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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to internationally recognised test methods.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Also compliant with Guideline for Screening Mutagenicity Testing of Chemicals Japan
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trioctyl benzene-1,2,4-tricarboxylate
EC Number:
201-877-4
EC Name:
Trioctyl benzene-1,2,4-tricarboxylate
Cas Number:
89-04-3
Molecular formula:
C33H54O6
IUPAC Name:
1,2,4-trioctyl benzene-1,2,4-tricarboxylate
Test material form:
liquid
Specific details on test material used for the study:
- Lot/batch No.: C-120

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:
S9 fraction (mixed induction rat liver preparation)
Test concentrations with justification for top dose:
Without S9 mix 0 313 625 1250 2500 &5000 ug/plate
With S9 mix 0 313 625 1250 2500 & 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Substance is soluble in acetone.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
TA100, TA98 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
WP2 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48h at 37 degrees C
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: plates/test: 3

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not observed at doses of up to 5000 ug/plate with or without S9

Evaluation criteria:
An increase in colony count of more than 2 fold when compared with concurrent controls and/or a dose dependent increase in the colony count which is statistically significantly higher than the count for the controls.
Statistics:
No statistical analyses were done.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: N/A
- Precipitation: No
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES: yes range of doses tested 1.22-5000ug/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity was not observed at 313-5000 ug/plate
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 - Results of reverse mutation test I

With (+) or

Dose

(µg/plate)

Number of revertants Mean (±SD)

without(-)

Base-pair substitution type

Frameshift type

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

 

0

105 (±3)

15 (±5)

34 (±4)

23 (±4)

9 (±2)

 

313

106 (±6)

17 (±4)

33 (±7)

23 (±4)

10 (±2)

S9 mix

625

113 (±7)

20 (±5)

38 (±2)

25 (±4)

8 (±1)

(-)

1250

124 (±16)

19 (±3)

38 (±6)

28 (±5)

10 (±1)

 

2500

123 (±12)

21 (±2)

36 (±6)

26 (±2)

8 (±2)

 

5000

116 (±9)

23 (±1)

36 (±3)

25 (±8)

8 (±3)

 

0

107 (±8)

16 (±1)

29 (±5)

35 (±3)

11 (±3)

 

313

114 (±8)

17 (±2)

35 (±2)

31 (±3)

13 (±3)

S9 mix

625

115 (±12)

20 (±2)

42 (±2)

35 (±2)

11 (±1)

(+)

1250

116 (±10)

20 (±1)

43 (±5)

39 (±3)

12 (±0)

 

2500

125 (±18)

21 (±3)

34 (±3)

36 (±3)

11 (±1)

 

5000

131 (±24)

21 (±3)

37 (±3)

39 (±3)

12 (±2)

+ve control

Chemical

AF-2

SA

ENNG

AF-2

9-AA

S9 mix(-)

Doseµg/plate

0.01

0.5

2

0.1

80

 

Colonies/plate

514 (±14)

441 (±30)

833 (± 42)

549 (±40)

313 (±6)

+ve control

Chemical

2-AA

2-AA

2-AA

2-AA

2-AA

S9 mix(+)

Doseµg/plate

1

2

10

0.5

2

 

Colonies/plate

1301 (±82)

228 (±3)

1092 (±28)

461 (±10)

189 (±13)

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SA = sodium azide; ENNG = N-ethyl-n’-nitro-N-nitrosoguanidine;  9-AA = 9-aminoacridine; 2 -AA = 2 -aminoanthracene

 

Table 2 - Results of reverse mutation test II

With (+) or

Dose

(µg/plate)

Number of revertants Mean (±SD)

without(-)

Base-pair substitution type

Frameshift type

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

 

0

151 (±5)

11 (±2)

33 (±2)

20 (±3)

8 (±2)

 

313

130 (±6)

10 (±1)

41 (±5)

18 (±2)

8 (±1)

S9 mix

625

135 (±3)

10 (±1)

39 (±2)

21 (±5)

7 (±2)

(-)

1250

135 (±13)

10 (±3)

39 (±7)

17 (±2)

9 (±3)

 

2500

142 (±6)

10 (±1)

42 (±1)

19 (±2)

11 (±1)

 

5000

140 (±20)

9 (±1)

41 (±1)

18 (±1)

8 (±1)

 

0

111 (±6)

10 (±1)

40 (±3)

28 (±2)

17 (±2)

 

313

135 (±13)

10 (±2)

48 (±3)

26 (±3)

13 (±2)

S9 mix

625

151 (±17)

10 (±1)

45 (±8)

29 (±5)

16 (±3)

(+)

1250

135 (±24)

12 (±1)

45 (±7)

30 (±4)

13 (±4)

 

2500

146 (±8)

10 (±1)

41 (±4)

31 (±10)

16 (±4)

 

5000

140 (±5)

12 (±2)

39 (±2)

29 (±3)

13 (±3)

+ve control

Chemical

AF-2

SA

ENNG

AF-2

9-AA

S9 mix(-)

Doseµg/plate

0.01

0.5

2

0.1

80

 

Colonies/plate

633 (±27)

485 (±8)

929 (±39)

531 (±53)

436 (±15)

+ve control

Chemical

2-AA

2-AA

2-AA

2-AA

2-AA

S9 mix(+)

Doseµg/plate

1

2

10

0.5

2

 

Colonies/plate

1230 (±38)

250 (±2)

1305 (±68)

510 (±19)

190 (±27)

 

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SA = sodium azide; ENNG = N-ethyl-n’-nitro-N-nitrosoguanidine;  9-AA = 9-aminoacridine; 2 -AA = 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on results under the conditions of this test the substance was not considered to be mutagenic to Salmonella typhimurium TA100, TA 1535, TA98, TA1537 & E coli WP2 uvrA.
Executive summary:

A bacterial reverse mutation assay (Ames test) has been undertaken following OECD test methods.

The substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli.