Reactive Orange 143

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is mutagenic in the bacterial reverse mutation assay and in the mouse lymphoma L5178Y test system under the experimental conditions but was found not clastogenic in human lymphocytes.

Based on the these studies the test substance should be considered as non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

L5178Y Mouse Lymphoma Cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2012 to 14 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at WIL Research Europe)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Stability in vehicle: Dimethyl sulfoxide
Solubility in vehicle: Dimethyl sulfoxide

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000 and 3330 µg/mL

Mutation experiment:
Without S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 33, 100 and 200 µg/mL
With S9-mix, 3 hours treatment: 30, 100, 500, 600, 700, 800 and 850 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD, EC) and literature (see chapter 9 for references).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196 °C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 E+6 cells/ml.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: With and without S9-mix: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120 %. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32.
d) The mutation frequency of MMS should not be below 500 per 10E6 survivors, and for CP not below 700 per 10E6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Dose range finding test

In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 33.3 to 3330 μg/ml in the absence and presence of S9-mix with a 3 hour treatment period. In the absence of S9-mix, the relative suspension growth was 14 % at the test substance concentration of 333 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 1000 μg/ml and above. In the presence of S9-mix, the relative suspension growth was 8 % at the test substance concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 3330 μg/ml.

 

Mutagenicity test

Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test:

Without S9-mix: 0.3, 1, 3, 10, 33, 100, 200, 250, 300, 325, 350, 400 and 450 μg/ml exposure medium.

 

With 8 % (v/v) S9-mix: 10, 30, 100, 300, 500, 600, 700, 800, 900, 1000, 1100 and 1200 μg/ml exposure medium.

 

Evaluation of toxicity

In the absence of S9-mix, the dose levels of 250 μg/ml and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, too many dose levels showed severe cytotoxicity, this part of the experiment was repeated (experiment 1A): the following dose range was selected: 0.3, 1, 3, 10, 33, 100, 300, 400, 500, 600, 700, 750 and 800 μg/ml. In this repeat, no dose level with a cell survival below 23 % was reached, therefore this experiment was rejected. In the repeat experiment (1B), the following dose range was selected: 30, 100, 300, 500, 600, 700, 750, 800, 850, 900 and 1000 μg/ml. The dose levels of 750, 900 and 1000 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 0.3, 1, 3, 10, 33, 100 and 200 μg/ml exposure medium.

 

With S9-mix: 30, 100, 300, 500, 600, 700, 800 and 850 μg/ml exposure medium.

 

In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 88 % compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 94 % compared to the total growth of the solvent controls.

 

Evaluation of the mutagenicity

In the absence of S9-mix, FAT 40854/A induced an up to 5.7-fold increase in the mutation frequency at the TK locus. FAT 40854/A showed up to 5.1- and 5.4-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

In the presence of S9-mix, FAT 40854/A induced an up to 11-fold increase in the mutation frequency at the TK locus. FAT 40854/A showed up to 13- and 6.3-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

Conclusions:

FAT 40854/A is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Executive summary:

In a GLP-compliant study, evaluation of the mutagenic activity of FAT 40854/A in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was conducted according to OECD guideline 476 and EU method B.17. This report describes the effects of FAT 40854/A on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). FAT 40854/A was tested up to concentrations of 200 and 850 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. FAT 40854/A was tested up to cytotoxic levels of 88 and 94 in the absence and presence of S9-mix, respectively.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of one of the solvent control cultures in the absence of S9-mix. However, since this response was just below the lower limit of the range and clear positive results were obtained, the validity of the test was considered to be not affected. Mutation frequencies in cultures treated with positive control chemicals were increased by 15-fold for MMS in the absence of S9-mix, and by 35-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, FAT 40854/A TE induced an up to 5.7-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls)(174 per 106survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A is considered mutagenic in the absence of S9-mix. In the presence of S9-mix, FAT 40854/A induced an up to 11-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls) (207 per 10E6 survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A is considered mutagenic in the presence of S9-mix.

It is concluded that FAT 40854/A is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January 2012 to 20 March 2012

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:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at NOTOX)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Stability in vehicle Water: Stability for at least 6 hours at room temperature
Solubility in vehicle Water: More than 80 g/L

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Exp 1: Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone / exp2 and 3: hamster liver S9-mix, uninduced male Golden Syrian Hamster

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2: TA1535, TA1537, TA98, TA100 and WP2uvrA
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 3:
TA100 and WP2uvrA
With S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle:
Test compound was stable in water and water has been accepted and approved by authorities and international guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9: TA100 at 650 µg/plate in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9: 10 µg/plate in DMSO for WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9: 5 µg/plate in saline for TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO

Remarks:

with S9: TA98, TA100, TA1535 and TA1537 and WP2uvrA.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2 and 3: preincubation for 30 minutes at 37 °C

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in the experiment using a preincubation step

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in the experiment using a preincubation step

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the top dose of 5000 µg/plate

First experiment: Direct plate assay

FAT 40854/A was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of rat liver S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 100, 333, 1000, 3330 and 5000 μg/plate. The dose range finding test and mutation test were reported together as one mutation test. Precipitation of FAT 40854/A on the plates was not observed at the start or at the end of the incubation period in all tester strains.

To determine the toxicity of FAT 40854/A, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed.

 

Mutagenicity

In the direct plate test, no increase in the number of revertants was observed upon treatment with FAT 40854/A under all conditions tested.

 

7.2. Second experiment: Pre-incubation assay

To obtain more information about the possible mutagenicity of FAT 40854/A, a pre-incubation experiment was performed in the absence and presence of hamster liver S9-mix. Based on the results of the first mutation assay, FAT 40854/A was tested up to the dose level of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitation of FAT 40854/A on the plates was not observed at the start or at the end of the incubation period. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

 

Mutagenicity:

In tester strain TA100, FAT 40854/A induced an up to 4.4-fold dose related increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In tester strain WP2uvrA, FAT 40854/A induced up to 3.3- and 4.8-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In the other tester strains, no biologically relevant increase in the number of revertants was observed upon treatment with FAT 40854/A.

 

Third experiment: Pre-incubation assay

Since the response of the solvent control of tester strain TA100 was below the limit of the range and to verify the results obtained in the pre-incubation assay, an additional third mutation experiment was performed with the tester strains TA100 and WP2uvrA in presence of S9-mix. Precipitation of FAT 40854/A on the plates was not observed at the start or at the end of the incubation period.

No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed.

 

Mutagenicity:

In tester strain TA100, FAT 40854/A induced an up to 6.8-fold dose related increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In tester strain WP2uvrA, FAT 40854/A induced an up to 6.4-fold dose related increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix.

Conclusions:

FAT 40854/A is mutagenic in the bacterial reverse mutation assay.

Executive summary:

In a GLP-compliant study, evaluation of the mutagenic activity of FAT 40854/A in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat) was carried out according to OECD guideline 471 and EU method B.13/14. FAT 40854/A was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed with rat liver S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone) and secondly a pre-incubation assay was performed with hamster liver S9-mix (uninduced male Golden Syrian Hamster liver S9-mix). To obtain more information about the possible mutagenicity of FAT 40854/A, an additional experiment was performed with the strains TA100 and WP2uvrA in presence of S9-mix in the pre-incubation assay. The test substance was dissolved in Milli-Q water. In a dose range finding study, FAT 40854/A was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. FAT 40854/A did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. In the first mutation experiment, FAT 40854/A was again tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation experiment, FAT 40854/A was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. To verify the results obtained in the pre-incubation assay, an additional third mutation experiment was performed with the tester strains TA100 and WP2uvrA in presence of S9-mix. FAT 40854/A was tested up to the dose level 5000 µg/plate. No toxicity was observed at this dose level. In the absence of S9-mix, FAT 40854/A induced a 3.3-fold dose related increase in tester strain WP2uvrA after the pre-incubation treatment. The increase observed was above the laboratory historical control data range and was more than three times the concurrent control. In the presence of S9-mix, FAT 40854/A induced dose related increases in two tester strains (TA100 and WP2uvrA) after the pre-incubation treatment. The increases observed in tester strain WP2uvrA were above the laboratory historical control data range, in two independently repeated experiments and were up to 6.4-fold the concurrent controls. The increases observed in tester strain TA100 were above the laboratory historical control data range in two independently repeated experiments and were up to 6.8-fold the concurrent controls. The increases observed in tester strain TA100 were only in the third experiment above the laboratory historical control data range, however the increase was more than two times the concurrent control in both experiments. Since 3.3- to 6.8-fold, dose related increases were observed in two tester strains, both in the absence and presence of S9-mix (WP2uvrA) and in the presence of S9-mix (TA100) and the results obtained in the presence of S9-mix were reproducible in the repeat assay, these increases are considered biologically relevant, FAT 40854/A is mutagenic in the absence and presence of S9-mix. All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except for TA100 in the presence of S9-mix (second and third experiment; solvent control) and WP2uvrA in the presence of S9-mix (third experiment; positive control). Although these values were without the limit of the range, clear mutagenic responses after treatment with FAT 40854/A were observed in these tester strains, therefore the validity of the test was considered to be not affected. Based on the results of this study it is concluded that FAT 40854/A is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with a pre-incubation step.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2012 to 19 April 2012

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at NOTOX)
Batch: TZ 5719 / BOP 02-11
Purity: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Stability in vehicle: Dimethyl sulfoxide
Solubility in vehicle: Dimethyl sulfoxide

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River (Sulzfeld, Germany). In the second cytogenetic assay the rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been injected intraperitoneal with Aroclor 1254 (500 mg/kg).

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 3330 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000 and 3330 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 33, 333 and 1000 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 100, 1000 and 2000 µg/ mL

Second cytogenetic test:
Without S9-mix, 24/48 hr exposure; 24 and 48 hr fixation: 33, 333 and 1000 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 33, 333 and 1000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9: in Hank's Balanced Salt Solution: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p <0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 3330 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 333 µg/ml and above in the absence and presence of S9, 3 hour treatment/24 hour fixation; at dose levels of 333 µg/ml and above in the absence of S9-mix at a continuous treatment of 24 and 48 hours.


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Dose range finding test

Since FAT 40854/A formed a very viscous liquid at a concentration of 500 mg/ml, a concentration of 333 mg/ml was tested for precipitate in the solubility experiment. The final concentration of 3330 μg/ml showed no precipitation in the culture medium. Therefore, a concentration of 3330 μg/ml was used as the highest concentration of FAT 40854/A. In the dose range finding test blood cultures were treated with 33, 100, 333, 1000 and 3330 μg FAT 40854/A/ml culture medium with and without S9-mix.

 

First cytogenetic assay

The pH and osmolarity of a concentration of 3330 μg/ml were 7.81 and 424 mOsm/kg, respectively, (compared to 7.68 and 448 mOsm/kg in the solvent control). Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Without and with S9-mix: 33, 100, 333, 1000, 2000 and 3330 μg/ml culture medium (3 h exposure time, 24 h fixation time). The following dose levels were selected for scoring of chromosome aberrations:

Without S9-mix : 33, 333 and 1000 μg/ml culture medium (3 h exposure time, 24 h fixation time).

With S9-mix : 100, 1000 and 2000 μg/ml culture medium (3 h exposure time, 24 h fixation time).

 

Both in the absence and presence of S9-mix, FAT 40854/A did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix, FAT 40854/A did not show a biologically relevant increase the number of polyploid cells and cells with endoreduplicated chromosomes.

 

Second cytogenetic assay

To obtain more information about the possible clastogenicity of FAT 40854/A, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to FAT 40854/A in the absence of S9-mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to FAT 40854/A. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix : 33, 100, 333, 1000, 2000 and 3000 μg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).

With S9-mix : 33, 100, 333, 1000, 2000 and 3000 μg/ml culture medium (3 h exposure time, 48 h fixation time).

 

Based on these observations the following doses were selected for scoring of chromosome aberrations:

Without S9-mix: 33, 333 and 1000 μg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).

With S9-mix: 33, 333 and 1000 μg/ml culture medium (3 h exposure time, 48 h fixation time).

 

Both in the absence and presence of S9-mix, FAT 40854/A did not induce a statistically significant

or biologically relevant increase in the number of cells with chromosome aberrations.Both in the absence and presence of S9-mix, FAT 40854/A did not show a biologically relevant increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Conclusions:

FAT 40854/A is not clastogenic in human lymphocytes.

Executive summary:

In a GLP-compliant study, evaluation of the ability of FAT 40854/A to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment) was carried out according to OECD guideline 473 and EU method B.10. This report describes the effect of FAT 40854/A on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone or araclor induced rat liver S9-mix). The possible clastogenicity of FAT 40854/A was tested in two independent experiments. FAT 40854/A was soluble in dimethyl sulfoxide at concentrations of 333 mg/ml and below. In the first cytogenetic assay, FAT 40854/A was tested up to 1000 and 2000 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8 % (v/v) S9-fraction, respectively. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, FAT 40854/A was tested up to 1000 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix FAT 40854/A was also tested up to 1000 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at this dose level. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. FAT 40854/A did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No biologically relevant effects of FAT 40854/A on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that FAT 40854/A does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions. Based on the study results, FAT 40854/A is not clastogenic in human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg under the experimental conditions andconcluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral dose at up to 2000 mg/kg bw. Based on the these studies the test substance should be considered as non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2012 to 11 May 2012

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

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder
Batch: TZ 5719 / BOP 02-11
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Stability in vehicle: Water: Stability for at least 6 hours at room temperature
Solubility in vehicle: Water: More than 80 g/L

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI BR mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 31 – 40 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: free access to tap-water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 – 21.7
- Humidity (%): 44 - 67
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: Test compound was stable in water and a solution could be obtained in physiological saline. Physiological saline has been accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 43.8, 87.5, 150 and 175 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight

Details on exposure:

The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of FAT 40854/A. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

Duration of treatment / exposure:

Treatment:
Solvent, positive control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours

Frequency of treatment:

Once

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Vehicle

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

Group B,C

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Group D

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Group E

Dose / conc.:

40 mg/kg bw (total dose)

Remarks:

Group F- Cyclophosphamide

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p <0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p <0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Statistics:

Wilcoxon Rank Sum Test, one-sided, p <0.05

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
The animals showed no treatment related clinical signs or mortality after dosing.

RESULTS OF DEFINITIVE STUDY

- Clinical signs of toxicity in test animals:
The animals showed no treatment related clinical signs or mortality after dosing.
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with FAT 40854/A.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis.

Dose range finding study

The animals showed no treatment related clinical signs or mortality after dosing.

 

Micronucleus main test

Mortality and toxic signs

Before use, animal numbers 27 to 30 (group F) had fighting marks. The animals of the groups treated with 2000, 1000 and 500 mg FAT 40854/A /kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

 

Micronucleated polychromatic erythrocytes

The mean number of micronucleated polychromatic erythrocytes scored in FAT 40854/A treated groups were compared with the corresponding vehicle control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of FAT 40854/A treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

 

Ratio polychromatic to normochromatic erythrocytes

The animals of the groups, which were treated with FAT 40854/A and the positive control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis.

Conclusions:

FAT 40854/A is not clastogenic or aneugenic in the bone marrow micronucleus test in male mice up to a dose of 2000 mg/kg.

Executive summary:

A GLP-compliant micronucleus test in bone marrow cells of the mouse with FAT 40854/A was carried out according to OECD guideline 474 and EU method B.12 to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. In the dose range finding study 3 males and 3 females were dosed via oral gavage with 2000 mg FAT 40854/A per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no substantial differences in toxicity between sexes only males were used in the main study. In the main study male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg FAT 40854/A per kg body weight. A positive control group was dosed via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with FAT 40854/A or control animals receiving vehicle or cyclophosphamide. Bone marrow of the groups treated with FAT 40854/A was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with FAT 40854/A. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. The groups that were treated with FAT 40854/A and the group treated with cyclophosphamide showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. It is concluded that FAT 40854/A is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg under the experimental conditions.

Reference 2

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2014 to 16 April 2014

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Lot Number: FAT 40854/A TE
Batch Number: TZ5719/BOP 02-11
Purity: It is a UVCB substance, with 46.22 % main component (per Protocol)
Description: Blackish red powder
Storage Conditions: Room temperature; protected from light with desiccant

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague Dawley (Hsd:SD) male rats were obtained from Harlan (Frederick, MD).
Justification for the Test System: The rodents have been routinely used as an animal model of choice for the UDS assay. This strain is an outbred strain that maximized genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (Frederick, MD).
- Animal: Virus antibody-free (VAF)
- Age at study initiation: 8 weeks
- Weight at study initiation: 259.2-286.5 grams.
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: Animals were housed in an AAALAC-accredited facility
- Diet: free access to Harlan 2018C Certified Global Rodent Diet
- Water: free access -Water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 23.9
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10 changes of fresh HEPA-filtered air every hour.
- Photoperiod (hrs dark / hrs light): 12- hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle: 0.9 % NaCl injection, USP.
- Lot/batch no. (if required): C907535
- CAS number: 7647-14-5
- Supplier: Baxter Healthcare
- Expiration date: 30 September 2014

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of Test Article Dose Formulations
The test article dose formulations were prepared on each day of dosing for 2 to 4 hour exposure and 12 to 16 hour exposure. Dose formulations were prepared in amber glass vials and stored at room temperature. The preparation was performed under yellow light as follows:
• An appropriate amount of the test article was weighed out and placed into the calibrated formulation container.
• The vehicle was added to the QS line and mixed magnetically until homogenous in appearance. The test article dose formulations were dark red solutions.

Duration of treatment / exposure:

- 2 to 4 hours exposure
- 12 to 16 hours exposure

Frequency of treatment:

Once

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low test dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mild test dose

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Highest test dose

No. of animals per sex per dose:

8
-( 4 rats evaluated for UDS after 2 to 4 hour dose administration)
-( 4 rats evaluated for UDS after 12 to 16 hour dose administration)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Dimethylnitrosamine (DMN)
- Route of administration: Oral gavage
- Doses / concentrations: 10 ml/kg / 35 mg/kg bw.

Tissues and cell types examined:

Primary cultures of hepatocytes obtained from test article-treated rats.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A DRF was not performed. According to the information provided by the sponsor, a 28-day oral toxicity study with FAT 40854 by daily gavage in Wistar rats, resulted in a NOAEL of at least 1000 mg/kg. In an acute oral toxicity study with FAT 40854 in Wistar rats, the LD50 was 2000 mg/kg body weight. The high dose for the definitive UDS assay was 2000 mg/kg. Two additional doses of 1000 and 500 mg/kg were also evaluated.

TREATMENT AND SAMPLING TIMES :
Animals were dosed 2 to 4 and 12 to 16 hours prior to hepatocytes preparation by oral gavage. This route has been routinely used and is widely-accepted for use in mammalian genetic toxicology testing. Animals were observed for clinical signs of toxicity prior to dose; approximately 1 and 2 hours following each dose administration and at the time of euthanasia.

DETAILS OF SLIDE PREPARATION:
For preparation of hepatocyte cultures, each rat 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). The cell viability for the vehicle control hepatocytes should be at least 70 %. Approximately 5 x 10E5 cells were seeded into each of six 35 mm tissue culture dishes containing 25mm 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 rat. The hepatocyte cultures were maintained in a humidified atmosphere of 5±1 % CO2 and 37±1 °C. The vehicle control hepatocyte viability was at least 70 % at the time of viable cell count prior to seeding for the assay. Ninety to 180 minutes after plating, the cells were washed once with complete WME and refed with 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 completion of the 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 hour before mounting cell side up on glass slides. The slides were labeled with the study number and a randomly selected code to identify the animal number. Initially 3 of the 6 slides for each rat were dipped in Kodak-NTB emulsion at 43.8 – 44.8 °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. The remaining three slides were dipped because the initial three slides were not scorable. Slides were developed in Kodak D-19 developer (diluted 1:1 in deionized water), fixed in Kodak fixer, and stained with hematoxylin-eosin stain.

Evaluation criteria:

All conclusions are based on sound scientific judgment; however, the following is offered as a guide to interpretation of the data. These data may also be used by the Study Director in making a final evaluation of the activity of the test article.
Positive Results
• Any mean net nuclear grain count (MNNGC) which was increased by at least five counts over the vehicle control is considered significant (Butterworth et al., 1987).
• The test article would be judged positive if it induced a dose-related increase with no less than one dose significantly elevated above the vehicle control.
• A significant increase in the MNNGC in at least two successive doses in the absence of a dose response would 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 with no evidence of a dose response would be judged to be equivocal.

Vehicle Results
• The test article 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) would also be reported.

Sex:

male

Genotoxicity:

not determined

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Clinical Signs of Toxicity

FAT 40854 was administered to 4 male rats per dose at two time points (2 to 4 hours and 12 to 16 hours) at dose levels of 500, 1000 and 2000 mg/kg bw once. In the 2 to 4 hour exposure, no mortality or clinical signs were observed in any of the vehicle control and test article-treated animals. The positive control group had the clinical sign diarrhea at sacrifice. In the 12 to 16 hour exposure, neither mortality nor clinical signs were observed in any of the vehicle and positive control-treated animals and the test article-treated animals.

 

In Vivo UDS Assay

2 to 4 hour exposure

The mean net nuclear grain count for the vehicle control group was -0.7 with 2 % of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were -0.7, -1.0 and -1.1 with 2 %, 4 % and 1 % of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 8.4 with 76 % of cells in repair. The mean net nuclear grain counts from the positive control or test article-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. None of the test article doses caused a significant increase in the mean net nuclear counts. The positive control compound, DMN, at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +9.1 over that of the vehicle control. According to the criteria set for evaluating the test results, the induced increase was considered to be significant in the DMN-treated animals.

 

12 to 16 hour exposure

The mean net nuclear grain count for the vehicle control group was -0.6 with 3% of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were -1.2, -0.7 and -0.8 with 3 %, 2 % and 4 % of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 9.2 with 74 % of cells in repair. The mean net nuclear grain counts from the positive control or test article-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. None of the test article doses caused a significant increase in the mean net nuclear counts. Thepositive control compound, DMN, at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +9.8 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.

Conclusions:

FAT 40854 is considered as to be negative in the Unscheduled DNA Synthesis (UDS) Assay with Mammalian Cells In Vivo.

Executive summary:

In a GLP-compliant study, FAT 40854, was tested in the unscheduled DNA synthesis (UDS) assay with rat liver cells in vivo. The UDS assay was used to evaluate the potential of the test article to induce unscheduled DNA synthesis in primary cultures obtained from test article-treated Sprague-Dawley rats. This experiment was done according to the OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo). FAT 40854 was administered to 4 male rats per dose at two time points (2 to 4 hours and 12 to 16 hours) 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 0.9 % NaCl Injection, USP or 35 mg/kg dimethylnitrosamine (DMN) which served as the vehicle and positive control, respectively. The test and control articles were administered at a constant volume of 10 mL/kg by a single oral gavage. In the definitive assay, no mortality was observed. However, clinical signs were observed in the positive control group at the 2 to 4 hour harvest. The animals (3/group) were euthanized 2 to 4 hours pos tdose and 12 to 16 hours post dose to harvest hepatocytes. Hepatocytes were exposed to culture media containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours, and then examined 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 40854 increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair. FAT 40854 did not cause a significant increase in average mean MNNGC at any dose level or 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. Based on these results, all criteria for a valid study were met, and FAT 40854 was concluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral dose at up to 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-vitro Bacterial Reverse Mutation Assay:

In a GLP-compliant study, evaluation of the mutagenic activity of FAT 40854/A in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat) was carried out according to OECD guideline 471 and EU method B.13/14. FAT 40854/A was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). In a dose range finding study, FAT 40854/A was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. FAT 40854/A did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. In the first mutation experiment, FAT 40854/A was again tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, FAT 40854/A TE was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. To verify the results obtained in the pre-incubation assay, an additional third mutation experiment was performed with the tester strains TA100 and WP2uvrA in presence of S9-mix. FAT 40854/A was tested up to the dose level 5000 µg/plate. No toxicity was observed at this dose level. In the absence of S9-mix, FAT 40854/A induced a 3.3-fold dose related increase in tester strain WP2uvrA after the pre-incubation treatment. The increase observed was above the laboratory historical control data range and was more than three times the concurrent control. In the presence of S9-mix, FAT 40854/A induced dose related increases in two tester strains (TA100 and WP2uvrA) after the pre-incubation treatment. The increases observed in tester strain WP2uvrA were above the laboratory historical control data range, in two independently repeated experiments and were up to 6.4-fold the concurrent controls. The increases observed in tester strain TA100 were above the laboratory historical control data range in two independently repeated experiments and were up to 6.8-fold the concurrent controls. The increases observed in tester strain TA100 were only in the third experiment above the laboratory historical control data range, however the increase was more than two times the concurrent control in both experiments. Since 3.3- to 6.8-fold, dose related increases were observed in two tester strains, both in the absence and presence of S9-mix (WP2uvrA) and in the presence of S9-mix (TA100) and the results obtained in the presence of S9-mix were reproducible in the repeat assay, these increases are considered biologically relevant, FAT 40854/A TE is mutagenic in the absence and presence of S9-mix. All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except for TA100 in the presence of S9-mix (second and third experiment; solvent control) and WP2uvrA in the presence of S9-mix (third experiment; positive control). Although these values were without the limit of the range, clear mutagenic responses after treatment with FAT 40854/A were observed in these tester strains, therefore the validity of the test was considered to be not affected.

Based on the results of this study it is concluded that FAT 40854/A is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with a pre-incubation step.

 

In-Vitro Chromosomal Aberration:

In a GLP-compliant study, evaluation of the ability of FAT 40854/A to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment) was carried out according to OECD guideline 473 and EU method B.10. In the first cytogenetic assay, FAT 40854/A was tested up to 1000 and 2000 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8 % (v/v) S9-fraction, respectively. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, FAT 40854/A was tested up to 1000 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix FAT 40854/A was also tested up to 1000 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at this dose level. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. FAT 40854/A did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No biologically relevant effects of FAT 40854/A on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that FAT 40854/A does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions. Based on the study results, FAT 40854/A is not clastogenic in human lymphocytes.

 

In-vitro Muse Lymphoma Assay

In a GLP-compliant study, evaluation of the mutagenic activity of FAT 40854/A in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was conducted according to OECD guideline 476 and EU method B.17. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of one of the solvent control cultures in the absence of S9-mix. However, since this response was just below the lower limit of the range and clear positive results were obtained, the validity of the test was considered to be not affected. Mutation frequencies in cultures treated with positive control chemicals were increased by 15-fold for MMS in the absence of S9-mix, and by 35-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, FAT 40854/A induced an up to 5.7-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls) (174 per 10E6 survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A is considered mutagenic in the absence of S9-mix.

In the presence of S9-mix, FAT 40854/A induced an up to 11-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls) (207 per 10E 6 survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A is considered mutagenic in the presence of S9-mix.

It is concluded that FAT 40854/A TE is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

 

In-vivo Micronucleus Test:

A GLP-compliant micronucleus test in bone marrow cells of the mouse with FAT 40854/A was carried out according to OECD guideline 474 and EU method B.12 to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. In the dose range finding study 3 males and 3 females were dosed via oral gavage with 2000 mg FAT 40854/A per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no substantial differences in toxicity between sexes only males were used in the main study. In the main study male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg FAT 40854/A per kg body weight. A positive control group was dosed via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with FAT 40854/A or control animals receiving vehicle or cyclophosphamide. Bone marrow of the groups treated with FAT 40854/A was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with FAT 40854/A. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. The groups that were treated with FAT 40854/A and the group treated with cyclophosphamide showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. It is concluded that FAT 40854/A is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg under the experimental conditions.

 

In-vivo UDS Assay:

In a GLP-compliant study, FAT 40854, was tested in the unscheduled DNA synthesis (UDS) assay with rat liver cells in vivo. The UDS assay was used to evaluate the potential of the test article to induce unscheduled DNA synthesis in primary cultures obtained from test article-treated Sprague-Dawley rats. This experiment was done according to the OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo). FAT 40854 was administered to 4 male rats per dose at two time points (2 to 4 hours and 12 to 16 hours) 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 0.9 % NaCl Injection, USP or 35 mg/kg dimethylnitrosamine (DMN) which served as the vehicle and positive control, respectively. FAT 40854 did not cause a significant increase in average mean MNNGC at any dose level or 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. Based on these results, all criteria for a valid study were met, and FAT 40854 was concluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral dose at up to 2000 mg/kg bw.

 

Conclusion on genotoxicity:

Reactive Orange 143 was found to be mutagenic in the bacterial reverse mutation assay and in the mouse lymphoma L5178Y test system under the experimental conditions but was found not clastogenic in the in vitro and in vivo studies. It returned negative results from the Unscheduled DNA Synthesis (UDS) assay conducted to investigate if it has any DNA damaging potential that may lead to expression of mutagenicty in the in vivo conditions. Hence, based on the these studies, Reactive Orange 143 should be considered as non-genotoxic.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance is not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.