Addition reaction products of conjugated sunflower-oil fatty acids and tall-oil fatty acids with maleic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (induction of rat liver enzymes by Phenobarbital and β-naphthoflavone)

Test concentrations with justification for top dose:

Assay 1, 3h treatment with metabolic activation:
5000, 3750, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL
Assay 1, 3 h treatment without metabolic activation:
2000, 1000, 750, 500, 375, 250, 125, 62.5 and 31.25 μg/mL
Assay 2, 3h with treatment with metabolic activation:
2500, 2187.5, 1875, 1562.5, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL
Assay 2: 20h treatment without metabolic activation:
1000, 500, 375, 312.5, 250, 187.5, 125, 62.5 and 31.25 μg/mL
Assay 3, 3h with treatment with metabolic activation:
2000, 1500, 1000, 875, 750, 625, 500, 375, 250, 125, 62.5 and 31.25 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: appropriate for formulation and its dilution; compatible to the test system

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulfonate (EMS) dissolved in DMEM, final concentration of 0.4 μL/mL (28-hour harvesting time) or 1.0 μL/mL (20-hour harvesting time)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP) dissolved in sterile physiological saline solution (0.9% NaCl infusion), final concentration of 6.0 μg/mL.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 20 hours
- Fixation time (start of exposure up to harvest of cells): 20 and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.2 μg/mL)
STAIN (for cytogenetic assays): 5 % Giemsa solution

NUMBER OF CELLS EVALUATED: 200 cells (metaphases)/dose

DETERMINATION OF CYTOTOXICITY
- Method: determination of cell concentration by use of a haemocytometer

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see Table 1 and 2 ("Additional information on results")

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no changes
- Effects of osmolality: changes observed ≥ 3750 μg/mL in the experiment with metabolic activation (only in Assay 1).
- Insolubility: ≥ 312,5 and ≥ 375 μg/mL with and without metabolic acitvation, respectively. Insolubility was detected at the end of the treatment period in the final treatment medium.

Table 1: Summarized results of the concentration selection cytotoxicity assays A and B

	Dose(μg/mL)	Testgroup without S9-mix Relative survival (%) (Assay A*/B**)	Testgroup with S9-mix Relative survival (%) (Assay A*/B**)
Untreated control		92/119	112/119
Vehicle control 1%(v/v) DMSO		100/100	100/100
Vehicle control 2% v/v DMSO		100/100	100/100
WS400104	5000	1/0	18/3
	2500	23/0	62/2
	1250	34/0	52/51
	625	12/0	39/44
	312,5	52/7	47/72
	156,25	71/75	69/96
	78,13	88/97	81/93
	39,06	83/91	95/102

*Time of Treatment/Sampling: 3h/20h with and without activation

**Time of Treatment/Sampling: 20h/28h without activation, 3h/28h with activation

Table 2: Summary table of Chromosome Aberration Assay 1

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival # (%)	Mean aberrant cells### %
WS400104 without metabolic activation (-S9)
Vehicle(solvent)control	3h/20h	100	3.0
2000	3h/20h	19	NE
1000	3h/20h	25	NE
750	3h/20h	10	NE
500	3h/20h	32	5,0
375	3h/20h	58	NE
250	3h/20h	64	4,5
125	3h /20h	93	3,5
62.5	3h/20h	92	NE
31.25	3h/20h	110	NE
Positive control	3h/20h	84	11,0**

WS400104 with metabolic activation (+S9)
Vehicle(solvent)control	3h/20h	100	3.0
5000	3h/20h	0	NE
3750	3h/20h	27	2,7
2500	3h/20h	0	NE
1250	3h/20h	0	NE
625	3h/20h	40	12,4**
312,5	3h/20h	87	3,5
156,25	3h/20h	87	5,0
78,13	3h/20h	94
39,06	3h/20h	88
Positive control	3h/20h	66	96.8***

Vehicle (solvent) control: 1% (v/v) DMSO without (Assay –S9), 2% (v/v) DMSO (Assay +S9)

Positive control (-S9): Ethyl methanesulfonate, 1μL/mL; Positive control (+S9): Cyclophosphamide, 6μg/mL

NE: not evaluated

#: compared to the vehicle (solvent control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Table 3: Summary table of Chromosome Aberration Assay 2

Concentration(μg/mL)	TimeofTreatment/Sampling	Relative # Survival (%)	Mean%aberrantcells###
WS400104withoutmetabolicactivation(-S9)
Vehicle(solvent)control	20h/28h	100	3,0
1000	20h/28h	0	NE
500	20h/28h	0	NE
375	20h/28h	0	NE
312,5	20h/28h	0	NE
250	20h/28h	15	NE
187,5	20h/28h	13	2.0
125	20h/28h	57	1.0
62,5	20h / 28h	91	2,0
31.25	20h/28h	91	NE
Positivecontrol	20h/28h	63	21,4***
WS400104withmetabolicactivation(+S9)
Vehicle(solvent)control	3h/28h	100	3.0
2500	3h/28h	59	NE
2187,5	3h/28h	53	NE
1875	3h/28h	37	NE
1562,5	3h/28h	46	NE
1250	3h/28h	38	1,5
625	3h/28h	47	6,0
312,5	3h/28h	49	2,5
156,52	3h/28h	87	2,0
78,13	3h/28h	94	3,0
39,06	3h/28h	75	NE
Positivecontrol	3h/28h	58	12,6***

Vehicle (solvent) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 0.4μL/mL; positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

#: compared to the negative (solvent control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

In Assays 1 and 2, none of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation except for 625 μg/mL concentration in Assay 1 with metabolic activation, in one replicate. This was not repeatable in Assay 2 (although elevated aberration frequency was observed at the same concentration), nor did the higher concentration assessed in Assay 1 with metabolic activation give a positive response. There was no evidence of any dose response in either assay.

Because of the equivocal results observed in Assay 1 with metabolic activation, an additional experiment with metabolic activation (Assay 3) was performed - with a more closely spaced concentration range, but otherwise using the same treatment and harvesting time as in Assay 1.

Table 4: Summary table of Chromosome Aberration Assay 3 with metabolic activation

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival#(%)	Mean % aberrant cells###
Vehicle(solvent)control	3h/20h	100	3.0
2000	3h/20h	19	NE
1500	3h/20h	14	NE
1000	3h/20h	8	NE
875	3h/20h	4	NE
750	3h/20h	7	NE
625	3h/20h	34	2,5
500	3h/20h	61	3,5
375	3h/20h	81	2,0
250	3h/20h	96	1,5
125	3h/20h	89	NE
62,5	3h/20h	92	NE
31,25	3h/20h	88	NE
Positive control	3h/20h	59	85,7***

Vehicle (solvent) control: 1% (v/v) DMSO

Positive control (+S9): Cyclophosphamide, 6μg/mL

NE: not evaluated

#: compared to the vehicle (solvent control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

It was concluded that the single result at 625 μg/mL with metabolic activation seen in a single replicate (not in the other replicate) was not reproducible in a repeat assay and did not show a dose response relationship. Hence this single result was not considered to represent an adverse effect of the test item.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid (1-11) and endoreduplicated (1-4) metaphases were found in some cases in the vehicle (solvent) control, positive control or test item treated samples in the performed experiments.

Conclusions:

negative without and with metabolic activation (-/+ S9)

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone for enzyme induction.

Test concentrations with justification for top dose:

Range-finding test: 0; 10; 31.6; 100; 316; 1000; 2500 and 5000 μg/plate
Experiment 1: 0; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate
Experiment 2: 0; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate
Complementary to Experiment 2: 0; 0.1581; 0.5; 1.581; 5; 15.81; 50; 158.1; and 500 μg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)
Justification for choice of solvent/vehicle:
DMSO was a suitable vehicle for exposure to the test substance up to the maximum guideline recommended test substance concentration of 5000 μg/plate.

Untreated negative controls:

yes

Remarks:

without and with S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine; sodium azide; 9-aminoacridine; methyl-methanesulfonate.

Remarks:

Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.

Untreated negative controls:

yes

Remarks:

without and with S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; Activity of S9 was additionally tested with Benzo[a]pyrene

Remarks:

Positive control substances for tests with metabolic activation (S9 mix). All of them are well established reference mutagens.

Details on test system and experimental conditions:

Range-finding test & Experiment 1: Standard Plate Incorporation Tests
Experiment 2 & Complementary to Experiment 2: Pre-incubation Test

All tests, except the Complementary to Experiment 2, were performed without and with metabolic activation (S9 mix).
The Complementary to Experiment 2, was performed without metabolic activation.
Proportion of S9 fraction in the S9 mix was 10% v/v and of S9 fraction in the final medium ca. 2% v/v in all tests with metabolic activation.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

4-nitro-1,2-phenylene-diamine: - 4 μg/plate, dissolved in DMSO: - strain: TA 98

Sodium azide: - 2 μg/plate, dissolved in distilled water: - strains: TA 100, TA 1535

9-Aminoacridine: - 50 μg/plate, dissolved in DMSO: - strain: TA 1537

Methyl-methanesulfonate: - 2 μL/plate, dissolved in distilled water: - strain: WP2 uvrA


With metabolic activation (S9 mix):

2-Aminoanthracene: - 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 1537, TA 98, TA 100

2-Aminoanthracene: - 50 μg/plate, dissolved in DMSO: - strain: WP2 uvrA


In addition, adeqaute biological activity of the S9 batch used in the present study was confirmed by use of two mutagens, Aminoanthracene and Benzo(a)pyrene, that require metabolic activation by microsomal enzymes.

Evaluation criteria:

The test substance is considered to exhibit mutagenic activity in this assay if the following criteria are met:
- a dose–related increase in the number of revertants and/or;
- a reproducible, biologically relevant positive response for at least one of the dose groups in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if in at least one of the five tested strains the number of reversion was more than twice higher than the reversion rate of the vehicle (solvent) control.

A test substance is considered non-mutagenic in this test if:
Neither a dose-related increase in the number of revertants nor a reproducible, biologically relevant positive response is evident in any of the dose groups, with or without metabolic activation.

The study was considered valid if:
- the number of revertant colonies of the vehicle (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Statistics:

The data were not statistically analysed. The study result was unequivocal.

Key result

Species / strain:

S. typhimurium, other: TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

but confined to strain TA 100, 5000 µg/plate without metabolic acitvation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the recommended maximum concentration of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Experiment 1 confined to 5000 µg/plate & 3 strains, in Experiment 2 confined to 1581 & 5000 µg/plate, except strain TA 1537 with reduced background lawn down to 158.1 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the recommended maximum concentration of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

confined to 158.1 and 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Viability checked by a plating experiment in each test was satisfactory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: in the Range-finding test

Precipitate / slight precipitate was observed in Experiment 2 in all tested bacterial strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2) at 5000 µg/plate concentration with and without metabolic activation.

Conclusions:

negative without and with metabolic activation (S9 mix)

Reference 3

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-5, RPMI-10 and RPMI-20 containing 5% v/v, 10% v/v and 20% v/v heat inactivated horse serum, respectively
and each medium containing: Antibiotic-antimycotic solution (containing penicillin, streptomycin & amphotericin-B),
Pyruvic acid and
NaHCO3.
RPMI-5 and RPMI-10 additionally contained Pluronic-F68.

- Type and identity of media, in general used for cell culture: RPMI-10
- Type of media used for Treatment media: RPMI-5, culture incubated for 3 or 24 hours at 37± 1 °C (approximately 5% CO2 in air)
- Plating for survival: Dilution with RPMI-10 followed by dilution with RPMI-20
- 3-day mutation expression period: RPMI-10
- Plating for viability after the expression period: Dilution with RPMI-10 followed by dilution with RPMI-20

Management of mouse lymphoma cell line L5178Y TK+/- 3.7.2 C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone (80 mg/kg/day over 3 consecutive days) for enzyme induction. Final concentration of liver homogenate in the test system was 2%.

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TESTING (plating efficiency relative to that of vehicle controls)
Test concentrations at 3 h exposure with (+S9) and without (–S9) metabolic activation and at 24 h exposure without metabolic activation (–S9):
39.06, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 12.5, 25, 50, 75, 100, 112.5, 125, 137.5, 150 and 200 μg/mL
Mutant phenotype determination at: 12.5, 25, 50, 75, 100, 112.5, 125, 137.5 and 150 μg/mL

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 12.5, 25, 50, 100, 150, 200, 250, 300 and 400 μg/mL
Mutant phenotype determination at: 12.5, 25, 50, 100 and 150 μg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 6.25, 12.5, 25, 50, 75, 100, 112.5, 125, 137.5, 150 and 200 μg/mL
Mutant phenotype determination at: 6.25, 12.5, 25, 50, 75, 100, 112.5, 125 and 137.5 μg/mL

Experiment 2, 3 h exposure (+S9):
Exposure concentrations: 12.5, 25, 50, 100, 120, 140, 160, 180, 200 and 250 μg/mL
Mutant phenotype determination at: 12.5, 25, 50, 100, 120, 140 and 160 μg/mL

Experiment 3, 24 h exposure (–S9):
Exposure concentrations: 25, 50, 75, 100, 110, 120, 130, 140, 150, 160 and 170 μg/mL
Mutant phenotype determination at: 25, 50, 75, 100 and 110 μg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
Fluctuation in osmolality and pH between treated groups and the vehicle control group should not exceed 50 mOsm/kg and 0.5, respectively. The presence of precipitate should not interfere with mutant phenotype determination. In addition, for a toxic test material, at least 4 analysable concentrations should be achieved which ideally span the toxicity range of 100 to 10-20% harmonised relative survival.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Justification for choice of solvent/vehicle:
DMSO was chosen as a vehicle, because it is compatible with the test system. In a preliminary solubility trial a suitable dilution of WS400104 in DMSO was achieved at 250 mg/mL. This concentration produced test material concentrations in culture medium of 2500 and 5000 µg/mL when administering the DMSO test material dilution to the culture medium at 1% v/v and 2% v/v, respectively. Adequate miscibility and/or solubility of the test material in the compatible vehicle facilitates maximum exposure of the cells in the test system to the test material.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Remarks:

0.15 µg/mL for 3 h treatment, 0.10 µg/mL for 24 h treatment, vehicle DMSO

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Positive control substance for tests without metabolic activation (-S9) in Experiments 1, 2 and 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Remarks:

4 µg/mL, vehicle DMSO

Positive control substance:

cyclophosphamide

Remarks:

Positive control substance for tests with metabolic activation (+S9) in Experiments 1 and 2

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment 1: 3 h exposure with (+S9) and without (–S9) metabolic activation
Experiment 2: 3 h exposure with (+S9) and 24 h exposure without metabolic activation (–S9)
Experiment 3: 24 h exposure without metabolic activation (–S9)

- Selection time: Approximately 3 days after the end of exposure addition of the selection agent trifluorothymidine (TFT)
then allowing approximately 2 weeks for cells to grow with TFT.

SELECTION AGENT: Trifluorothymidine (TFT), at 3 µg/mL in final medium

NUMBER OF REPLICATIONS: 2 cultures at each test concentration, negative control, vehicle control or positive control
- from each test concentration, negative, vehicle or positive control (per culture):
2 flasks for assessment of growth in suspension,
two 96-well plates for assessment of survival,
two 96-well plates for assessment of cloning efficiency (viable clones)
and four 96-well plates for assessment of mutant potential.

NUMBER OF CELLS EVALUATED: 2000 cells/well x 384 wells = 768000 cells per test concentration, negative, vehicle or positive control (per culture)
corresponding to 1536000 cells per test concentration, negative, vehicle or positive control (sum from both cultures).

DETERMINATION OF CYTOTOXICITY: Relative survival corrected with the post treatment cell concentrations;
[in preliminary toxicity test relative survival after treatment (Day 0) and on Days 1 and 2]

Evaluation criteria:

Mutagenicity of the test material was considered to be evident if all of the following criteria were met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle (solvent) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 10^6 viable cells
(Global Evaluation Factor, GEF according to Moore et al. 2006) higher than the corresponding vehicle (solvent) control value.

Reference for GEF:
Moore, M.M., Honma, M., Clements, J., Bolcsfoldi, G., Burlinson, B. Cifone, M., Clarke, J., Delongchamp, R., Durward, R., Fellows, M., Gollapudi, B., Hou, S., Jenkinson, P., Lloyd, M., Majeska, J., Myhr, B., O’Donovan, M, Omori, T, Riach, C., San, R., Stankowski. JR. L.F., Thakur, A.K., Van Goethem, F., Wakuri, S. and Yoshimura, I. (2006). Mouse lymphoma thymidine kinase gene mutation assay: Follow-up meeting of the international workshop on Genotoxicity testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis. 47, 1-5.

Statistics:

Microsoft Excel 2000 software was used for statistical analysis of mutant frequencies (total wells with clones). Log mutant frequency (LMF) of the vehicle control group was compared with the LMF of each treatment group, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore any negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

Preliminary Toxicity Testing: 3 h exposure (–/+S9) and 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not applicable

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment 1, 3 h exposure (–/+S9) & Experiment 2, 3 h exposure (+S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiments 2 & 3, 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
In the present study variations in osmolality and pH between vehicle control and test material treated culture media were within acceptable limits, but cytotoxicity of the test material was a confounding factor. Precipitate in test material treated medium was only evident in the preliminary toxicity test, but this did not affect the selection of test concentrations for the subsequent main assays.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

negative without and with metabolic activation (-/+S9)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative results attained in all in vitro genotoxicity studies, WS400104 is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].