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Genetic toxicity in vitro

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

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  

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.

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:
1990-08-30 to 1990-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
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
- Storage condition of test material: stored in the dark at room temperature
Other: Chemical names: di-n-C8-C10-alkylphthalate; octyl-decylphthalate
Species / strain / cell type:
lymphocytes: obtained from human blood
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 tissue culture medium
- Properly maintained: not applicable
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
other: stable karyotype with 46 chromosomes, stimulated to cell division in vitro by addition of the mitogen PHA (phytohaemogglutinin)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 of Arochlor 1254 induced Sprague-Dawley rats
Test concentrations with justification for top dose:
Range finding: 0.4, 0.8, 1.6, 3.2, 6.3, 12.5, 25, 50, 100, 200 µg/mL
Dose levels selected for metaphase analysis in the main tests: 25, 100 and 200 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test substance was miscible with ethanol at 500 mg/mL, all final concentrations greater than 200 µg/mL were unmiscible with the tissue culture medium. 200 µg/mL was, therefore, regarded as the maxium achievable concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol, 1% in tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 750 µg/mL; dissolved in dimethylsulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol, 1% in tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 15 µg/mL, dissolved in sterile distilled water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cells were suspended at a concentation of 1x10E6 cells/mL in culture medium with addition of 20 % foetal bovine serum and 5 mL aliquots were incubated in multiwell plates at 37°C with 5 % CO2 for approx. 48 hours. The cells were treated with different test substance concentrations or positive controls and for tests with metabolic activation system the rat liver S9 mix were added. Three hours after dosing the cultures containing metabolic activation were resuspended with fresh medium and incubated for further 21 hours. 22 hours after treatment with test item mitotic activity was arrested by addition of colchicine (final concentration 0.25 µL/mL). After 2 hours cells were removed from the culture dishes, incubated under hypotonic conditions for 10 minutes. The cell suspensions were centrifuged and the cell pellets fixed by addition of fresh methanol/glacial (3:1 v/v). Prior to slide preparation the pellets were aspirated through a hypodermic needle, then centrifugated and finally resuspended in a small volume of fixative. The cells suspensions were dropped onto each of five slides where they allowed to air-dry and stained afterwards with Giemsa solution, dried and mounted in DPX. The prepared slights were examined by light microscopy.


DURATION
- Preincubation period: not applicable
- Exposure duration: 3 h in the presence of S9 mix; 24 h in the absence of S9 mix
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colchicine (final concentration 0.25 µL/mL)
STAIN (for cytogenetic assays): Giemsa solution


NUMBER OF REPLICATIONS: two cultures per dose level, four replicates for control


NUMBER OF CELLS EVALUATED: 200 metaphases per dose level (with normal a maximum of 25 from each slide)


DETERMINATION OF CYTOTOXICITY
- Method: The proportion of metaphase figures were examined. From these results the dose level causing a decrease in mitotic index of 50 - 80 % of the solvent control value or, if there was no decrease, the maximum achievable (or advisable) concentration was used as the highest dose level for the metaphase analysis. The intermediate and low doses were 50 % and 12.5 % of the highest concentration.


OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other: Cells were examined for the presence of the following aberrations:
Gaps (chromatid and isochromatid), breaks (chromatid and isochromatid), interchanges, dicentric chromosomes, acentric chromosomal fragments, chromosome rings, complete metaphase pulverisation


OTHER: no repeat experiment performed
Evaluation criteria:
not specified
Statistics:
Statistical analysis used was Fisher's test.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
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 examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: All final concentrations greater than 200 µg/mL were immiscible with the tissue culture medium
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: No cytotoxicity at concentrations up to 200 µg/ml in the presence and absence of metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA: no data


ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Table #1: Summary of data obtained in chromosomal aberration test              

    Treatment                Aberrant Cell Frequency   
  Conc. [µg/mL]   S9 Mix Mitotic index[%] No. of CellsScored    Including Gaps          Excluding Gaps
           %  Judge  %  Judge
Vehicle Control  1% Ethanol  +  8.9 400   0.0  -  0.0  -
Test substance  25 +  7.3 200  0.0 neg.  0.0  neg.
Test substance   100 +  8.0 200   0.5 neg.  0.0  neg.
Test substance 200 + 9.4 200   0.0  neg.  0 .0  neg.
 Cyclophosphamide 15 +  n.d. 151  15.9 pos. ***  15.9  pos.***
 Vehicle Control 1% Ethanol  -  8.5 400   1.5  -  1.5  -
Test substance  25 - 6.3 200   2.0  neg.  2.0  neg.
Test substance  100 8.1 200  1.0  neg.  1.0  neg.
Test substance  200 -  6.0 200   1.5  neg.  1.5  neg.
Ethylmethanesulphonate 750  -  n.d. 200   12.5   pos. ***  12.5  pos.***

neg. = negative aberration frequency pos.*** = positive aberration frequency, statistically significant (p<0.001) n.d. = not determined

neg. = negative aberration frequency pos.*** = positive aberration frequency, statistically significant (p<0.001)

Conclusions:
It was concluded that the target substance 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters had shown no evidence of clastogenic activity in this in-vitro cytogenetic test system.
Executive summary:

It was concluded that 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters had shown no evidence of clastogenic activity in this in-vitro cytogenetic test system.

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:
1990-08-20 to 1990-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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 tested
- Storage condition of test material: in the dark at room temperature
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
H5: Ham¿s F12 medium with 5% heat-inactivated foetal bovine serum (FCS), 200 mM L-Glutamine, 50 µg/mL Gentamycin
H0: see above without foetal bovine serum
exposure medium: H0 and conditioned H5 medium (medium in which the CHO cells have grown for 24 hours prior to treatment) in a ratio of 1 in 6
H5-TG: H5 medim supplemented with 10 µg/mL 6-thioguanine
HAT: H5 containing 15µg/mL hypoxanthine, 0.3 µg/mL amethopterin, and 4 µg/mL thymidine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, for one week with HAT medium
Additional strain / cell type characteristics:
other: deficient in HPRT, selected by resistance to 6-thioguanine (TG)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9-microsomal liver enzymes, obtained from Sprague-Dawley rats
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 1, 10, 50, 100, 150, 200, 250, 300, 350 µg/mL with and without S9 mix
Main tests: 0, 25, 50, 75, 100, 150, 200 µg/mL with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not stated
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
H0 medium/ 1% ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
ethylmethanesulphonate
Remarks:
vehicle: ethanol Migrated to IUCLID6: 250 µg/mL final concentration in the test
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
H0 medium/ 1% ethanol plus S9 mix
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 20-Methylcholanthrene, 5 µg/mL final concentration in the test
Remarks:
vehicle: Dimethyl sulfoxid
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days, subculturing every 3-4 days at appropriate cell density
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable


SELECTION AGENT (mutation assays): 10 µg/mL 6-thioguanine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable


NUMBER OF REPLICATIONS: two incubated cultures per test concentration or positive control seeded in 3 dishes each, for negative control four incubated cultures seeded in 3 dishes each


NUMBER OF CELLS EVALUATED: 2x10E5 cells were seeded in selective agent


DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency of the treated population relative to the control cultures


OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: none


OTHER: Two independent experiments in the absence as well as in the presence of exogenous metabolic activation were carried out.
Evaluation criteria:
A positive response is claimed if three criteria are fulfilled:
1. statistically significant dose-related increase in mutant frequency at concentrations of test compound which result in greater than 10% cell survival
2. reproducible response
3. the mean mutant frequency in treated cultures reaches a value of twice the highest acceptable mean spontaneous mutant frequency found in the testing laboratory
Statistics:
analysis by linear regression (mutant frequency v concentration)
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: at a concentration of 250 µg/mL and above
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test cells were treated with test substance at concentrations ranging from 0 - 350 µg/mL. Cytotoxicity was not observed either in the absence or presence of S9-mix at any dose level. However, following the incubation period of 4 hours after dosing a precipitate was seen in the media of cultures treated with 250 µg/mL or above, both in the absence and presence of S9-mix. It was therefore decided to use 200 µg/mL as the top dose in the main tests.


COMPARISON WITH HISTORICAL CONTROL DATA: The 97.5% upper confidence limit for the mean spontaneous mutant frequency found in the laboratory was 15 mutants/10E6 viable cells. No tested concentration reached a value twice the highest acceptable mean spontaneous mutant frequency found in the laboratory.


ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity in all tested concentrations was observed either with or without S9 mix
Remarks on result:
other: strain/cell type: CHO-K1-BH4

Table #1: Plating efficiency of dose finding

concentration [µg/mL]   S9-mix Number of colonies on viability plates  Mean % survival (relative to control)
     test #1  test #2  test #1  test #2
 0 (solvent control)  -  91  74  100  100
 25  -  66  93  72  126
 50  -  78  71  86  96
 75  -  91  77  100  104
 100  -  70  69  76  93
 150  -  97  74  106  100
 200  -  62  79  68  106
 EMS (250 µg/mL)  -  60  38  66  51
 0 (solvent control)  +  84  78  100  100
 25  +  81  65  96  83
 50  +  88  60  104  77
 75  +  102  65  121  83
 100  +  84  49  100  63
 150  +  90  37  107  47
 200  +  79  57  94  73
 20-MCA (5 µg/mL)  +  90  82  107  105

Table #2: Mutation frequencies of test substance and controls

concentration [µg/mL]  mutant frequency (x10E6)          
  test #1 -S9   test #1 +S9  test #2 -S9  test #2 +S9
 0 (solvent control)  3  4  10  4
 50  1  4  2  1
 75  6  4  5  6
 100  0  3  4  1
 150  2  5  8  1
200  2  1  3  2
EMS (250 µg/mL)  189   n.p.  423   n.p.
 20-MCA (5 µg/mL) n.p.  222  n.p.  312

n.p. = not performed

Conclusions:
It was concluded that the target substance 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters did not demonstrate mutagenic potential in this in-vitro mammalian cell assay.
Executive summary:

It was concluded that 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters did not demonstrate mutagenic potential in this in-vitro mammalian cell assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A set of genotoxicity tests has been conducted on the target substance 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters and structurally related phthalic acid esters. Bacterial reverse mutation assays (Ames test) have been undertaken using methods similar to or equivalent to OECD test methods. The substances do not induce reverse mutation in Salmonella typhimurium.

No effects were observed in a mammalian cell gene mutation test (HPRT).

 

The ability to cause chromosomal damage in cultured human lymphocytes, followingin vitro treatment in the absence and presence of S9 metabolic activation has been investigated according to OECD/EU test methods. No statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level or any sampling time.

 

Mutagenic activity has been examined by assaying for the induction mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method according to OECD/EU test methods. No relevant increases in mutant frequencies were observed following treatment.

 

Mutagenic activity has also been examined by the mouse Balb/3T3 cell transformation assay. No activity was apparent even at concentrations higher than the aqueous solubility of the phthalates.


Justification for classification or non-classification

The Ames-tests of the target substance and the source substances were negative with and without metabolic activation. Furthermore, no cytogenicity in mammalian cells in-vitro and no mutagenicity in mammalian cells in-vitro were found in the target substance. All available data investigating the genetic toxicity indicate that the target substance has no genotoxic potential and classification according to EU classification criteria for genetic toxicity is not required.