Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

C1618FA-TEPA-compound is not genotoxic based on the following results:
- not mutagenic in the Salmonella typhimurium reverse mutation assay (OECD guideline 471; GLP)
- not mutagenic in the mammalian cell gene mutation assay (OECD guideline 476; thymidine kinase L5178Y mouse lymphoma cells; GLP)
- not clastogenic in an in vitro chromosome aberration test (OECD guideline 473; V79 cells; GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 21, 2013 - March 04, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 6.9; 13.8; 27.5; 55.0; 110.0 (p); 165.0 (p) µg/mL
with S9 mix: 6.9; 13.8; 27.5; 55.0; 110.0 (p); 165.0 (p) µg/mL
Experiment II:
without S9 mix: 0.9; 1.7; 3.4; 6.9; 13.8; 27.5; 55.0; 110.0 µg/mL
with S9 mix: 6.9; 13.8; 27.5; 55.0; 82.5; 110.0 µg/mL
(p) = precipitation visible to the unaided eye

Vehicle / solvent:

deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1E07 (3E06 during 24 h exposure) cells/flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 4 h / 24 h

FOR GENE MUTATION:
- Expression time: 48 h
- Selection time: 10 - 15 days
- Method used: microwell plates
- selective agent: trifluorothymidine, 5 μg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 4E03 cells in selective medium
- Criteria for small (slow growing) and large (fast growing) colonies: Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- viability (cloning efficiency), relative total growth (RTG)

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1E06 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 1E06 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		-	100.0	40	166	100.0	53	179
Pos. control with MMS	19.5	-	28.5	240	166	27.9	305	179
Test item	6.9	-	112.8	24	166	69.0	24	179
Test item	13.8	-	65.1	26	166	38.1	94	179
Test item	27.5	-	36.5	21	166	22.6	43	179
Test item	55.0	-	4.2	28	166	1.9	55	179
Test item	110.0 (P)	-	culture was not continued^#			culture was not continued^#
Test item	165.0 (P)	-	culture was not continued^#			culture was not continued^#
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	69	195	100.0	95	221
Pos. control with CPA	3.0	+	31.3	331	195	65.9	207	221
Pos. control with CPA	4.5	+	33.3	475	195	59.6	279	221
Test item	6.9	+	90.4	81	195	84.8	58	221
Test item	13.8	+	72.7	83	195	114.7	66	221
Test item	27.5	+	69.0	90	195	123.4	42	221
Test item	55.0	+	99.6	34	195	106.8	56	221
Test item	110.0 (P)	+	20.9	44	195	11.3	43	221
Test item	165.0 (P)	+	culture was not continued^#			culture was not continued^#
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	72	198	100.0	91	217
Pos. control with MMS	13.0	-	9.1	491	198	12.9	571	217
Test item	0.9	-	culture was not continued^##			culture was not continued^##
Test item	1.7	-	culture was not continued^##			culture was not continued^##
Test item	3.4	-	71.1	39	198	92.8	32	217
Test item	6.9	-	62.6	54	198	49.8	130	217
Test item	13.8	-	40.7	68	198	53.8	60	217
Test item	27.5	-	21.2	43	198	20.0	58	217
Test item	55.0	-	0.0	72	198	0.0	39	217
Test item	110.0 (P)	-	culture was not continued^#			culture was not continued^#
Experiment II / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	45	171	100.0	53	179
Pos. control with CPA	3.0	+	52.7	163	171	61.2	154	179
Pos. control with CPA	4.5	+	45.9	239	171	31.6	315	179
Test item	6.9	+	culture was not continued^##			culture was not continued^##
Test item	13.8	+	155.1	44	171	157.4	48	179
Test item	27.5	+	98.2	50	171	134.2	24	179
Test item	55.0	+	70.4	68	171	99.5	33	179
Test item	82.5 (P)	+	43.0	44	171	42.7	37	179
Test item	110.0 (P)	+	19.5	50	171	18.2	31	179

Threshold = number of mutant colonies per 1E06 cells of each solvent control plus 126

(p)  precipitation visible to the unaided eye

#    culture was not continued due to exceedingly severe cytotoxic effects

##   culture was not continued as a minimum of four concentrations is required by the guidelines

 

Conclusions:

Interpretation of results: negative

The test substance C16-18FA-TEPA-compound was concluded to be not mutagenic with and without metabolic activation in the L5178Y/TK Mouse Lymphoma Mutagenesis Assay when tested up to cytotoxic concentrations.

Executive summary:

In a mammalian cell gene mutation assay thymidine kinase locus according to OECD guideline 476, L5178Y mouse lymphoma cells cultured in vitro were exposed to C16-18FA-TEPA-compound at the following concentrations in the presence and absence of mammalian metabolic activation (S9- mix):

Experiment I:

without S9 mix: 6.9; 13.8; 27.5; and 55.0 µg/mL
with S9 mix: 6.9; 13.8; 27.5; 55.0; and 110.0 µg/mL

Experiment II:

without S9 mix: 3.4; 6.9; 13.8; 27.5; and 55.0 µg/mL
with S9 mix: 13.8; 27.5; 55.0; 82.5; and 110.0 µg/mL

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours and in the presence of metabolic activation with a treatment time of 4 hours.The experimental part of the second experiment without metabolic activation was terminated prior to the generation of any data on mutagenicity as strong cytotoxic effects completely inhibited cell growth down to low concentrations. This experimental part was repeated in a lower concentration range. The data are reported as experiment II without metabolic activation.

The test medium was checked for precipitation visible to the naked eye at the end of the 4 hours treatment just before the test item was removed. Precipitation was observed at 110.0 and 165.0 µg/mL in experiment I with and without metabolic activation. In experiment II precipitation occurred at 110.0 µg/mL without metabolic activation and at 82.5 and 110.0 µg/mL with metabolic activation.

Relevant toxic effects indicated by a relative total growth of less than 50% of survival in both parallel cultures were observed in experiment I at 27.5 µg/mL and above without metabolic activation and at 110.0 µg/mL with metabolic activation following 4 hours of treatment. In experiment II cytotoxic effects as described above occurred at 27.5 µg/mL and above without metabolic activation (24 hours treatment) and at 82.5 µg/mL and above with metabolic activation (4 hours treatment). The recommended cytotoxic range of approximately 10-20% RTG was covered with and without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation up to cytotoxic concentrations. The positive control substances induced the appropriate responses.

There was no evidence of induced mutant colonies over background. Under the conditions of the study, the test substance was negative for mutagenic potential.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 19, 2005 - May 03, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(21 July 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)

Target gene:

not applicable, chromosome aberration assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-test for cytotoxicity: 39.1 to 5000 µg/mL (with and without S9 mix)
Experiment I: 3.1, 6.3, 12.5, 25.0, 50.0, 100.0 µg/mL (with and without S9 mix)
Experiment II: 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 µg/mL (with and without S9 mix)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1E04 - 6E04 cells per chamber
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
without S9 mix with S9 mix
Experiment I Experiment II Experiment I Experiment II
Exposure period 4 h 18 h 28 h 4 h 4 h
Recovery 14 h - - 14 h 24 h
Preparation interval 18 h 18 h 28 h 18 h 28 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemid (0.2 µg/mL culture medium), 15.5 hrs and 25.5 hrs, respectively after the start of the treatment, 2.5 hrs later, the cells on the slides were treated with hypotonic solution
- Methods of slide preparation and staining technique: cells were fixed with a mixture of methanol and glacial acetic acid and stained with Giemsa
- Number of cells spread and analysed per concentration: 100 well spread metaphase plates per culture were scored; 200 per concentration

Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations
gaps were recorded as well but not included in the calculation of the aberration rates
only metaphases with characteristic chremosome numbers of 22 + 1 were included in the analysis.
number of polyploid cells in 500 metaphase cells per culture was determined

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Evaluation criteria:

A test item is classified as non-clastogenic if:
— the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
— no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
— the number of induced structural chromosome aberrations is not in the-range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
— either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher’s exact test (9) (p < 0.05).
However, both biological and statistical significance should be considered together If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory's historical centrol data (0.0 - 8.5 % polyploid cells).

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

treatment		mitotic index	polyploidy cells [%]	aberrant cells [%]
				incl. gaps	excl. gaps	with exchanges
Experiment I, without metabolic activation, 4 h exposure, 18 h preparation time
Negative control		100.0	2.1	1	1	0
Solvent control		100.0	2.7	2.5	1.5	0
Positive control		80.1	2.7	12	11*	1
Test item [µg/mL]	3.1	115.5	-	-	-	-
	6.3	108.5	1.6	1	0	0
	12.5	99.1	1.9	2	1	0
	25.0	100.6	1.6	1.5	0.5	0
	50.0	81.1	-	-	-	-
	100.0	n.e.	-	-	-	-
Experiment I, with metabolic activation, 4 h exposure, 18 h preparation time
Negative control		100.0	2.1	2	1	0.5
Solvent control		100.0	1.6	1.5	1.	0.5
Positive control		89.6	2.1	14	12*	2.5
Test item [µg/mL]	3.1	94.3	1.7	3.5	3*	1.5
	6.3	82.3	2.5	2.5	2.5	1.5
	12.5	109.0	2.1	3	2	0.5
	25.0	105.4	-	-	-	-
	50.0	88.6	-	-	-	-
	100.0	n.e.	-	-	-	-
Experiment II, without metabolic activation, 18 h exposure, 18 h preparation time
Negative control		100.0	0.6	2.5	2.0	0.5
Solvent control		100.0	1.5	1.5	1.5	0.0
Positive control		106.4	1.3	12.0	11.0*	1.5
Test item [µg/mL]	1.6	92.7	-	-	-	-
	3.1	98.2	0.7	1.5	1.5	0.0
	6.3	82.2	0.6	0.5	0.5	0.0
	12.5	101.8	1.3	2.5	1.5	1.0
	25.0	6.5	-	-	-	-
	50.0	0.0	-	-	-	-
Experiment II, without metabolic activation, 28 h exposure, 28 h preparation time
Negative control		100.0	1.9	0.5	0.5	0.0
Solvent control		100.0	1.5	1.0	1.0	0.0
Positive control		87.9	2.6	13.5	12.5*	3.5
Test item [µg/mL]	6.3	94.2	-	-	-	-
	12.5	90.3	2.8	1.5	1.0	0.0
	25.0	n.e.	-	-	-	-
	50.0	n.e.	-	-	-	-
Experiment II, with metabolic activation, 4 h exposure, 28 h preparation time
Negative control		100.0	1.1	1.0	1.0	0.5
Solvent control		100.0	0.9	0.5	0.0	0.0
Positive control		96.1	1.7	9.5	9.0*	2.0
Test item [µg/mL]	1.6	99.3	-	-	-	-
	3.1	95.7	1.1	2.0	2.0	0.0
	6.3	100.3	1.0	0.5	0.5	0.5
	12.5	99.0	0.5	1.5	1.0	0.0
	25.0	106.3	-	-	-	-
	50.0	102.7	-	-	-	-

 

n.e. = not evaluated

- = not determined

* =aberration frequency statistically significant higher than corresponding control values

 

A single significant (p < 0.05) increase was observed in Experiment I, in the presence of S9 mix after 4 h treatment with 3.1 µg/mL. The response was not dose-related and it was within the laboratory's historical control data range. Therefore, the statistical significance has to be regarded as being biologically irrelevant.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Based on the results of this study, it is concluded that C16-18FA-TEPA-compound is not clastogenic in the in vitro mammalian chromosome aberration test.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473 (21 July 1997), Chinese hamster lung fibroblast (V79) cultures were exposed to C16-18FA-TEPA-compound in the following concentrations in the absence and presence of metabolic activation (S9 mix):

Experiment I (4 h exposure time, 18 h fixation time): 3.1, 6.3, 12.5, 25.0, 50.0, 100.0 µg/mL

Experiment II (18 h and 28 h exposure time, 18 h and 28 h fixation time without S9 mix/ 4 h exposure time, 28 h fixation time with S9 mix): 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 µg/mL

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells in the solvent control cultures was within the laboratory historical control data range. The positive controls produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

C16-18FA-TEPA-compound was tested up to precipitating concentrations. A single significant (p < 0.05) increase was observed in Experiment I, in the presence of S9 mix after 4 h treatment with 3.1 µg/mL. The response was not dose-related and it was within the laboratory's historical control data range. Therefore, the statistical significance has to be regarded as being biologically irrelevant.

Both in the absence and presence of S9-mix C16-18FA-TEPA-compound did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of C16-18FA-TEPA-compound on the number of polyploid cells were observed both in the absence and presence of S9-mix. Therefore it can be concluded that C16-18FA-TEPA-compound does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

There was no evidence of chromosome aberrations induced over background.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 01, 2005 - Feb 10, 2005

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:

(21 July 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)

Target gene:

Salmonella typhimurium: histidine (his); E. coli: tryptophan (trp)

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:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II (pre-incubation): 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene diamine (without metabolic activation)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) (experiment I); preincubation (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

E. coli WP2

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

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Based on the results of this study it is concluded that C16-18FA-TEPA-compound is not mutagenic in the reverse mutation assay in bacteria.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (21 July 1997), strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to C16-18FA-TEPA-compound (90% a.i.) at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in the first experiment (plate incorporation) and 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate in the second experiment (pre-incubation) in the presence and absence of metabolic activation (S9 mix).

C16-18FA-TEPA-compound was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. 

There was no evidence of induced mutant colonies over background.

Based on the results of this study it is concluded that C16-18FA-TEPA-compound is not mutagenic in the reverse mutation assay in bacteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the assessment of the mutagenic potential of C1618FA-TEPA-compound reliable (RL=1), relevant and adequate studies are available: a reverse gene mutation assay in bacteria (Ames test), a mammalian cell gene mutation assay (mouse lymphoma assay), and an in vitro mammalian cell chromosome aberrations test. Also the source substances used for read-across (partially unsaturated IQAC, DMS quaternised and oleic-acid based IQAC; DMS quaternised) were negative for genotoxicty. The supporting data on the source substance have only been taken into the dossier in order to justify the read-across approach for systemic toxicity endpoints.

 

Bacterial reverse gene mutation assays

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (21 July 1997), strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to C1618FA-TEPA-compound (90% a.i.) at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in the first experiment (plate incorporation) and 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate in the second experiment (pre-incubation) in the presence and absence of metabolic activation (S9 mix). C1618FA-TEPA-compound was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background. Based on the results of this study it is concluded that C1618FA-TEPA-compound is not mutagenic in the reverse mutation assay in bacteria.

 

In a reverse gene mutation assay in bacteria according to OECD Guideline 471, 1984 and US EPA-TSCA Guidelines, strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 of S. typhimurium were exposed to the partially unsaturated IQAC, DMS quaternised (75 %) at concentrations of 0.050 to 8.0 µL/plate in the presence and absence of mammalian metabolic activation (plate incorporation).

The test material exhibited varying degrees of toxicity with all the strains at higher doses in the non-activation and activation assays.

No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Since TA 1537 and TA 100 strains were contaminated in the independent repeat assay, the tests with these two strains were repeated in the non-activation and activation assays.

The positive control treatments in both the non-activation and S9 activation assays induced large increases in the revertant numbers with all the indicator strains, which demonstrated the effectiveness of the S9 activation system and the ability of the test system to detect known mutagens.

There was no evidence of induced mutant colonies over background.

E. coli WP2 or S. typhimurium TA102 have not been tested. It is known that the tested strains (TA 100, TA 1535, TA 1537, TA 1538 and TA 98) may not detect certain oxidising mutagens, cross-linking agents and hydrazines. The adopted OECD TG 471 (1997) requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the partially unsaturated IQAC, DMS quaternised is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to OECD TG 471 (1984) and US Toxic Substances Control Act (TSCA) Final Rule, EPA (1985) without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the test substance in this bacterial test system.

 

Mammalian cell gene mutation assays

In a mammalian cell gene mutation assay thymidine kinase locus according to OECD guideline 476, L5178Y mouse lymphoma cells cultured in vitro were exposed to C1618FA-TEPA-compound at the following concentrations in the presence and absence of mammalian metabolic activation (S9- mix):

Experiment I:

without S9 mix: 6.9; 13.8; 27.5; and 55.0 µg/mL
with S9 mix: 6.9; 13.8; 27.5; 55.0; and 110.0 µg/mL

Experiment II:

without S9 mix: 3.4; 6.9; 13.8; 27.5; and 55.0 µg/mL
with S9 mix: 13.8; 27.5; 55.0; 82.5; and 110.0 µg/mL

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours and in the presence of metabolic activation with a treatment time of 4 hours. The experimental part of the second experiment without metabolic activation was terminated prior to the generation of any data on mutagenicity as strong cytotoxic effects completely inhibited cell growth down to low concentrations. This experimental part was repeated in a lower concentration range. The data are reported as experiment II without metabolic activation.

The test medium was checked for precipitation visible to the unaided eye at the end of the 4 hours treatment just before the test item was removed. Precipitation was observed at 110.0 and 165.0 µg/mL in experiment I with and without metabolic activation. In experiment II precipitation occurred at 110.0 µg/mL without metabolic activation and at 82.5 and 110.0 µg/mL with metabolic activation.

Relevant toxic effects indicated by a relative total growth of less than 50% of survival in both parallel cultures were observed in experiment I at 27.5 µg/mL and above without metabolic activation and at 110.0 µg/mL with metabolic activation following 4 hours of treatment. In experiment II cytotoxic effects as described above occurred at 27.5 µg/mL and above without metabolic activation (24 hours treatment) and at 82.5 µg/mL and above with metabolic activation (4 hours treatment). The recommended cytotoxic range of approximately 10-20% RTG was covered with and without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation up to cytotoxic concentrations. The positive control substances induced the appropriate responses.

There was no evidence of induced mutant colonies over background. Under the conditions of the study, the test substance was negative for mutagenic potential.

 

In a mammalian gene mutation assay, L5178 Y (mouse lympoma thymidine kinase locus) cellscultured in vitro were exposed to the oleic-acid based IQAC; DMS quaternised (98 % a.i.) at the following concentrations:

Experiment I

- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/mL

- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/mL

Experiment II

- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/mL

- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/mL

The oleic-acid based IQAC, DMS quaternised was tested up to cytotoxic concentrations(>/= 16.0 µg/mL without metabolic activation and >/= 40.0 µg/mL with metabolic activation). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increased number of induced mutant colonies over background.

 

In vitro cytogenetics assays

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473 (21 July 1997), Chinese hamster lung fibroblast (V79) cultures were exposed to C1618FA-TEPA-compound in the following concentrations in the absence and presence of metabolic activation (S9 mix):

Experiment I (4 h exposure time, 18 h fixation time): 3.1, 6.3, 12.5, 25.0, 50.0, 100.0 µg/mL

Experiment II (18 h and 28 h exposure time, 18 h and 28 h fixation time without S9 mix/ 4 h exposure time, 28 h fixation time with S9 mix): 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 µg/mL

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells in the solvent control cultures was within the laboratory historical control data range. The positive controls produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

C1618FA-TEPA-compound was tested up to precipitating concentrations. A single significant (p < 0.05) increase was observed in Experiment I, in the presence of S9 mix after 4 h treatment with 3.1 µg/mL. The response was not dose-related and it was within the laboratory's historical control data range. Therefore, the statistical significance has to be regarded as being biologically irrelevant.

Both in the absence and presence of S9-mix C1618FA-TEPA-compound did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of C1618FA-TEPA-compound on the number of polyploid cells were observed both in the absence and presence of S9-mix. Therefore it can be concluded that C1618FA-TEPA-compound does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.There was no evidence of chromosome aberrations induced over background.

 

In a mammalian cell cytogenetics assay according to OECD Guideline 473, 1983 and US EPA-TSCA Guidelines, CHO cell cultures were exposed to the partially unsaturated IQAC, DMS quaternised (75 %) at concentrations of 15.0 to 199 µL/mL with metabolic activation and at 3.74 to 74.8 µL/mL without metabolic activation. Evaluation for aberrant cells was performed at for aberrant cells at 49.9, 99.7, 150 and 199 µL/mL with metabolic activation and at 24.9, 37.4, 49.9 and 74.8 µL/mL without metabolic activation.

Cytotoxic effects of the test item were observed with and without metabolic activation; at the highest evaluated concentrations reduction in the cell monolayer confluency was 40% in both experiments, without and with metabolic activation.

Neither without metabolic activation nor with metabolic activation a significant increase in chromosomally aberrant cells was observed at the concentrations analysed.

As positive controls reference mutagens Mitomycin C and cyclophosphamide were tested in parallel to the test item. They induced distinct and biologically relevant increases in cells with structural chromosomal aberration.There was no evidence of chromosome aberration induced over background.

 

Based on the available reliable, relevant and adequate data, there was no evidence of genotoxicity for C1618FA-TEPA-compound. There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

Justification for classification or non-classification

Based on the available data, C1618FA-TEPA-compound does not need to be classified for mutagenicity according to the criteria given in regulation (EC) 1272/2008.Thus, no labelling is required.