Mono Dimer Trimer FA TETA PAA

Genetic toxicity: in vitro

Administrative data

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:

28th May 2012 to 19th November 2012.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to GLP and relevant testing guidelines.

Justification for type of information:

Please refer to the read-across justification document enclosed in chapter 13 for more details.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

TETA – Fatty acids adducts (Mw 600-1000Da)

IUPAC Name:

TETA – Fatty acids adducts (Mw 600-1000Da)

Constituent 2

Reference substance name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 3

Reference substance name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 4

Reference substance name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

EC Number:

292-587-7

EC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Cas Number:

90640-66-7

IUPAC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): TOFA_DimerFA_TETA_PAA
- Physical state: Yellow liquid with a brown hue.
- Analytical purity: 100%
- Lot/batch No.: BB001030V1
- Expiration date of the lot/batch: 30 May 2013
- Storage condition of test material: When not in use the test article was stored in a sealed container, at room temperature in the dark.

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cells were stored as frozen stock in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Range-finder: 0.7324, 1.465, 2.930, 5.859, 11.72, 23.44, 46.88, 93.75 and 187.5 μg/mL
Experiment 1 without S9 mix: 1, 2, 4, 6, 8, 10, 12, 15, 18, 25 and 30 μg/mL
Experiment 1 with S9 mix: 2, 4, 8, 12, 16, 20, 25, 30 and 40 μg/mL
Experiment 2 without S9 mix: 2.5, 5.0, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30 and 40 μg/mL
Experiment 2 with S9 mix: 5, 10, 15, 20, 25, 30, 40, 50, 60, 75 and 90 μg/mL

Vehicle / solvent:

- Vehicle used: acetone

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

acetone diluted 100-fold in the treatment medium.

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

0.15 and 0.20 μg/mL

Positive control substance:

other: 4-nitroquinoline 1-oxide

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

acetone diluted 100-fold in the treatment medium.

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Remarks:

2 and 3 μg/mL

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A preliminary range-finding study was conducted prior to the main experiment. In this range-finding test, single cultures were used and positive controls were not included. The final treatment volume was 20 mL. Following treatment, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 8-9 days. Wells containing viable clones were identified by microscope/by eye using background illumination and counted.

In the mutation assay, at least 10E7 cells in a volume of 18.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL test article, vehicle, untreated or positive control solution was added. S9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1ºC with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed and resuspended in 20 mL RPMI 10. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 10E5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies. Osmolality and pH measurements on post-treatment media were taken in the initial cytotoxicity Range-Finder Experiment.

Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL.

Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of not exceeding 1 x 10E6 cells/mL and, where possible, retaining at least 6 x 106 cells/flask.

At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10E5 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10E5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10E4 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12 days) and wells containing clones were identified as above and counted.

DETERMINATION OF CYTOTOXICITY
- Method: viability, relative survival, mutant frequency

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05).
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical analysis of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Toxicity:
In the second cytotoxicity Range-Finder Experiment, nine concentrations were tested in the absence and presence of S9 ranging from 0.7324 to 187.5 μg/mL. Following the 3 hour treatment incubation period, precipitate was observed at the highest four concentrations in the absence and presence of S9 (23.44 to 187.5 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentration to provide >10% RS in the absence of S9 was 11.72 μg/mL, which gave 24% RS. In the presence of S9, cultures treated at 23.44 μg/mL gave 85% RS but post-treatment precipitate was observed at this concentration.
No marked changes in osmolality or pH were observed in the first cytotoxicity Range-Finder at the highest concentrations analysed (187.5 μg/mL in the absence of S9 or 93.75 μg/mL in the presence of S9), compared to the concurrent vehicle controls.

In the first main mutation assay, eleven concentrations, ranging from 1 to 30 μg/mL, were tested in the absence of S9 and nine concentrations, ranging from 2 to 40 μg/mL, were tested in the presence of S9. Seven days after treatment, the highest two concentrations in the absence of S9 (25 and 30 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance and the lowest concentration (1 μg/mL) in the absence of S9 was not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S9. The highest concentrations analysed were 18 μg/mL in the absence of S9 and 40 μg/mL in the presence of S9, which gave 16% and 41% RS, respectively.
In the 2nd main experiment, eleven concentrations, ranging from 2.5 to 40 μg/mL in the absence of S9 and from 5 to 90 μg/mL in the presence of S-9, were tested. Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the presence of S9 (60 to 90μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the presence of S9 was retained and higher concentrations were discarded. Seven days after treatment, the highest three concentrations in the absence of S9 (25 to 40 μg/mL) and the highest concentration in the presence of S9 (60 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, concentrations of 7.5 and 12.5 μg/mL in the absence of S9 and 5 and 25 μg/mL in the presence of S9 were not selected as there were sufficient non-toxic concentrations to define appropriate toxicity profiles. All other concentrations were selected in the absence and presence of S9. The highest concentrations analysed were 20 μg/mL in the absence of S9 and 50 μg/mL in the presence of S9, which gave 19% and 16% RS, respectively.

Mutation:
In Experiments 1 and 2, no statistically significant increases in mutant frequency (MF) were observed following treatment with TOFA_DimerFA_TETA_PAA at any concentration tested in the absence and presence of S9. The vehicle control MF values in the absence and presence of S9 were less than three times the current historical mean MF values at the time of both experiments and were considered acceptable. A weak but statistically significant linear trend was observed in the presence of S9 in Experiment 1. However, as there were no significant increases in mutant frequency at any concentration tested in this experiment, this isolated observation was considered not biologically relevant.
These data are considered sufficiently robust because the optimum upper limit for toxicity (10-20% RS) was achieved in both the absence and presence of S9 during the course of the study, albeit only once in the presence of S9. However, the data showed no evidence of mutagenic activity up to and including toxic concentrations under both treatment conditions.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1 (3 hour treatment in the absence and presence of S9) - Mutation Data

Treatment (µg/mL)	Presence of S9 mix		Treatment (µg/mL)	Absence of S9 mix
	% RS	MF		% RS	MF
HVC	-	4.15	HVC	-	4.46
0	100	3.66	0	100	5.09
UTC	99	3.62    NS	UTC	109	4.07    NS
2	96	3.11    NS	2	108	4.91    NS
4	79	2.95    NS	4	107	4.35    NS
6	70	4.18    NS	8	97	6.45    NS
8	59	5.40    NS	12	100	5.99    NS
10	41	4.94    NS	16	112	6.48    NS
12	36	0.65    NS	20	91	4.80    NS
15	20	3.03    NS	25	80	7.55    NS
18	16	4.91    NS	30	62	6.45    NS
			40	41	7.65    NS
Linear trend                          NS			Linear trend                  *
NQO			B[a]P
0.15	66	23.89	2	104	22.14
0.2	46	23.08	3	55	41.96

Experiment 2 (3 hour treatment in the absence and presence of S9) - Mutation Data

Treatment (µg/mL)	Presence of S9 mix		Treatment (µg/mL)	Absence of S9 mix
	% RS	MF		% RS	MF
HVC	-	4.37	HVC	-	4.69
0	100	1.62	0	100	2.08
UTC	113	3.14    NS	UTC	96	1.43    NS
2.5	94	3.28    NS	10	100	1.11    NS
5	89	1.32    NS	15	99	0.45    NS
10	64	1.31    NS	20	81	1.37    NS
15	21	1.47    NS	30	66	3.41    NS
17.5	16	1.94    NS	40	69	2.27    NS
20	19	2.67    NS	50	16	1.34    NS
Linear trend                                      NS			Linear trend                                       NS
NQO			B[a]P
0.15	44	26.28	2	53	14.67
0.2	40	33.83	3	28	36.12

UTC Untreated control

§ 6-TG resistant mutants/10⁶viable cells 7 days after treatment

HVC Historical vehicle control MF at the time of this experiment

% RS Percent relative survival adjusted by post treatment cell counts

NS Not significant

*, **, *** Test for linear trend: χ²(one-sided), significant at 5%, 1% and 0.1% level respectively

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study, TOFA_DimerFA_TETA_PAA did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells, in the presence and absence of S9 mix in two independent experiments.

Executive summary:

The mutagenic potential of TOFA_DimerFA_TETA_PAA was evaluated in an in vitro mammalian cell gene mutation study, conducted according to OECD Test Guideline 476 and GLP. The study was conducted in L5178Y mouse lymphoma cells. The study consisted of two cytotoxicity Range-Finder Experiments followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S9). The test article was formulated in acetone. A 3 hour treatment incubation period was used for all experiments.

In the first cytotoxicity Range-Finder Experiment, concentrations ranging from 46.88 to 1500 μg/mL were tested in the absence and presence of S9 but extreme toxicity was observed at all concentrations plated for survival in the absence of S9 (46.88 to 187.5 μg/mL) and post-treatment precipitate was observed at all concentrations tested in the presence of S9.

In the second cytotoxicity Range-Finder Experiment, nine concentrations were tested in the absence and presence of S9, ranging from 0.7324 to 187.5 μg/mL. The highest concentration to provide >10% relative survival (RS) in the absence of S9 was 11.72 μg/mL, which gave 24% RS. In the presence of S-9, cultures treated at 23.44 μg/mL gave 85% RS but post-treatment precipitate was observed at this concentration.

In Experiment 1 eleven concentrations, ranging from 1 to 30 μg/mL, were tested in the absence of S9 and nine concentrations, ranging from 2 to 40 μg/mL, were tested in the presence of S9. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 18 μg/mL in the absence of S9 and 40 μg/mL in the presence of S9, which gave 16% and 41% RS, respectively.

In Experiment 2 eleven concentrations, ranging from 2.5 to 40 μg/mL in the absence of S9 and from 5 to 90 μg/mL in the presence of S9, were tested. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 20 μg/mL in the absence of S9 and 50 μg/mL in the presence of S9, which gave 19% and 16% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S9) and benzo(a)pyrene (with S9). Therefore the study was accepted as valid.

In Experiments 1 and 2, no statistically significant increases in mutant frequency (MF) were observed following treatment with TOFA_DimerFA_TETA_PAA at any concentration tested in the absence and presence of S9. The vehicle control MF values in the absence and presence of S9 were less than three times the current historical mean MF values at the time of both experiments and were considered acceptable. A weak but statistically significant linear trend was observed in the presence of S9 in Experiment 1. However, as there were no significant increases in mutant frequency at any concentration tested in this experiment, this isolated observation was considered not biologically relevant.

Under the conditions of this study, TOFA_DimerFA_TETA_PAA did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells, in the presence and absence of S9 mix in two independent experiments.