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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:
1990-09-03 to 1990-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1991

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Phthalic acid, di-n-octyl, n-decyl ester
- Substance type: product
- Physical state: liquid
- Stability under test conditions: not determined
- Storage condition of test material: room temperature in the dark

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced liver S9 mix
Test concentrations with justification for top dose:
Dose range finding test: 5000, 500, 50, 5 µg/plate
Mutation tests: 5000, 1500, 500, 150, 50 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not mentioned
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
buffer
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-Aminoacredine, at 80 µg/plate with TA 1537; N-Ethyl-N'-nitro-N-nitrosoguanidine at 3 µg/plate with TA 100 and at 5 µg/plate with TA 1535; 2-Nitrofluorene at 1 µg/plate with TA 98 and at 2 µg/plate with TA 1538
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
S9 mix
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 0.5 µg/plate with TA 1538 and TA 98; at 1 µg/plate with TA 100; at 2 µg/plate with TA 1535 and TA 1537
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation, two independent tests were performed: Bacterial culture, test substance dissolved in Ethanol, sterile sodium othophosphate buffer (pH 7.4) or S9-mix were given into sterile bijou bottles, histidine deficient agar was added, thoroughly mixed and then overlaid onto minimal agar plates.


DURATION
- Preincubation period: not applicable
- Exposure duration: at least 3 days


NUMBER OF REPLICATIONS: 3 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects were detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn


OTHER EXAMINATIONS:
- Other: A preliminary toxicity test was conducted for range finding purposes
Evaluation criteria:
The test compound is considered to show evidence of mutagenic activity if it induces an at least 2-fold increase in revertant colony numbers compared to solvent control with some evidence of a positive dose-relationship in two seperate experiments with any bacterial strain either in the presence or in the absence of metabolic activation. The test substance is considered to show no evidence of mutagenic activity if it does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain. If the results fail to satisfy the criteria for a clear "positive" or "negative" response the following approach is taken: Repeat test may be performed using modifications (e. g. in dose range). If no clear "positive" response can be obtained the test data may be subjected to statistical analysis to determine the statistical significance of any observed increases.
Statistics:
analysis of variance followed by Student's T test

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not mentioned
- Precipitation: no
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: No cytotoxicity up to the highest concentation of 5000 µg/plate was seen in the tester strains.


COMPARISON WITH HISTORICAL CONTROL DATA: not performed


ADDITIONAL INFORMATION ON CYTOTOXICITY: none
Remarks on result:
other: strain/cell type: Salmonella typhimurium

Any other information on results incl. tables

Table #1: Plate incorporation test #1: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 1535]

[Strain TA 1537]

[Strain TA 1538]

Conc.
[unit]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

0*

10±1.5

 11±3.5

 no

 9±1.2

 9±1.5

 no

 10±0.6

 14±1.2

 no

50

 12±1.5

 11±3.2

no

 9±2.1

 9±2.1

 no

 8± 1.2

 12±0.6

 no

150

 9±3.0

 11±3.2

 no

 9±1.0

 9±1.5

 no

 8±1.7

 9±3.0

 no

500

 9±2.6

 9±1.0

 no

 11±2.3

 12±1.2

 no

 9±3.1

 11±0.6

 no

1500

 9±1.2

 10±2.5

 no

 10±2.5

 12±0.6

 no

 8±1.5

 12±2.3

 no

5000

 7±0.6

 11±1.0

 no

 10±2.1

 10±0.6

 no

 11±2.1

 14±2.5

 no

Positive control

 986±85.7

 70±4.2

 no

  X

 47±3.2

 no

 59±8.5

 56±1.5

 no

*solvent control with Ethanol X = too many colonies to count accurately  

Table #2: Plate incorporation test #1: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 98]

[Strain TA 100]

[-]

Conc.
[unit]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)


0*

 20±3.5

 23±1.5

 no

 98±3.5

 110±6.0

 no

 

 

 

50

 18±2.6

 23±5.0

 no

 82±5.5

 101±2.6

 no

 

 

 

150

 18±2.1

 19±1.5

 no

 84±6.5

 104±5.2

 no

 

 

 

500

 20±3.2

 21±5.0

 no

 96±9.3

 99±1.2

 no

 

 

 

1500

 19±0.6

 21±4.6

 no

 83±6.2

 90±16.6

 no

 

 

 

5000

 20±3.5

 23±5.3

 no

 69±2.1

 104±1.7

 no

 

 

 

Positive control

 64±4.0

 67±11.2

 no

 484±17.5

 324±20.6

 no

 

 

 

*solvent control with Ethanol  

Table #3: Plate incorporation test #2: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 1535]

[Strain TA 1537]

[Strain TA 1538]

Conc.
[unit]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

0*

 5±1.5

 11±4.6

 no

 10±4.0

 14±1.7

 no

 16±3.5

 16±0.0

 no

50

 5±1.5

 12±2.1

 no

 42.1

 6±1.2

 no

 7±1.5

 13±2.1

 no

150

 10±2.5

 12±1.5

 no

 4±2.5

 6±1.5

 no

 14±2.6

 12±2.0

 no

500

 8±1.7

 11±1.5

 no

 9±3.0

 9±1.5

 no

 6±1.5

 11±2.1

 no

1500

 5±1.5

 12±2.3

 no

 4±1.7

 5±0.6

 no

 8±1.5

 12±3.0

 no

5000

 8±4.2

 9±1.0

 no

 8±2.5

 6±2.0

 no

 7±2.0

 16±1.7

 no

Positive control

 329± 108.6

 111±9.5

 no

 2261± 38.3

 57±2.5

 no

 44±4.6

 42±3.0

 no

*solvent control with Ethanol  

Table #4: Plate incorporation test #2: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 98]

[Strain TA 100]

[-]

Conc.
[unit]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)


0*

 21±3.8

 22±4.0

 no

 91±8.0

 99±5.8

 no

 

 

 

50

 19±2.1

 22±2.0

 no

 84±7.6

 87±9.5

 no

 

 

 

150

 22±2.3

 21±4.0

 no

 86±7.2

 84±5.3

 no

 

 

 

500

 22±2.8

 24±3.2

 no

 90±7.0

 98±4.0

 no

 

 

 

1500

 19±2.1

 24±1.5

 no

 86±2.3

 90±14.8

 no

 

 

 

5000

 20±3.1

 19±4.0

 no

 91±1.0

 89±14.7

 no

 

 

 

Positive control

 60±2.1

 87±15.9

 no

 353±12.4

 336±11.5

 no

 

 

 

*solvent control with Ethanol  

Applicant's summary and conclusion

Conclusions:
It was concluded that the target substance 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters showed no evidence of mutagenic activity when tested in this bacterial system.
Executive summary:

It was concluded that 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters showed no evidence of mutagenic activity when tested in this bacterial system.