Fatty acids, tall-oil, reaction products with triethanolamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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)

Version / remarks:

of 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau, November 24, 2000

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 and TA 100

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: essential amino acid requiring strain

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

Experiment 1: Doses: 0; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate
Experiment 2: Doses: 0; 50; 150; 500; 1500 and 5000 μg/plate

Vehicle / solvent:

Acetone

Untreated negative controls:

other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide; 9-aminoacridine; 2-Nitrofluorene; 4-nitroquinoline-1-oxide.

Remarks:

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

Untreated negative controls:

other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; Benzo[a]pyrene

Remarks:

Positive control substances for tests with metabolic activation (S9 mix)

Details on test system and experimental conditions:

Standard Plate Incorporation Tests were performed in both experiments (Experiments 1 and 2) and both experiments were conducted without and with metabolic activation (S9 mix). Test procedures varied in that the proportion of S9 fraction in the S9 mix was 10% in Experiment 1 whereas 20% in Experiment 2.

In both experiments, precipitate was observed on all plates containing WS400128 at 5000 μg/plate.

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

Without metabolic activation (S9 mix):

Sodium azide (CAS No. 26628-22-8):
- 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100

9-Aminoacridine (CAS No. 90-45-9):
- 50 μg/plate, dissolved in DMSO: - strain: TA 1537

2-Nitrofluorene (CAS No. 607-57-8):
- 2 μg/plate, dissolved in DMSO: - strain: TA 98

4-Nitroquinoline-1-oxide (CAS No. 56-57-5):
- 2 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

With metabolic activation (S9 mix):

2-Aminoanthracene (CAS No. 613-13-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100
- 10 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

Benzo[a]pyrene (CAS No. 50-32-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1537, TA 98

Evaluation criteria:

The test chemical is considered to exhibit mutagenic activity in this assay if the following criteria are met:
A reproducible increase in revertant colony number, (i.e. at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 the concurrent vehicle controls), with some evidence of a positive dose-response relationship. Such positive response in at least one tester strain without or with metabolic activation (S9 mix.) is sufficient for concluding mutagenic activity.

A test substance is considered non-mutagenic in this test if:
Exposure to a test substance does not produce a reproducible increase in revertant colony numbers.

Statistics:

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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All sterility control plates were colony free. Hence the absence of microbial contamination of the S9 mix, buffer and test substance formulation was confirmed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation (S9 mix)

Reference 2

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)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

of 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human, cultured in vitro in whole blood culture

Details on mammalian cell type (if applicable):

- Source of lymphocytes: Human blood collected aseptically from two healthy, non-smoking male donors and pooled.
- Type and identity of media:
RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

EXPERIMENT 1:
Concentrations prepared without and with metabolic activation (S9): 0, 10.08, 16.80, 27.99, 46.66, 77.76, 129.60, 216, 360, 600 and 1000 μg/mL .
Because of low positive control values following metaphase analysis of cultures without S9, an additional test was conducted.
Concentrations prepared without S9 (additional test): 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 μg/mL.
Microscopically examined (metaphase analysis) without S9: 0, 100, 300 and 400 μg/mL.
Microscopically examined (metaphase analysis) with S9: 0, 360, 600 and 1000 μg/mL.

EXPERIMENT 2:
Concentrations prepared without metabolic activation (S9): 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 and 700 μg/mL
Because the appropriate toxicity profile was not achieved without S9, an additional test was conducted.
Concentrations prepared without S9 (additional test): 0, 50, 100, 115, 130, 145, 160, 175, 190, 205, 220, 235 and 250 μg/mL
Concentrations prepared with S9: 0, 125, 250, 500, 750 and 1000 μg/mL
Microscopically examined (metaphase analysis) without S9: 0, 220, 235 and 250 μg/mL
Microscopically examined (metaphase analysis) with S9: 0, 125, 500 and 1000 μg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR METAPHASE ANALYSIS:
The concentration causing a decrease in mitotic index of at least 50% (where possible) of the solvent control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected. Where no decrease in mitotic index was observed, the highest three concentrations tested were selected for metaphase analysis.

Vehicle / solvent:

Ethanol

Justification for choice of solvent/vehicle:
No precipitate was observed at the limit concentration of 5000 µg/mL WS400128 in aqueous tissue culture medium when ethanol was used as a vehicle, thus facilitating exposure of the cultures to quite high test substance concentrations. However, at concentrations > 1000 µg/mL fluctuation in osmolality was > 50 mOsm/kg leading to the choice of 1000 µg/mL as the maximum concentration tested.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C tested at 0.2 μg/mL (3 hour treatment) and 0.1 μg/mL (21 hour continuous treatment), vehicle sterile purified water

Migrated to IUCLID6: without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide tested at 5 μg/mL (3 hour treatment), vehicle sterile purified water

Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

CELL DIVISION STIMULANT:
Phytohaemagglutinin

METHOD OF APPLICATION:
in cell culture medium;

DURATION
- Exposure duration:
3 hours in Experiment 1 (without and with metabolic activation) and in Experiment 2 (with metabolic activation).
21 hours in Experiment 2 (without metabolic activation)
[After the 3 h treatment the cells were cultivated with fresh media for 18 h].
- Final concentration of S9 mix:
Experiment 1: 2 % v/v
Experiment 2: 5 % v/v
- Fixation time (start of exposure up to fixation or harvest of cells):
21 hours in each of both experiments

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® was added to the cultures (0.1 µg/mL culture medium) 19 hours after treatment start.
2 h later, the cells were treated with hypotonic solution (0.075 M KCl) for 10 min at 37 °C. After incubation in the hypotonic solution, the cells were fixed with 3 + 1 methanol + glacial acetic acid.

STAIN (for cytogenetic assays):
After fixation the cells were stained with 10% Giemsa.

NUMBER OF REPLICATIONS:
Duplicate cultures were treated at each concentration.

NUMBER OF CELLS EVALUATED:
100 metaphases per culture, amounting to a total of 200 metaphases per dose concentration, were scored for structural chromosomal aberrations.
This number was reduced In cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) determined by counting the number of mitotic cells in 1000 cells;

Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (including and excluding gaps),
- Number of gaps,
- Types of aberrations
Chromatid break, Chromosome break, Chromatid gap, Chromatid exchange, Chromosome exchange, Chromosome gap,
Others: Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes

Determination of polyploidy:
- Polyploid and endoreduplicated cells were noted when seen.

Evaluation criteria:

An assay is considered to be acceptable if the vehicle and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
-Significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) at one or more test concentration.
-The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
-Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.

Statistics:

One-tailed Fisher exact test (Fisher 1973) for comparison of the number of aberrant metaphase cells in each test substance group with the solvent control value.

In addition, a Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

ARMITAGE, P. (1955) Tests for linear trends in proportions and frequencies. Biometrics, 11, 375-386. (Cochran-Armitage test).
FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In both experiments, following 3 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With metabolic acitviation (S9 at 5% v/v final concentration) and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

At one cytotoxic concentration (250 µg/mL), following 21 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At concentrations > 1000 µg/mL fluctuation in osmolality was > 50 mOsm/kg leading to the choice of 1000 µg/mL as the maximum concentration tested.

Remarks on result:

other: strain/cell type: human lymphocytes with 44- 48 chromosomes

Remarks:

Migrated from field 'Test system'.

 

Table 1:    Experiment 1 Chromosome Aberration and Cytotoxicity Results Based on Duplicate Cultures/Concentration
Treatment period	S9 mix	Nominal concentration of WS400128	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative mitotic index (%)	Polyploidy mean incidence (%)
(hours)	(v/v)	(µg/mL)	Individual values (%)		Mean (%)	Individual values (%)		Mean (%)
3	-	0 (Ethanol)	0.0	1.0	0.5	0.0	1.0	0.5	100	0.0
		100	1.0	1.0	1.0	2.0	1.0	1.5	99	0.0
		300	2.0	0.0	1.0	2.0	0.0	1.0	64	0.5
		400	0.0	1.0	0.5	1.0	2.0	1.5	46	0.5
		0.2 (Mitomycin C)	27.8	19.2	22.7***	30.6	19.2	23.9***	-	0.0
3	+	0 (Ethanol)	2.0	2.0	2.0	2.0	3.0	2.5	100	0.5
	(2%)	360	1.0	1.0	1.0	2.0	3.0	2.5	115	1.5
		600	1.0	2.0	1.5	2.0	2.0	2.0	106	2.5
		1000	1.0	4.0	2.5	3.0	7.0	5.0	118	3.5
		5 (Cyclophosphamide)	19.2	25.6	22.0***	19.2	28.2	23.1***	-	0.0

 

One-tailed Fisher's exact test

***                 p<0.001

Otherwise         p>0.01

 

 

Table 2:    Experiment 2 Chromosome Aberration and Cytotoxicity Results Based on Duplicate Cultures/Concentration
Treatment period	S9 mix	Nominal concentration of WS400128	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative mitotic index (%)	Polyploidy mean incidence (%)
(hours)	(v/v)	(µg/mL)	Individual values (%)		Mean (%)	Individual values (%)		Mean (%)
21	-	0 (Ethanol)	1.0	2.0	1.5	2.0	2.0	2.0	100	0.5
		220	3.0	3.0	3.0	5.0	6.0	5.5	124	0.5
		235	3.0	5.0	4.0	6.0	6.0	6.0	78	2.5
		250	5.0	7.0	6.0**	5.0	9.0	7.0**	52	1.0
		0.1 (Mitomycin C)	13.2	17.2	14.9***	17.1	22.4	19.4***	-	0.0
3	+	0 (Ethanol)	1.0	0.0	0.5	2.0	0.0	1.0	100	0.0
	(5%)	125	0.0	0.0	0.0	0.0	0.0	0.0	92	0.5
		500	3.0	0.0	1.5	4.0	0.0	2.0	76	1.0
		1000	3.0	1.0	2.0	4.0	1.0	2.5	52	0.0
		5 (Cyclophosphamide)	21.7	4.0	9.6***	21.7	5.0	10.3***	-	0.0

 

One-tailed Fisher's exact test

***                  p<0.001

**                   p<0.01

Otherwise         p>0.01

 

Conclusions:

Interpretation of results (migrated information):
negative 3 h treatment without and with metabolic activation (S9)
ambiguous without metabolic activation 21 h treatment

The ambiguous result after 21 hour exposure without metabolic activation is considered as artifactual result. Based on the very low water solubility of the test substance it is to be assumed that cells were not exposed to true solutions of the substance but to dispersions. It is known that exposure of cells to precipitates or dispersions, i.e. concentrated test substance, can lead to artifactual, false positive results.

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:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

of 1998

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

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.

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:
Medium R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v, whereby medium R0 is RPMI 1640,
buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
- 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 Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TESTING (suspension growth 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):
6.35, 12.7, 25.39, 50.78, 101.56, 203.13, 406.25, 812.5, 1625 and 3250 µg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 50, 100, 200, 400, 450, 500, 550, 600, 650, 700, 750 and 800 µg/mL
Mutant phenotype determination at: 100, 400, 450, 500 and 550 µg/mL

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 25, 50, 100, 150, 200, 250, 300, 350 and 400 µg/mL
Mutant phenotype determination at: 25, 100, 200, 300, 350 and 400 µg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 50, 200, 300, 400, 450, 500, 550, 600, 650, 700, 750 and 800 µg/mL
Mutant phenotype determination at: 200, 300, 400, 450 and 500 µg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (i.e. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Vehicle / solvent:

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

Justification for choice of solvent/vehicle:
Ethanol was chosen as a vehicle to maximise exposure of cultures in the test system to WS400128. No precipitate was observed at the limit concentration of 5000 µg/mL WS400128 in aqueous tissue culture medium when ethanol was used as a vehicle, thus facilitating exposure of the cultures to quite high test substance concentrations. At this concentration, the pH of the medium was also acceptably similar to that of vehicle control medium. However, at 5000 µg/mL fluctuation in osmolality was > 50 mOsm/kg compared with the vehicle control. At 3250 µg/mL fluctuation in osmolality was within accepptable limits leading to the choice of 3250 µg/mL as the maximum concentration tested in the preliminary toxicity test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

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

Migrated to IUCLID6: 3h exposure: 10 µg/mL; 24 h exposure: 5 µg/mL, vehicle DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

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

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/mL, vehicle DMSO

Positive control substance:

benzo(a)pyrene

Remarks:

Positive control substance for tests with metabolic activation (+S9) in Experiment 1

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: 24 h exposure without metabolic activation (–S9)

- Selection time: At 48 h after the end of exposure addition of the selection agent trifluorothymidine (TFT)
then allowing 10-14 days for cells to grow with TFT.

SELECTION AGENT: Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 cultures at each concentration,
[from each culture two vials for assessment of growth in suspension, two 96-well plates for assessment of cloning efficiency
and two 96-well plates for assessment of mutant potential].

NUMBER OF CELLS EVALUATED: 2000 cell/well x 192 wells = 384000 cells per culture

DETERMINATION OF CYTOTOXICITY: Relative total growth

Evaluation criteria:

The mutation test result was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF = 126 x 10^–6, Moore et al. 2006, detailed reference see below).

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied: If the linear trend test was negative, the result was regarded as negative. If the linear trend test was positive, this indicated a positive, biologically relevant response.

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:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. 1989.
Robinson, W.D., Green, M.H.L., Cole, J., Healy, M.J.R., Garner, R.C., and Gatehouse, D. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: Kirkland, D. J. (Ed). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part 111. Statistical Evaluation of Mutagenicity Test Data, p.102-140. Cambridge University Press, Cambridge.

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

Remarks:

Detailed in Table 1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment 1, 3 h exposure (–/+S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Detailed in Table 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment 2, 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Detailed in Table 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

At concentrations > 3250 µg/mL fluctuation in osmolality was > 50 mOsm/kg leading to the choice of 3250 µg/mL as the maximum concentration in the preliminary toxicity test. The concentrations chosen for mutagenicity testing In both main mutation experiments (Experiments 1 and 2) were considerably lower than 3250 µg/mL because of cytotoxicity (detailed in Table 2).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 

Table 1: Precipitation and Cytotoxicity Expressed as Relative Suspension Growth (RSG) in the Preliminary Toxicity Test
3 h exposure (–S9)		3 h exposure (+S9)		24 h exposure (–S9)
Treatment / Concentration (µg/mL)	RSG (%)	Treatment / Concentration (µg/mL)	RSG (%)	Treatment / Concentration (µg/mL)	RSG (%)
Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100
WS400128 (6.35)	75	WS400128 (6.35)	106	WS400128 (6.35)	96
WS400128 (12.7)	88	WS400128 (12.7)	83	WS400128 (12.7)	90
WS400128 (25.39)	68	WS400128 (25.39)	90	WS400128 (25.39)	98
WS400128 (50.78)	80	WS400128 (50.78)	105	WS400128 (50.78)	100
WS400128 (101.56)	120	WS400128 (101.56)	70	WS400128    (101.56)(p)	101
WS400128    (203.13)(p)	61	WS400128 (203.13)	19	WS400128    (203.13)(p)	86
WS400128    (406.25)(p)	55	WS400128    (406.25)(p)	2	WS400128    (406.25)(p)	40
WS400128     (812.5)(p)	1	WS400128     (812.5)(p)	0	WS400128     (812.5)(p)	1
WS400128     (1625)(p)	0	WS400128     (1625)(p)	0	WS400128     (1625)(p)	0
WS400128    (3250)(p)	0	WS400128    (3250)(p)	0	WS400128    (3250)(p)	0

 

(p):  Precipitate observed by eye at the end of treatment  

Table 2: Precipitation and Cytotoxicity Expressed as Mean Relative Total Growth (RTG) in Both Main Mutation Experiments
Experiment 1, 3 h exposure (–S9)		Experiment 1, 3 h exposure (+S9)		Experiment 2, 24 h exposure (–S9)
Treatment / Concentration (µg/mL)	RTG (%)	Treatment / Concentration (µg/mL)	RTG (%)	Treatment / Concentration (µg/mL)	RTG (%)
Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100
		WS400128 (25)	125
WS400128 (100)	105	WS400128 (100)	94
		WS400128 (200)	71	WS400128 (200)	133
		WS400128     (300)(p)	41	WS400128    (300)(p)	80
		WS400128     (350)(p)	39
WS400128 (400)	58	WS400128     (400)(p)	18	WS400128    (400)(p)	33
WS400128     (450)(p)	52			WS400128    (450)(p)	23
WS400128     (500)(p)	20			WS400128    (500)(p)	15
WS400128    (550)(p)	16

 

(p):  Precipitate observed by eye at the end of treatment

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation (-/+S9)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2014

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WS400128, batch no. 0001591644
- Expiration date of the lot/batch: February 2020
- Purity test date: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

OTHER SPECIFICS:

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd
- Age at study initiation: ca. 41-48 days old
- Weight at study initiation: 29-34 g
- Assigned to test groups randomly: yes
- Housing: group housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 0, 50, 100, 200 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: mixing of WS400128 with corn oil

Duration of treatment / exposure:

The test item was administered on two ccasions approx. 24 hours apart.

Frequency of treatment:

two times

Post exposure period:

18-24 hours after the second dose

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 males per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

bone marrow smears previously prepared from mice administered Mitomycin C (12 mg/kg bw/day) were stained and coded along with the bone marrow smears prepared in the study summarised .

Tissues and cell types examined:

bone marrow from femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: preliminary study at max. dose of 2000 mg/kg/day in male and female mice

DETAILS OF SLIDE PREPARATION:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature in the dark until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water

METHOD OF ANALYSIS: fluorescence microscopy

Evaluation criteria:

Acceptance Criteria
The following criteria were applied for assessment of assay acceptability:
The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
Concurrent positive control or scoring controls should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
A maximum tolerated dose or maximum feasible dose has been achieved.
Adequate number of cells and doses have been analysed.
Each treated and control group should include at least 5 analysable animals.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative, if in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) There is no dose-related increase at any sampling time when evaluated by an appropriate trend test,
c) All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and d) Bone marrow exposure to the test item(s) occurred.
Evidence of exposure of the bone marrow to a test item may include a depression of the polychromatic to normochromatic erythrocyte ratio or measurement of the plasma or blood levels of the test item. In case of intravenous administration, evidence of exposure is not needed. Alternatively, ADME data, obtained in an independent study using the same route and same species can be used to demonstrate bone marrow exposure. Negative results indicate that, under the test conditions, the test chemical does not produce micronuclei in the polychromatic erythrocytes of the test species.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see attached "Table 2 Individual animal data" for details of results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In the in vitro chromosomal aberration test the ambiguous result after 21 hour exposure without metabolic activation is considered as artifactual result. Based on the very low water solubility of the test substance it is to be assumed that cells were not exposed to true solutions of the substance but to dispersions. It is known that exposure of cells to precipitates or dispersions, i.e. concentrated test substance, can lead to artifactual, false positive results.

In the in vivo micronucleus test in mice induction of chromosomal aberration was not detected up to and including the highest dose of 2000 mg/kg/day.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There were no statistically significant increases in three in vitro and one in vivo genetic toxicity studies. Therefore, the attained results do not necessitate any labelling regarding mutagenicity according to EU classification rules [REGULATION (EC) 1272/2008].