Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in all strains tested (OECD TG 471)

Cytogenicity in mammalian cells: negative in Chinese hamster ovary (CHO) cells (OECD TG 473)

Mutagenicity in mammalian cells: read across from analogous substance Crude Tall Oil: negative in L5178Y mouse lymphoma cells (OECD TG 476)

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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.
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.
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'.

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

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.
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:
2001-04-17 to 2001-12-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD guideline 473, and in compliance with GLP.
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
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: basic medium (Ham's F-10) containing HEPES buffer, supplemented with antibiotic minocycline.

- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver enzymes (S9 mix)
Test concentrations with justification for top dose:
Experiment 1 without S9 mix: 9 dose levels up to 5000 μg/ml.
Experiment 1 with S9 mix: 10, 20, 40, 60 and 80 μg/ml.
Experiment 2 without S9 mix: 50, 55, 60, 62.5, 65, 67.5 and 70 μg/ml.
Experiment 2 with S9 mix: 5, 10, 20, 30 , 40, 50 and 60 μg/ml.
The presence of S9 mix increased the cytotoxicity of the test substance and the test had to be repeated with more suitable concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item was freely soluble in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix Migrated to IUCLID6: 10-40 mg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix Migrated to IUCLID6: 20-50 μg/ml
Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension.


DURATION

- Preincubation period: 20h, see Table 1.

- Exposure duration: 6h (Experiment 1); 22h (Experiment 2), see Table 2.

- Expression time (cells in growth medium): recovery period Experiment 1 = 6-22 h; recovery period Experiment 2 - 22-46 h, see Table 2.

- Fixation time (start of exposure up to fixation or harvest of cells): Experiment 1 - 24 h; Experiment 2 - 48 h , see Table 2.


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: from 2 slides per culture, up to 50 metaphase cells per slide, a total of 100 metaphase cells per culture were examined.


DETERMINATION OF CYTOTOXICITY

- Method: evidence of metaphase cells and signs of cellular necrosis.


OTHER EXAMINATIONS:

- Determination of polyploidy: yes.

- Determination of endoreplication: yes.

- Other: 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.


OTHER: anueploidy, multiple aberation, double minutes, uncondensed chromosome, reciprocal translocation, robertsonian translocation, pericentric inversion, pulverised chromosome, multiple aberration.
Evaluation criteria:
A negative response was recorded if the results from the item treated cultures were within 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 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 above. The test item was positive if both Experiment 1 and 2 were positive, as described above, or if the second test was positive after the first gave indications of activity. These indications may be suspicious levels of aberrant cells (95-99% confidence limits).
Statistics:
The cells and aberrations were counted manually.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
60 μg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Weakly clastogenic results were recorded at 30μg/L in Experiment 2, however the concentration tested was deemed overtly toxic to the cells.

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: no pH measurements were made because the culture medium did not change colour.

- Effects of osmolality: no

- Evaporation from medium:

- Water solubility: the test item was freely soluble in DMSO.

- Precipitation: slightly cloudy at 78 μg/ml, cloudy between 156 and 5000 μg/ml

- Other confounding effects: substance was highly cytotoxic with S9 mix.


RANGE-FINDING/SCREENING STUDIES: A range finding study was conducted to examine the change in osmotic pressure and precipitation with addition of the test substance.


COMPARISON WITH HISTORICAL CONTROL DATA: in Experiment 1 all the cultures treated with Tall Oil had levels of structural aberrations within the 95% confidence limits of the historical negative control data. In Experiment 2 in the absence of S9 mix all culture conformed with historical data, in the presence of S9 mix


ADDITIONAL INFORMATION ON CYTOTOXICITY: in Experiment 1 none of the concentrations assessed for aberrations were deemed toxic to the cells. In Experiment 2 cultures treated with 30 μg/ml had <50% reduced cell count.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 3a. Result of chromosome analysis Experiment 1 with activation (per 200 cells scored from 2 replicates, except for positive control where 100 cells were scored from 1 plate), 6 h treatment, 24 h harvest.

   Solvent Control (1%)*  Positive control 40 μg/ml Positive Control 50 μg/ml  Low dose 10 μg/ml Medium dose 20 μg/ml   High dose 40 μg/ml  
 Cytotoxicity    N  N  N  N  N  N
 Chromatid aberrations        gaps  0  0  0  0  0  0
 deletions  0  1  7  2  0  0
 fragment  0  0  3  0  1  0
 Chromosome        gaps  0  0  0  0  0  0
 deletions  0  0  0  0  0  0
 fragment  0  0  0  0  0
Complex  exchange  0  2  7  0  0  1
Aberrant Cell Frequency  excluding gaps (%)  0  1.5 6.5  0.5  0.5  0.5
 Aneuplody

 (% of cells

with)

 0  0  1.5  0  0  0
Endoreplication  0  0  0  0  0  5.5
 Polyploidy  2  0.5  0.5  0.5  0  0

Table 3b. Result of chromosome analysis Experiment 1 without activation (per 200 cells scored from 2 replicates, except for positive control where 100 cells were scored from 1 plate), 6 h treatment, 24 h harvest.

 

 

 Solvent Control

(1%)*

 Positive control

20 μg/ml

Positive Control

30 μg/ml

 Low dose

20 μg/ml

Medium dose

39 μg/ml  

High dose

78 μg/ml  

 Cytotoxicity

 

 N

 N.R.

 N.R.

 N

 N

 N

 Chromatid aberrations      

 gaps

 0

 1

 1

 0

 0

 1

 deletions

 0

 0

 8

 0

 0

 0

 fragment

 0

 0

 0

 0

 0

 0

 Chromosome      

 gaps

 0

 0

 0

 0

 0

 0

 deletions

 0

 0

 0

 0

 0

 0

 fragment

 0

 0

 

 0

 0

 0

Complex

 exchange

 0

 1

 5

 0

 0

 0

Aberrant Cell Frequency

 excluding gaps (%)

 0

 0.5

6

 0

 0

 0

 Aneuplody

 (% of cells

with)

 0

 0

 0

 0

 0

 0.5

Endoreplication

 0

 0

 0

 0

 0

 0

 Polyploidy

 0.5

 0.5

 0

 0.5

 0.5

 0

Table 3c. Result of chromosome analysis Experiment 2 with activation (per 200 cells scored from 2 replicates, except for positive control where 100 cells were scored from 1 plate), 6 h treatment, 24 h harvest.

 

 

 Solvent Control (1%)*

 Positive control 20 μg/ml

Positive Control 30 μg/ml

 Low dose 10 μg/ml

Medium dose 20 μg/ml  

High dose 30 μg/ml  

 Cytotoxicity

 

 N

 

 

 

 

 

 Chromatid aberrations      

 gaps

 0

 3

 5

 0

 1

 5

 deletions

 0

 11

 20

 0

 0

 7

 fragment

 0

 1

 2

 3

 1

 2

 Chromosome      

 gaps

 0

 0

 0

 0

 0

 1

 deletions

 0

 0

 0

 0

 0

 0

 fragment

 0

 0

 

 0

 0

 0

Complex

 exchange

 0

 2

 5

 0

 1

 6

Aberrant Cell Frequency

 excluding gaps (%)

 0

 5

 10

 0.5

 1

 5

 Aneuplody

 (% of cells

with)

 0.5

 0

 0

 0.5

 0.5

 1

Endoreplication

 0

 0

 0

 0

 2

 3.5

 Polyploidy

 0

 0

 0

 1

 1

 1.5

Table 3d. Result of chromosome analysis Experiment 2 without activation (per 200 cells scored from 2 replicates, except for positive control where 100 cells were scored from 1 plate), 22 h treatment, 24 h harvest.

 

 

 Solvent Control

(1%)*

 Positive control

20 μg/ml

Positive Control

30 μg/ml

 Low dose

65 μg/ml

Medium dose

67.5 μg/ml  

High dose

70 μg/ml  

 Cytotoxicity

 

 N

 N.R.

 N.R.

 N

 N

 N

 Chromatid aberrations      

 gaps

 0

 1

 3

 0

 0

 0

 deletions

 0

 2

 15

 0

 0

 1

 fragment

 0

 1

 7

 1

 0

 0

 Chromosome      

 gaps

 0

 0

 0

 0

 0

 0

 deletions

 0

 0

 0

 0

 0

 0

 fragment

 0

 0

 

 0

 0

 0

Complex

 exchange

 0

 2

 11

 0

 0

 0

Aberrant Cell Frequency

 excluding gaps (%)

 0

 2.5

 11.5

 0.5

 0

 0.5

 Aneuplody

 (% of cells

with)

 0.5

 0

 0.5

 0.5

 0

 0

Endoreplication

 0

 0

 0

 0

 0

 0

 Polyploidy

 1

 0

 0

 0

 0.5

 0.5

* = in this test the solvent control is used as a control.

N.R. = not reported.

Conclusions:
Interpretation of results: negative

The test substance was tested in Chinese Hamster Ovary cells (CHO) under GLP to OECD 473 (2000) and was weakly clastogenic only at overtly toxic concentrations. It is the reviewer's opinion that the test material should be considered negative under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 April 2010 to 05 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
Reason / purpose for cross-reference:
read-across: supporting information
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 Migrated to IUCLID6: 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 Migrated to IUCLID6: 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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
Interpretation of results: negative with and without metabolic activation

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)

Genetic toxicity in vivo

Description of key information

No information is available from in vivo studies. In view of the lack of genotoxic effects seen in the in vitro studies, testing in vivo is not required.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reliable studies are available for in vitro mutagenicity to bacteria and cytogenicity to mammalian cells. No studies are available on the mutagenicity of Distilled Tall Oil to mammalian cells, but information for this endpoint is available for the related substance Crude Tall Oil (CAS 8002 -26 -4).

Distilled Tall Oil is derived from Crude Tall Oil by distillation, and so contains only constituents that are also present in Crude Tall Oil (see section 1.4 for more detail on composition and read-across justification). The concentration of components varies between these products, but as no effects were seen in Crude Tall Oil it is justifiable to read-across the mammalian mutagenicity result to Distilled Tall Oil. None of the constituents of CTO and DTO is known to be genotoxic. Functional groups are considered to be of importance when evaluating genetic toxicology, as a number of reactive groups and molecular substructures are associated with mutagenic and carcinogenic properties of chemicals (Benigni and Bossa, 2006). None of the constituents of CTO and DTO contains functional groups that are linked to genotoxicity (Benigni et al 2008). The results of all the studies on Distilled Tall Oil and Crude Tall Oil are in agreement.

BACTERIAL MUTAGENICITY

Distilled Tall Oil (CAS No. 8002-26-4) was shown to be non-mutagenic in a GLP study, conducted according to OECD test guideline 471 (Stevenson, 2001). Mutagenic activity was tested using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and Escherichia coli WP2uvrA. The tests were conducted with and without metabolic activation (Araclor 1254-induced rat liver S9 mix). The test substance was diluted in dimethylsulfoxide, which was also used as the vehicle control. The dose levels used in toxicity testing were 17, 50, 167, 500, 1667 and 5000 µg per plate. Based on the results of toxicity testing, the same concentrations were used in the mutagenicity tests. Independent tests were conducted using these concentrations and bacteria, one using the direct plate method, and the other the pre-incubation method.

Each dose level and strain was tested in triplicate. Precipitation of the test substance was observed in both mutation assays at 5000 µg per plate. Toxicity was observed only in strain TA 1537 in the pre-incubation assay at 1667 and 5000 µg per plate. In the other strains, precipitation at 5000 µg obscured the condition of the background lawns. No mutagenicity was observed in any strain used in the presence or absence of metabolic activation.

 

MAMMALIAN MUTAGENICITY

The related substance Crude Tall Oil, from which Distilled Tall Oil is derived, has been tested for the induction of mutations in mammalian cells according to OECD test guideline 476 and under GLP. The result of this study is negative.

 

Distilled Tall Oil is derived from Crude Tall Oil by distillation, and so contains only constituents that are also present in Crude Tall Oil (see Section 1.4 for more detail on composition and read-across justification). The concentration of components varies between these products, but as no effects were seen in Crude Tall Oil it is justifiable to read-across the mammalian mutagenicity result to Distilled Tall Oil. None of the constituents of CTO and DTO is known to be genotoxic. Functional groups are considered to be of importance when evaluating genetic toxicology, as a number of reactive groups and molecular substructures are associated with mutagenic and carcinogenic properties of chemicals (Benigniand Bossa, 2006). None of the constituents of CTO and DTO contains functional groups that are linked to genotoxicity (Benigni et al 2008). The results of all the studies on Distilled Tall Oil and Crude Tall Oil are in agreement.

 

MAMMALIAN CYTOGENICITY

Distilled Tall Oil (CAS No 8002-26-4) was shown to be non-clastogenic in a GLP chromosomal aberration study in Chinese hamster ovary (CHO) cells, conducted according to OECD test guideline 473 (Murie, 2001) in the presence and absence of metabolic activation (Araclor 1254-induced rat liver S9 mix). The test substance was diluted in dimethylsulfoxide, which was also used as the vehicle control. Two independent tests were conducted, with and without metabolic activation. In the first test, the dose levels selected were 20 - 5000 µg/ml with and without S9. The test in the presence of S9 was repeated at 10 - 80 µg/ml due to toxicity. Toxicity was observed at 156 µg/ml and above without S9 and at 60 µg/ml and above with S9. In the second test, the dose levels selected were 50 - 70 µg/ml without S9 and 5 - 60 µg/ml with S9. Toxicity was observed at

70 µg/ml and above without S9 and at 30 µg/ml and above with S9. The test conditions and results are summarised in Table 5.10.

Table5.10: Conditions and results of in vitro cytogenicity test on Tall Oil

 

Test 1

Test 2

 

With metabolic activaton

Without metabolic activation

With metabolic activaton

Without metabolic activation

Dose

20 to 5000 µg/ml, repeated at 10 to 80 µg/ml

20 to 5000 µg/ml

5 to 60 µg/ml

50 to 70 µg/ml

Toxicity observed at

60 µg/ml and above

156 µg/ml and above

30 µg/ml and above

70 µg/ml and above

Result

negative

negative

positive at 30 µg/ml

negative

Clastogenicity was reported in Test 2 in the presence of S9 at 30 µg/ml. This concentration was judged to be overtly toxic to cells, so this result is regarded as an artefact.

Justification for classification or non-classification

The available information for the substance indicates that when tested in vitro, Distilled Tall Oil does not induce mutations in bacterial cells or chromosome aberrations in mammalian cells. The related substance, Crude Tall Oil (CAS 8002 -26 -4), did not induce mutation in mammalian cells. There is no justification from the in vitro results for testing in vivo. Therefore it is considered that classification for mutagenicity is not required.