N,N-dibutylformamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen.

The test substance is considered to be non-mutagenic in the in vitro mircronucleus test, when tested up to cytotoxic or to the higest evaluable concentrations.

The test substance is consicdered to be non-mutagenic in the HPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(July 21,1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(December 1992)

Deviations:

no

GLP compliance:

yes

Remarks:

Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Probe 527
- Purity test date: 18 January 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

OTHER SPECIFICS:
- main component + 1.76% formic acid

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9 mix

Test concentrations with justification for top dose:

20 µg - 5000 µg/plate (standard plate test)
4 µg - 2500 µg/plate (preincubation test)

Vehicle / solvent:

Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation, for all strains

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix: strain TA 1535, TA 100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S-9 mix: strain TA 98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix: strain TA 1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S-9 mix: strain E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Exposure duration: Preincubation Test: 20 min
- Expression time: Standard plate test and Preincubation Test: 48-72 hours in the dark at 37°C

SELECTION AGENT (mutation assays): Standard plate test: Salmonella typhimurium, 0.5 mM histidine + 0.5 mM biotin; Escherichia coli 0.5 mM tryptophan

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
Method:
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer

Evaluation criteria:

Generally, the experiment is to be considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination .
- The positive control articles both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical control
- The titer of viable bacteria was >10E9/ mL

The test chemical is considered positive in this assay if the following criteria are met :
- A dose-related and reproducible increase in the number of revertant colonies, i .e . about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system .
A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. No test substance precipitation was found .

Table 1: Number of revertants per strain (mean from three plates)- Standard Plate Test

	Strain TA 1535		Strain TA 100		Strain TA 1537		Strain TA 98		E.coli WP2 uvrA
Conc. [µg/plate]	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix
Solvent control	18	18			10	10	38	29
Positive control*	131	525	1210	649	173	525	1130	1040	252	607
20	18	20	115	114	9	10	38	29	37	37
100	16	19	108	106	8	7	35	26	39	34
500	11	18	109	102	9	9	32	26	38	29
2500	8	12	75	75	6	6	24	19	29	24
5000	6	7	59	55	3	3	15	12	24	19

*without S9 mix: MNNG (5 µg/plate): TA1535, TA 100; AAC (100 µg/plate): TA 1537; NOPD (10 µg/plate): TA98, 4-NQO (5 µg/plate): E.coli WP2 uvrA

*with S9 mix: 2-AA (2.5 µg/plate): TA1535, TA 100; TA 1537, TA 98, E.coli WP2 uvrA (60 µg/plate)

 

Table 2: Number of revertants per strain (mean from three plates)- Preincubation Test

	Strain TA 1535		Strain TA 100		Strain TA 1537		Strain TA 98		E.coli WP2 uvrA
Conc. [µg/plate]	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix
Solvent control	17	18	120	106	9	10	31	27	41	31
Positive control*	126	834	942	609	162	443	941	1063	218	550
4	16	19	118	110	9	9	30	26	37	29
20	15	16	111	110	9	9	26	25	36	27
100	15	15	107	106	7	7	32	20	36	33
500	10	14	111	91	4	4	22	15	33	27
2500	7	9	22	74	1	1	17	6	31	25

*without S9 mix: MNNG (5 µg/plate): TA1535, TA 100; AAC (100 µg/plate): TA 1537; NOPD (10 µg/plate): TA98, 4-NQO (5 µg/plate): E.coli WP2 uvrA

*with S9 mix: 2-AA (2.5 µg/plate): TA1535, TA 100; TA 1537, TA 98, E.coli WP2 uvrA (60 µg/plate)

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy non-smoking donors not receiving medication.
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Cell cycle length, doubling time or proliferation index:Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
- Sex, age and number of blood donors if applicable: blood was collected from a male donor (23 years old) for the pre-experiment, from a male donor (34 years old) for Experiment I and from a male donor (24 years old) for Experiment II
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (induced with phenobarbital/ß-naphthoflavone)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: domecolcine

Details on test system and experimental conditions:

Reason for the choice of human lymphocytes:
Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
Cell cultures:
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (23 years old) for the pre-experiment, from a male donor (34 years old) for Experiment I and from a male donor (24 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
Culture conditions:
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Test Item Preparation:
Stock formulations of the test item and serial dilutions were made in DMSO. The final concentration of DMSO in the culture medium was 0.5 %. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. All formulations were prepared freshly before treatment and used within two hours of preparation.

Evaluation criteria:

The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval).
− The concurrent positive controls should produce a statistically significant increase in the micronucleus frequency and should be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described, were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item N,N-Dibutylformamide, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group, two parallel cultures were analyzed. 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 1613 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the content (97.52%, preliminary information at the beginning of the study) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In Experiment I, phase separation of the test item in the culture medium was observed at 1613 μg/mL in the presence of S9 mix at the end of treatment. In all other experimental parts, neither phase separation nor precipitation was observed at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In Experiment I in the absence of S9 mix, moderate cytotoxicity (39.0% cytostasis) was observed at the highest evaluated concentration. In the presence of S9 mix, cytotoxicity (48.0% cytostasis) was observed at the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix after continuous treatment, clear cytotoxicity (53.4% cytostasis) was observed at the highest evaluated concentration.
In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.
Demecolcine (100 ng/mL), MMC (0.8 μg/mL) or CPA (15.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, N,N-Dibutylformamide is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or to the highest evaluable concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: HPRT

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix (phenobarbital/ß-naphthoflavone

Test concentrations with justification for top dose:

1st Exp.: 12.6, 25.2, 50.4, 100.8, 201.6, 403.3, 806.5, 1613.0 µg/mL (4 h, without S9 mix)
25.2, 50.4, 100.8, 201.6, 403.3, 605.0, 806.5, 1613.0 µg/mL (4 h, with S9 mix)
2nd Exp.: 25.2, 50.4, 100.8, 201.6, 403.3, 806.5, 1209.8, 1613.0 µg/mL (24 h, without S9 mix)
25.2, 50.4, 100.8, 201.6, 403.3, 605.0, 705.8, 806.5 µg/mL (4 h, with S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 h and 24 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 6 - 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): 6-thioguanine (11 μg/mL)

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

Statistics:

A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
The highest evaluated concentration in both experiments with and without metabolic activation was limited by cytotoxicity.
Cytotoxic effects indicated by an adjusted cloning efficiency I (referring to the mean values) below 50% in both cultures was observed in experiment I at 806.5 μg/mL of both cultures without metabolic activation and at 605.0 μg/mL and above with metabolic activation. In experiment II without metabolic activation cytotoxic effects as described above were observed at 403.3 μg/mL and above. In experiment II in the presence of metabolic activation a higher cytotoxicity was observed at 605.0 μg/mL (cell density 7.7%) than in the next higher concentration of 705.80 μg/mL. This observation might be based on a technical error. Since the next lower evaluated concentration with 403.3 μg/mL shows a spacing factor of 1.75 to the highest reported concentration, the reported concentrations meet the recommended spacing factor and therefore, the test is judged as valid.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, N,N-Dibutylformamide is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No data available.

Additional information

In vitro

The test substance was tested according OECD TG 471 for its mutagenic potential in Salmonella typhimurium and Escherichia coli strains in a reverse mutation assay. Following stains were tested: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. In the standard plate corporation test, the dose range of 20-5000 µg/plate was used and in the preincubation test a dose range of 4-2500 µg/plate. Both tests were with and without metabolic activation (Aroclor-induced rat liver S-9 mix). No precipitation of the test substance was found. A weak bacteriotoxic effect was observed under all test conditions. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test, either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay under the experimental conditions chosen (2000, reliability score: 1).

The study was performed to investigate the potential of N,N-Dibutylformamide to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (OECD TG 476). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with a treatment period of 4 hours with and without microsomal activation. The second main experiment was performed with a 24 hours treatment period without microsomal activation and a 4 hours treatment period with microsomal activation. The maximum test item concentration of the pre-experiment (1613.0 μg/mL) was chosen with respect to the current OECD guideline 476 regarding the content (97.52%, preliminary information at the beginning of the study) of the test item. The test item was dissolved in DMSO. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, N,N-Dibutylformamide is considered to be non-mutagenic in this HPRT assay (2018).

The test item N,N-Dibutylformamide, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes (OECD TG 487) in vitro in two independent experiments. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1613 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the content (97.52%, preliminary information at the beginning of the study) of the test item and with respect to the current OECD Guideline 487. In Experiment I in the absence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration. In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.Therefore, N,N-Dibutylformamide is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or to the highest evaluable concentrations (2018).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the study results, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.