Tall oil, potassium salt

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

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.

Additional strain / cell type characteristics:

other: Histidine mutation G46; rfa; uvrB

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.

Additional strain / cell type characteristics:

other: Histidine mutation D3052; rfa; uvrB; pkM101

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.

Additional strain / cell type characteristics:

other: Histidine mutation G46; rfa; uvrB; pkM101

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (derived from rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

Experiment 1
0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of metabolic activation for all strains tested.

Experiment 2
0, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of metabolic activation in strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was not sufficiently soluble in water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: The results of the first experiment were verified by a second, independent experiment. Triplicate repetitions were run for each dose group in each of the two separate experiments that were conducted and for the positive controls. For the vehicle control groups, six-fold repetitions were run.

PERFORMANCE OF THE TEST
- Conditions of cultivation: One day prior to the test, a small amount from each of the frozen bacterial cultures was transferred to nutrient broth. The liquid cultures were incubated in a shaker overnight at 37 °C and then used for the exposure.
- Exposure technique: For each sample the following solutions were combined: 0.1 mL of the overnight culture of the bacteria, 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation), 0.1 mL of the appropriate test or reference material solution and 2 mL of top agar
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
- Colony counting: The plates with less than about 50 revertant colonies, with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
- Determination of the toxicity: The bacterial background of the plates was inspected visually. The following signs of toxicity, if present, were recorded: a reduced bacterial background lawn (mottled instead of homogeneous), microcolonies of bacteria instead of a homogeneous background lawn, no background lawn or clearly reduced numbers of revertant colonies.

Evaluation criteria:

1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response.
5. Statistical analysis of data as determined by UKEMS.

Species / strain:

S. typhimurium, other: TA1535, TA1537,TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions of this study, the test material was non mutagenic to the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2uvrA both in the presence and absence of metabolic activation.

Executive summary:

The mutagenic activity of the test material was investigated in a reverse mutation test in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli WP2uvrA were exposed to the test material in DMSO using the direct plate incorporation method both in the presence and absence of exogenous metabolic activation (S9-mix derived from rat liver). The bacteria were also exposed to vehicle and appropriate positive controls. The concentrations tested were 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. An independent, repeat experiment was conducted with pre-incubation method at 0, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results. Under the conditions of this study, the test substance was non mutagenic to the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA both in the presence and absence of metabolic activation (Wisher, 2019).

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

Study period:

5 June 2001 to 26 September 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cells: The cells were grown as monolayers and have a generation time of approximately 12 hours. The modal chromosome number has been determined for these cells to be 21.
- Type and identity of media: The basic medium (Ham's F-10) containing HEPES buffer, was supplemented with the antibiotic minocycline. For cell growth and treatment in the absence of S9 mix, foetal bovine serum (10 % v/v) was added. The medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment, was serum free.
- Periodically checked for Mycoplasma contamination: yes; tested (in house) on a regular basis.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 enzymes prepared from the livers of adult, male Fischer rats.

Test concentrations with justification for top dose:

Experiment 1 without S9: 20, 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/mL
Experiment 1 with S9: 10, 20, 40, 60, 80 µg/mL
The presence of S9 mix increased the toxicity and therefore the test was repeated with lower concentrations.

Experiment 2 without S9: 50, 55, 60, 62.5, 65, 67.5 and 70 µg/mL
Experiment 2 with S9: 5, 10, 20, 30, 40, 50 and 60 µg/mL

Vehicle / solvent:

- Vehicle: DMSO
- Justification for choice of solvent/vehicle: The test material was freely soluble in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

EXPERIMENTAL PROCEDURE
All experimental procedures, up to harvesting, were conducted using aseptic technique and under amber light.

CELL CULTURES
Cells were trypsinised from stock flasks and resuspended in fresh culture medium at densities of 0.1 x 10E6 or 0.05 x 10E6 cells/mL. These cells, in 5 mL volumes, were dispensed into 25 cm2 tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 hours post treatment respectively. Test cultures were established from the stock flask about 20 hours before testing.

S9 MIX
Aroclor 1254-induced S9 enzymes (supernatant post-mitochondrial fraction obtained after centrifugation at 9000 x gravity) were prepared from the livers of adult, male Fischer rats. S9 was stored, as 2 or 5 mL samples, in sterile plastic tubes immersed in liquid nitrogen (-196 °C). Enzymic activity of each batch of S9 was characterised by testing selected pre-mutagens in an Ames test with S. typhimurium TA 1538.
S9 batches used demonstrated, within each test, a satisfactory clastogenic response in cells treated with cyclophosphamide.

To prepare the S9 mix, Ham's F-10 was added to pre-weighed cofactors:
Nicotinamide adenine dinucleotide phosphate (NADP) disodium salt: 4 mM
Glucose-6-phosphate (G-6-P) disodium salt: 25 mM
This solution was immediately filter-sterilised by passage through a 0.2 µm disposable filter assembly and mixed 9:1 (v/v) with the S9.

TREATMENT OF CULTURES
Tests were conducted both in the presence and absence of S9 mix. Treatments with the test material or vehicle control substances were performed on duplicate cell cultures. Several concentrations of the positive controls were tested using single cultures.
Cultures to be treated in the presence of S9 mix were washed before treatment with serum free medium.
Exposure medium was prepared, immediately before dosing, in sterile containers.
After treatment, cells were washed twice with serum free medium, then full growth medium added, for the recovery period and colcemid treatment. The volume of medium for the recovery period was 5 mL.
Living cultures were examined for evidence of changes to cell morphology, once at the end of the treatment period and again before harvesting of cultures.

HARVESTING OF CULTURES AND SLIDE PREPARATION
Colcemid was added to all cultures at a final concentration of 0.1 µg/mL. Culturing the cells in medium containing colcemid for 2 hours accumulated cells in metaphase.
Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation (approximately 190 x gravity), and treated with hypotonic solution (1 % trisodium citrate) for 15 minutes at room temperature. The cells were then fixed (after sedimentation as before) using 4 mL of freshly prepared fixative (methanol:glacial acetic acid, 3:1). Two further changes (after sedimentation as before) of fixative were made.

Monolayer cells were trypsinised, counted and discarded. This provided a quantitative measure of toxicity.

For both experiments, 3 slides per culture were made. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. The slides were stained with Giemsa, then made permanent by mounting coverslips with DPX mountant.

SLIDE ASSESSMENT
Screen for Toxicity
Slides were examined for evidence of metaphase cells and signs of cellular necrosis.

Chromosomal Aberrations
Based on the toxicity (i.e. cell counts and slide/culture observations) 3 concentration levels were selected for assessment of chromosomal aberrations.
From 2 slides per culture, up to 50 metaphase cells per slide (a total of 100 metaphase cells per culture) were examined.
A Leitz-Dialux 20 microscope was used for this assessment, the magnification used being x 1000 or x 1250 (achieved with x 10 or x 12.5 eyepieces and x 100 objective).

The number of chromosomes in each metaphase cell and all abnormalities was recorded.

Evaluation criteria:

EVALUATION OF RESULTS
Toxicity: from the cell counts, the number of cells recovered per culture was calculated and compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures.

Structural and Numerical Chromosomal Aberrations: 4 parameters were calculated, and judged as negative, suspicious or positive:
-Lesions per cell
-Percentage of aberrant cells including cells with gaps only
-Percentage of aberrant cells excluding cells with gaps only
-Percentage of aneuploid cells
The third parameter is considered the most important in judging the true clastogenicity of a test material.

ACCEPTANCE CRITERIA
The experiments were deemed to be valid because they fulfilled the following criteria:
-There was no evidence of contamination
-Cells in vehicle control cultures had normal growth
-The results of vehicle and positive control cultures were typical
-The test material had 3 acceptable dose levels for assessment.

Interpretation of Toxicity
A dose level was considered toxic if the cell count was reduced to less than 50 % of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.

Interpretation of Clastogenicity
-The results for the test material and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data.
-A negative response was recorded if responses from the test material treated cultures are within the 95 % confidence limits for the historical negative control data.
-The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99 % confidence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate.
-A test was positive if the response in at least one acceptable dose level is significant by the criterion described above.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TOXICITY DATA
In Experiment 1, in the presence of S9 mix, toxicity was noted in the cultures treated with 60 and 80 µg/mL, where there were no cells available for assessment. In the absence of S9 mix (6 hour treatment), where the cultures were treated with concentrations up to 5000 µg/mL, toxicity was noted in the cultures treated with 156 to 5000 µg/mL. There were no cells available for assessment in these cultures. In this test, none of the concentrations assessed for aberrations were deemed toxic to the cells.
In Experiment 2, in the presence of S9 mix, there was a slight shift in toxicity. Toxicity was noted in the cultures treated with 30 to 60 µg/mL, with no metaphase cells available for analysis in the cultures treated with 40 to 60 µg/mL.
In Experiment 1, 40 µg/mL could be assessed for aberrations and was deemed non toxic. In Experiment 2, the cultures treated with 30 µg/mL had reduced cell counts (below 50 % of the vehicle control cultures).
In Experiment 2, in the absence of S9 mix (which had a 22 hour treatment in this test), the cultures were treated with concentrations up to 70 µg/mL. Reduced cell counts were noted in the top concentration tested (70 µg/mL).

CHROMOSOMAL ABERRATIONS
The results are included in Tables 1 to 4.
Cultures treated with the following dose levels were selected for assessment of chromosomal aberrations:
-Experiment 1, with S9 mix: 10, 20 and 40 µg/mL. No toxicity was seen at these dose levels.
-Experiment 1, without S9 mix: 20, 39 and 78 µg/mL. No toxicity was seen at these dose levels.
-Experiment 2, with S9 mix: 10, 20 and 30 µg/mL. Toxicity was seen at the 30 µg/mL dose level; this was evidenced by a reduced cell count (<50 % of that of the vehicle). The culture contained patches of round cells and the slide prepared for evaluation contained very sparse numbers of metaphase cells with a large number of interphase cells. The average index for the two slides scored at this level was 0.10.
-Experiment 2, without S9 mix: 65, 67.5 and 70 µg/mL. Toxicity was seen at the 70 µg/mL dose level; this was evidenced by a reduced cell count (<50 % of that of the vehicle). The average index for the two slides scored at this level was 0.41.

INTERPRETATION
In Experiment 1, all the cultures treated with the test material had levels of structural aberrations within the 95 % confidence limits of the historical negative control data. In this test, however, a toxic concentration could not be assessed in the presence of S9 mix.

In Experiment 2, in the absence of S9 mix, all the cultures had levels of structural aberrations within the 95 % confidence limits of the historical negative control data, even when assessed into the toxic range.
In the presence of S9 mix, where concentrations that were deemed toxic to the cells could be assessed for aberrations, a positive response was noted in the cultures treated with 30 µg/mL.
Due to this positive response at the 24 hour harvest, it was not deemed necessary by the laboratory to assess the slides from the 48 hour harvest (absence of S9 mix) or carry out the extra assessment of polyploidy in 300 metaphase cells.

However, it is judged that the overall result should be interpreted as negative.
The positive response was observed only at a single concentration, which was too toxic: at 30 µg/mL only 10 % of the cells survived to treatment (where generally approximately 50 % is required).
In accordance with the interpretation criteria, a response at a single dose was classified as significant if the percent of aberrant cells was consistently greater than the 99 % confidence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate.
A test was classed as positive if the response in at least one acceptable dose level was significant by the criterion described above.
The aberrant cell frequency at 30 µg/mL was 5 % and the 99 % confidence limit was >4 (i.e. the value was just slightly above). Based on toxicity, the dose level should have been considered to be not acceptable.

CONTROL CULTURES
-The vehicle control cultures had levels of structural and numerical aberrations within the 95 % confidence limits of the historical negative control data.
-The positive control substances, cyclophosphamide in the presence and methyl methanesulphonate in the absence of S9 mix, induced positive frequencies of structural aberrations in at least one concentration level per section. These results demonstrated the sensitivity of the test system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Key

- = Negative aberration frequency

+- = Suspicious aberration frequency

+ = Positive aberration frequency

100 cells were scored per slide

 

Table 1 Aberration Data, Test 1: With S9 Mix, 6 Hour Treatment, 24 Hour Harvest

Treatment Group	Conc. (µg/mL)	Structural Aberrations									Aberration Frequency		Aberrant Cell Frequency				Numerical Aberrations
		Chromatid			Chromosome			Complex			Lesions/Cell		Including Gaps		Excluding Gaps		% of Cells With
		G	B	F	G	B	F	E	D	R		Judge	%	Judge	%	Judge	AE	ER	PP
DMSO	1%	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.00	- -	0 0	- -	0 0	- -	0 0	0 0	4 1
Test Material	10	0 0	2 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.02 0.00	- -	1 0	- -	1 0	- -	0 0	0 0	1 0
	20	0 0	0 0	0 1	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.01	- -	0 1	- -	0 1	- -	0 0	0 0	0 0
	40	0 0	0 0	0 0	0 0	0 0	0 0	0 1	0 0	0 0	0.00 0.02	- -	0 1	- -	0 1	- -	0 0	6 5	0 0
Cyclophosphamide	40 50	0 0	1 7	0 3	0 0	0 0	0 0	2 7	0 0	0 0	0.05 0.24	- +	3 13	- +	3 13	+- +	0 3	0 0	1 1

 

Table 2 Aberration Data, Test 1: Without S9 Mix, 6 Hour Treatment, 24 Hour Harvest

Treatment Group	Conc. (µg/mL)	Structural Aberrations									Aberration Frequency		Aberrant Cell Frequency				Numerical Aberrations
		Chromatid			Chromosome			Complex			Lesions/Cell		Including Gaps		Excluding Gaps		% of Cells With
		G	B	F	G	B	F	E	D	R		Judge	%	Judge	%	Judge	AE	ER	PP
DMSO	1%	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.00	- -	0 0	- -	0 0	- -	0 0	0 0	0 1
Test Material	20	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.00	- -	0 0	- -	0 0	- -	0 0	0 0	0 1
	39	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.00	- -	0 0	- -	0 0	- -	0 0	0 0	0 1
	78	1 0	0 0	0 0	0 0	0 0	0 0	0 1	0 0	0 0	0.01 0.00	- -	1 0	- -	0 0	- -	0 1	0 0	0 0
Methyl Methanesulphonate	20 30	1 1	0 8	0 0	0 0	0 0	0 0	1 5	0 0	0 0	0.03 0.19	- +	2 13	- +	1 12	- +	0 0	0 0	1 0

 

Table 3 Aberration Data, Test 2: With S9 Mix, 6 Hour Treatment, 24 Hour Harvest

Treatment Group	Conc. (µg/mL)	Structural Aberrations									Aberration Frequency		Aberrant Cell Frequency				Numerical Aberrations
		Chromatid			Chromosome			Complex			Lesions/Cell		Including Gaps		Excluding Gaps		% of Cells With
		G	B	F	G	B	F	E	D	R		Judge	%	Judge	%	Judge	AE	ER	PP
DMSO	1%	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.00	- -	0 0	- -	0 0	- -	1 0	0 0	0 0
Test Material	10	0 0	0 0	2 1	0 0	0 0	0 0	0 0	0 0	0 0	0.02 0.01	- -	1 1	- -	1 1	- -	1 0	0 0	0 2
	20	1 0	0 0	0 1	0 0	0 0	0 0	1 0	0 0	0 0	0.03 0.01	- -	2 1	- -	1 1	- -	1 0	2 2	2 0
	30	3 2	5 2	2 0	1 0	0 0	0 0	3 3	0 0	0 0	0.17 0.10	+ +	8 7	+ +	5 5	+ +	1 1	3 4	1 2
Cyclophosphamide	20 30	3 5	11 20	1 2	0 0	0 0	0 0	2 5	0 0	0 0	0.19 0.37	+ +	12 23	+ +	10 20	+ +	0 0	0 0	0 0

 

Table 4 Aberration Data, Test 2: Without S9 Mix, 22 Hour Treatment, 24 Hour Harvest

Treatment Group	Conc. (µg/mL)	Structural Aberrations									Aberration Frequency		Aberrant Cell Frequency				Numerical Aberrations
		Chromatid			Chromosome			Complex			Lesions/Cell		Including Gaps		Excluding Gaps		% of Cells With
		G	B	F	G	B	F	E	D	R		Judge	%	Judge	%	Judge	AE	ER	PP
DMSO	1%	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.00	- -	0 0	- -	0 0	- -	0 1	0 0	1 1
Test Material	65	0 0	0 0	1 0	0 0	0 0	0 0	0 0	0 0	0 0	0.01 0.00	- -	1 0	- -	1 0	- -	0 0	0 0	0 0
	67.5	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.00 0.00	- -	0 0	- -	0 0	- -	0 0	0 0	1 0
	70	0 0	1 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0.01 0.00	- -	1 0	- -	1 0	- -	0 0	0 0	0 1
Methyl Methanesulphonate	20 30	1 3	2 15	1 7	0 0	0 0	0 0	2 11	0 0	0 0	0.08 0.47	+- +	6 2	+- +	5 23	+ +	0 1	0 0	0 0

 

Conclusions:

Under the conditions of this study, the test material was judged to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation.

Executive summary:

The clastogenic potential of the test substance was investigated in vitro in accordance with OECD Guideline 473 and EU Method B.10 under GLP conditions. Chromosomal aberrations assays were performed with duplicate, Chinese hamster ovary (CHO) cell cultures. This study was conducted incorporating two independent tests. Both tests were conducted in the presence and absence of metabolic activation. Cultures, established approximately 20 hours before testing, were treated for 6 hours in the presence, or 6 and 22 hours in the absence of S9 mix. Cultures were harvested at either 24 or 48 hours post treatment. Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used in both tests. The test material was toxic to CHO cells in vitro, in both the presence and absence of S9 mix. In Experiment 1, it was tested to the maximum permitted concentration of 5000 µg/mL in the absence of S9 mix and up to 80 µg/mL in the presence of S9 mix. Toxicity was noted at 60 and 80 µg/mL in the presence of S9 mix and at 156 to 5000 µg/mL in the absence of S9 mix. In Experiment 2, it was tested up to 60 µg/mL in the presence of S9 mix and up to 70 µg/mL in the absence of S9 mix. Toxicity was noted in cultures treated with 30 to 60 µg/mL (presence of S9 mix) and in cultures treated with 70 µg/mL (absence of S9 mix). The test material did not cause chromosome aberrations at any of the dose levels assessed, with the exception of the 30 µg/mL dose level in the presence of S9 mix in Experiment 2. However, it was deemed that this concentration was overtly toxic to the cells and therefore the overall result was negative. Under the conditions of this study, the test substance was considered to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation (Murie, 2001).

Reference 3

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

Study period:

9 May 2001 to 17 June 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented, acceptable study according to OECD guideline and GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

see below

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K,) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20. The freeze lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced S-9 activation system

Test concentrations with justification for top dose:

217.5, 435, 870, and 1740 μg/mL

Vehicle / solvent:

Sterile Water

Negative solvent / vehicle controls:

yes

Remarks:

The solvent for the test article (Sterile water) was used as the solvent control at the same concentration as that found in the test article-treated groups

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The test article was soluble in water at a concentration of 17.4 mg/mL, the maximum concentration tested.

For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 10E5 cells/25 cm2 flask and were incubated at 37± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air for 16-24 hours. Treatment was carried out by refeeding duplicate flasks with 4.5 mL complete medium (McCoy's 5A medium supplemented with 10 % FBS, 100 units penicillin and 100 µg streptomycin/mL, and 2 mh4 L-glutamine) for the non-activated study or 4.5 mL S9 reaction mixture for the S9 activated study, to which was added 500 µL of dosing solution of test or control article in solvent or solvent alone. The osmolality of the highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.
In the non-activated study, the cells were exposed to the test article for 4 hours or continuously for 20 hours up to the cell harvest at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2, in air (Swierenga et ai., 1991). In the 4 hour exposure group, after the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, re-fed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks returned to the incubator until cell collection.
In the S9 activated study, the cells were exposed for 4 hours at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air (Swierenga et al., 1991). After the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks were returned to the incubator until cell collection.

A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.

Evaluation criteria:

The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (P50.05, Fisher's exact test) relative to the solvent control.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test.
Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity conc: With metabolic activation: None
Cytotoxicity conc: Without metabolic activation: None
Precipitation conc: None
Genotoxic effects: With metabolic activation: negative
Genotoxic effects: Without metabolic activation: negative

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material was judged to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation.

Executive summary:

The clastogenic potential of the test substance was investigated in vitro in accordance with the standardised guideline OECD 473 under GLP conditions. Cells were treated with the test material at concentrations of 217.5, 435, 870, and 1740 μg/mL in sterile water. In the non-activated study, the cells were exposed to the test material for 4 hours or continuously for 20 hours up to the cell harvest. In the S9 activated study, the cells were exposed for 4 hours. A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems and appropriate vehicle and positive controls were used. Under the conditions of this study, the test substance was judged to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation (Gudi, 2001).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 April 2010 to 5 July 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % CO₂ in air. RPMI 1640 with 20 % donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Properly maintained: Yes. Stocks of cells are stored at approximately -196 °C in liquid nitrogen.
- Periodically "cleansed" against high spontaneous background: Yes. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours (Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test
0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 500 μg/mL in the absence and presence of metabolic activation

Mutagenicity Test Experiment 1
4 hour exposure: 0, 5, 10, 20, 30, 40, 50, 60 and 70 μg/mL in the absence of metabolic activation
4 hour exposure: 0, 6.25, 12.5, 25, 50, 75, 100, 125 and 150 μg/mL in the presence of metabolic activation

Mutagenicity Test Experiment 2
24 hour exposure: 0, 10, 20, 40, 60, 80, 100, 120 and 160 μg/mL in the absence of metabolic activation
4 hour exposure: 0, 20, 40, 50, 60, 70, 80, 90 and 100 μg/mL in the presence of metabolic activation

Vehicle / solvent:

- Vehicle used: Vehicle (DMSO) treatment groups were used as the vehicle controls.
- Justification for choice of vehicle: Formed a solution suitable for dosing at the required concentration. There was no marked change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

MUTAGENICITY TEST
Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation at eight dose levels of the test material, vehicle and positive controls.
To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.
The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10E6 cells/mL in 10 mL duplicate cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours; therefore 0.3 x 10E6 cells/mL in 10 mL duplicate cultures were established in 25 cm2 tissue culture flasks.
To each culture 2 mL of S9-mix was added if required, 50 μL of the treatment dilutions, 0.2 mL for the positive control and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24 hour exposure group).
The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
At the end of the treatment period for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10E5 cells/mL.

On Day 2 of the experiment, the cells were counted, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (% V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (% RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (% V) data a Relative Total Growth (RTG) value.

PLATE SCORING
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days' incubation at 37 °C with 5 % CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately 0.25 to 0.75 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

Evaluation criteria:

INTERPRETATION OF RESULTS
The normal range for mutant frequency per survivor is 50 to 200 x 10E-6 at the test laboratory. Vehicle control results should ideally be within this range, although minor errors may cause this to be slightly elevated. Experiments where the vehicle control values are markedly >250 x 10E-6 mutant frequency per survivor are not normally acceptable and will be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Both % RSG and RTG values are used either individually or combined to designate the level of toxicity achieved by the test material for any individual dose level. Dose levels that have survival values <10 % are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10E-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance.
Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value, scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria.

Statistics:

The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY TEST
A summary of the results are presented in Tables 1 and 2.

Experiment 1
There was evidence of marked toxicity following exposure to the test material in the presence of metabolic activation, as indicated by the % RSG and RTG values. The expected levels of toxicity were not achieved in the absence of metabolic activation. However, based on the levels of toxicity observed in the preliminary test and the very steep toxicity curve of the test material, it is considered that a slightly higher dose level of 80 μg/mL would have resulted in an excessive level of toxicity.
Therefore, with no evidence of any toxicologically significant increases in mutant frequency in the 24 hour exposure group in the absence of metabolic activation in Experiment 2, where very near optimum levels of toxicity were achieved at a higher dose concentration, it is considered that the test material had been adequately tested. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation due to the very steep toxicity curve of the test material, despite using a narrow dose interval, a dose level that exceeded the usual upper limit of acceptable toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. A slightly elevated mutant frequency was observed at this dose level. However, whilst the GEF was exceeded, the mutant frequency value observed only marginally exceeded the upper acceptable limit for vehicle controls, there was no evidence of any marked increase in absolute numbers of mutant colonies and there was no shift towards small colony formation that would have indicated a clastogenic response.
It was considered that the increase in mutant frequency observed at this dose level was due to a cytotoxic mechanism and, therefore, of no toxicological significance. The heterogeneity observed at this dose level was also considered to be due to the very high level of toxicity. It should also be noted that a marked decrease in viability was also observed at this dose level, indicating that marked residual toxicity had occurred. The excessive levels of toxicity observed at and above 125 μg/mL in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were outside the acceptable range and both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in either the absence or presence of metabolic activation. Precipitate of test material was not observed at any of the dose levels.


Experiment 2
As was seen in the preliminary test, there was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the % RSG and RTG values. The levels of toxicity observed in the 24 hour exposure group in the absence of metabolic activation were very similar to those of the preliminary toxicity test and very near optimum levels of toxicity were achieved.
There was also evidence of a modest decrease in viability (% V) in the absence of metabolic activation indicating that some residual toxicity had also occurred. However, it should be noted that the decrease was only observed at a dose level that had been excluded from the statistical analysis due to excessive toxicity. Once again optimum or near optimum levels of toxicity were not achieved in the presence of metabolic activation due to the very steep toxicity curve of the test material, despite using an even narrower dose range than that of Experiment 1. Due to the nature of the toxicity exhibited by the test material, it was considered that to achieve optimum toxicity in the presence of metabolic activation would be incredibly difficult.
Therefore, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels in either the first or second experiment, including dose levels that induced near optimum levels of toxicity or exceeded the upper limit of acceptable toxicity, it was considered that the test material had been adequately tested. The excessive toxicity observed at 160 μg/mL in the absence of metabolic activation, and at 100 μg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 120 μg/mL in the absence of metabolic activation exceeded the upper acceptable limit of 90 %, therefore, this dose level was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The 24 hour exposure without metabolic activation demonstrated that the extended time point had no effect on the toxicity of the test material.
Neither of the vehicle control mutant frequency values were outside the acceptable range and both the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in either the absence or presence of metabolic activation. Precipitate of test material was not observed at any of the dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Summary of Results - Experiment 1

Dose Level (μg/mL)	4 hours -S9			Dose Level (μg/mL)	4 hours +S9
	% RSG	RTG	MF		% RSG	RTG	MF
0 5* 10* 20 30 40 50 60 70	100 103 100 103 108 105 105 81 69	1.00     1.04 1.00 1.08 0.98 0.82 0.72	89.47     84.23 79.90 87.43 77.52 100.51 82.68	0 6.25 12.5 25 50 75 100** 125* 150*	100 107 113 110 111 78 2 0 0	1.00 0.93 1.03 0.97 0.84 0.63 (<0.01)	91.08 106.59 88.73 120.32 109.32 119.76 (278.35)
Linear Trend - Not Significant				Linear Trend - Not Significant
EMS 400	75	0.52	936.58	CP 2	71	0.23	1294.25

% RSG = Relative suspension growth

RTG = Relative total growth

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

*Not plated for viability or TFT resistance

**Evidence of heterogeneity (poor correlation between A and B cultures) and treatment excluded from the statistics due to toxicity

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

 

Table 2 Summary of Results - Experiment 2

Dose Level (μg/mL)	24 hours -S9			Dose Level (μg/mL)	4 hours +S9
	% RSG	RTG	MF		% RSG	RTG	MF
0 10* 20 40 60 80 100 120† 160*	100 107 104 99 75 55 28 1 0	1.00   0.96 0.97 0.78 0.56 0.24 0.02	93.64   117.64 105.72 105.74 93.50 106.79 162.70	0 20* 40 50 60 70 80 90 100*	100 98 102 100 96 93 76 55 2	1.00   0.98 1.06 0.97 0.87 0.67 0.50	126.36   115.39 98.11 73.88 114.54 142.67 116.35
Linear Trend - Not Significant				Linear Trend - Not Significant
EMS 150	67	0.41	1292.13	CP 2	74	0.52	730.73

% RSG = Relative suspension growth

RTG = Relative total growth

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

*Not plated for viability or TFT resistance

†Treatment excluded from the statistics due to toxicity

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

The mutagenic potential of the test substance was investigated in vitro in accordance with the standardised guidelines OECD 476 and EU Method B.17 under GLP conditions.

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material in two independent experiments. In Experiment 1, the cells were treated with the test material at up to eight dose levels, in duplicate, together with vehicle and positive controls using 4 hour exposure periods both in the absence and presence of 2 % S9 metabolic activation (5 to 70 μg/mL in the absence of metabolic activation, 6.25 to 150 μg/mL in the presence of metabolic activation). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4 hour exposure period in the presence of 1 % S9 metabolic activation and a 24 hour exposure period in the absence of metabolic activation (10 to 160 μg/mL in the absence of metabolic activation, 20 to 100 μg/mL in the presence of metabolic activation). The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The vehicle controls had acceptable mutant frequency values and the positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Under the conditions of this study, the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2010).

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 April 2010 to 2 June 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The recommendations of the "International Workshop on Genotoxicity Tests Workgroup" (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of growth media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10 % (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: Yes. Stocks of cells are stored at -196 °C in liquid nitrogen. The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10⁶ cells/mL.
- Periodically "cleansed" against high spontaneous background: Yes. The cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period, cells were returned to R10 medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test
0, 33, 100, 333, 666 and 1011 μg/mL in the absence and presence of metabolic activation

Mutagenicity Test Experiment 1
3 hour exposure: 0, 1, 3, 10, 33, 100, 333, 666 and 1011 μg/mL in the absence and presence of metabolic activation

Mutagenicity Test Experiment 2
24 hour exposure: 0, 1, 3, 10, 33, 100, 333, 666 and 1011 μg/mL in the absence of metabolic activation
3 hour exposure: 0, 1, 3, 10, 33, 100, 333, 666 and 1011 μg/mL in the presence of metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium
The test material was dissolved in RPMI 1640 (Hepes buffered medium (Dutch modification) and filter (0.22 µm)-sterilised. Test material concentrations were used within 50 minutes of preparation. The pH and osmolarity of a concentration of 1011 µg/mL were 7.37 and 0.315 Osm/kg respectively (compared to 7.39 and 0.300 Osm/kg in the solvent control).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment with and without S9-mix: 3 hours
Prolonged treatment period without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days. During this period at least 4 x 10⁶ cells (if possible) were sub-cultured every day in order to maintain log phase growth.
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10⁵ cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent mutant frequency (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test material is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

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:

FIRST MUTAGENICITY TEST
No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.
No significant increase in the mutation frequency at the TK locus was observed either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

SECOND MUTAGENICITY TEST
No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.
No significant increase in the mutation frequency at the TK locus was observed either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Summary of Results - Experiment 1

Dose Level (μg/mL)	3 hours -S9			Dose Level (μg/mL)	3 hours +S9 (8 % v/v)
	% RSG	RTG	MF x 10^-6		% RSG	RTG	MF x 10^-6
0 1 3 10 33 100 333 666 1011	100 103 93 101 105 100 91 91 92	100 101 77 90 97 87 95 83 77	54 56 64 46 38 61 6 53 56	0 1 3 10 33 100 333 666 1011	100 97 97 92 91 90 100 99 94	100 98 92 107 99 119 91 90 102	43 51 77 42 48 52 67 62 49
MMS 15	66	50	881	CP 7.5	52	42	894

% RSG = Relative suspension growth

RTG = Relative total growth

MF = Mutation frequency

MMS = Methylmethanesulphonate

CP = Cyclophosphamide

 

Table 2 Summary of Results - Experiment 2

Dose Level (μg/mL)	24 hours -S9			Dose Level (μg/mL)	3 hours +S9 (12 % v/v)
	% RSG	RTG	MF x 10^-6		% RSG	RTG	MF x 10^-6
0 1 3 10 33 100 333 666 1011	100 116 119 123 128 110 104 113 107	100 116 126 139 111 109 109 96 103	94 85 82 74 78 89 75 98 75	0 1 3 10 33 100 333 666 1011	100 110 103 94 111 101 88 87 93	100 123 101 108 122 97 110 103 85	89 62 94 61 63 76 66 68 103
MMS 5	70	59	803	CP 7.5	47	32	1466

% RSG = Relative suspension growth

RTG = Relative total growth

MF = Mutation frequency

MMS = Methylmethanesulphonate

CP = Cyclophosphamide

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

The mutagenic potential of the test substance to cause gene mutation in mammalian cells was investigated in vitro in accordance with the standardised guidelines OECD 476 and EU Method B.17 under GLP conditions. L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material in two independent experiments. In Experiment 1, the cells were treated with the test material at concentrations up to 1011 µg/mL together with vehicle and positive controls using 3 hour exposure periods both in the absence and presence of 8 % S9 metabolic activation. In Experiment 2, the cells were treated with the test material at concentrations up to 1011 µg/mL together with vehicle and positive controls using a 24 hour exposure period in the absence of metabolic activation and a 3 hour exposure in the presence of 12 % S9 metabolic activation. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The vehicle controls had acceptable mutant frequency values and the positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Under the conditions of this study, the test substance was considered to be non-mutagenic to L5178Y cells (Verspeek-Rip, 2010).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation in bacteria

 

Tall oil K salt

The mutagenic activity of the test material was investigated in a reverse mutation test in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and E.coli WP2uvrA were exposed to the test material in DMSO using the direct plate incorporation method both in the presence and absence of exogenous metabolic activation (S9-mix derived from rat liver). The bacteria were also exposed to vehicle and appropriate positive controls. The concentrations tested were 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. An independent, repeat experiment was conducted with pre-incubation method at 0, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results. Under the conditions of this study, the test substance was non mutagenic to the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA both in the presence and absence of metabolic activation (Wisher, 2019).

 

In vitro mammalian cell cytogenicity

Distilled tall oil

The clastogenic potential of the test substance was investigatedin vitroin accordance with OECD Guideline 473 and EU Method B.10 under GLP conditions. Chromosomal aberrations assays were performed with duplicate, Chinese hamster ovary (CHO) cell cultures. This study was conducted incorporating two independent tests. Both tests were conducted in the presence and absence of metabolic activation. Cultures, established approximately 20 hours before testing, were treated for 6 hours in the presence, or 6 and 22 hours in the absence of S9 mix. Cultures were harvested at either 24 or 48 hours post treatment. Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used in both tests. The test material was toxic to CHO cellsin vitro, in both the presence and absence of S9 mix. In Experiment 1, it was tested to the maximum permitted concentration of 5000 µg/mL in the absence of S9 mix and up to 80 µg/mL in the presence of S9 mix. Toxicity was noted at 60 and 80 µg/mL in the presence of S9 mix and at 156 to 5000 µg/mL in the absence of S9 mix. In Experiment 2, it was tested up to 60 µg/mL in the presence of S9 mix and up to 70 µg/mL in the absence of S9 mix. Toxicity was noted in cultures treated with 30 to 60 µg/mL (presence of S9 mix) and in cultures treated with 70 µg/mL (absence of S9 mix). The test material did not cause chromosome aberrations at any of the dose levels assessed, with the exception of the 30 µg/mL dose level in the presence of S9 mix in Experiment 2. However, it was deemed that this concentration was overtly toxic to the cells and therefore the overall result was negative. Under the conditions of this study, the test substance was considered to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation (Murie, 2001).

Potassium sulfate

The clastogenic potential of the test substance was investigatedin vitroin accordance with the standardised guideline OECD 473 under GLP conditions. Cells were treated with the test material at concentrations of 217.5, 435, 870, and 1740 μg/mL in sterile water. In the non-activated study, the cells were exposed to the test material for 4 hours or continuously for 20 hours up to the cell harvest. In the S9 activated study, the cells were exposed for 4 hours. A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems and appropriate vehicle and positive controls were used. Under the conditions of this study, the test substance was judged to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation (Gudi, 2001).

In vitro mammalian cell gene mutation

Crude tall oil

The mutagenic potential of the test substance was investigatedin vitroin accordance with the standardised guidelines OECD 476 and EU Method B.17 under GLP conditions.

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material in two independent experiments. In Experiment 1, the cells were treated with the test material at up to eight dose levels, in duplicate, together with vehicle and positive controls using 4 hour exposure periods both in the absence and presence of 2 % S9 metabolic activation (5 to 70 μg/mL in the absence of metabolic activation, 6.25 to 150 μg/mL in the presence of metabolic activation). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4 hour exposure period in the presence of 1 % S9 metabolic activation and a 24 hour exposure period in the absence of metabolic activation (10 to 160 μg/mL in the absence of metabolic activation, 20 to 100 μg/mL in the presence of metabolic activation). The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The vehicle controls had acceptable mutant frequency values and the positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Under the conditions of this study, the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2010).

Potassium nitrate

The mutagenic potential of the test substance to cause gene mutation in mammalian cells was investigatedin vitroin accordance with the standardised guidelines OECD 476 and EU Method B.17 under GLP conditions. L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material in two independent experiments. In Experiment 1, the cells were treated with the test material at concentrations up to 1011 µg/mL together with vehicle and positive controls using 3 hour exposure periods both in the absence and presence of 8 % S9 metabolic activation. In Experiment 2, the cells were treated with the test material at concentrations up to 1011 µg/mL together with vehicle and positive controls using a 24 hour exposure period in the absence of metabolic activation and a 3 hour exposure in the presence of 12 % S9 metabolic activation. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The vehicle controls had acceptable mutant frequency values and the positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Under the conditions of this study, the test substance was considered to be non-mutagenic to L5178Y cells (Verspeek-Rip, 2010).

Justification for classification or non-classification

Based on the results of the in vitro mutagenicity and clastogenicity assays with the test substance and th read-across substances, distilled and crude tall oil, the test substance does not warrant require classification for genetic toxicity according to EU CLP Regulation (EC) no. 1272/2008.