1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

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.

Reference 2

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

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.

Reference 3

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

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.