Tall oil

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-04-17 to 2009-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Tall Oil

- Stability under test conditions: stable

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver (S9 mix)

Test concentrations with justification for top dose:

17, 50 167, 500, 1667 and 5000 μg per plate.

Vehicle / solvent:

- Vehicle/solvent used: DMSO.

Controls

Controls 1

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:

TA 1535, TA 1537, TA 98 , TA 100 and E. coli with activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

E. coli without activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA 100 without activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DSMO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Experiment 1) and preincubation (Experiment 2).


DURATION

- Preincubation period: 20 min at 37 degC

- Exposure duration: 2 or 3 days


NUMBER OF REPLICATIONS: 3 plated per concentration and strain in the mutagenicity test, and one replicate in the toxicity test.


DETERMINATION OF CYTOTOXICITY

- Method: other: thinning of the background lawn of microcolonies and large size of resistant colonies.

Evaluation criteria:

For S. typhimurium strains TA 1535, TA 1537, TA 98 and E.coli at least a doubling of the mean concurrent vehicle control values at some consideration of the test item. For S. typhimurium strain TA 100, a 1.5 fold increase over the control value was considered significant. If the mean colony count on the vehicle control plates was <10, then a value of 10 was assumed and minimum count of 20 was required before a significant mutagenic response was registered.

Statistics:

The number of revertant colonies was counted by hand.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

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

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: A high level of precipitate was visible at 1667 and 5000 μg/plate in both the presence and absence of S9 mix.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity to bacteria was observed although the precipitation at 5000 μg/plate was too dense to assess the condition of background lawn accurately. See table 4 for details.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2a. Experiment 1 - Mutagenicity Assay. Number of revertants per plate (mean of 3 plates).

Strain	TA 1535			TA 1537			TA 98
Conc. (μg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	10	11	no	5	7	no	11	17	no
17	8	14	no	2	9	no	9	20	no
50	9	11	no	4	10	no	12	16	no
167	10	10	no	5	10	no	15	18	no
500	10	9	no	7	5	no	7	17	no
1667	10	9	no	4	5	no	14	15	no
5000	7**	11**	no	3**	4**	no	6**	11**	no
Positive control	325	378	no	2531	258	no	387	716	no

* = solvent control with DMSO

** = with precipitation, precipitation too dense to assess background lawn accurately - assumed to be normal.

Table 2b. Experiment 1 - Mutagenicity Assay. Number of revertants per plate (mean of 3 plates).

Strain	TA 100			WP2uvrA
Conc. (μg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	85	118	no	8	11	no
17	99	110	no	11	10	no
50	109	101	no	8	10	no
167	105	129	no	9	6	no
500	92	100	no	6	9	no
1667	91	100	no	10	15	no
5000	102**	104**	no	6**	5**	no
Positive control	1025	1090	no	355	643	no

* = solvent control with DMSO

** = with precipitation, precipitation too dense to assess background lawn accurately - assumed to be normal.

Table 3a. Experiment 2 - Mutagenicity Assay. Number of revertants per plate (mean of 3 plates).

Strain	TA 1535			TA 1537			TA 98
Conc. (μg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	12	14	no	12	10	no	21	22	no
17	14	20	no	4	10	no	14	23	no
50	11	21	no	4	7	no	13	21	no
167	14	16	no	6	11	no	19	22	no
500	17	10	no	9	11	no	14	18	no
1667	10	11	no	7^STLP	9 ^P	no	15^P	15^P	no
5000	14**	16**	no	2^TLP	5**	no	14**	17**	no
Positive control	417	223	no	219.1	252	no	374	452	no

* = solvent control with DMSO

** = with precipitation, precipitation too dense to assess background lawn accurately - assumed to be normal.

STLP - slightly thin lawn, with precipitation

TLP = thin lawn, precipitation

P = with precipitation

Table 3b. Experiment 2 - Mutagenicity Assay. Number of revertants per plate (mean of 3 plates).

Strain	TA 100			WP2uvrA
Conc. (μg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	102	123	no	16	12	no
17	99	117	no	11	14	no
50	107	136	no	11	14	no
167	101	128	no	12	13	no
500	110	101	no	11	12	no
1667	105	97^P	no	9^P	11^P	no
5000	99**	108**	no	8**	11**	no
Positive control	1142	586	no	355	874	no

* = solvent control with DMSO

** = with precipitation, precipitation too dense to assess background lawn accurately - assumed to be normal.

P = with precipitation.

Table 4. Toxicity test results with strain TA 100.

Concentration (μg/plate)	Cell count - MA	Cell count + MA
0*	119	108
17	107	104
50	123	105
167	125	102
500	97	103
1667	126	108^P
5000	98**	86**

* = solvent control with DMSO

** = precipitation too dense to assess background lawn accurately - assumed to be normal.

P = precipitation

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

The test substance did not produce an increase in the number of revertants in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A when tested under GLP to OECD 471 (2000). The test material was therefore considered to be non-mutagenic under the conditions of this test.