N-{[Benzyl(ethyl)amino]-[(chloro-cyano-nitrophenyl)substituted]phenyl}acetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 41047/A TE did not show mutagenic potential with or without metabolic activation in the bacterial reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Source: Health Protection Agency The National Collection of Industrial National Collection of Type and Marine Bacteria Ltd. (NCIMB) Cultures (NCTC) Ferguson Building 61, Colindale A venue Craibstone Estate, Bucksburn London NW9 SEQ Aberdeen, AB2 l 9Y A, Great Britain.

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB),
Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB2 l 9Y A, Scotland, U.K.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 ml/rat ready to use solution), 5 days prior to sacrifice. The S9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 °C. S9 homogenate was thawed immediately before use and mixed with the cofactor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCh and 33 mM KCl in PBS.

S9 mix was prepared by mixing 1 and 5.5 mL of the S9 homogenate with 9 and 49.5 mL of the co-factors solution for the preliminary toxicity and mutation assay respectively, kept in an ice bath and used within one hour.

Test concentrations with justification for top dose:

Concentration: 50, 100, 200, 400, 800, 1600, 3200 and 5000 µg/plate
The test item did not cause precipitation on the basal agar plates at any of the tested doses.
Also, no toxicity to tester strains at any test dose. The Intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates, both in the presence and absence of metabolic activation.
Based on these observations, it was decided to test the OECD 471 recommeded top dose of 5000 µg/plate in the mutation assay.

Vehicle / solvent:

Dimethyl formamide (DMF)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-Aminoanthracene: with S9

Remarks:

TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-Aminoanthracene: with S9

Remarks:

TA100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-Aminoanthracene: with S9

Remarks:

TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-Aminoanthracene: with S9

Remarks:

TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-Aminoanthracene: with S9

Remarks:

WPA2uvrA (pKM101)

Details on test system and experimental conditions:

Three replicate plates were maintained for initial as well as confirmatory mutation assays.

Plating procedure:
Initial mutation assay was conducted using the direct plate incorporation method as follows:

Presence of Metabolic Activation:
a) 2.0 mL soft agar containing histidine-biotin/tryptophan
b) 100 μL test dose/vehicle/appropriate positive control
c) 100 μL bacterial culture
d) 500 μL S9 mix

B. Absence of Metabolic Activation :
a) 2.0 mL soft agar containing histidine-biotin/tryptophan
b) 100 μL test dose /vehicle/appropriate positive control
c) 100 μL bacterial culture
d) 500 μL of PBS

These test constituents were transferred into sterile test tubes, mixed and overlaid onto VB agar plates and allowed to set. The plates were then incubated at 37 ± 1 °C for 67 hours. Revertant colonies were counted manually and the plates were examined for bacterial background lawn.

The confirmatory mutation assay was conducted using the pre-incubation method as follows:
A. Presence of Metabolic Activation
a) 100 µL test dose /vehicle/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL S9 mix

B. Absence of Metabolic Activation
a) 100 µL test dose/vehicle/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL PBS

These test constituents were transferred into sterile test tubes and kept in an incubator shaker for 20 minutes at 37 °C. After this period, 2 mL soft agar containing histidine-biotin / tryptophan were added to each of the tubes. The tube content were mixed and overlaid onto VB agar plates and allowed to set. The plates were then incubated al 37 ± 1 °C for 67 hours. Revertant colonies were counted manually and the plates were examined for bacterial background lawn.

Evaluation criteria:

- To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.
- The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TAI00 and WP2uvrA (pKMI0l) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.
- 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 will be evaluated as negative, if it is neither positive nor equivocal.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: E.coli WP2uvrA (pKM101)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

FAT 41047/A TE was not mutagenic in this bacterial reverse mutation test.

Executive summary:

The test item, FAT 41047/A TE was tested for its mutagenic potential in the bacterial reverse mutation assay according to OECD test guideline 471.The study was conducted using TA98,TA 100,TA 1535 and TA 1537 strains of Salmonella typhimurium and WP2uvrA(pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay.

 

The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). FAT 41047/A TE was suspended in Dimethyl formamide (DMF) at 50 mg/mL and was found stable and re-suspendable in DMF for 24 hours at room temperature, at the fortification levels of 25 and 50000 μg /mL.

 

In a preliminary toxicity test, the test item did not precipitate on the basal agar plates at any of the test doses. The test item did not cause toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, a top dose of 5000 μg/plate was tested in the mutation assay.

 

The bacterial tester strains were exposed to FAT 4104 7 / A TE in triplicate at 50, 158, 500, 1581 and 5000 μg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas the confirmatory mutation assay was carried out using the pre-incubation mode of exposure.

The vehicle control (DMF) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains. The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation during initial as well as confirmatory assays. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

Hence based on the findings of the study, it can be concluded that FAT 41047/A TE did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item, FAT 41047/A TE was tested for its mutagenic potential in the bacterial reverse mutation assay according to OECD test guideline 471.The study was conducted using TA98,TA 100,TA 1535 and TA 1537 strains ofSalmonella typhimuriumand WP2uvrA(pKM101) strain ofEscherichia coliin three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay.

 

The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). FAT 41047/A TE was suspended in Dimethyl formamide (DMF) at 50 mg/mL and was found stable and re-suspendable in DMF for 24 hours at room temperature, at the fortification levels of 25 and 50000 μg /mL.

 

In a preliminary toxicity test, the test item did not precipitate on the basal agar plates at any of the test doses. The test item did not cause toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, a top dose of 5000 μg/plate was tested in the mutation assay.

 

The bacterial tester strains were exposed to FAT 41047/A TE in triplicate at 50, 158, 500, 1581 and 5000 μg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas the confirmatory mutation assay was carried out using the pre-incubation mode of exposure.

The vehicle control (DMF) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains. The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation during initial as well as confirmatory assays. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

Hence based on the findings of the study, it can be concluded that FAT 41047/A TE did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation.

Justification for classification or non-classification

FAT 41047/A was found to be not genotoxic, hence it does not warrant classification for mutagenicity as per the Regulation (EC) No. 1272/2008.