Tall oil, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro
Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from analogous substance Crude Tall Oil CAS 8002-26-4: negative with and without activation in all strains tested (OECD TG 471)

Cytogenicity in mammalian cells: read-across from analogous substance Crude Tall Oil CAS 8002-26-4: negative in cultured human lymphocytes (OECD TG 473)

Mutagenicity in mammalian cells: read-across from analogous substance Crude Tall Oil CAS 8002-26-4: negative in L5178Y cells (OECD TG 476)

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

Study period:

2005-08-22 to 2005-09-15

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

Species / strain / cell type:

S. typhimurium, other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

see Table 1

Vehicle / solvent:

- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: DMSO is a common vehicle for the Ames test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine

Remarks:

TA97a without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

TA97a with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA98, TA100 and TA1535 with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: t-Butyl-hydroperoxide

Remarks:

TA102 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxy-anthraquinone

Remarks:

TA102 with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 2 days


NUMBER OF REPLICATIONS: triplicate for each dose group, six replicates for the solvent controls and three replicates for positive control.



DETERMINATION OF CYTOTOXICITY

- Method: other: reduced or no bacterial background lawn, microcolonies of bacteria instead of a homogenous background lawn, clearly reduced number of revertant colonies.

Evaluation criteria:

A result would be considered positive if there was a reproducible increase in the number of revertants to more than 2.5 fold for strains TA98 and TA1538 and more than 1.33 fold for TA97a, TA100 and TA102.

Statistics:

The number of revertant colonies was counted by hand in strains TA98 and TA1535 and by a computer program in strains TA97a, TA100 and TA102. The mean and standard deviation of replicate results were calculated.

Key result

Species / strain:

other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

185 μg/plate to strain TA97a

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: A precipitate was visible at 556 μg/plate samples and above. The precipitate was still visible at 5000 μg/plate when the colonies were counted but did not impede the counting.

- Other confounding effects: the substance is known to be ready biodegradable.

RANGE-FINDING/SCREENING STUDIES: see Table 2


COMPARISON WITH HISTORICAL CONTROL DATA: numbers of spontaneous revertants are comparable with the historic control data for the negative controls.

Table 2: Dose range-finding study. Number of revertants per plate (average of 2 plates).

	TA 100
Concentration (μg/Plate)	- MA	Cytotoxic (Yes/No)
21	93	No
62	93	No
185	81	No
556	58	No
1667	59	No
5000	75	No

 

 

Table 3: Experiment 1 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates in solvent control, mean of 3 plates in the other exposure).

	TA97a			TA98			TA100
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
5000	-	-	Yes	6.0	9.0	No	42.7	46.3	No
1667	0.0	9.7	Yes	8.7	9.3	No	44.3	48.0	No
556	0.0	54.7	Yes	6.3	8.0	No	52.0	56.3	No
185	67.7	110.3	No	11.0	11.0	No	66.3	69.7	No
62	119.3	120.3	No	9.3	9.3	No	66.0	79.0	No
0*	607.0	140.5	No	8.0	12.8	No	73.5	77.0	No
Positive control	374.0	607.0	No	109.7	415.7	No	422.3	1955.0	No

*solvent control with DMSO

 

Table 3 (continued): Experiment 1 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates in the solvent control, mean of 3 plates in the other exposures).

	TA102			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
5000	66.3	127.0	Yes	7.0	4.3	Yes
1667	85.0	152.7	Yes	7.3	7.7	Yes
556	102.7	198.0	No	11.0	10.7	No
185	117.0	244.3	No	22.0	16.3	No
62	170.3	260.7	No	17.7	18.7	No
0*	181.7	247.8	No	20.0	18.2	No
Positive control	487.7	595.0	No	207.0	196.7	No

*solvent control with DMSO

Table 4: Experiment 2 Mutagenicity Assay. Number of revertants per plate) mean of 6 plates per solvent control, mean of 3 plates in other exposures).

	TA97a
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)
556	4.3	8.3	Yes
185	32.7	102.7	No
62	102.3	108.3	No
21	110.0	103.7	No
7	109.3	135.0	No
2.3	118.0	122.7	No
0*	85.8	110.7	No
Positive control	229.7	351.7	No

*solvent control with DMSO

Table 4 (continued): Experiment 2 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates per solvent control, mean of 3 plates in other exposures).

	TA98			TA100			TA100
Conc. (µg/plate)	— MA	+MA	Cytotoxic (yes/no)	—MA	+MA	Cytotoxic (yes/no)	—MA	+MA	Cytotoxic (yes/no)
5000	6.3	7.3	No	54.7	56.0	No	54.7	56.0	No
1667	5.7	6.7	No	57.3	61.3	No	57.3	61.3	No
556	5.3	8.0	No	59.0	68.0	No	59.0	68.0	No
185	5.7	10.7	No	69.0	79.0	No	69.0	79.0	No
62	5.3	11.3	No	69.0	77.0	No	68.0	77.0	No
0*	7.7	11.8	No	86.5	69.5	No	86.5	69.5	No
Positive control	207.3	359.3	No	253.0	1372.0	No	253.0	1372.0	No

*solvent control withDMSO

Table 4 (continued): Experiment 2 Mutagenicity Assay. Number of revertants per plate (mean of 6 plates per solvent control, mean of 3 plates in other exposures).

	TA102			TA1535
Conc. (µg/plate)	—MA	+MA	Cytotoxic (yes/no)	—MA	+MA	Cytotoxic (yes/no)
5000	34.7	85.3	Yes	5.0	4.7	Yes
1667	45.3	100.3	Yes	6.3	7.3	Yes
556	51.3	125.7	Yes	10.7	12.0	No
185	80.0	149.7	No	15.0	17.0	No
62	86.0	166.3	No	183	19.7	No
0*	112.5	147.8	No	15.7	17.7	No
Positive control	344.0	615.0	No	158.0	163.0	No

 

Conclusions:

The test substance did not produce an increase in the number of revertants in S. typhimurium (strains TA97a, TA98, TA100, TA102 and TA1535) when tested under GLP to OECD 471 (2000). The test material was therefore considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I: 0.003, to 5.00 µL/mL (with S9), 0.003 to 0.53 µL/mL (without S9)
Experiment II: 0.025 to 5.00 µL/mL (with S9), .001 to 0.200 µL/mL (without S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours (about 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: two cultures per test concentration, two independent experiments

NUMBER OF CELLS EVALUATED: At least 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

M.I, reduced at 0.114 µL/m (-S9), 22h exp.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item CTO (Crude Tall Oil) CAS 8002-26-4, dissolved in acetone, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphase plates per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 5.0 µL/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 0.019 µL/mL and above in the absence of S9 mix and at 0.099 µL/mL and above in the presence of S9 mix. In Experiment II, precipitation occurred at the end of treatment at 0.037 µL/mL in the absence of S9 mix and at 0.10 µL/mL in the presence of S9 mix. No relevant increase in the osmolarity or pH value was observed (Exp. I: solvent control: 369 mOsm, pH 7.3 versus 372 mOsm and pH 7.2 at 0.53 µL/mL; Exp. II: solvent control: 386 mOsm, pH 7.4 versus 389 mOsm and pH 7.4 at 0.20 µL/mL).
In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. However, in Experiment II after continuous treatment in the absence of S9 mix the mitotic index was clearly reduced to 63.7 % at the highest evaluated concentration. In Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data. A statistically significant increase was observed after treatment with 0.099 µL/mL (3.0 % aberrant cells, excluding gaps). This value is in the range of the laboratory’s historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660 or 770 µg/mL) or CPA (7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Summary of results of the chromosomal aberration study with CTO (Crude Tall Oil) CAS 8002-26-4

Table 1 Experiment 1 Results: 4 h exposure without metabolic activation

Test item concentration in µL/mL	Mitotic indices in % of control	Aberrant cells in %
		incl. gaps*	excl. gaps*	carrying exchanges
Solvent control^1	100.0	0.5	0.5	0.0
Positive control^2	58.4	8.5	7.5^S	0.5
0.011	105.1	2.5	2.5	0.0
0.019^P	100.2	2.0	2.0	0.0
0.099^P^#	91.9	3.3	3.0^S	0.0

 

Table 2 Experiment 1 Results: 4 h exposure with metabolic activation

Test item concentration in µL/mL	Mitotic indices in % of control	Aberrant cells in %
		incl. gaps*	excl. gaps*	carrying exchanges
Solvent control^1	100.0	1.0	1.0	0.0
Positive control^2	59.9	8.0	8.0^S	0.0
0.057	82.8	1.0	1.0	0.0
0.099^P	88.0	1.0	0.5	0.0
1.630^P	104.2	2.5	2.0	0.0

 

 Table 3 Experiment 2 Results: 22 h exposure without metabolic activation

Test item concentration in µL/mL	Mitotic indices in % of control	Aberrant cells in %
		incl. gaps*	excl. gaps*	carrying exchanges
Solvent control^1	100.0	1.5	1.5	0.0
Positive control^2	72.0	11.0	9.5^S	1.0
0.021	102.2	0.0	0.0	0.0
0.037^P	120.7	1.0	1.0	0.0
0.065^P	97.1	1.0	0.5	0.0
0.114^P	63.7	0.5	0.5	0.0

 

Table 4 Experiment 2 Results: 4 h exposure with metabolic activation

Test item concentration in µL/mL	Mitotic indices in % of control	Aberrant cells in %
		incl. gaps*	excl. gaps*	carrying exchanges
Solvent control^1	100.0	0.5	0.5	0.0
Positive control^2	64.3	8.5	8.5^S	1.0
0.025	118.2	1.5	1.5	0.0
0.050^P	98.1	1.0	1.0	0.0
0.100^P	116.6	1.0	1.0	0.0

*  Including cells carrying exchanges

^#   Evaluation of 200 metaphases per culture

^P  Precipitation occurred at the end of treatment

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Acetone 0.5 % (v/v)

²     EMS 770.0 µg/mL

³     EMS 660.0 µg/mL

Conclusions:

Crude tall oil has been tested in a valid study according to OECD TG 473 under GLP. Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro in either the initial experiment or the repeat with longer exposure. Vehicle and positive controls gave expected results.
Therefore, CTO (Crude Tall Oil) CAS 8002-26-4 is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Executive summary:

The test item CTO (Crude Tall Oil) CAS 8002-26-4, dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphase plates were scored for structural chromosomal aberrations.

The highest applied concentration in this study (5.0 µL/mL of the test item) was chosen with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. However, in Experiment II after continuous treatment in the absence of S9 mix the mitotic index was clearly reduced to 63.7 % at the highest evaluated concentration. In Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the absence of S9 mix, one statistically significant increase was observed after treatment with 0.099 µL/mL (3.0 % aberrant cells, excluding gaps). This value is in the range of the laboratory’s historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 April 2010 to 05 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitol/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: 5 to 70 μg/ml (-S9); 6.25 to 150 μg/ml (+S9)
Experiment 2: 10 to 160 μg/ml (-S9); 20 to 100 μg/ml (+S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given in draft report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation, 400 μg/ml (expt. 1); 100 μg/ml (expt. 2)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; 2 μg/ml

Details on test system and experimental conditions:

METABOLIC ACTIVATION: final concentration S9: 2% experiment 1; 1% experiment 2. cofactors used: NADP; Glucose-6-phosphate

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none

- Exposure duration: 4 h (experiment 1 + and - S9, experiment 2 +S9); 24 h (experiment 2 -S9)

- Expression time (cells in growth medium): 2 days

- Selection time (if incubation with a selection agent): 10-14 days



SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: duplicate treatments, experiment repeated

NUMBER OF CELLS EVALUATED: 2000 cells / well evaluated for mutant frequency

DETERMINATION OF CYTOTOXICITY

- Method: relative total growth


OTHER EXAMINATIONS:

- Other: small and large colonies

OTHER: microtitre plates used

Evaluation criteria:

A mutagenic response is a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value, by an amount that is greater than the Global Evaluation Factor of 126 x 10E-06. The increase must be reproducible or part of a dose-related response.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

80 μg/ml (+ 1% S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: no marked change in pH was observed

- Effects of osmolality: osmolality did not increase by more than 50 mOsm

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: results of range finding experiment showed a steep toxicity curve

COMPARISON WITH HISTORICAL CONTROL DATA: control results were within the range of the historical data

Table 1 Preliminary toxicity test

Dose (μg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0*	100	100	100
19.53	103	97	107
39.06	83	93	96
78.13	0	42	38
156.25	0	0	0
312.5	0	0	0
625	0	0	0
1250	0	0	0
2500	0	0	0
5000	0	0	0

* Solvent control with DMSO

 

Table 2 Results of mutagenicity study, experiment 1 (mean of 2 treatments)

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

* Solvent control with DMSO

** Treatment excluded from test statistics due to toxicity

RSG: Relative Suspension Growth

RTG: Relative Total Growth

MF: 5-TFT resistant mutants/10E06 viable cells 2 days after treatment

 

Table 3 Results of mutagenicity study, experiment 2 (mean of 2 treatments)

Concentration (μg/ml)	24 hours -S9			Concentration (μg/ml)	4 hours + S9
	% RSG	RTG	MF		% RSG	RTG	MF
0*	100	1.00	93064	0	100	1.00	126.36
10	107	not plated		20	98	not plated
20	104	0.96	117.64	40	102	0.98	115.39
40	99	0.97	105.72	50	100	1.06	98.11
60	75	0.78	105.74	60	96	0.97	73.88
80	55	0.56	93.50	70	93	0.87	114.54
100	28	0.24	106.79	80	76	0.67	142.67
120**	1	0.02	162.70	90	55	0.50	116.35
160	0	not plated		100	2	not plated
Linear trend	Not significant			Linear trend	Not significant
Positive control	75			Positive control	74	0.52	730.73

* Solvent control with DMSO

** Treatment excluded from test statistics due to toxicity

RSG: Relative Suspension Growth

RTG: Relative Total Growth

MF: 5-TFT resistant mutants/10E06 viable cells 2 days after treatment

Conclusions:

Crude Tall Oil has been tested according to OECD 476 and under GLP. 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 and positive controls produced expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No information is available for Tall Oil Soap, therefore good quality studies for the related substance Crude Tall Oil CAS 8002-26-4 have been read across. Data are available for all the required in vitro studies. The results of all the studies were in agreement.

Read-across justification:

Justification for classification or non-classification

Based on reliable in vitro studies with the analogue substance Crude Tall Oil, Tall Oil Soap does not induce mutations in bacterial or mammalian cells, nor chromosome aberrations in mammalian cells. There is no justification from in vitro results for testing in vivo. Therefore, it is considered that classification for mutagenicity is not required according to Regulation (EC) No 1272/2008.