Diisodecyl adipate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 487, 22 Jul 2010, “In vitro Mammalian Cell Micronucleus Test”

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Diisononyl adipate

EC Number:

251-646-7

EC Name:

Diisononyl adipate

Cas Number:

33703-08-1

IUPAC Name:

bis(7-methyloctyl) adipate

Details on test material:

- Name of test material (as cited in study report): Hexanedioic acid, diisononyl ester
- Physical state: Liquid, colorless, clear
- Analytical purity: Complex mixture out of many isomeres Water content: 0.03 g/100 g
- Lot/batch No.: Tank B 4306 Probenahme 26.04.2012

Method

Target gene:

V 79 cells

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line (1, 2) is a permanent cell line derived from the Chinese hamster and has a, high proliferation rate (doubling time of about 12 - 14 hours), high plating efficiency (≥ 90%), stable karyotype (modal number of 22 chromosomes)
- Type and identity of media: MEM
- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from liver of male Wistar rats

Test concentrations with justification for top dose:

1st Experiment
4 hours exposure; 24 hours harvest time; without S9 mix: 0; 31.25; 62.5; 125; 250; 500; 1 000 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix: 0; 31.25; 62.5; 125; 250; 500; 1 000 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix 0; 62.5; 125; 250; 500; 1 000 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix 0; 46.88; 93.75; 187.5; 375; 750 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: insolubility of the test substance in water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest recommended dose, 5 mg/mL, at which distinct test substance precipitation was observed.

After the attachment period, about 6 hours after seeding, the medium was removed from the slides and the treatment medium was added (see table below). The cultures were incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% humidity.
At the end of the exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10% [v/v] FCS) was added and incubated at 37°C, 5% (v/v) CO2 and ≥ 90% humidity for the respective recovery time. In the case of continuous treatment, the cell preparation was started directly at the end of exposure.
At the harvest time, 24 hours after start of exposure, the medium was completely removed. For hypotonic treatment, 5 mL of prewarmed 1.5% (w/v) Sodium citrate solution (37°C) was added for about 5 minutes. Then the hypotonic solution was removed and 5 mL cold fixative (ethanol:glacial acetic acid, ratio 3:1; +4°C) was added. After 5 minutes the fixative was removed and 5 mL of fresh cold fixative was added. Then the dishes were kept at room temperature for at least another 5 minutes for complete fixation.
A sample of 1 000 cells for each culture were analyzed for micronuclei, i.e. 2 000 cells for each test group.







Posive control
Without metabolic activation: 500 and 600 μg/mL ethyl methanesulfonate (EMS; SIGMA, M-0880)
With metabolic activation: 2.5 μg/mL cyclophosphamide (CPP; Baxter Oncology GmbH, E 432-1)

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
• The number of cells containing micronuclei in the vehicle control was within the range of the historical negative control data
• The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
• The number of micronucleated cells exceeds both the value of the concurrent vehicle control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE).

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.
The test substance was soluble in the most suitable vehicle acetone, but it was poorly soluble in culture medium. Due to missing cytotoxicity either in the pretest or in the main experiments the dose selection for the evaluation of cytogenetic damage was based on the
solubility properties of the test substance.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary table - experimantal parts without S9 mix

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity Micronucleated cells** [%]	Cytotoxicity Proliferation index (PI)	Cytotoxicity RICC*** [%]
1	4 hrs	Vehicle control1	-	n.d.	1.0	2.66	100.0
		31.25 μg/mL	-	-	n.d.	n.d.	100.0
		62.50 μg/mL	-	-	n.d.	n.d.	12.1
		125.00 μg/mL	-	-	0.5	2.47	90.9
		250.00 μg/mL	-	+	0.9	2.69	87.5
		500.00 μg/mL	-	+	n.d.	n.d.	108.2
		1 000.00 μg/mL	-	+	0.8	2.61	100.3
		Positive control2	-	n.d.	3.6^S	2.39	n.t.
2	24 hrs	Vehicle control1	-	n.d.	0.8	2.42	100.0
		62.50 μg/mL	-	-	0.9	2.42	98.2
		125.00 μg/mL	-	-	0.8	2.43	111.7
		250.00 μg/mL	-	+	1.3	2.35	115.9
		500.00 μg/mL	-	+	n.d.	n.d.	89.0
		1 000.00 μg/mL	-	+	n.d.	n.d.	101.1
		Positive control2	-	n.d.	5.9^S	2.21	n.t

 *         Precipitation in culture medium at the end of exposure period (macroscopic)

**       Relative number of micronucleated cells per 2 000 cells scored per test group

***     Relative increase in cell count (RICC)

S         Frequency statistically significant higher than corresponding control values

n.d.     Not determined

n.t.     Not tested

1        Acetone 1% (v/v)

2        EMS 500 μg/mL

Summary table - experimental parts with S9 mix

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity Micronucleated cells** [%]	Cytotoxicity Proliferation index (PI)	Cytotoxicity RICC*** [%]
1	4 hrs	Vehicle control1	+	n.d.	0.7	2.47	100.0
		31.25 μg/mL	+	-	n.d.	n.d.	103.5
		62.50 μg/mL	+	-	n.d.	n.d.	128.9
		125.00 μg/mL	+	-	0.5	2.23	121.7
		250.00 μg/mL	+	+	1.1	2.23	117.9
		500.00 μg/mL	+	+	n.d.	n.d.	102.3
		1 000.00 μg/mL	+	+	1.0	1.92	114.2
		Positive control2	+	n.d.	3.3^S	1.84	n.t.
2	24 hrs	Vehicle control1	+	n.d.	1.3	2.24	100.0
		46.88μg/mL	+	-	1.8	2.22	126.1
		93.75μg/mL	+	-	1.6	2.09	129.8
		187.50μg/mL	+	+	1.4	2.06	116.6
		375.00μg/mL	+	+	n.d.	n.d.	127.1
		750.00 μg/mL	+	+	n.d.	n.d.	99.0
		Positive control2	+	n.d.	4.2^S	1.90	n.t

*         Precipitation in culture medium at the end of exposure period (macroscopic)

**        Relative number of micronucleated cells per 2 000 cells scored per test group

***      Relative increase in cell count (RICC)

S            Frequency statistically significant higher than corresponding control values

n.d.    Not determined

n.t.     Not tested

1            Acetone 1%(v/v)      2            CPP 2.5μg/mL