N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 36156 has been tested for mutagenic effects in the Ames test according to OECD 471 involving Salmonella tester stains TA98, TA100, TA102, TA1535 and E. coli WP2/uvra with and without metabolic activation. In addition, the substance has been investigated for clastogenic effects in eukaryotic cells using the chromosome aberration test according to OECD guideline 473. Incubation of bacteria with the test substance showed mutagenic effects, results in eukaryotic cells raised concerns for clastogenic effects.

Link to relevant study records

Reference

Reference 1

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:

12 October 2015 to 27 January 2016

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 20140804 (China)
- Expiration date of the lot/batch: 21 August 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 0, 200, 600, 2000 μg/mL
Without S9 (4 h): 5, 10, 20, 40, 60, 80, 100 μg/mL
Without S9 (20 h): 0.25, 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10 μg/mL
Without S9 (4 h): 5, 10, 20, 40, 60, 80, 100 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethylsulfoxide (DMSO) was used as the vehicle based on the solubility tests and compatibility with the target cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Toxicity of FAT 36156/D TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 30% at 40 μg/mL, the highest test dose level evaluated for chromosome aberrations.

Toxicity of FAT 36156/D TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 46% at 40 μg/mL, the highest test dose level evaluated for chromosome aberrations

DETERMINATION OF CYTOTOXICITY
Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 600 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 6 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.

Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:
Non-activated 4 hr: 5, 10, 20, 40, 60, 80, 100 μg/mL
Non-activated 20 hr: 0.25, 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10 μg/mL
S9-activated 4 hr: 5, 10, 20, 40, 60, 80, 100 μg/mL

Evaluation criteria:

Criteria for Determination of a Valid Test:

Vehicle Controls:
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).

Positive Controls:
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).

Cell Proliferation:
The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.

Test Conditions:
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 20-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

Analyzable Concentrations:
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.

Evaluation of Test Results:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were 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.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

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:

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: The pH of the highest dose level of test substance in treatment medium was 7.4.
- Effects of osmolality: The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numerical aberrations in the 4-hr treatment were statically positive without a dose response. The induced values were in the line of the historical control range (0-5%, in the non-activated 4-hour exposure group and 0-9.5%, in the S9-activated 4-hour exposure group), therefore, it was considered biologically irreverent.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The results of the evaluation of cell growth inhibition are presented in Tables under any other information section. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 600 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 6 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.

Remarks on result:

other: strain/cell type: Chinese hamster ovary (CHO-K1) cells

Preliminary Toxicity Assay

In the preliminary toxicity assay, CHO cells were exposed to nine dose levels of FAT 36156/D TE, ranging from 0.2 to 2000 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 h, or continuously for 20 h in the absence of S9 activation. The test substance formed clear solutions in DMSO from 0.02 to 0.06 mg/mL and workable suspensions in DMSO from 0.2 to 200 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

 

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 h	≥ 600 µg/mL	≥ 60 µg/mL
	20 h	≥ 600 µg/mL	≥ 60 µg/mL
S9-activated	4 h	≥ 600 µg/mL	≥ 60 µg/mL

 

The osmolality in treatment medium was measured as follows:

The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%. The pH of the highest dose level of test substance in treatment medium was 7.4.

 

 

Dose tested	Dose levels (µg/mL)	Osmolality (mmol/kg)
Vehicle	0	430
Highest soluble	200	438
Lowest precipitating	600	440
Highest	2000	417

 

In the chromosome aberration assay, the test substance formed clear solutions in DMSO from 0.025 to 0.05 mg/mL and workable suspensions in DMSO from 0.1 to 10 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 h	None	≥ 40 µg/mL
	20 h	None	None
S9-activated	4 h	None	≥ 40 µg/mL

 

Conclusions:

FAT 36156/D TE was concluded to be positive for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

FAT 36156/D TE, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells following OECD guideline 473 in compliance to GLP 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 cells were treated for 4 and 20 h in the non-activated test system and for 4 h in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dimethylsulfoxide (DMSO) was used as the vehicle.

 

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 600 μg/mL in the non-activated 4-h exposure group and at dose levels ≥ 6 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-h exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 60 μg/mL in all three treatment conditions. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 5 to 100 µg/mL for the non-activated and S9-activated 4-h exposure groups, and from 0.25 to 10 µg/mL for the non-activated 20-h exposure group.

 

In the chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 3.5 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the non-activated and S9-activated 4-h exposure groups. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 40 μg/mL in the non-activated and S9-activated 4-h exposure groups. The dose levels selected for microscopic analysis were 10, 20, and 40 µg/mL for the non-activated and S9-activated 4-hour exposure groups, and 1, 2, and 4.5 µg/mL for the non-activated 20-h exposure group.

 

The percentage of cells with structural and numerical aberrations in the non-activated 4-h exposure group was statistically significantly increased relative to vehicle control at dose levels 10, 20, and 40 μg/mL (p ≤ 0.05 or p ≤ 0.01, 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-h exposure group was statistically significantly increased relative to vehicle control at 40 μg/mL (p ≤ 0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose-response (p ≤ 0.05).

 

The percentage of cells with numerical aberrations in the S9-activated 4-h exposure group was statistically significantly increased relative to vehicle control at dose levels 10 and 20 μg/mL (p ≤ 0.01, 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 non-activated 20-h exposure group was statistically significantly increased relative to vehicle control at dose levels 2 and 4.5 μg/mL (p ≤ 0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose-response (p ≤ 0.05). The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

 

The numerical aberrations in the 4-h treatment were statically positive without a dose response. The induced values were in the line of the historical control range (0-5%, in the non-activated 4-h exposure group and 0-9.5%, in the S9-activated 4-h exposure group), therefore, it was considered biologically irreverent. All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

 

Under the conditions of the assay described in this report, FAT 36156/D TE was concluded to be positive for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. Although there was a significant induction in numerical aberrations, it was considered biologically irrelevant.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2015 to 03 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 20140804 (China)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

Target gene:

Histidine auxotophs - Salmonella strains, tryptophan auxotrophs - E. coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Preliminary toxicity assay: The dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate.
Mutagenicity assay: The dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate with tester strain TA98, and 50.0, 150, 500, 1500 and 5000 μg per plate with the remaining tester strains.

Vehicle / solvent:

The vehicle used to deliver FAT 36156/D TE to the test system was DMSO.
- Vehicle: Dimethyl sulfoxide (DMSO)
- CAS Number: 67-68-5
- Supplier: Sigma-Aldrich
- Lot Number: BCBJ4366V
- Purity/ Grade: 99.99 %
- Expiration Date: February 2018

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. Following sonication at 24.7 °C for 30 minutes, the test substance formed a workable suspension in DMSO at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation. Aroclor 1254-induced rat liver S9 activation (oxidative). (TA98 and TA1535: 2-AA at 1.0 μg/plate; TA100 and TA1537: 2-AA at 2.0 μg/plate and WP2 uvrA: 2-AA at 15μg/plate.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

With uninduced hamster liver S9 activation (reductive). Red congo at 100 μg/plate for TA98.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation. 2-nitrofluorene at 1.0 μg/plate for TA98.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation. Sodium azide at 1.0 μg/plate for TA100 and TA 1535.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation. 9-aminoacridine at 75.0 μg/plate for TA1537.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation. Methylmethanesulfonate at 1000 μg/plate for WP2 uvrA.

Details on test system and experimental conditions:

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

NUMBER OF REPLICATIONS: Three

After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2 °C. Plates that were not counted immediately following the incubation period were stored at 2-8 °C until colony counting could be conducted.

Evaluation criteria:

Evaluation of Test Results:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below :
- Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
- Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

Standard deviation

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Solubility Test

DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. Following sonication at 24.7 °C for 30 minutes, the test substance formed a workable suspension in DMSO at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance.

 

Sterility Results

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

 

Preliminary Toxicity Assay

In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 50.0 mg/mL and a 100 µL plating aliquot. The dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Dose-responsive increases in revertant counts (ranging from 4.2- to 50.7-fold maximum increases) were observed with all Salmonella tester strains in the absence of S9 activation and in the presence of oxidative and reductive S9 activation, as well as tester strain WP2uvrA in the presence of oxidative S9 activation. Precipitate was observed beginning at 667 or 3333 µg per plate. No toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

 

Mutagenicity Assay

In Experiment B1 (Mutagenicity Assay), positive mutagenic responses (ranging from 5.6- to 47.1-fold maximum increases) were observed with all Salmonella tester strains in the absence of S9 activation and in the presence of oxidative and reductive S9 activation. No positive mutagenic responses were observed with tester strain WP2uvrA under any activation condition. The dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with testerstrain TA98, and 50.0, 150, 500, 1500 and 5000 µg per plate with the remaining tester strains. Precipitate was observed beginning at 500 µg per plate. No toxicity was observed.

Conclusions:

FAT 36156/D TE did cause positive mutagenic responses with all Salmonella tester strains in the absence of S9 activation and in the presence of both oxidative and reductive S9 activation.

Executive summary:

FAT 36156/D TE, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the absence of S9 activation and in the presence of both Aroclor-induced rat liver S9 activation (oxidative) and uninduced hamster liver S9 activation (reductive). The assay was performed in two phases, using the preincubation method. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test substance. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. Following sonication at 24.7°C for 30 minutes, the test substance formed a workable suspension in DMSO at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance. In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 50.0 mg/mL and 100 µL plating aliquot. The dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Dose-responsive increases in revertant counts (ranging from 4.2- to 50.7-fold maximum increases) were observed with all Salmonella tester strains in the absence of S9 activation and in the presence of oxidative and reductive S9 activation, as well as tester strain WP2 uvrA in the presence of oxidative S9 activation. Precipitate was observed beginning at 667 or 3333 µg per plate. No toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the mutagenicity assay, positive mutagenic responses (ranging from 5.6- to 47.1-fold maximum increases) were observed with all Salmonella tester strains in the absence of S9 activation and in the presence of oxidative and reductive S9 activation. No positive mutagenic responses were observed with tester strain WP2 uvrA under any activation condition. The dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with tester strain TA98, and 50.0, 150, 500, 1500 and 5000 g per plate with the remaining tester strains. Precipitate was observed beginning at 500 µg per plate. No toxicity was observed. All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, FAT 36156/D TE did cause positive mutagenic responses with all Salmonella tester strains in the absence of S9 activation and in the presence of both oxidative and reductive S9 activation. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

FAT 36156 caused cytogenetic damage but did not lead to increased DNA damage at and up to 2000 mg/kg bw/day. 

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 30 July 2022; Experiment start date - 03 August 2022; Experiment completion date - 16 November 2023; Study completion date - 10 January 2023.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Name of Test Item: FAT 36156
Chemical Name (IUPAC): N-{2-[-(2-cyano-4,6-dinitrophenyl)diazenyl]-5-(diethylamino) phenyl}acetamide
CAS No. : 24170-60-3
Physical Appearance (with colour): green powder
Batch No.: FAT 36156 D/TE (20140804 (China))
Purity (as per certificate of analysis): 96.9 %
Batch Produced by (Name and address) : Third party (confidential)
Date of Manufacture: August 2014
Date of Expiry : April 28th, 2023
Date of Retest: May 3rd, 2021
Storage Conditions: Ambient (21 to 29 °C)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.

Sex:

male

Details on test animals or test system and environmental conditions:

Name of Test Item: FAT 36156
CAS No : 24170-60-3
Physical Appearance (with colour): green powder
Batch No: D/TE (20140804 (China))
Purity (as per certificate of analysis): 96.9 %
Date of Manufacture: August 2014
Date of Expiry: April 28th, 2023
Date of Retest: May 3rd, 2021
Storage Conditions: Ambient (21 to 29 °C)

Route of administration:

oral: gavage

Vehicle:

0.5 % carboxymethyl cellulose

Details on exposure:

The test item was administered through oral route. The oral route is one of the probable routes of human exposure. The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula. All the doses were administered in an equal volume of 10 mL/kg bw/day the dose of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg bw/day as low, mid and high dose, respectively. Vehicle control group (G1) animals were administered with vehicle. Positive control group G5 and G6 animals were administered with ethyl methanesulfonate and cyclophosphamide monohydrate, respectively, at the dose volume of 10 mL/kg bw/day. Ethyl methanesulfonate and cyclophosphamide monohydrate were dissolved in distilled water and administered at a dose of 250 and 100 mg/kg bw/day, respectively.

Duration of treatment / exposure:

oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula.

Frequency of treatment:

oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula

Post exposure period:

Post 3 hours of the last day dosing, terminal sacrifice was done for all animals and all the animals were subjected to gross pathological examination.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

6 males per group

Control animals:

yes

Positive control(s):

Ethyl methanesulfonate

Tissues and cell types examined:

The animals were subjected to gross pathological examination.

Details of tissue and slide preparation:

Animals were euthanized by cervical dislocation 3 hours after the third treatment and subjected to gross pathological examination. Bone marrow was collected for micronucleus test. Liver, glandular stomach, duodenum and testicles were collected for the comet assay.

The liver, glandular stomach and duodenum for the comet assay was placed into ice-cold mincing and/or homogenization buffer (DPBS) and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and stored in ice-cold mincing buffer until processed. A portion of the liver, glandular stomach and duodenum were collected and preserved in 10 % Neutral Buffered Formalin fixative and testes were preserved in modified Davidson’s fixative for 24 to 48 hours and then transferred to 10 % NBF for histopathology.

All the slides were coded before evaluation to avoid group bias during evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes and stain with ethidium bromide, covered with a fresh coverslip and cells were scored under 400 X magnification.

Evaluation criteria:

At least 150 cells were analysed per sample. The open comet software was used for analysis. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs was determined for at least 150 cells per sample.

Statistics:

Body weight of day 1, 2 and 3 was analysed by SPSS, at a 95 % level (p ≤0.05) of significance. Inter group comparison of body weight of Day 1, 2 and 3 were done.

The clinical chemistry examination was analysed by SPSS at a 95% level (p ≤0.05) of significance. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gamma-glutamyl transpeptidase (GGT) were subjected to statistical analysis.
The percentage of tail DNA was analysed by SPSS at a 95 % level of confidence (p <0.05) of significance*.
The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95 % level (p ≤0.05) of significance. All analysis and comparisons were evaluated at the 95 % level of confidence (p <0.05). Statistically significant changes obtained were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (p <0.05).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The dose formulation samples were analysed for dose concentration by HPLC and the results are found within the range of ± 15 % (i.e. 85 % to 115 %) recovery to the nominal concentration.

There was no statistically significant variation in body weight for treated animals.
No test item related gross or histopathological changes were seen in any of the treated animals.
No adverse treatment-related changes were observed in clinical chemistry parameters.

Clinical Signs of Toxicity and Mortality
The administration with the test item resulted in no clinical signs or mortality in any of the treated animals.

Body Weight
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

Gross Pathology
No gross pathological findings were observed in any of the animals dosed with the test item at the doses of 500, 1000 and 2000 mg/kg bw/day.

HISTOPATHOLOGY FINDINGS
There were no test item-related histopathological findings observed in the study. Few microscopic findings were observed in the study such as infiltration of mononuclear cells in liver and all other findings were considered incidental as they occurred randomly across the groups and/or were expected for laboratory rats.

Clinical Chemistry
No adverse treatment related variation was noted in clinical chemistry parameters.
There were no statistically significant variations noted.

EVALUATION OF DNA DAMAGE
All the slides were coded before evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes, stained with ethidium bromide, covered with a fresh coverslip and cells were then scored under 400X magnification.
At least 150 cells were analyzed per sample. The comet endpoint collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs was determined in at least 150 cells per sample bw/day. The average % tailing for DNA from liver cells was 3.66, 3.74, 3.69 and 3.76 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. The average % tailing for DNA from glandular stomach cells were 3.28, 3.35, 3.22 and 3.13 respectively and in duodenum, the % tailing observed were 3.77, 3.74, 3.81, and 3.65, at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose-dependent or statistically significant increase in the % tail DNA in any of the test item dosed animals, in comparison with the vehicle control group. The average % tail DNA observed in the liver, glandular stomach and duodenum of the positive control (at 250 mg/kg bw/day of ethyl methyl sulfonate) dosed animals were 8.25, 8.90 and 8.03, respectively.

Conclusions:

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 36156 not lead to increased DNA damage at and up to 2000 mg/kg bw/day.

Executive summary:

The test item, FAT 36156, was evaluated in the “In vivo Mammalian Alkaline Comet Assay” as per OECD Guideline No. 489, adopted on 29 July 2016. This study was conducted to determine if the test item, FAT 36156, can cause an increase in DNA damage in cells from specific organs and also to detect damage. The comet assay detects single and double stranded breaks when DNA is analyzed under alkaline conditions (pH >13). These strand breaks, when they occur in vivo, may be repaired, resulting in no persistent effect, or may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. This study used 6 groups of rats and each group consisted of 6 males. The animals received designated treatment for 3 consecutive days by oral route using gavage canula. The animals designated as group G1 animals were administered with 0.5 % carboxymethyl cellulose as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 36156, respectively. The animals in group G5 were administered 250 mg/kg bw/day of the positive control ethyl methanesulfonate (for comet assay). Approximately 3 hours after the last dosing, all rats were sacrificed by cervical dislocation and the designated organs (liver, duodenum, glandular stomach and testis) were collected for G1 to G5 groups. The collected tissues were processed, single cells were isolated, and slides were prepared. Slides were run through submarine-type electrophoresis and drained. Drained slides were stained with ethidium bromide and evaluated for % tailing of DNA, i.e. tail length in microns measured from the estimated edge of the head region closest to the anode. In the comet assay, the average % tailing for DNA from male liver cells was 3.66, 3.74, 3.69 and 3.76 at 0, 500, 1000 and 2000 mg/kg bw/day respectively. The average % tailing for DNA from glandular stomach cells were 3.28, 3.35, 3.22 and 3.13 and in duodenum, the % tailing observed were 3.77, 3.74, 3.81 and 3.65 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any of the organs for any of the FAT 36156 treated groups when compared to the vehicle control group. The average % tail DNA observed in the liver, glandular stomach and duodenum of the positive control (at 250 mg/kg bw/day of ethyl methyl sulfonate) dosed animals were 8.25, 8.90 and 8.03 respectively. The positive control [G5], ethyl methanesulfonate at a dose of 250 mg/kg bw/day produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (liver, duodenum and glandular stomach) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the comet assay for this test of FAT 36156 were fully adequate. There was no statistically significant variation in body weight for treated animals. No test item related gross or histopathological changes were seen in any of the treated animals. No adverse treatment-related changes were observed in clinical chemistry parameters. The dose formulation samples were analyzed for dose concentration by HPLC and the results are found within the range of ±15 % (i.e. 85 % to 115 %) recovery to the nominal concentration. Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 36156 not lead to increased DNA damage at and up to 2000 mg/kg bw/day.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 30 July 2022; Experiment start date - 03 August 2022; Experiment completion date - 16 November 2023; Study completion date - 10 January 2023

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Name of Test Item: FAT 36156
CAS No : 24170-60-3
Physical Appearance (with colour): green powder
Batch No: D/TE (20140804 (China))
Purity (as per certificate of analysis): 96.9 %
Date of Manufacture: August 2014
Date of Expiry: April 28th, 2023
Date of Retest: May 3rd, 2021
Storage Conditions: Ambient (21 to 29 °C)

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Test System:
Animal species: Rat (Rattus norvegicus)
Strain: Wistar
Justification for selection of species: The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.
Source of supply: Inhouse bred animals
No. of animals / group and sex: 6 males/group (36 males)
Age at treatment: 9 weeks
Body weight range at receipt (g): 194.45 to 243.5 g
Acclimatization period: All the animals were identified by tail marking using a black marker pen. Additionally, a cage card was displayed which included study no., cage no., sex, animal no. (temporary), start date and end date of acclimatization period.
Treatment period: All the animals were identified by body marking using turmeric solution, potassium permanganate and additionally, a cage card was displayed which included at least study no., cage no., sex, animal no. (permanent), treatment date and date of necropsy.

Husbandry:
Environmental Conditions: Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.7 to 22.9 ºC and relative humidity 45 % to 64 % in main study, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Housing: Maximum of three animals of same sex and group were housed in a standard polysulphonate rat cage (L 430 × B 280 × H 210 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized corn cob was used as a bedding material.
Feed: Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

0.5 % carboxy methyl cellulose

Duration of treatment / exposure:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula. All the doses were administered in an equal volume of 10 mL/kg bw/day the dose of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg bw/day as low, mid and high dose, respectively. Vehicle control group (G1) animals were administered with vehicle. Positive control group G6 animals were administered cyclophosphamide monohydrate, at the dose volume of 10 mL/kg bw/day. cyclophosphamide monohydrate were dissolved in distilled water and administered at a dose of100 mg/kg bw/day,
The dose volume administered to each animal was calculated for individual animals on the day of treatment based on the individual animal body weight.

Frequency of treatment:

The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula

Post exposure period:

Post 3 hours of the last day dosing, terminal sacrifice was done for all animals and all the animals were subjected to gross pathological examination.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

6 males/group (36 males)

Control animals:

yes

Positive control(s):

Yes, Cyclophosphamide Monohydrate.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

The femurs were isolated from each animal for bone marrow collection. Bone marrow cells were obtained by cut opening the epiphyses of femur bone immediately following sacrifice. The marrow was flushed out into a centrifuge tube using the Fetal Bovine Serum (FBS). The femur bone marrow cells were centrifuged at about 2700±100 rpm for 10 minutes. Prior to smear preparation, the supernatant were discarded and the cell pellet was then resuspended in approximately 50 µL of Fetal Bovine Serum (FBS).

Evaluation criteria:

All the slides including those of positive and vehicle controls were coded before microscopic evaluation to avoid group bias during evaluation. For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCEs: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).

Statistics:

Body weight of day 1, 2 and 3 was analysed by SPSS, at a 95 % level (p ≤0.05) of significance. Inter group comparison of body weight of Day 1, 2 and 3 were done. The slides from main study were decoded after analysis, the number of PCE (Polychromatic erythrocytes), RBC (Red blood corpuscles), MNPCE (Micronucleated Polychromatic erythrocytes) and PCEs/ total erythrocytes ratio (Polychromatic erythrocytes/ total erythrocytes) and frequency of MNPCEs was calculated. The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95 % level (p ≤0.05) of significance. All analysis and comparisons were evaluated at the 95 % level of confidence (p <0.05).

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Clinical Signs of Toxicity and Mortality:
The administration with the test item resulted in no clinical signs or mortality in any of the treated animals.

Body Weight:
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

Gross Pathology:
No gross pathological findings were observed in any of the animals dosed with the test item at the doses of 500, 1000 and 2000 mg/kg bw/day.

HISTOPATHOLOGY FINDINGS:
There were no test item-related histopathological findings observed in the study. Few microscopic findings were observed in the study such as infiltration of mononuclear cells in liver and all other findings were considered incidental as they occurred randomly across the groups and/or were expected for laboratory rats.

Clinical Chemistry:
No adverse treatment related variation was noted in clinical chemistry parameters. There were no statistically significant variations noted.

PCEs: Total RBC Ratio and Incidence of Micronucleus:
The average numbers of micronucleated polychromatic erythrocytes (MNPCEs) were observed for a minimum of 4000 polychromatic erythrocytes (PCEs). The average percentage of MNPCEs in animals dosed with vehicle was 0.08. In the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.16, 0.64 and 1.22. There was statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at 1000 and 2000 mg/kg bw/day, when compared with vehicle control. In the positive control animals, the incidence of percent MNPCEs was 0.83.

Conclusions:

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 36156 can cause cytogenetic damage, at and up to 2000 mg/kg bw/day.

Executive summary:

This study was conducted according to OECD test guideline 474 in a GLP certified laboratory, to determine the genotoxic potential of FAT 36156 (the test item) in the micronucleus test using bone marrow cells of Wistar rats. The test item, vehicle control and the positive control were dosed for three consecutive days by oral route using oral gavage cannula. After the last dosing, animals were sacrificed and bone marrow cells were collected. The slides of bone marrow cells were stained with May-Gruenwald and Giemsa stain and observed for incidences of micronucleated polychromatic erythrocytes (MNPCEs). This study used 6 groups of rats and each group consisted of 6 males. The animals received designated treatment for 3 consecutive days by oral route using gavage canula. The animals designated as group G1 animals were administered with 0.5 % carboxymethyl cellulose as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 36156, respectively. The animals in group G6 were administered 100 mg/kg bw/day of the positive control cyclophosphamide monohydrate (for micronucleus test). Approximately 3 hours after the last dosing, all rats were sacrificed by cervical dislocation. Bone marrow was collected for G6 group. In the micronucleus test, the average percentage of MNPCEs was 0.08 in males dosed with vehicle. For the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.16, 0.64 and 1.22 respectively. There was statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at the doses of 1000 and 2000 mg/kg bw/day of the test item, in comparison with the vehicle control. The positive control group (G6), cyclophosphamide monohydrate at 100 mg/kg bw/day exhibited statistically significant increase in the numbers of MNPCEs when compared to vehicle control and the average percentage of MNPCEs (per 4000 PCEs scored) in positive control was 0.83. This demonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. There was no statistically significant variation in body weight for treated animals. No test item related gross or histopathological changes were seen in any of the treated animals. No adverse treatment-related changes were observed in clinical chemistry parameters. The dose formulation samples were analyzed for dose concentration by HPLC and the results are found within the range of ±15 % (i.e. 85 % to 115 %) recovery to the nominal concentration. Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 36156, can cause cytogenetic damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

In vitro bacterial reverse mutation assay:

The test substance, FAT 36156/D, was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the absence of S9 activation and in the presence of both Aroclor-induced rat liver S9 activation (oxidative) and uninduced hamster liver S9 activation (reductive). The assay was carried out according to OECD Guideline 471. The assay was performed in two phases, using the preincubation method. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test substance.

In the mutagenicity assay, positive mutagenic responses (ranging from 5.6- to 47.1-fold maximum increases) were observed with all Salmonella tester strains in the absence of S9 activation and in the presence of oxidative and reductive S9 activation. No positive mutagenic responses were observed with tester strain WP2 uvrA under any activation condition. The dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with tester strain TA98, and 50.0, 150, 500, 1500 and 5000 g per plate with the remaining tester strains. Precipitate was observed beginning at 500 µg per plate. No toxicity was observed. All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, FAT 36156/D did cause positive mutagenic responses with all Salmonella tester strains in the absence of S9 activation and in the presence of both oxidative and reductive S9 activation. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted. Under the conditions of this study, FAT 36156/D was concluded to be positive with all Salmonella tester strains in the absence of S9 activation and in the presence of both oxidative and reductive S9 activation in the bacterial reverse mutation assay.

 

In-vitro chromosomal aberration test:

A second key study, FAT 36156/D, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells following OECD Guideline 473 in compliance to GLP 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 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. Dimethylsulfoxide (DMSO) was used as the vehicle.

In the chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥3.5 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the non-activated and S9-activated 4-hour exposure groups. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥40 μg/mL in the non-activated and S9-activated 4-hour exposure groups. The dose levels selected for microscopic analysis were 10, 20, and 40 µg/mL for the non-activated and S9-activated 4-hour exposure groups, and 1, 2, and 4.5 µg/mL for the non-activated 20-hour exposure group. The percentage of cells with structural and numerical aberrations in the non-activated 4-hour exposure group was statistically significantly increased relative to vehicle control at dose levels 10, 20, and 40 μg/mL (p ≤0.05 or p ≤0.01, 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 group was statistically significantly increased relative to vehicle control at 40 μg/mL (p ≤0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose-response (p ≤0.05). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was statistically significantly increased relative to vehicle control at dose levels 10 and 20 μg/mL (p ≤0.01, 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 non-activated 20-hour exposure group was statistically significantly increased relative to vehicle control at dose levels 2 and 4.5 μg/mL (p ≤0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose-response (p ≤0.05). The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The numerical aberrations in the 4-hr treatment were statically positive without a dose response. The induced values were in the line of the historical control range (0-5 %, in the non-activated 4-hour exposure group and 0-9.5 %, in the S9-activated 4-hour exposure group), therefore, it was considered biologically irreverent. All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤0.01). Thus, all criteria for a valid study were met.

 

Under the conditions of the assay described in this report, FAT 36156/D was concluded to be positive for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. Although there was a significant induction in numerical aberrations, it was considered biologically irrelevant.

 

As there were positive results in the in vitro genotoxicity studies that needs to be conducted in accordance with Annex VII and VIII and there are no results available from an in vivo study already, an in vivo mammalian alkaline comet assay combined with micronucleus test was conducted as per ECHA recommendations. 

In vivo mammalian alkaline assay combined with micronucleus test

The test item, FAT 36156, was evaluated in the “In vivo Mammalian Alkaline Comet Assay” as per OECD Guideline No. 489, adopted on 29 July 2016 and “Mammalian Erythrocyte Micronucleus Test” as per OECD Guideline No. 474, adopted on 29 July 2016. This study used 6 groups of rats and each group consisted of 6 males. The animals received designated treatment for 3 consecutive days by oral route using gavage canula. The animals designated as group G1 animals were administered with 0.5 % carboxymethyl cellulose as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 36156, respectively. The animals in group G5 were administered 250 mg/kg bw/day of the positive control ethyl methanesulfonate (for comet assay) and the animals in group G6 were administered 100 mg/kg bw/day of the positive control cyclophosphamide monohydrate (for micronucleus test). Approximately 3 hours after the last dosing, all rats were sacrificed by cervical dislocation and the designated organs (liver, duodenum, glandular stomach and testis) were collected for G1 to G5 groups. Bone marrow was collected for G6 group. The collected tissues were processed, single cells were isolated, and slides were prepared. Slides were run through submarine-type electrophoresis and drained. Drained slides were stained with ethidium bromide and evaluated for % tailing of DNA, i.e. tail length in microns measured from the estimated edge of the head region closest to the anode. In the micronucleus test, the average percentage of MNPCEs was 0.08 in males dosed with vehicle. For the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.16, 0.64 and 1.22, respectively. There was a statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at the doses of 1000 and 2000 mg/kg bw/day of the test item, in comparison with the vehicle control. The positive control group (G6), cyclophosphamide monohydrate at 100 mg/kg bw/day exhibited statistically significant increase in the numbers of MNPCEs when compared to vehicle control and the average percentage of MNPCEs (per 4000 PCEs scored) in positive control was 0.83. This demonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. In the comet assay, the average % tailing for DNA from male liver cells was 3.66, 3.74, 3.69 and 3.76 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. The average % tailing for DNA from glandular stomach cells were 3.28, 3.35, 3.22 and 3.13 and in duodenum, the % tailing observed were 3.77, 3.74, 3.81 and 3.65 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose related or statistically significant increase in the % tailing of DNA from cells of any of the organs for any of the FAT 36156 treated groups when compared to the vehicle control group. The average % tail DNA observed in the liver, glandular stomach and duodenum of the positive control (at 250 mg/kg bw/day of ethyl methyl sulfonate) dosed animals were 8.25, 8.90 and 8.03, respectively. The positive control [G5], ethyl methanesulfonate at a dose of 250 mg/kg bw/day produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (liver, duodenum and glandular stomach) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the comet assay for this test of FAT 36156 were fully adequate. Based on the findings of the study, it was concluded that FAT 36156 caused cytogenetic damage but did not lead to increased DNA damage at and up to 2000 mg/kg bw/day. 

Conclusion on genetic toxicity potential:

As the chemical was found to have clastogenic effect in the in vivo micronucleus test, it needs to be classified as mutagenic category 2 in accordance with Regulation (EC) No. 1272/2008.

Justification for classification or non-classification

As the chemical was found to have clastogenic effectcs in the in vivo micronucleus test, it needs to be classified as mutagenic 2 in accordance with Regulation (EC) No. 1272/2008.