TE (mg/kg) -500 mg/kg	259	33	150	9.3±4.0	11.5±3.8	-2.2±3.2	3%	-2.8±0.5	2%
	260	29	150	8.2±3.5	11.4±3.5	-3.2±3.3	2%
	261	20	150	10.3±4.3	13.1±4.4	-2.9±3.3	2%
FAT 40868/A TE (mg/kg) -1000 mg/kg	267	19	150	11.1±4.6	14.0±5.1	-2.9±3.8	3%	-2.7±0.2	3%
	268	40	150	12.5±5.3	15.1±5.0	-2.6±4.2	5%
	269	15	150	9.5±4.0	12.1±3.9	-2.6±3.3	1%
FAT 40868/A TE (mg/kg) -2000 mg/kg	275	22	150	8.0±3.3	10.2±3.4	-2.3±3.0	1%	-2.1±0.2	2%
	276	34	150	9.3±4.0	11.6±4.0	-2.3±3.6	3%
	277	17	150	9.8±4.4	11.7±4.1	-1.9±2.9	2%
Positive Control: Dimethylnitrosamine -35 mg/kg	286	34	150	28.9±8.2	8.8±3.1	20.1±7.8	99%*	20.8±1.2	99%
	284	36	150	28.1±10.1	8.0±4.1	20.1±9.5	99%*
	285	31	150	30.1±10.9	7.8±3.6	22.2±10.4	99%*

1Standard deviation reflecting slide to slide variation

2S.D.#: Standard deviation reflecting variation between animals

* Significant (see protocol Section 10, Criteria for Determination of a Valid Test)

Conclusions:

FAT 40868/A TE did not cause unscheduled DNA synthesis (UDS) in primary cultures of hepatocytes obtained from test substance-treated rats relative to the concurrent vehicle control. Therefore, FAT 40868/A TE was concluded to be negative.

Executive summary:

The test substance, FAT 40868/A TE, was evaluated for its genotoxic potential to induce unscheduled DNA synthesis in primary cultures obtained from test substance-treated rats. Deionized water was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg by oral gavage once at each of the two time points (12 to 16 hours and 2 to 4 hours prior to hepatocyte preparation). FAT 40868/A TE was administered to 4 male rats per dose at two time points (2 to 4 hours and 12 to 16 hours prior to hepatocyte preparation) in single oral doses of 500, 1000 and 2000 mg/kg bw. Two additional groups of 4 rats each received a single oral dose of deionized water or 35 mg/kg dimethylnitrosamine (DMN) which served as the vehicle and positive control, respectively. No mortality was observed. However, clinical signs were observed at the time of euthanasia at both time points. The animals were euthanized 2 to 4 hours post-dose or 12 to 16 hours post-dose to harvest hepatocytes. Hepatocytes (from 3 animals/group – hepatocytes from the first three successful perfusions) were seeded on the coverslips in culture media for one and half to two hours and then treated with culture media containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours. After the incubation period, the coverslips were fixed, mounted on the glass slides and processed for autoradiography using photographic emulsion. These coverslips were scored using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if FAT 40868/A TE increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair. Test item administration did not cause a significant increase in average mean MNNGC at either harvest time. At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15%, the average mean MNNGC of the vehicle control group was less than 1, and the average mean MNNGC of the positive control group was at least 5 counts over that of the vehicle control group. Thus, all criteria for a valid assay were met for the UDS assay. Hence, under the conditions of this study, FAT 40868/A TE up to and including 2000 mg/kg bw did not cause an increase in the net nuclear grain counts relative to the concurrent vehicle control. Therefore, FAT 40868/A TE was concluded to be negative in the in vivo mammalian cell unscheduled DNA synthesis (UDS) assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

None

Additional information

Four genetic toxicity studies are available for the substance and results are discussed below:

Bacterial reverse mutation assay:

The test substance, FAT 40868/A TE, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the absence of S9 activation and in the presence of both Aroclor-induced rat liver S9 activation (oxidative) and uninduced hamster liver S9 activation (reductive). The assay was performed in two phases, using the preincubation method. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test substance. Water was selected as the solvent of choice based on information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water for injection-quality, cell culture grade water at a concentration of approximately 50 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Increases in revertant counts (3.0- and 3.1-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, positive mutagenic responses (2.0- and 2.6-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. No positive mutagenic responses were observed with the remaining test conditions. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed; however, reductions in revertant counts were observed beginning at 3000 or at 5000 μg per plate with a few test conditions. Although the reductive S9 used in the mutagenicity assay was within the expiration date provided by the supplier, it was more than six months beyond its preparation date as required by the study protocol. Therefore, the reductive metabolic activation condition was retested.

In the retest of the mutagenicity assay, a positive mutagenic response (2.5-fold maximum increase) was observed with tester strain WP2 uvrA in the presence of reductive S9 activation. No positive mutagenic responses were observed with the remaining test conditions in the presence of reductive S9 activation. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Hence, based on the findings of this study, FAT 40868/A TE was concluded to be positive with tester strain WP2 uvrA in the presence of both oxidative and reductive S9 activation in the Bacterial Reverse Mutation Assay.

Unscheduled DNA synthesis assay (In vivo):

The test substance, FAT 40868/A TE, was evaluated for its genotoxic potential to induce unscheduled DNA synthesis in primary cultures obtained from test substance-treated rats. Deionized water was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg by oral gavage once at each of the two time points (12 to 16 hours and 2 to 4 hours prior to hepatocyte preparation). FAT 40868/A TE was administered to 4 male rats per dose at two time points (2 to 4 hours and 12 to 16 hours prior to hepatocyte preparation) in single oral doses of 500, 1000 and 2000 mg/kg bw. Two additional groups of 4 rats each received a single oral dose of deionized water or 35 mg/kg dimethyl nitrosamine (DMN) which served as the vehicle and positive control, respectively. No mortality was observed. However, clinical signs were observed at the time of euthanasia at both time points. The animals were euthanized 2 to 4 hours post-dose or 12 to 16 hours post-dose to harvest hepatocytes. Hepatocytes (from 3 animals/group – hepatocytes from the first three successful perfusions) were seeded on the coverslips in culture media for one and half to two hours and then treated with culture media containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours. After the incubation period, the coverslips were fixed, mounted on the glass slides and processed for autoradiography using photographic emulsion. These coverslips were scored using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if FAT 40868/A TE increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair. Test item administration did not cause a significant increase in average mean MNNGC at either harvest time. At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15%, the average mean MNNGC of the vehicle control group was less than 1, and the average mean MNNGC of the positive control group was at least 5 counts over that of the vehicle control group. Thus, all criteria for a valid assay were met for the UDS assay. Hence, under the conditions of this study, FAT 40868/A TE up to and including 2000 mg/kg bw did not cause an increase in the net nuclear grain counts relative to the concurrent vehicle control. Therefore, FAT 40868/A TE was concluded to be negative in the in vivo mammalian cell unscheduled DNA synthesis (UDS) assay.

In vitro Chromosomal aberration Assay:

The test substance, FAT 40868/A TE, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, the CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Water was used as the vehicle based on the information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a clear solution in sterile water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 μg/mL. Substantial toxicity (at least 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. Substantial toxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 1500 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 500 to 5000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 100 to 1500 μg/mL for the non-activated 20-hour exposure group.

In the initial chromosome aberration assay, substantial toxicity was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. Substantial toxicity was observed at dose levels ≥ 700 μg/mL in the non-activated 20-hour exposure group. However, due to lack of requisite number of non-precipitating doses for scoring chromosome aberrations, the assay was repeated in the non-activated and S9-activated 4-hour exposure groups at dose levels ranging from 100 to 2000 μg/mL. In the repeat assay, substantial toxicity was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced an approximately 50% reduction in cell growth index which met the dose limit as recommended by testing guidelines for this assay. No significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). 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, all criteria for a valid study were met.

Under the conditions of the assay described in this report, FAT 40868/A TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

In vivo micronucleus assay:

The test substance, FAT 40868/A TE, 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. Deionized water was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg by oral gavage. In the definitive assay, dose levels tested were 500, 1000 or 2000 mg/kg body weight. Groups 1 and 4 consisted of 10 animals each designated for either 24 or 48 hour bone marrow collections and Groups 2, 3 and 5 consisted of 5 animals each designated for 24 hour bone marrow collection. Following scheduled euthanasia times, femoral bone marrow was collected; bone marrow slides were prepared and stained with acridine orange. Bone marrow cells [polychromatic erythrocytes (2000 PCEs/animal)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MnPCEs) and statistical analysis of data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05). The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes (EC) in the test substance groups relative to the vehicle control groups was also evaluated to reflect the test substance’s cytotoxicity. Under the conditions of this study, the administration of FAT 40868/A TE at doses up to and including a dose of 2000 mg/kg was concluded to be negative in the Micronucleus assay.

The substance was determined to be positive with tester strain WP2 uvrA in the presence of both oxidative and reductive S9 activation in an in vitro Ames test, so an in vivo UDS test (genotoxicity tests in somatic cells) was done to check whether the effect can be found in the in vivo system. And the results showed that the substance is negative in this in vivo UDS assay and this finding can overrule the positive result observed in Ames test. So the substance was concluded to have no mutagenic effect. Further, the substance can be considered to have no clastogenic potential as negative results are obtained in both in vitro chromosome aberration test and in vivo micronucleus test. Hence based on the above results, it was concluded that the substance can be considered to be not genotoxic.

Justification for classification or non-classification

Based on the above mentioned results, the substance was concluded to have no genotoxic effect. Hence, no classification is required according to CLP regulation (Regulation EC No.1272/2008).