N,N-bis(2-ethylhexyl)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:

09 - 23 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

duplicate plating without scientific justification, no standard deviations reported

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-ethylhexyl)formamide

EC Number:

860-695-9

Cas Number:

88247-41-0

Molecular formula:

C17H35NO

IUPAC Name:

N,N-bis(2-ethylhexyl)formamide

Method

Target gene:

His operon (S. typhimurium strains) and trp operon (E. coli strain)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- source of S9 : Tsukuba Research Institute, BoZo Research Center, Tokyo, Japan
- method of preparation of S9 mix: S9 fraction was prepared from the livers of male Sprague-Dawley rats. The animals were 7 weeks of age, weighed 222.2 - 269.7 g and were induced with phenobarbital and 5,6-benzoflavone by intraperitoneal injection. Phenobarbital was administered at 30, 60, 60 and 60 mg/kg bw/day on Day 1, 2, 3 and 4, 5,6-benzoflavone was administered at 80 mg/kg bw on Day 3. S9 mix was prepared by addition of cofactors and contained 8 µmol MgCl2, 33 µmol KCl, 5 µmol glucose-6-phosphate, 4 µmol NADPH, 4 µmol NADH and 100 µmol sodium phosphate buffer.
- concentration or volume of S9 mix: 10%

Test concentrations with justification for top dose:

Pre-experiment:
all strains: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate

Main test:
strains TA98 and TA100: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate -S9 mix; 313, 625, 1250, 2500 and 5000 µg/plate +S9 mix
strains TA1535 and TA1537: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate -S9 mix; 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate +S9 mix
strain WP2 uvrA: 313, 625, 1250, 2500 and 5000 µg/plate ± S9 mix

Supplemental test:
strains TA98 and TA100: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate -S9 mix
strains TA1535 and TA1537: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate -S9 mix; 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate +S9 mix

Justification for top dose: Concentrations were selected based on the results of a preliminary range-finding test (maximum concentration recommended by the guidline for non-cytotoxic substances or based on cytotoxicity).

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: AF-2: -S9, in DMSO, 0.01 µg/plate for TA100 and WP2uvrA, 0.1 µg/plate for TA98; ICR-191: -S9, in DMSO, 1 µg/plate for TA1537; 2AA: +S9, in DMSO, 2 µg/plate for TA1535 and 10 µg/plate for Wp2uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 3 (preliminary test, main test and supplementary test)

METHOD OF APPLICATION: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period: 20 min at 37 °C
- Exposure duration: 48 h for the preliminary test and 49 h for the main test and supplementary test

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants and clearing of the bacterial background lawn

Evaluation criteria:

If the number of revertant colonies in the test article treatment group was increased to at least two-fold that of the naturally revertant colonies (negative control value) and there were dose-relationship and reproducibility, or if there was no clear dose-relationship but the number of revertant colonies showed clear increase of at least 2-fold that of the naturally revertant colonies and showed reproducibility, the test article was judged to be positive.

Statistics:

Not performed in this study

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 78.1 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 78.1 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 19.5 µg/plate -S9 mix and at 78.1 µg/plate +S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 19.5 µg/plate -S9 mix and at ≥ 78.1 µg/plate +S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitate was noted at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test substance was observed at 5000 µg/plate for all strains in the presence and absence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: Please refer to Table 1 under "Any other information on results incl. tables".
A preliminary test was conducted at concentrations in the range of 1.22 - 5000 µg/plate with and without S9 mix using all strains. Cytotoxicity, evident as a reduction in the number of spontaneous revertants or growth inhibition was observed for strain TA1535 at ≥ 19.5 µg/plate -S9 mix and at ≥ 78.1 µg/plate +S9 mix, for strain TA98 at ≥ 1250 µg/plate -S9 mix and for strain TA1537 at ≥ 78.1 µg/plate -S9 mix and at ≥ 1250 µg/plate +S9 mix. For these strains, concentrations for the main mutagenicity tests were selected based on cytotoxicity. For strains TA100 and WP2uvrA no cytotoxicity was observed up to the highest concentrations tested. The concentrations selected for the main study for these strains corresponded to the limit concentration of 5000 /plate recommended by the current OECD guideline.

STUDY RESULTS : Please refer to Tables 2 and 3 under "Any other information on results incl. tables".
For the number of revertant colonies by the treatment with the test article, there was no increase for any bacterial strain irrespective of the presence/absence of metabolic activation of at least 2-fold that of the negative control value, and there was no dose-relationship.
It was judged that the study was conducted appropriately since the positive control values showed increased numbers of the revertant colonies of at least 2-fold that of the negative control group values, the mean values for the negative control group values and the positive control group values were within the acceptable range of the historical background data of the test facility, and there were no abnormalities such as bacterial contamination in the sterility test or in the study procedure.

CYTOTOXICITY:
Growth inhibition of bacteria was observed at 78.1 µg/plate and above for strain TA98 and TA100 without metabolic activation, at 19.5 µg/plate and above for strain TA1535 and TA1537 without metabolic activation, and at 78.1 µg/plate and above for strain TA1535 and TA1537 with metabolic activation.

HISTORICAL CONTROL DATA: Please refer to Table No. 4 under "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Results of the preliminary test: mean values of two plates (full result tables can be found attached as pdf document under "Attached background material")

Preliminary experiment
Strain	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
DMSO mean	110	111	7	7	23	23	17	27	9	9
Test item [µg/plate]
1.22	118	126	7	7	20	24	15	27	6	9
4.88	100	118	5	4	24	23	13	28	8	8
19.5	105	126	0x	6	21	22	14	22	4x	8
78.1	90x	101	0x	3x	24	32	14x	28	2x	6x
313	106x	125	0x	3x	22	26	13x	26	2x	6x
1250	97x	100	0x	3x	22	19	7x	29	4x	3x
5000^P	88x	95	0x	1x	19	18	6x	23	2x	2x
Positive control (µg/plate)
	AF-2 (0.01)	B[a]P (5.0)	SAZ (0.5)	2AA (2.0)	AF-2 (0.01)	2AA (10.0)	AF-2 (0.1)	B[a]P (5.0)	ICR-191 (1.0)	B[a]P (5.0)
mean	545	1000	206	238	102	562	282	285	1751	97
Positive controls: AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; SAZ: Sodium azide; ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine-2HCl; B[a]P: Benzo[a]pyrene; 2AA: 2-Aminoanthracene
^P= precipitation observed;^x: growth inhibition of tester strain observed

Table 2: Results of the main test (full result tables can be found attached as pdf document under "Attached background material")

Main experiment
Strain	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
DMSO mean	103	104	10	9	24	32	19	24	9	9
Test item
0.61	not performed	not performed	8	not performed	not performed	not performed	not performed	not performed	10	not performed
1.22	not performed	not performed	6	not performed	not performed	not performed	not performed	not performed	5	not performed
2.44	88	not performed	7	8	not performed	not performed	25	not performed	8	8
4.88	90	not performed	7	9	not performed	not performed	15	not performed	8	9
9.77	96	not performed	6	6	not performed	not performed	20	not performed	7	9
19.5	84	not performed	6x	12	not performed	not performed	16	not performed	8x	7
39.1	86	not performed	not performed	4	not performed	not performed	16	not performed	not performed	11
78.1	74x	not performed	not performed	6x	not performed	not performed	19x	not performed	not performed	3x
156	not performed	not performed	not performed	not performed	not performed	not performed	not performed	not performed	not performed	not performed
313	not performed	115	not performed	not performed	26	32	not performed	27	not performed	not performed
625	not performed	111	not performed	not performed	24	31	not performed	22	not performed	not performed
1250	not performed	124	not performed	not performed	24	26	not performed	32	not performed	not performed
2500	not performed	118	not performed	not performed	26	29	not performed	22	not performed	not performed
5000^P	not performed	121	not performed	not performed	28	22	not performed	17	not performed	not performed
Positive control (µg/plate)
	AF-2 (0.01)	B[a]P (5.0)	SAZ (0.5)	2AA (2.0)	AF-2 (0.01)	2AA (10.0)	AF-2 (0.1)	B[a]P (5.0)	ICR-191 (1.0)	B[a]P (5.0)
mean	662	1065	346	195	127	525	420	290	1469	97
Positive controls: AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; SAZ: Sodium azide; ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine-2HCl; B[a]P: Benzo[a]pyrene; 2AA: 2-Aminoanthracene
^P= precipitation observed;^x: growth inhibition of tester strain observed

Table 3: Results of the supplementary test (full result tables can be found attached as pdf document under "Attached background material")

Supplementary experiment
Strain	TA 100		TA 1535		TA 98		TA 1537
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
DMSO mean	108	not performed	7	11	16	not performed	7	8
Test item [µg/plate]
0.61	not performed	not performed	6	not performed	not performed	not performed	5	not performed
1.22	not performed	not performed	10	not performed	not performed	not performed	8	not performed
2.44	104	not performed	12	7	15	not performed	7	12
4.88	97	not performed	7	6	15	not performed	5	14
9.77	106	not performed	5	9	14	not performed	8	6
19.5	93	not performed	7x	11	13	not performed	4x	5
39.1	106	not performed	not performed	7	16	not performed	not performed	13
78.1	96x	not performed	not performed	10	16x	not performed	not performed	5
Positive control (µg/plate)
	AF-2 (0.01)	B[a]P (5.0)	SAZ (0.5)	2AA (2.0)	AF-2 (0.1)	B[a]P (5.0)	ICR-191 (1.0)	B[a]P (5.0)
mean	676	not performed	321	208	363	not performed	1485	103
Positive controls: AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; SAZ: Sodium azide; ICR-191 : 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine-2HCl; B[a]P: Benzo[a]pyrene; 2AA: 2-Aminoanthracene
^P :precipitation observed; ^x: growth inhibition of tester strain observed

Table 4: Historical control data, generated in the testing facility 16 Nov 2018 - 22 Mar 2019

Tester Strains	S9 Mix (-) or (+)	Classification	Mean	S.D.	Management ranges		N umber of plates
					Lower limit	Upper limit
TA 100	-	Solvent control	115	11	83	146	102
		Positive control AF-2 (0.01 pg/plate)	605	46	467	742	102
	+	Solvent control	120	11	89	152	102
		Positive control B[a]P (5.0 pg/plate)	1241	113	903	1578	102
TA1535	-	Solvent control	9	2	3	16	102
		Positive control SAZ (0.5 pg/plate)	269	53	109	429	102
	+	Solvent control	9	2	2	16	102
		Positive control 2AA (2.0 pg/plate)	244	27	163	325	102
WP2uvrJ	-	Solvent control	24	4	12	35	102
		Positive control AF-2 (0.01 pg/plate)	103	15	57	149	102
	+	Solvent control	26	5	12	40	102
		Positive control 2AA (10.0 pg/plate)	596	54	433	759	102
TA98	-	Solvent control	21	4	9	34	102
		Positive control AF-2 (0.1 pg/plate)	376	49	228	524	102
	+	Solvent control	30	5	15	45	102
		Positive controlB[a]P (5.0 pg/plate)	320	29	234	406	102
TA1537	-	Solvent control	8	2	2	14	102
		Positive control ICR-191 (1.0 pg/plate)	1442	198	849	2035	102
	+	Solvent control	10	2	3	16	102
		Positive control B[a]P (5.0 pg/plate)	95	15	51	140	102
Solvent controls: Water, Dimethyl sulfoxide(DMSO), Acetone, 1,4-Dioxane Positive controls: AF-2 : 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; SAZ : Sodium azide; ICR-191 : 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine-2HCl; B[a]P : Benzo[a]pyrene; 2AA : 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the test item was not mutagenic in S. typhimuirum strains TA98, TA100, TA1535 and TA1537 and in E. coli strain WP2uvrA with and without metabolic activation.