Diisononyl adipate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is considered to be non-mutagenic in vitro in the Ames test (OECD 471) with and without metabolic activation tested up to the highest guideline defined concentration.

The test substance is not mutagnic in vitro in the micronucleus test (OECD 487) in the absence or presence of metabolic activation.

The test material is considered to be non-mutagenic in vitro in the mouse lymphoma mammalian cell mutation assay (similar to OECD 476).

There are also studies available to the structural analogue diethylhexyl adipate (DEHA):

The test substance is considered to be non-mutagenic in vitro in the Ames test (similar to OECD 471).

The test material is not mutagenic in vitro in the mouse lymphoma mammalian cell mutation assay (similar to OECD 476).

Inconclusive and contradicting results were found in the in vitro assays: chromosome aberration assay and sister chromatide exchange assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

no

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): DINA, Cas-No. 33703-08-1
- Analytical purity: 99 %

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9-mix

Test concentrations with justification for top dose:

without S9-mix: 0, 7.5, 10, 13, 18, 24, 56, 75, 100 µl/mL
with S9-mix: 0, 5.6, 10, 13, 18, 24, 32, 42, 56, 75, 100 µl/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: due to solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The mouse lymphoma gene mutation assay was conducted essentially as described by Clive and co-workers [1973, 1979; Clive and Spector, 1975]. Suspension cultures of mouse lymphoma cells, heterozygous for thymidine kinase activity, were grown in Fisher medium for leukemic mouse cells supplemented with 0.1% pluronic and 10% heat-inactivated horse serum (Fl0P) and were exposed to test substances in the same medium. Treated cells were grown in Fl0P for 48 hr to allow mutation expression. Approximately 3 x l0^6 cells from each culture were then plated in a selective medium containing 3 µg/ml trifluorothymidine (TFT) to select for mutant clones. Appropriately diluted cells from each culture were also seeded in plates without TFT for viability determinations (200 cells/plate). Mutant and total colony counts at each dose point were determined by triplicate plating. Colonies were counted with an automatic colony counter; differential sizing was not performed.

Evaluation criteria:

The validity of the experimental assay was verified through the use of concurrent positive, control materials, 7,12dimethyl-benzanthracene (DMBA) and ethylmethane sulfonate (EMS). Increased mutation frequencies induced by the positive control materials under appropriate conditions demonstrated both the integrity of the metabolic activation system and the responsiveness of the assay system as compared to its historical performance in the laboratory.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Conclusions:

The provided mammalian cell mutation assay in mouse lymphoma cells provided an overall negative result for the testes substance.

Executive summary:

In an mouse lymphoma test (similar to OECD 476) DINA was tested with and without S9-mix in a dose range of 5.6 - 1000 µl/ml. The test substance was dissolved in acetone. There was no evidence of a mammalian cell mutation in the mouse lymphoma assay (McKee, 1985)

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

-S9-mix positive controls missing for TA 98, TA 100, and TA 1538 no E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102

GLP compliance:

no

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): DINA, Cas-No. 33703-08-1
- Analytical purity: 99 %

Target gene:

detects mutation in an amino-acid requiring strain (histidine)

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-induced S9-mix

Test concentrations with justification for top dose:

Pre-test: up to 2000 µg/plate
Main test: 0, 10, 50, 100, 500, 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: was used due to good solubility of test item

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: without S9-mix: N-Methyl-N'-nitro-N-nitrosoguanidin

Details on test system and experimental conditions:

The Salmonella/mammalian microsome mutagenicity test was conducted in accordance with published techniques [Ames et al, 1975; Haworth et al, 1983; Maron and Ames, 1983]. Bacterial tester strains were characterized to verify mutagen responsiveness and antibiotic sensitivity [Ames et al,1975]. For test material evaluation, fresh bacterial stocks were exposed to graded doses of test chemicals both in the presence and absence of exogenous activation mixtures (0.1 ml S9 per 1.0 ml S9 mix or 50 µl S9 per plate) prepared from the livers of Aroclor 1254-induced Sprague-Dawley rats. Revertants were scored 72 hr after exposure to test chemicals with a Biotran-III Colony Counter (New Brunswick Scientific Company). The validity of the assay was verified with concurrent positive control materials, benzo(a)pyrene (BAP) and N-methyl-N-nitro-N-nitrosoguanidin (MNNG). The spontaneous reversion frequency of each tester strain was determined from concurrent untreated and solvent (acetone) controls.

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

other: valid with restriction because further positive controls should have been used to cover all bacterial strains; MNNG does not work for TA98 and TA 100

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

other: valid with restriction because further positive controls should have been used; MNNG does not work for TA 1538

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Yes
COMPARISON WITH HISTORICAL CONTROL DATA: no data
ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity occured in the pre-test at a concentration of 2000 µg/plate

Conclusions:

Across all tested strains no potential mutagenic effect was observed along with negative results for the cytotoxicity.

Executive summary:

The genotoxic potential of the test substance was evaluated in the Salmonella/mammalian microsome mutagenicity assay. The test substance did not exhibit any evidence of mutagenic or transforming potential in any of the assays utilized.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

purity: >99% (GC)
test substance name: Di-iso-nonyladipat
Charge: 14626

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 1538 and TA 98

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix prepared from liver homogenate of Aroclor 1254-pretreated male Sprague-Dawley rats

Test concentrations with justification for top dose:

1st and 2nd experiment
TA 1535, TA 100, TA 1537, TA 1538, TA 98
0, 20, 100, 500, 2500 and 5000 ug/plate

Vehicle / solvent:

ethanol, DMSO for the positive controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9-mix, DMSO as a vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix, DMSO as vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix, DMSO as vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S-9 mix, DMSO as vehicle

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfil the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Species / strain:

other: TA 1535, TA 100, TA 1537, TA 1538, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

No increase in the number of revertants and no bacteriotoxicity was observed under any test condition. The test substance was completely soluble in DMSO up to the highest dose. Positive controls gave the expected increase in the number of revertants.

Conclusions:

Across all tested strains no potential mutagenic effect was observed along with negative results for the cytotoxicity.

Executive summary:

For DINA an Ames test (comparable to OECD guideline 471) was performed. DINA was incubated with TA 98, TA 100, TA 1535, TA 1537 and TA 1538 strains of Salmonella with and without metabolic activation.

The dose range was 20 µg - 5000 µg/plate. No bacteriotoxic effect (reduced his- background growth) was observed.

According to the results of the present study, the substance diisononyl adipate is thus not mutagenic in the Ames test under the experimental conditions chosen here. (BASF,1984).

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

22 Jul 2010

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- 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

Target gene:

V 79 cells

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; 1000 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix: 0; 31.25; 62.5; 125; 250; 500; 1000 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix 0; 62.5; 125; 250; 500; 1000 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix 0; 46.88; 93.75; 187.5; 375; 750 μg/mL

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.

Vehicle / solvent:

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

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).

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).

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

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

Conclusions:

Under the experimental conditions described, Hexanedioic acid, diisononyl ester is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There are studies available to the structural analogue diethylhexyl adipate (DEHA):

The test substance is considered to be non-mutagenic in vivo in the mouse micronucleus assay (similar to OECD 474).

In a dominant lethal test in mice (similar to OECD 478), the test material is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

Corn oil

Duration of treatment / exposure:

Three consecutive days.

Frequency of treatment:

Three daily treatments.

Post exposure period:

A single sample was taken 24 hours after the final treatment (injection).

Dose / conc.:

375 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 male mice per dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-dimethylbenzanthracene (DMBA).

Tissues and cell types examined:

Tissue: bone marrow.
Cells: erythrocytes.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The provided mouse micronucleus assay did not reveal a potential DNA interaction of the test substance after intraperitoneal exposure up to the limit dose of 2000 mg/kg.
Besides, no cytotoxicity was observed.

Executive summary:

The mouse micronucleus assays did not reveal evidence of DNA interactions after intraperitoneal exposure to 2000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro test:

Ames

For diisononyl adipate (DINA) an Ames test (comparable to OECD guideline 471) was performed. DINA was incubated with TA 98, TA 100, TA 1535, TA 1537 and TA 1538 strains of Salmonella with and without metabolic activation. The dose range was 20 µg - 5000 µg/plate. No bacteriotoxic effect (reduced his- background growth) was observed. According to the results of the present study, the substance diisononyl adipate is thus not mutagenic in the Ames test under the experimental conditions chosen here. (BASF,1984).

In vitro MNT

The substance Hexanedioic acid, diisononyl ester was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) (OECD 487). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). The exposure period was 4 hrs or 24 hrs. In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage.

Test item concentrations ranging from 31.25 to 1000 μg/mL (with and without S9 mix). 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. Under the experimental conditions described, Hexanedioic acid, diisononyl ester is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation (BASF, 2013).

Mammalian Cell Gene mutation

In an mouse lymphoma test (similar to OECD 476) DINA was tested with and without S9-mix in a dose range of 5.6 - 1000 µl/ml. The test substance was dissolved in acetone. There was no evidence on of mammalian cell mutation in the mouse lymphoma assay (McKee, 1985)

For the structural analogue diethylhexyl adipate (DEHA) several in vitro and in vivo test are available.

In vitro test:

Ames

No mutations were found when Diethylhexyl adipate (DEHA) was incubated with TA-98, -100, -1535, or - 1537 Salmonella strains with and without metabolic activation at concentrations up to 10000µg/plate (Zeiger et al. 1985). This result was confirmed in a further unpublished Ames assay, which additionally included TA1538 (CMA 1982; for details, please refer to the registration dossier for DEHA).

Mammalian Cell Gene Mutation

There was no evidence of mammalian cell mutation in mouse lymphoma assays conducted with and without metabolic activation (McGregor et al. 1998; CMA unpublished studies, 1982) up to 5000µg/mL. Cytotoxicity was observed in both assays and precipitation occured at and above 1000µg/mL.

Chromosome aberration

In a chromosome aberration assay an ambiguous result was obtained without S9, while in an SCE assay treatment in the presence of S9 led to ambiguous results (Galloway 1987). These inconclusive and contradicting results were clarified in vivo.

Sister chromatid exchange

In a sister chromatid exchange assay (similar to OECD 479) with chinese hamster ovary cells DEHA was tested with and without metabolic activation in a dose range of 40 - 400 µg/mL.

The assay provided negative results without activation and equivocal with activation.

In vivo test:

In vivo MNT

A mouse micronucleus assays did not reveal evidence of DNA interactions after intraperitoneal exposure to 2000 mg/kg (Shelby et al., 1993).

The same result was obtained in an unpublished report after i.p. exposure to 5000mg/kg (CMA 1982).

Dominant lethal assay

In a dominant-lethal study using mice, DEHA did not demonstrate decreases in litter size that might suggest adverse effects on spermatogenesis up to doses of close to 10000mg/kg (Singh et al ., 1975).

Justification for classification or non-classification

Based on the available data, classification as a genotoxic substance is not triggered according EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.