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Genetic toxicity in vitro

Description of key information

Three in vitro mutagenicity tests are performed. All tests showed no evidence of genetic toxicity in vitro. No classification according to CLP regulation (EC) No 1272/2008 is needed.

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.09.2016 to 26.01.2017
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Chromosome aberration test in mammalian cells (in vitro cytogenetic study)
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254
Test concentrations with justification for top dose:
125, 250, 500, 1000, 2000 µg/mL medium of DINCD
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
yes
Remarks:
ethanol (vehicle)
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h without S9 mix; 4h with S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells):
Two hours before termination the cell division was arrested by the addition of 0.5 mL of the spindle poison Colcemid® to each culture (10 µg/mL solution). The tubes were capped and left to incubate for a further two hours.
The cells were harvested by low speed centrifugation (80 - 100 x g) and the pellets of cells collected were resuspended in hypotonic potassium chloride solution (0.56%) and fixed in freshly prepared methanol : glacial acetic acid fixative (4:1, v/v).

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid®

STAIN (for cytogenetic assays):
Giemsa stain (1:10 in WEISE's buffer12 pH 6.8)

NUMBER OF REPLICATIONS:
duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The tubes were centrifuged, the fixative removed and the cell pellet resuspended in a few drops of 60% acetic acid. Single drops of the cell suspension were spread on clean, grease-free glass slides on a hot plate (approx. 50°C) and the slides were left to air-dry. Two slides were prepared per culture, stained for 30 minutes in Giemsa stain (1:10 in WEISE's buffer12 pH 6.8), washed in buffer and left to air-dry. Permanent slides were made using CONSUL MOUNT mountant after clearing in xylene.


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
For each culture (duplicate) 150 metaphases were examined, yielding a total of 300 metaphases for each concentration analysed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:To examine the toxicity of the test item, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.
Rationale for test conditions:
Acceptance of the test was based on the following criteria:
o The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
o Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
o All three experimental conditions were tested unless one resulted in positive results (see paragraph 28 of the OECD 473).
o Adequate number of cells and concentrations are analysable (paragraphs 31 and 21 of the OECD 473).
o The criteria for the selection of top concentration are consistent with those described in paragraphs 22, 23 and 24 of the OECD 473.
o Both replicate cultures lead to similar results.
o Adequate numbers of cells (i.e. at least 1000 countable cells) and concentrations re-analyzable.
Evaluation criteria:
The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
o The number of chromosomal aberrations is significantly (at p  0.05) increased compared with the solvent control in at least one of the test concentrations
o The increase observed is concentration-dependent
o The increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
o Any of the results are outside the distribution of the historical negative control data
o A reproducible increase in the number of cells with chromosomal aberrations
o All three experimental conditions were tested unless one resulted in positive results
Statistics:
The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p  0.05) as recommended by the UKEMS guidelines (1989).
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Under the present test conditions, Diisononyl 1 ,4-cyclohexanedicarboxylate (DINCD) tested up to a concentration of 2000 μg /ml medium in the absence and
presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay .
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.12.2015 to 07.07.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro forward mutation assay in mammalian cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA
-clone: 3.7.2C

MEDIA USED
- growth medium: RPMI 1640 with glutamaxTM medium supplemented with Pluronic® F68 , gentamycin , amphotericin B1 and horse serum1 (10% by volume)
- Treatment medium: growth medium with a reduced horse serum content (5% by volume)
-Plating medium: growth medium with increased horse serum content but without Pluronic F68
-selection medium: plating medium that contains 3 µg/mL of TFT
Additional strain / cell type characteristics:
other: heterozygous at the TK locus (+/-)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
preliminary study for cytoxicity testing: 31.6, 100, 316, 1000, 3160 and 5000 µg Diisononyl 1,4-cyclohexanedicarboxylate (DINCD)/mL medium
mutagenicity study: 10.0, 31.6, 100, 316 and 1000 µg Diisononyl 1,4-cyclohexanedicarboxylate (DINCD)/mL medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) was not soluble in dimethylsulfoxide (DMSO) the solvent recommended by the OECD guideline, but the test item was completely soluble in ethanol.
Untreated negative controls:
yes
Remarks:
ethanol (vehicle)
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8 cells/mL

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays):
The Selection medium contains 3 µg/mL of TFT2 (AppliChem GmbH 64291 Darmstadt, Germany)

NUMBER OF REPLICATIONS:
Single cultures were used for each test item concentration level.

NUMBER OF CELLS EVALUATED:
The mutant frequency is expressed as mutants/10^6 viable cells.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:
Cytotoxicity is defined as the Relative Total Growth (RTG) which includes the Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Plating Efficiency (RPE2) obtained at the time of mutant selection
Rationale for test conditions:
An assay is considered acceptable for evaluation of the test results only if all of the criteria given below are satisfied.
The activation and non-activation portions of the mutation assays are usually performed concurrently, but each portion is in fact an independent assay with its own positive and negative controls. The activation or non-activation assays would be repeated independently, as needed, to satisfy the acceptance and evaluation criteria.
- Adequate number of cells and concentrations should be analysable

a) Data of the untreated/solvent control (Mutant Frequency, Cloning Efficiency and the Suspension Growth meet the IWGT MLA Workgroup acceptance criteria (M.M. Moore et al. 2006).
b) The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF, or
the positive control has an increase in the small colony MF of at least 150 x 10 6 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10-6).
c) Mutation Frequencies of both, negative and positive controls fall within the normal range (historical data).

Evaluation criteria:
For the MLA, significant work on biological relevance and criteria for a positive response has been conducted by The Mouse Lymphoma Expert Workgroup of the IWGT (M.M. Moore et al., 2006). Therefore, the interpretation of test chemical results is based on those recommendations:
To define positive and negative results and to assure that the increased MF is biologically relevant instead of a statistical analysis (generally used for other tests), the interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10-6) is based on the analysis of the distribution of the negative control MF data from participating laboratories (M.M. Moore et al., 2006).
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data is evaluated by expert judgement and/or further investigations.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the present test conditions, Diisononyl 1,4-cyclohexanedicarboxylate (DINCD), tested up to the concentration
of 1000 μg per mL medium, that led to test item precipitation, in two independent experiments was negative with
respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions
the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Diisononyl 1,4-cyclohexanedicarboxylate (DINCD)
also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay,
these findings indicate that Diisononyl 1,4-cyclohexanedicarboxylate (DINCD), tested up to theconcentration of 1000 μg per mL medium,
that led to test item precipitation, did neither induce mutations nor have any chromosomal aberration potential.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.11.2015 to 19.02.2016
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
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMF
- Justification for choice of solvent/vehicle:
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
Strains: TA 1535, TA 100 Name: sodium azide, NaN3 Purity: at least 99 % Dissolved in: deionised water Concentration: 10 μg/plate
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
Strains: TA 1537, TA 98 Name: 4-nitro-o-phenylene-diamine, 4-NOPD Purity: > 99.9 % Dissolved in: DMSO (purity >99 %) Concentration: 10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
Strain: WP2 uvrA Name: methyl methane sulfonate, MMS Purity: > 99.0 % Dissolved in: deionised water Concentration: 2.0 μL/plate
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
Strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA Name: 2-aminoanthracene, 2-AA Purity: 97.5 % Dissolved in: DMSO (purity >99 %) Concentration: 2.5 μg/plate (10.0 μg/plate in WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), pre-incubation
- Cell density at seeding (if applicable): 10^8-10^9 cells/mL

DURATION
- Preincubation period:
In the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- Exposure duration:
In the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification
- Expression time (cells in growth medium):
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

NUMBER OF REPLICATIONS:
triplicates

NUMBER OF CELLS EVALUATED:
The number of colonies were counted.
Rationale for test conditions:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported,
the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames-Test:

This study was performed to investigate the potential of DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Mouse lymphoma forward mutation assay:

In order to investigate the mutagenic potential on mammalian cells, the Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.

Under the present test conditions, Diisononyl 1,4-cyclohexanedicarboxylate (DINCD), tested up to the concentration of 1000 μg per mL medium, that led to test item precipitation, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.

In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that Diisononyl 1,4-cyclohexanedicarboxylate (DINCD), tested up to the concentration of 1000 μg per mL medium, that led to test item precipitation, did neither induce mutations nor have any chromosomal aberration potential.

In vitro assessment of the clastogenic activity in cultured human peripheral lymphocytes:

The potential ofDiisononyl 1,4-cyclohexanedicarboxylate (DINCD) to induce chromosomal aberrationswas examined in anin vitrocytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out in three independent experiments. In experiment 1, the cells were exposed to the test item for 4 hours without S9 mix. In experiment 2, cells were exposed to the test item for 24 hours without S9 mix, and in experiment 3, the cells were exposedto the test itemfor 4 hours with S9 mix. The harvesting time was 24 hours after initiation of exposure. Two replicate cultures were used at each concentration tested.

Under the present test conditions, Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) tested up to a concentration of 2000 µg/mL mediumin the absence and presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.

Justification for classification or non-classification

No classification for genetic toxicity is indicated according to the classification, labeling, and packaging (CLP) regulation (EC) No 1272/2008.