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Toxicological information

Genetic toxicity: in vitro

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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

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 / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
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.
Controlsopen allclose all
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
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
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
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
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 resultsopen allclose all
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
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

 7STLP

 9 P

 no 

 15P

 15P

 no

 5000

 14**

 16**

 no

 2TLP

 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

 97P

 no

 9P

 11P

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
 108P
 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.

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