Amides, C16-C18 (even) , N,N'-ethylenebis

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 (Ames), similar to OECD 471): negative with and without metabolic activation in S. typhimurium TA 1538, TA 1537, TA 1535, TA 100 and TA 98 and Saccharomyces cerevisiae D4

In vitro cytogenicity in mammalian cells (chromosome aberration, similar to OECD 473): negative with and without metabolic activation in Chinese hamster lung (CHL/IU) cells

In vitro gene mutation in mammalian cells (mouse lymphoma assay, OECD 476): negative with and without metabolic activation in mouse lymphoma L5178Y cells

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Strain to detect cross-linking mutagens missing, analytical purity of test substance not specified.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Strain to detect cross-linking mutagens missingy, analytical purity of test substance not specified.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon (S. typhimurium), ade/trp-loci (S. cerevisiae)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

Saccharomyces cerevisiae

Details on mammalian cell type (if applicable):

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 activation; 0.1, 1.0, 10.0, 100.0 and 500.0 µg/plate with 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

Positive control substance:

ethylmethanesulphonate

Remarks:

TA1535, TA100, D4 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: QM, 10 µg/plate

Remarks:

TA1537 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA1538, TA98 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramin, 2.5 µg/plate

Remarks:

TA1535, TA1537, TA1538, TA98, TA 100 with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

D4 with S9

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.

Statistics:

no data

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

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slightly toxic to TA 1537 at 500 µg per plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

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

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:

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

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

True 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 TA 1537 at 500 µg per plate.
TA 98 was repeated at 100, 500 and 1000 µg/plate because of the increased number of revertants at 500 µg/plate over the background level. The repeat test was negative.

Conclusions:

Interpretation of results: negative

Executive summary:

The test substance did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration 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:

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:

Chinese hamster lung (CHL/IU)

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

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24 h
- 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:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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: A precipitate of the test material was seen at and above 78.13 µg/mL in both exposure groups and at and above 39.06 µg/mL in the 24 h 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 exposure 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

Executive summary:

The test material 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 liver enzyme metabolising system or after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March - 27 April 2010

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)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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-glutamin + 10% (v/v) heat-inactivated horse serum (24-h exposure); for 3-h 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:
In the absence and presence of 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 3 h

Experiment 2:
In the absence of S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 24 h
In the presence of 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 3 h

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:

cyclophosphamide

methylmethanesulfonate

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 AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT) (Sigma)
- Selection time (if incubation with a selection agent): 11 - 12 days

NUMBER OF REPLICATIONS: 5 exposure plates in 2 experiments

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 (10E6) 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 h treatment) and between 32 and 180 (24 h treatment).
d) The mutation frequency of MMS should not be below 500 x 10E-6, and for 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 frequeny (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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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 h treatment at the 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 Ending 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 h 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 h 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 h 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 h 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

Conclusions:

Interpretation of results: negative

Executive summary:

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. The test substance did not induce gene mutation in mammalian cells in vitro under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is data from in vitro assays available addressing gene mutation in bacteria, clastogenic activity in mammalian cells and gene mutation in mammalian cells.

In vitro gene mutation in bacteria

Gene mutation in bacteria was assessed by the Reverse Mutation Assay (Ames test) conducted with 5 Salmonella typhimurium strains (TA1535, TA1537, TA1538, TA98 and TA 100) and the yeast strain Saccharomyces cerevisiae D4, which was comparable to OECD guideline 471 (Litton Biometics, 1978). All assays were conducted in presence and absence of a metabolic activation system consisting of Aroclor 1254-induced rat liver S9 with test substance concentrations ranging from 0.1 to 500 µg/plate; additionally 1000 µg/plate was included in a repetition test with TA98. 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 used when the study was conducted. In this study no mutagenic effects on bacteria or yeast were observed with or without metabolic activation at any of the concentrations tested.

In vitro chromosome aberration

The clastogenic activity of the test substance was investigated by a Chromosomal Aberration Assay conducted with a Chinese Hamster lung cell line according to OECD guideline 473 (Safepharm, 2006). Duplicate cell cultures were exposed for either 6 h to concentrations up to 156.25 µg/mL in presence or absence of a metabolic activation system consisting of 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 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 presence or absence of a metabolic activation system after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.

In vitro gene mutation in mammalian cells

Mutagenicity of the test substance in mammalian cells in vitro was determined in the L5178Y mouse lymphoma cell line according to OECD guideline 476 (NOTOX, 2010). The number of mutants derived from 5 exposure plates was determined in 2 independent exeriments. In the first experiment the mouse lymphoma cells were exposed for 3 h to test substance concentrations in the range of 0 to 1.0 µg/mL in the absence and presence of a metabolic activation system consisting of 8% (v/v) Phenobarbital/β-Naphtoflavone-induced rat liver S9-mix. In this setup 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. In conclusion the test material did not induce gene mutations in mammalian cells in vitro.

According to Regulation (EC) No. 1907/2006, Annex IX, Item 8.4., Column 2, testing for genetic toxicity in vivo is not indicated as the test substance did not demonstrate any genotoxic activity in bacteria or mammalian cells in vitro.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.