N,N-bis(1-methylethyl)formamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Jan.13, 2021 to Jan.22, 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

June 26, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(1-methylethyl)formamide

EC Number:

220-284-1

EC Name:

N,N-bis(1-methylethyl)formamide

Cas Number:

2700-30-3

Molecular formula:

C7H15NO

IUPAC Name:

N,N-Diisopropylformamide

Specific details on test material used for the study:

Batch No.: M-O303190723301F
Purity: 99.68%

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

In this study, rat liver S9 prepared in-house was used as the metabolic activation system.
Preparation of S9: The S9 was prepared from the livers of the rats induced with Aroclor1254, and the details were shown below: Male SD (Sprague-Dawley) rats were treated by a single intraperitoneal injection of Aroclor 1254 at the dose of 500 mg/kg. body weight five days prior to the S9 preparation. The livers of the rats were taken out under aseptic conditions, homogenized in a 0.15 mol/L KCl solution (1g liver: 3ml KCl solution) and centrifuged at 11000rpm for 10 min with AllegraTM64R Auto Freeze Centrifuge. The supernatant of S9 was stored in the liquid nitrogen (about-196℃) not more than two years.

Test concentrations with justification for top dose:

5000, 1500, 500, 150, 50 and 15 μg/plate

Vehicle / solvent:

Name: Ultra pure water (H2O)
CAS No.: 7732-18-5
Purpose: Used as the solvent for test item preparation and as the concurrent solvent control for all tester strains.
Reason to choose: H2O are accredited by OECD Guideline for Testing of Chemicals, No. 471 (Corrected: June 26, 2020). Furthermore, according to the test results of test item solubility performed within preliminary test, H2O was used as the solvent.
Supplier: Sartorius arium@ mini ultra-pure water machine in this center (Z-9)
Batch No.(Preparation date): Jan. 14, 2021 and Jan. 20, 2021
Appearance: Colorless transparent liquid
Grade: Sterile (0.22μm aqueous membrane filtration)
Storage condition: RT
Expiry date: Ultra-pure water was prepared just before use and was not saved after using.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Used in the tester strain TA1535 in S9+.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Used in the tester strain TA102 in S9+.

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used in the tester strains TA100 and TA1535 in S9-.

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Used in the tester strains TA97a, TA98 and TA100 in the presence of metabolic activation system (S9+).

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Used in the tester strains TA97a, TA98 and TA102 in the absence of metabolic activation system (S9-).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: One day before the plate-incorporation, the stored tester strains were thawed and100μl of bacterium suspension was cultivated in nutrient broth medium (20ml) for approximate 16 hours at 36.2 ~37.4℃.
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times):

METHODS FOR MEASUREMENTS OF GENOTOXICIY
After incubation, the number of revertant colonies in each plate was counted, and the signs of background lawn were observed microscopically.

Evaluation criteria:

1) When there is a dose-related increase in the mean number of revertant colonies (equal to or greater than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and/or equal to or greater than three times of that of the concurrent solvent control in TA1535) over the range tested in at least one tester strain with or without metabolic activation, the test item should be evaluated as positive.
2) When there is a reproducible increase in the mean number of revertant colonies (equal to or greater than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and/or equal to or greater than three times of that of the concurrent solvent control in TA1535) at one or more dose levels in the mean number of revertant colonies in at least one tester strain with or without metabolic activation, the test item should be evaluated as positive.

Statistics:

In the verified Microsoft Office Excel (2007) (Bacterial Reverse Mutation Test-the statistical results table for the number of colonies 2.0), for three plates at each dose and control group, the mean number and the standard deviation of the number of revertant colonies were calculated. At the same time, the ratio of the mean number of revertant colonies between the each group and the concurrent solvent control was calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the viable count in two tests showed that the density of the cultures for each tester strain was within the acceptable range of 0.9~9×109 CFU/ml. The results met the test requirement.
In the first test and the validation test, the mean number of revertant colonies of the concurrent solvent control and positive controls were within the range of historical control data in this lab. In addition, the background lawn of the concurrent solvent control and positive controls had grown as densely packed microcolonies which formed a uniform layer observed with microscope. So the sensitivity of the test and the efficacy of the S9 mix were validated.
In the first test, no precipitate was observed on the surface of MGA plates inoculated with TA98 at any dose level before and after incubation, with and without the metabolic activation system. In the validation test, the same precipitate results were obtained as those in the first test.
In the first test, no cytotoxicity was observed at any dose level in any tester strain in two treated conditions. It was shown that the number of revertant colonies was not significantly decreased, and the signs of background lawn was not clearing or thinner than the concurrent solvent control groups. In the validation test, similar cytotoxicity results were obtained as those in the first test.
In the first test, the mean number of revertant colonies at each dose level was less than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and less than three times of that of the concurrent solvent control in TA1535. In the validation test, the similar results (the mean number of revertant colonies at each dose level was less than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and less than three times of that of the concurrent solvent control in TA1535) were obtained as in the first test.

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic in the bacterial reverse mutation test using the histidine requiring tester strains of Salmonella typhimurium.

Executive summary:

The study was performed to evaluate the capability of the test item to induce reverse mutations in the genome of the histidine requiring tester strains of Salmonella typhimurium with and without the metabolic activation system (S9 mix), and the method was designed in compliance with OECD Guideline 471.

Five histidine requiring (his-) mutant tester strains of Salmonella typhimurium including TA97a, TA98, TA100, TA102 and TA1535 were treated. The test item was considered to be non-mutagenic in the bacterial reverse mutation test using the histidine requiring tester strains of Salmonella typhimurium.