N,N'-ethane-1,2-diylbisoleamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:
Gene mutation in bacteria (Reverse Mutation Test, similar to OECD 471): negative, based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis
Cytogenicity in mammalian cells (Chromosomal Aberration, similar to OECD 473): negative, based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis
Gene mutation in mammalian cells (Mouse Lymphoma Assay, OECD 476): negative, based on read-across from Amides, C16-C18 (even), N,N’-ethylenebis

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. Strain to detect cross-linking mutagens missing.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

strain to detect cross-linking mutagens missing

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Species / strain / cell type:

other: Saccharomyces cerevisiae D4

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction of Aroclor 1254-pretreated rats

Test concentrations with justification for top dose:

0.1, 1.0, 10.0, 100.0, 500.0 and 1000.0 µg/plate without metabolic activation
0.1, 1.0, 10.0, 100.0 and 500.0 µg/plate with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water, dimethylsulfoxid (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

2-nitrofluorene

ethylmethanesulphonate

other: quinacrine mustard

Remarks:

ethyl methanesulphonate (-S9; 10 µL/plate, TA1535, TA100, D4); quinacrine mustard (-S9; 10 µg/plate, TA1537); 2-nitrofluorene (-S9; 10 µg/plate, TA1538, TA98); Migrated to IUCLID6: 10 µL/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

other: 2-anthramine

Remarks:

2-anthramine (+S9; 2.5 µg/plate, TA1535, TA1537, TA1538, TA98, TA 100); N-dimethylnitrosamine (+S9; 100 µmol/plate, D4)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h for TA 1535, TA 1537, TA 1538, TA 98, TA 100; 3-5 days for D4

DETERMINATION OF CYTOTOXICITY
- Method: other: direct revertant colony counts

Evaluation criteria:

Strains TA 1535, TA 1537, and TA 1538: if the solvent control value is within the normal range, a chemical that produces a positive dose-response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
Strains TA 98, TA 100, and D4: if the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA 100 and 2-3 times the solvent control value for strains TA 98 and D4 is considered to be mutagenic. For these strains, the dose-response increase should start at approximately the solvent control value.
If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data lose significance.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

cytotoxicity

Remarks:

slightly toxic at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

with and without

Genotoxicity:

negative

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:

ADDITIONAL INFORMATION ON CYTOTOXICITY: the compound was slightly toxic to the strain T-1537 at 500 µg per plate. TA-98 was repeated at 100, 500 and 1000 µg because of the increased number of revertants at 500 µg over the background level. The repeat test was negative.

Conclusions:

Interpretation of results:
negative

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

10 Mar – 27 Apr 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance N,N’-Ethylenebis(stearamide). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin, sodium pyruvate and L-glutamine + 10% (v/v) heat-inactivated horse serum (24-hour exposure); for 3-hour exposure only 5% (v/v) heat-inactivated horse serum were included. Selective medium consisted of the basic medium + 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphtoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1:
With and without 8% (v/v) S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 3h

Experiment 2:
Without S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 24h
With 12% (v/v) S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 3h

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: solubility/ability to suspend

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide

Remarks:

with S9-mix: 7.5 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate

Remarks:

without S9-mix: 15 µg/mL and 5 µg/mL for 3 h and 24 h treatment, respectively

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h, respectively
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT) (Sigma)

NUMBER OF REPLICATIONS: 5 exposure plates

NUMBER OF CELLS EVALUATED: not applicable, number of mutants per well counted; 2000 cells/well inserted, total sum of mutants given

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth, relative survival, suspension growth, relative suspension growth, growth rate

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells (10E+6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 x 10E-6 and ≤ 170 x 10E-6.
c) The growth rate (R) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32 and 180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 x 10E-6, and fro CP not below 700 x 10E-6.

The global evaluation factor (GEF) has been identified by the IWTG as the mean of the negative/solvent mutation frequency (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than the MF of the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if: a) none of the tested concentrations reaches a mutation frequency of the MF of the controls + 126. b) The results are confirmed in an independently repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility and precipitation: a slight precipitate of the test substance in the exposure medium was observed after 3 hours treatment at a concentration of 0.8 µg/mL and severe precipitate was observed at concentrations of 2.4 µg/mL and above. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range test was 24 µg/mL

RANGE-FINDING/SCREENING STUDIES:
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 24 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent treated control cultures were within the ranges of the historical controls.

Table 1: Cytotoxic and mutagenic responses

Treatment	Concentration [µg/mL]	Cloning efficiency [%]	Relative total growth [%]	Mutation frequency x 10^-6
				total	small colonies	large colonies
3 hours treatment without S9-mix
Solvent control (mean)	--	99	100	114	62	46
Test substance	0	116	126	110	59	43
	0.1	123	126	99	53	40
	0.2	113	111	110	63	41
	0.3	110	124	118	67	44
	0.4	113	139	128	60	59
	0.5	110	154	118	63	48
	0.6*	108	125	126	71	48
	0.8*	118	125	105	52	47
	1.0*	115	125	119	67	45
MMS	15	58	55	1227	675	354
3 hours treatment with 8% (v/v) S9-mix
Solvent control (mean)	--	106	100	102	55	42
Test substance	0	105	102	80	38	38
	0.1	94	89	119	66	46
	0.2	105	55	89	51	34
	0.3	93	76	113	64	43
	0.4	120	81	93	47	41
	0.5	121	127	97	55	37
	0.6*	101	86	87	53	30
	0.8*	97	94	72	44	25
	1.0*	121	112	79	45	30
CP	7.5	47	23	1431	876	351
24 hours treatment without S9-mix
Solvent control (mean)	--	93	100	58	29	27
Test substance	0	86	138	66	34	31
	0.1	72	108	77	43	32
	0.2	79	121	78	38	38
	0.3	98	150	47	26	19
	0.4	90	151	55	27	26
	0.5	81	133	69	37	30
	0.6*	80	125	67	35	31
	0.8*	90	143	61	37	22
	1.0*	88	131	63	34	27
MMS	5	66	81	629	357	211
3 hours treatment with 12% (v/v) S9-mix
Solvent control (mean)	--	106	100	82	50	29
Test substance	0	85	294	83	52	28
	0.1	107	58	109	48	55
	0.2	118	60	77	42	32
	0.3	91	161	88	50	34
	0.4	85	127	86	55	28
	0.5	101	146	74	49	23
	0.6*	86	42	103	66	32
	0.8*	93	155	67	40	25
	1.0*	62	66	130	55	70
CP	7.5	37	43	1905	1113	500

*precipitation of test substance in the exposure medium

The test substance did not induce a significant increase in mutation frequency in the absence or presence of metabolic activation in the first experiment. This result was confirmed in an independent experiment with a different concentration of the metabolic activation system or a longer exposure time without activation.

Conclusions:

Interpretation of results:
negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance N,N’-Ethylenebis(stearamide). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: CHL/IU (Chinese Hamster Lung cell line)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagles Mimimal Essential Media with 10% Foetal Bovine Serum
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/β-Naphtoflavone-induced rat liver S9

Test concentrations with justification for top dose:

6(18)-hour without S9: 0, 4.89, 9.77, 19.53, 39.06, 78.13, 156.25 µg/mL
6(18)-hour with S9: 0, 4.89, 9.77, 19.53, 39.06, 78.13, 156.25 µg/mL
24-hour without S9: 0, 2.44, 4.89, 9.77, 19.53, 39.06, 78.13 µg/mL

Concentrations selected for metaphase analysis, highest concentration was lowest precipitating concentration:
6(18)-hour without S9: 0, 19.53, 39.06, 78.13 µg/mL
6(18)-hour with S9: 0, 19.53, 39.06, 78.13 µg/mL
24-hour without S9: 0, 9.77, 19.53, 39.06 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C

Remarks:

without S9-mix: 0.1 µg/mL for 6 h and 0.05 µg/mL for 24 h treatment

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: cyclophosphamide

Remarks:

with S9-mix: 5 µg/mL for 6 h treatment Migrated to IUCLID6: 5 µg/mL for 6h treatment

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 (100 per plate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. If there was a dose-related increase in endoreduplicated cells then they are reported separately. The percentage of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported. The number of gap-type aberrations was recorded and reported.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary with the concurrent vehicle control value using Fisher‘s Exact test.

Key result

Species / strain:

other: CHL/IU (Chinese Hamster Lung cell line)

Metabolic activation:

with and without

Genotoxicity:

negative

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
- Precipitation: a precipitate of the test material was seen at and above 78.13 µg/mL in both pulse exposure groups and at and above 39.06 µg/mL in the 24 hours continuous exposure group

RANGE-FINDING/SCREENING STUDIES: In all cases the test material showed no evidence of cell toxicity. The dose selection for the main experiments was based on the lowest precipitating dose level, which was 78.13 µg/mL for both short term exposu.re groups and 39.06 µg/mL for the continuous exposure group. An additional dose above the lowest precipitating dose was included for all exposures.

Table 1: Chromosome aberrations and cell viability

Test item	Concentration	Cell viability	Mean	Aberrant cells in %
	in µg/mL	in %	mitotic index	Numerical	Structural
Exposure period 6 hrs without S9 mix
DMSO	--	100	100	0	0.5
MMC	0.1	78	97	0	52.0
EBS	19.53	93	149	0.5	2.0
	39.06	87	104	0	1.0
	78.13	90	110	0	0.5
Exposure period 24 hrs without S9 mix
DMSO	--	100	100	0	0
MMC	0.05	54	274	0	34.0
EBS	9.77	99	116	0	0.5
	19.53	106	179	0	1.5
	39.06	110	168	0	1.0
Exposure period 6 hrs with S9 mix
DMSO	--	100	100	0	0
CP	5.0	56	58	0	10.5
EBS	19.53	87	97	0	0
	39.06	84	112	0	0
	78.13	83	89	0	0

                    

MMC: Mitomycin C

CP: Cyclophosphamide

EBS: Ethylene Bis(stearamide)

Conclusions:

Interpretation of results:
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo:
Waiving - No in vivo testing required as none of the in vitro tests were positive for genetic toxicity.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no data available on the genetic toxicity of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis. In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance is conducted.

A detailed justification for the grouping of chemicals and read-across is provided in Sections 7.1 and 13.

In vitro

There exist three reliable studies on genetic toxicity in vitro of the structurally related substance Amides, C16-C18 (even), N,N'-ethylenebis. Therefore, read-across based on the analogue approach is performed to cover this endpoint.

- Gene mutation in bacteria

The gene mutation in bacteria was assessed by the Reverse Mutation Assay (Ames test) conducted with 5 Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and the yeast strain Saccharomyces cerevisiae D4, which was comparable to OECD guideline 471 (Jagannath, 1978). All assays were conducted with the test substance at concentrations ranging from 0.1 to 500 µg/plate in the presence and absence of a metabolic activation system (Aroclor1254-induced rat liver S9). Additionally, 1000 µg/plate was included as test concentration in a repetition test with TA 98. Although there was no strain included to detect cross-linking mutagens according to the actual version of the guideline, the study was acceptable according to the standards of the time of conduction. In this study no mutagenic effects on bacteria were observed with or without metabolic activation at any of the concentrations tested.

- Gene mutation in mammalian cells

The mutagenicity of the test substance in mammalian cells in vitro was determined in the L5178Y mouse lymphoma cell line according to OECD guideline 476 (Verspeek-Rip, 2010). The number of mutants derived from 5 exposure plates was determined in 2 independent experiments. In the first experiment, the mouse lymphoma cells were exposed for 3 h to the test substance at concentrations ranging from 0 to 1.0 µg/mL in the absence and presence of a metabolic activation system (8% (v/v) Phenobarbital/β-Naphtoflavone-induced rat liver S9-mix). Precipitation occurred at concentrations ≥ 0.6 µg/mL, no cytotoxicity was observed, and there was no significant increase of mutant frequencies at the TK locus compared to the control groups. These findings were confirmed by a second independent experiment conducted with the same test substance concentrations, but with 3-h exposure in the presence of 12% (v/v) rat liver S9-mix or 24-h exposure without metabolic activation. Therefore, it was concluded that under the conditions used in the study, the test material was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation.

- Chromosome aberrations

The clastogenic activity of the test substance was investigated using a Chromosomal Aberration Assay in a Chinese Hamster lung (CHL) cell line according to OECD guideline 473 and in compliance with GLP (Wright, 2006). Duplicate cell cultures were exposed for 6 h to concentrations up to 156.25 µg/mL in the presence or absence of a metabolic activation system (Phenobarbital/β-Naphtoflavone-induced rat liver S9-mix), followed by an 18-h recovery period, or for 24 h to concentrations up to 78.13 µg/mL in the absence of metabolic activation. Three concentrations (up to 78.13 µg/mL) and the vehicle control were chosen for analysis of 200 metaphases. The highest concentration chosen was the lowest one at which precipitation occurred. The chromosomes were analysed for structural aberrations comprising chromosome and chromatid breaks and exchanges, gaps, numerical aberrations and multiple aberrations. Under the conditions of this study the test substance did not induce any statistically significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in the presence or absence of a metabolic activation after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.

In vivo

No study investigating the genetic toxicity in vivo of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. However, there exist reliable data on genetic toxicity in vitro of the structurally related substance Amides, C16-C18 (even), N,N'-ethylenebis, which are used for read-across based on the analogue approach.

According to Regulation (EC) No 1907/2006, Annex IX, column 2, testing for genetic toxicity in vivo is not indicated as the structurally closely related source substance Amides, C16-C18 (even), N,N'-ethylenebis did not demonstrate any genotoxic activity in bacteria or mammalian cells in vitro.

Based on the negative results of the available studies on the structural analogue Amides, C16-C18 (even), N,N'-ethylenebis, it may be concluded that the substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis does not induce genetic toxicity in vitro and in vivo.

Justification for classification or non-classification

The available data on genetic toxicity of a substance structurally related to Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis according to Regulation (EC) No 1907/2006, Annex XI, 1.5 do not meet the criteria for classification according to Regulation (EC) No 1272/2008; therefore, Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not expected to exert a genotoxic potential, either, and the data are thus conclusive but not sufficient for classification.