Fatty acids, C12-18 and C18-unsatd., 2-sulfoethyl esters, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiated on 16 May 2007 and was completed (final report issued) on September 2007. Experimental work started on 22 May 2007 and was completed on 10 July 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted in accordance with current guideline (OECD 476) and GLP-compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): JORDAPON SCI (90)
- Physical state: cream coloured granular powder
- Analytical purity: 90.14%
- Purity test date: 15 May 2007
- Lot/batch No.: sample number S2789301
- Stability under test conditions: Stability of the test solutions was not assessed as this was a short term study and the test solutions were freshly prepeared for each experiment
- Storage condition of test material: desiccated at room temperature in the dark under nitrogen

Method

Target gene:

tk locus (5-trifluorothymidine [TFT] resistance

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178 TK +/- mouse lymphoma cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK- mutants, checked for spontaneous mutant frequency and that it was 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% v/v CO2 in air. When cells were growing well, sub-cultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Range-finder: 0, 5.625, 11.25, 22.5, 45, 90 and 180 µg/mL for 3-hour treatment; 0, 0.7031, 1.406, 2.813, 5.625, 11.25, 22.5, 45, 90 and 180 µg/mL for 24-hour treatment.
Experiment 1: 0, 15, 25, 35, 45, 52.5, 60, 67.5, 75, 82.5 and 90 µg/mL (-S-9); 0, 20, 40, 70, 85, 100, 115, 130, 145, 160 and 180 µg/ml (+S-9).
Experiment 2 (3-hour treatment): 0, 20, 40, 60, 80, 90, 100, 110, 120, 130, 140, 150 and 160 µg/mL (-S-9); 0, 40, 60, 80, 90, 100, 110, 120, 130, 140 and 150 µg/mL (+S-9).
Experiment 2 (3 and 24-hour treatment): 0, 60, 80, 90, 100, 110, 120, 130 and 140 µg/mL (-S-9 only).

Vehicle / solvent:

- Vehicle(s) / solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility data indicated that JORDAPON SCI (90) was soluble in dimethyl sulphoxide (DMSO) at concentrations up to approximately 18.55 mg/mL. The solubility limit in culture media was greater than 185.5 µg/mL. Concentrations for the maini experiments were selected based on the results of the cytotoxicity range-finding experiment.

Controls

Untreated negative controls:

no

Remarks:

See Solvent / Vehicle controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

True negative controls:

no

Remarks:

Not applicable for this assay.

Positive controls:

yes

Remarks:

4-nitroquinoline 1-oxide (NQO, -S-9), benzo(a)pyrene (BP, +S-9) (both prepared in DMSO)

Positive control substance:

other: 4-nitroquinoline 1-oxide (NQO, -S-9), benzo(a)pyrene (BP, +S-9) (both prepared in DMSO).

Remarks:

NQO at 0.15 and 0.2 µg/mL final concentration (0.05 and 0.1 µg/mL in Experiment 2); BP at 2.00 and 3.00 µg/mL final concentration.

Details on test system and experimental conditions:

Metabolic activation system:
Rat liver S-9 fraction from male Sprague Dawley rats induced with Aroclor 1254. Batches were stored at -80°C prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P450-catalyzed enzyme activities.
Treatment was carried out both in the absence and presence of S-9, prepared in the following way:
Glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), potassium chloride (KCl) (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2.
For all cultures treated in the presence of S-9, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL.
The final concentration of the liver homogenate in the test system was 2%. Cultures treated for 3 hours in the absence of S-9 received 1 mL KCl (150 mM).

Growth media:
RPMI 1640 media with antibiotics and horse serum.
Heat-activated horse serum was used in order to eliminate a factor that degrades TFT.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assys if the mutant frequency (MF) of any test concentration exceeded the sum of the mean control mutant frequency plus global evaluation factor (GEF) and the linear trend was positive. This indicated a positive, biologically relevant response.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Extreme caution would be exercised with positive results obtained at levels of RTG lower than 10%.
The test agent was regarded as giving a negative result if the MF of all test concentrations were less than the sum of the mean control mutant frequency plus GEF.

Statistics:

The significance of increases in mutant frequencies (total wells with clones), by comparison with concurrent controls and Global Evaluation factor (GEF) was assessed according to the recommendations of the Mouse Lymphoma Workgroup, Aberdeen, 2003. The control mutant frequency was compared with each test article treatment and the data were checked for a linear trend in mutant frequency with treatment concentration using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic at concentrations equal to or greater than 130 µg/mL in the absence of S-9, and 150 µg/mL in the presence of S-9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The mutant frequencies of the concentrations plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF), indicating a negative result. A significant linear trend was observed in the presence of S-9 in Experiment 1 but, in the absence of any marked increases in mutant frequency at all concentrations analysed in this experiment, this observation was not considered biologically relevant.
In addition, for the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony mutant frequencies could be estimated and the proportion of small mutant colonies could be calculated. For the negative controls (diluted DMSO), the proportion of small colony mutants in the absence and presence of S-9 ranged from 29% to 37% in Experiment 1 and from 33% to 42% in Experiment 2. Marked increases in the number of both small and large colony mutants were observed with the positive control chemicals NQO and BP.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Cytotoxicity Range-finder (3-hour treatment)

JORDAPON SCI (90)	-S-9	+S-9
(µg/mL)	% RTG	% RTG
0	100	100
5.625	97	103
11.25	84	98
22.5	69	116
45	117	91
90	0	70
180	0	0

% RTG  Relative Total Growth

 

 

Table 2: Cytotoxicity Range-finder (24-hour treatment)

JORDAPON SCI (90)	-S-9
(µg/mL)	% RTG
0	100
0.7031	112
1.406	90
2.813	89
5.625	104
11.25	124
22.5	97
45	133
90	62
180	0

% RTG  Percentage Relative Total Growth (adjusted by Day 0 factor)

 

Table 3: Experiment 1 (3-hour treatment -/+ S-9)

JORDAPON SCI (90)	-S-9		JORDAPON SCI (90)	+S-9
(µg/mL)	% RTG	MF§	(µg/mL)	% RTG	MF§
0	100	62.58	0	100	54.21
15	94	64.56	20	89	57.14
25	95	66.64	40	105	56.31
35	93	51.91	70	73	58.65
45	94	46.55	85	62	70.08
52.5	93	80.39	100	38	79.69
60	98	56.82	115	6	85.08
67.5	71	65.06
75	74	60.84
82.5	64	60.30
90	59	67.92
Linear trend		NS	Linear trend		**
NQO			BP
0.15	78	283.93	2	64	244.01
0.2	62	427.36	3	42	404.67

§                             5-TFT resistant mutants/10⁶viable cells 2 days after treatment

NS                         Not significant

% RTG                  Relative Total Growth

*,**,***                   Test for linear trend: x2 (one-sided), significant at 5%, 1% and 0.1% level, respectively

 

Experiment 2 (3-hour treatment -/+ S-9)

JORDAPON SCI (90)	-S-9		JORDAPON SCI (90)	+S-9
(µg/mL)	% RTG	MF§	(µg/mL)	% RTG	MF§
0	100	65.59	0	100	67.63
20	109	55.82	40	77	55.11
40	91	71.17	60	75	63.20
60	94	63.30	80	91	52.57
80	78	76.08	90	83	63.68
90	82	66.06	100	65	55.18
100	73	55.22	110	54	52.78
110	61	59.13	120	51	61.70
120	45	49.18	130	40	46.35
130	23	58.67	140	33	45.46
140	10	66.68	150	17	63.63
Linear trend		NS	Linear trend		NS
NQO			BP
0.15	72	385.06	2	82	350.51
0.2	60	481.31	3	43	703.75

§                             5-TFT resistant mutants/10⁶viable cells 2 days after treatment

NS                         Not significant

% RTG                  Relative Total Growth

 

Experiment 2 (24-hour treatment in the absence of S-9)

JORDAPON SCI (90)	-S-9
(µg/mL)	% RTG	MF§
0	100	56.37
60	104	45.95
80	92	51.06
90	70	60.42
100	64	48.80
110	56	68.65
120	46	55.37
130	37	65.65
140	14	65.65
Linear trend		NS
NQO
0.1	35	506.55

§                             5-TFT resistant mutants/10⁶viable cells 2 days after treatment

NS                         Not significant

% RTG                  Relative Total Growth (adjusted by Day 0 factor for 24-hour treatment)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative See Conclusions.

JORDAPON SCI (90) did not induce mutation at the tk locus of L5178Y mouse lymphoma cells under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

JORDAPON SCI (90) was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment 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 (S-9).

A 3 hour treatment incubation period was used for all experiments performed in the presence of S-9. In the absence of S-9, the range-finder was performed using 3 and 24 hour treatment incubation periods, Experiments 1 and 2 were performed using a 3 hour treatment incubation and a 24 hour treatment incubation was also performed in Experiment 2.

In the cytotoxicity range-finding experiment, 3 hour treatment, six concentrations of JORDAPON SCI (90) were tested in the absence and presence of S-9, ranging from 5.625 to 180 μg/mL (limited by solubility). The highest concentrations to give >10% relative total growth (RTG) were 45 μg/mL in the absence of S-9 and 90 μg/mL in the presence of S-9, which yielded 117% and 70% RTG, respectively.

In the cytotoxicity range-finding experiment, 24 hour treatment, nine concentrations of JORDAPON SCI (90) were tested in the absence of S-9, ranging from 0.7031 to 180 μg/mL (limited by solubility). The highest concentration to give >10% RTG was

90 μg/mL, which yielded 62% RTG.

Accordingly, in Experiment 1 (3 hour treatment), ten concentrations, ranging from 15 to 90 μg/mL in the absence of S-9 and from 20 to 180 μg/mL in the presence of S-9, were tested. The highest concentrations selected to determine viability and TFT resistance were 90 μg/mL in the absence of S-9, which yielded 59% RTG, and 100 and 115 μg/mL in the presence of S-9, which yielded 38% and 6% RTG, respectively.

Insufficient toxicity was achieved in the absence of S-9 in Experiment 1, therefore Experiment 2 included 3 and 24 hour treatments in the absence of S-9.

In Experiment 2, twelve concentrations, ranging from 20 to 160 μg/mL, were tested in the absence of S-9 (3 and 24 hour treatments) and eleven concentrations, ranging from 20 to 150 μg/mL, were tested in the presence of S-9 (3 hour treatment). The highest concentrations selected to determine viability and TFT resistance were 140 μg/mL (3 hour treatment in the absence of S-9), 150 μg/mL 3 hour treatment in the presence of S-9) and 140 μg/mL (24 hour treatment in the absence of S-9), which yielded 10%, 17% and 14% RTG, respectively.

Negative (vehicle) and positive control treatments were included in each mutation experiment in the absence and presence of S-9. 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 S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2, the mutant frequencies of the concentrations plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF), indicating a negative result. A significant linear trend was observed in the presence of S-9 in Experiment 1 but, in the absence of any marked increases in mutant frequency at all concentrations analysed in this experiment, this observation was not considered biologically relevant.

It is concluded that JORDAPON SCI (90) did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).