4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H-naphth[2,3-f]isoindole-1,3,5,10(2H)-tetrone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 36152/G induced point mutations by frameshifts in the genome of the strain TA 98 with metabolic activation. Source chemical, FAT 36152/F was not mutagenic in the Salmonella typhimurium reverse mutation assay, while source chemical FAT 36152/M 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Source chemical 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

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 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 at a dose of 500 mg/kg bw was negative in the in vivo mammalian cell unscheduled DNA synthesis (UDS) assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Target chemical FAT 36152/G was found to induce mutations in the genome of the strain TA 98 in presence of metabolic activation. No other studies are available either to fulfil the data requirements as required by Regulation (EC) No. 1272/2008 or to complete the assessment of genetic toxicity potentisal of the substance. Hence, genetic toxicity available with structurally similar substance (FAT 36152/F and FAT 36152/M) were used.

 

In vitro studies:

As stated earlier only a bacterial reverse mutation assay is available with the target chemical. Source chemical was evaluated in a bacterial reverse mutation assay and an in vitro mammalian chromosomal aberration assay.

 

Target chemical:

A study was performed to investigate the potential of target chemical, FAT 36152/G; Terasil Blau BGE Langamin roh feucht (laborgetrocknet) to induce gene mutations according to the plate incorporation test (experiment I and II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. No toxic effects occurred in experiment I and II with and without metabolic activation in all strains used. The plates incubated with the test article showed normal background growth up to 5000.0µg/plate with and without S9 mix in all strains used. Up to the highest investigated dose a clear reproducible dosedependent increase in revertant colony numbers was observed with metabolic activation in tester strain TA 98. In conclusion, it can be stated that the target chemical induced point mutations by frameshifts in the genome of the strain TA 98 with metabolic activation.

 

Source chemical

A study was performed to investigate the potential of source chemical 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 another study, source chemical FAT 36152/M 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 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:

Currently no in vivo genetic toxicity studies are available with target chemical, however source chemical was evaluated in an UDS assay as well as micronucleus assay.

 

Source Chemical:

FAT 36152/M, 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 at a dose of 500 mg/kg bw was negative in the in vivo mammalian cell unscheduled DNA synthesis (UDS) assay. 

FAT 36152/M, 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 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.

The target chemical was found to have mutagenic activity when tested with bacterial cells, while source chemical was positive in an in vitro mammalian chromosomal aberration assay. However, source chemical 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, source chemical and based on the principles of read across, target chemical are 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.