4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Target chemical was not mutagenic in the Salmonella typhimurium reverse mutation assay, while it was concluded to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the S9-activated test system in the in vitro mammalian chromosome aberration test using CHO cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- & his+ reversions

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation. (S9 mix )

Test concentrations with justification for top dose:

Exp. I: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate
Exp. II: 33.3; 100.0; 333.3; 666.6; 1000.0; and 2500.0 µg/plate
active ingredient

Vehicle / solvent:

No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Salmonella TA 1535, TA 100 ; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 1537, TA 98;without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 102;without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102 ; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Selective Agar
2. 0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilisations were performed at 121° C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar*
6.0 g NaCl*
10.5 mg L-histidine x HCl X H20*
12.2 mg biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121° C in an autoclave.

NUMBER OF REPLICATIONS:
Each concentration, including the controls, was tested in triplicate.

Evaluation criteria:

A test article is considered as positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate (4). Also, a dose-dependent and reproducible increase in the number of
revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA 1535 (without S9 mix) at 5000.0 µg/plate and in TA 102 with and without S9 at 2500.0 and 5000.0 µg/plate in experiment I. In experiment II toxicity was observed in strain TA 102 at the highest concentration (2500.0 µg/plate) with S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

FAT 36152/F did not induce point mutations by base pair changes or frameshifts in the genome of the strains used in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of FAT 36152/F; to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

Exp. I: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate

Exp. II: 33.3; 100.0; 333.3; 666.6; 1000.0; and 2500.0 µg/plate

Toxic effects evidenced by a reduction of revertants occurred in strain TA 1535 (without S9 mix) at 5000.0 µg/plate and in strain. TA 102 with and without metabolic activation at 2500.0 and 5000.0 µg/plate in experiment I. In experiment II toxicity was observed in strain TA 102 at the highest concentration (2500.0 µg/plate) with S9 mix. The plates incubated with the test article showed normal background growth up to 2500.0 (exp. II) and 5000.0 µg/plate (exp.I), respectively with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 36'152/F; Terasil Blau BGE Kurzamin roh feucht (laborgetrocknet) at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 36152/F is considered to be non-mutagenic in this Salmonella typhimurium 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:

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

Target gene:

None

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimal essential Medium; SEROMED; D-12247 Berlin)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix liver microsomal fraction

Test concentrations with justification for top dose:

- Test concentrations in the pre-test on toxicity (with and without S9 mix, exposure time 4 hours and 24 hours):
0.2 to 200 μg/mL

- Main experiment (Without S9-mix):
40, 80, 120, 160 µg/ml.

- Main experiment (With S9-mix):
30, 60, 90, 120 µg/ml.

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test article was dissolved in Dimethylformamide (DMF).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Positive control for non-activated studies, Mitomycin C (MMC). Positive control for S9-activated studies, cyclophosphamide (CP).

Details on test system and experimental conditions:

DURATION
- Exposure duration: 20 hours without S9 mix; 4 hours with S9 mix.

NUMBER OF CELLS EVALUATED: 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.

DETERMINATION OF TOTOXICITY
- In the pre-test the toxicity of the test article was examined using the determination of the cell number. Cell numbers of two cultures (10 coordinate defined fields per culture) were determined for each experimental group.

Evaluation criteria:

A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher's-exact-test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

other: positive for the induction of structural and negative for the induction of numerical chromosome aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dose levels 5, 20, 45, and 60 μg/mL in the S9-activated 4-hour exposure group.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 2000 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 600 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 200 μg/mL in the non-activated 4 and 20-hour exposure groups, and at dose levels ≥ 60 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 300 μg/mL for the non-activated 4-hour exposure group, from 0.5 to 60 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 300 μg/mL for the non-activated 20-hour exposure group.

In the chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose levels in the non-activated 4 and 20-hour exposure groups. Cytotoxicity was observed at dose levels 5, 20, 45, and 60 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 200 μg/mL in the non-activated 4-hour exposure group, at 60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 150 μg/mL in the non-activated 20-hour exposure group. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 55 ± 5% reduction in cell growth index relative to the vehicle control, which met the dose limit as recommended by testing guidelines for this assay.

The percentage of cells with structural aberrations in the non-activated 4-hour exposure condition was statistically significantly increased (3.3%) relative to vehicle control at 100 μg/mL (p ≤ 0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05).

The percentage of cells with structural aberrations in the S9-activated 4-hour exposure condition was statistically significantly increased (5.3% and 4.7%) relative to vehicle control at dose levels 10 and 45 μg/mL, respectively (p ≤ 0.05, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05).

The percentage of cells with structural aberrations in the non-activated 20-hour exposure condition was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

The percentage of cells with numerical aberrations in the test substance-treated groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

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.

None

Conclusions:

FAT 36152/M TE was concluded to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the S9-activated test system in the in vitro mammalian chromosome aberration test using CHO cells. FAT 36152/M TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated test system in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

FAT 36152/M 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, CHO the 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. Dose formulations were adjusted for the purity of the test substance (91%), using a correction factor of 1.09. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 2000 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 600 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 200 μg/mL in the non-activated 4 and 20-hour exposure groups, and at dose levels ≥ 60 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 300 μg/mL for the non-activated 4-hour exposure group, from 0.5 to 60 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 300 μg/mL for the non-activated 20-hour exposure group.

In the chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose levels in the non-activated 4 and 20-hour exposure groups. Cytotoxicity was observed at dose levels 5, 20, 45, and 60 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 200 μg/mL in the non-activated 4-hour exposure group, at 60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 150 μg/mL in the non-activated 20-hour exposure group. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 55 ± 5% reduction in cell growth index relative to the vehicle control, which met the dose limit as recommended by testing guidelines for this assay.

The percentage of cells with structural aberrations in the non-activated 4-hour exposure condition was statistically significantly increased (3.3%) relative to vehicle control at 100 μg/mL (p ≤ 0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). The percentage of cells with structural aberrations in the S9-activated 4-hour exposure condition was statistically significantly increased (5.3% and 4.7%) relative to vehicle control at dose levels 10 and 45 μg/mL, respectively (p ≤ 0.05, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05).

The percentage of cells with structural aberrations in the non-activated 20-hour exposure condition was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

The percentage of cells with numerical aberrations in the test substance-treated groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

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, FAT 36152/M TE was concluded to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the S9-activated test system in the in vitro mammalian chromosome aberration test using CHO cells. FAT 36152/M TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated test system in the in vitro mammalian chromosome aberration test using CHO cells.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

FAT 36152/M TE was concluded to be negative in the in vivo mammalian cell unscheduled DNA synthesis (UDS) assay as well as micronucleus assay.

Link to relevant study records

Reference

Reference 1

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:

2016

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:

yes

Remarks:

: The mortality check for animals was performed twice on 30 Nov 2015. This is a deviation from the protocol which required that the mortality check be performed once each day.

Principles of method if other than guideline:

Guideline followed

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ENVIGO, Frederick, MD
- Age at study initiation: 8 weeks
- Weight at study initiation: 222.3 - 259.8 g
- Housing: Micro barrier cage
- Diet (e.g. ad libitum): Envigo 2018C Certified Global Rodent Diet ad libitum
- Water (e.g. ad libitum): Tap water (U.S. EPA drinking water standards) ad libitum
- Acclimation period: 6-9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3 °F
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10 changes of fresh HEPA-filtered air per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

:

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1%w/v methylcellulose in deionized water
- Concentration of test material in vehicle: Dose formulations were adjusted to compensate for the purity (91%) of the test substance using a correction factor of 1.090. The dosing formulation was prepared at a concentration of 3.5 mg/mL, just prior to use.
- Amount of vehicle (if gavage or dermal): 70%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were adjusted to compensate for the purity (91%) of the test substance using a correction factor of 1.090.
Dose formulations were prepared fresh on each day of use. Each concentration was prepared by calibrating a suitable size amber vial with a PTFE stir bar to appropriate volume. An appropriate amount of the test substance was weighed out and then transferred into the calibrated formulation container and then the vehicle was added to the QS line and mixed magnetically until homogenous in appearance. The final dose formulation was stored at room temperature.
Residual dose formulations were discarded after use.

All dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the unscheduled DNA synthesis assay.

Duration of treatment / exposure:

Only the high dose of 500 mg/kg at 2 to 4 hours and 12 to 16 hours harvest timepoints was evaluated for the UDS along with vehicle and positive controls.

Frequency of treatment:

All dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the unscheduled DNA synthesis assay.

Post exposure period:

None

Remarks:

Doses / Concentrations:
150, 350, 500
Basis:
nominal conc.

No. of animals per sex per dose:

4 animals/dose

Control animals:

yes

Positive control(s):

- Dimethylnitrosamine (DMN)
- Doses / concentrations: 35 mg/kg bw

Tissues and cell types examined:

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 microg/mL gentamicin and 10% fetal bovine serum). The cell viability for the vehicle control hepatocytes should be at least 70% for use in the assay.

Approximately 5 x 10E5 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 was set up for each animal. The hepatocyte cultures were maintained in a humidified atmosphere of 5±1% CO2 and 37±1°C.

Details of tissue and slide preparation:

Ninety to 180 minutes after plating, the cells were washed once with complete WME and serum-free WME containing 10 µCi 3H-thymidine/mL was added. 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 hours before mounting cell side up on glass slides. Each slide was identified by the study number, group number and animal number. Slides were coded using a random number table by an individual not involved with the scoring process, whenever possible.
At least 3 of the 6 slides for each animal were dipped in photographic emulsion at 43 - 45 °C, allowed to drain and dry for at least 1.5 hours at room temperature and were stored for eight days at 2-8 °C in light tight boxes with desiccant. Slides were developed in Developer (diluted 1:1 in deionized water), fixed in Fixer, and stained with hematoxylin-eosin stain.

Evaluation criteria:

Once the criteria for a valid assay were met, the results were evaluated as follows:
• Any mean net nuclear count that was increased by at least five counts over the negative control was considered significant (Butterworth et al., 1987).
• Test article was considered to be positive if it induced a dose related increase with no less than one dose significantly elevated above the negative control or if a significant increase in the mean net nuclear grain count in at least two successive doses was observed in the absence of a dose-response.
• The test substance was considered to be negative if no significant increase in the net nuclear grain counts was observed.
• The test substance was considered to be equivocal if there was a significant increase in the net nuclear grain count at the high dose only with no evidence of a dose-response or there was a significant increase in the net nuclear grain count at one dose level without a dose-response.
• Other criteria may have been used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations

Statistics:

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 could be counted. The ProtoCOL system with accompanying support software was used for grain counts. First, the number of grains in a nucleus was counted. Then, the number of grains in 3 separate nuclear-size cytoplasmic areas (taken from the area adjacent to the nucleus and which appeared to have the highest grain counts) was counted. The counts were captured directly by the software and stored as raw data in an electronic data file created by the software.
Fifty nuclei were scored from each of three replicate cultures for a total of 150 randomly selected nuclei from each animal. Nuclei selected were surrounded by cytoplasm. 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.
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.
The slides will be discarded prior to the finalization of the report.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

The following clinical signs were observed in the 12 to 16 hour harvest:

Dose Level (mg/kg bw)	Males
Vehicle	Normal
50	Normal
350	Normal
500	Normal
Positive Control	Diarrhea

The results of the UDS assay using primary hepatocytes isolated 2 to 4 hours post-exposure are summarized:

Dose Level (mg/kg bw)		Net Nuclear Grain Count (Mean±SD)	Cells in Repair (%)
Vehicle: 1% w/v methylcellulose (medium viscosity) in deionized water		-1.9±0.3	2
Test Substance: FAT 36152/M TE	350	-1.6±0.5	2
	500	-2.7±0.5	2
Positive Control: DMN 35 mg/kg		18.9±0.4	98

The results of the UDS assay using primary hepatocytes isolated 12 to 16 hours post-exposure are summarized:

Dose Level (mg/kg bw)		Net Nuclear Grain Count (Mean±SD)	Cells in Repair (%)
Vehicle: 1% w/v methylcellulose (medium viscosity) in deionized water		-1.8±0.8	2
Test Substance: FAT 36152/M TE	350	-2.3±0.8	3
	500	-1.7±1.2	3
Positive Control: DMN 35 mg/kg		25.1±4.6	98

Conclusions:

FAT 36152/M was concluded to be negative for the induction of unscheduled DNA synthesis in primary cultures.

Executive summary:

FAT 36152/M TE was evaluated for its genotoxic potential to induce unscheduled DNA synthesis in primary cultures of hepatocytes obtained from test substance-treated rats according to OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo).

1% w/v methycellulose (medium viscosity) in 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 only once. 

The definitive assay dose levels tested were 150, 350 and 500 mg/kg. Two harvest timepoints, 2 to 4 hours and 12 to 16 hours after dose administration were used for hepatocyte collection. Cells were processed and scored for the UDS assay. Only the high dose of 500 mg/kg with vehicle and positive control were scored and evaluated.

No significant increase in average MNNGC ( mean net nuclear grain count) was observed 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 MNNGC of the vehicle control group was less than 1, and the average MNNGC of the positive control group was at least 5 counts over that of the vehicle control group. 

Under the conditions of this study, the administration ofFAT 36152/M TE at a dose of 500 mg/kg bw was negative in the in vivo mammalian cell unscheduled DNA synthesis (UDS) assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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

Species:

mouse

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Dublin, VA on 27 August 2015
- Age at study initiation: 6 weeks
- Weight at study initiation: 161.1 - 190.3 g
- Housing: Micro barrier cages with heat treated hardwood chips were used for bedding to absorb liquids
- Diet (e.g. ad libitum): Certified Global Rodent Diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +- 3 °F
- Humidity (%): 50 +- 20%
- Air changes (per hr): 10 changes of fresh HEPA-filtered air per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

Deionised water

Details on exposure:

All dose formulations were administered at a volume of 10 mL/kg via oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely.

Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose, as indicated. 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.

Duration of treatment / exposure:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Frequency of treatment:

Single dose

Post exposure period:

No data

Remarks:

Doses / Concentrations:
150, 350, 500 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide monohydrate (CP)
- Route of administration: Once / daily
- Doses / concentrations: 10 ml/kg b.w.

Tissues and cell types examined:

The femora were removed, the epiphyses were cut off and the marrow .

Details of tissue and slide preparation:

Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose, as indicated. 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.

Preparation of Micronucleus Slides
The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant 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 four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least 5 sets of two positive control slides) were stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup. Each slide was identified by the harvest date, study number, and animal number (or slide number for positive control slides). Slides were coded using a random number table by an individual not involved with the scoring process.

Evaluation criteria:

The test substance was considered to have induced a positive response if -

• at least one of the test substance doses exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• when multiple doses are examined at a particular sampling time, the increase is dose-related (p ≤ 0.01), and
• results of the group mean or of the individual animals in at least one group are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there is evidence that the bone marrow was exposed to the test substance (unless intravenous administration was used).

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

None

Micronucleus Assay

No mortality occurred at any dose level during the course of the definitive assay. All animals appeared normal throughout the observation period.

Bone Marrow Analysis

The incidence of MnPCEs per 20,000 PCEs scored (4000 PCEs/animal) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized.

The scoring results and a statistical analysis of data indicated the following:

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

• No statistically significant increase in the incidence of MnPCEs in the test substance treated groups was observed relative to the negative control group (ANOVA, p > 0.05, Dunnett’s post hoc analysis).

• The positive control, CP, induced a statistically significant increase in the incidence of MnPCEs (Student’s t-test, p ≤ 0.05).

• The number of MnPCEs in the vehicle control groups did not exceed the historical control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Based upon this, all criteria for a valid test were met as specified in the protocol.

Conclusions:

FAT 36152/M TE was concluded to be negative for the induction of MnPCEs in the In Vivo Mammalian Erythrocyte Micronucleus Assay in Rats.

Executive summary:

FAT 36152/M TE, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in male rat bone marrow. Methylcellulose (1% w/v) (medium viscosity) in deionized water was used as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg via oral gavage. In the definitive assay, the dose levels tested were 150, 350, and 500 mg/kg.

No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes scored (PCEs/EC ratio) was observed in the test substance groups compared to the vehicle control group, indicating the test did not induce cytotoxicity. No statistically significant increase in the incidence of micronucleated PCEs (MnPCEs) in the test substance treated groups was observed relative to the vehicle control groups. The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control groups did not exceed the historical control range.

Under the conditions of this study, the administration of FAT 36152/M TE at doses up to and including a dose of 500 mg/kg was concluded to be negative in the In Vivo Mammalian Erythrocyte Micronucleus Assay in Rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

Target chemical was evaluated in a bacterial reverse mutation assay and an in vitro mammalian chromosomal aberration assay.

Bacterial reverse mutation assay:

A study was performed to investigate the potential of FAT 36152/F to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 36152/F at any dose level, either in the presence or absence of metabolic activation (89 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Therefore, FAT 36152/F is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro mammalian chromosomal aberration assay:

In another study, FAT 36152/M TE was tested in the in vitro 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. Based on the findings of the study, FAT 36152/M TE was concluded to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the S9-activated test system in the in vitro mammalian chromosome aberration test using CHO cells. FAT 36152/M TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated test system in the in vitro mammalian chromosome aberration test using CHO cells.

In vivo studies:

Target chemical was evaluated in an UDS assay as well as micronucleus assay.

In vivo UDS assay

FAT 36152/M TE, was evaluated for its genotoxic potential to induce unscheduled DNA synthesis in primary cultures of hepatocytes obtained from test substance-treated rats according to OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo). 1% w/v methycellulose (medium viscosity) in 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 only once. The definitive assay dose levels tested were 150, 350 and 500 mg/kg. Two harvest time points, 2 to 4 hours and 12 to 16 hours after dose administration were used for hepatocyte collection.  At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15%, the average MNNGC of the vehicle control group was less than 1, and the average MNNGC of the positive control group was at least 5 counts over that of the vehicle control group.  Under the conditions of this study, the administration of FAT 36152/M TE at a dose of 500 mg/kg bw was negative in the in vivo mammalian cell unscheduled DNA synthesis (UDS) assay.

Micronucleus assay

FAT 36152/M TE, was evaluated in a micronucleus assay for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in male rat bone marrow. Methylcellulose (1% w/v) (medium viscosity) in deionized water was used as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg via oral gavage. In the definitive assay, the dose levels tested were 150, 350, and 500 mg/kg. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes scored (PCEs/EC ratio) was observed in the test substance groups compared to the vehicle control group, indicating the test did not induce cytotoxicity. No statistically significant increase in the incidence of micronucleated PCEs (MnPCEs) in the test substance treated groups was observed relative to the vehicle control groups. The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control groups did not exceed the historical control range. Under the conditions of this study, the administration of FAT 36152/M TE at doses up to and including a dose of 500 mg/kg was concluded to be negative in the In Vivo Mammalian Erythrocyte Micronucleus Assay in Rats.

Target chemical was found to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the S9-activated test system in the in vitro mammalian chromosome aberration test using CHO cells.

However, it was not mutagenic in a bacterial reverse mutation assay, did not induce DNA damage in an in vivo UDS assay and did not induce clastogenic activity in a micronucleus assay. Hence, the target chemical was considered to be neither mutagenic nor clastogenic and hence not genotoxic.

Justification for classification or non-classification

The substance under evaluation is considered to be not genotoxic, hence does not warrant classification for mutagenicity as per the Regulation (EC) No. 1272/2008 (CLP) criteria.