Registration Dossier
Registration Dossier
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EC number: 248-299-9 | CAS number: 27178-16-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
An Ames test conducted with diisodecyl adipate was negative.
Although diisodecyl adipate tested positive in an in vitro chromosome aberration test and equivocal in a mouse lymphoma test, the effects seen in the in vitro tests are not expected in view of the structure of substance.
For the following 2 reasons, we feel that the outcome of the chromosome aberration test can be considered of lower relevance in determining the overall genotoxic activity of Hatcol 2910:
Firstly, there was no increase in aberrations in the presence of metabolic activation (+S9).
For the non-activated (-S9) experiment, no significant increase in the frequency of cells with structural aberrations was noted at the 3-hour treatment. At the 20-hour treatment, a significant increase in the frequency of aberrant cells was noted at 2495 μg/mL (7% vs 0.5% for the vehicle control, p < 0.001). At the 44-hour treatment, a significant increase in the frequency of aberrant cells was noted at 2495 μg/mL for the male donor (14% vs 2% for the vehicle control, p < 0.001).The observed effect was at doses suspected to be highly toxic, even not always clearly demonstrated by the decrease of the mitotic index, as demonstrated inCIT/Study No. 23464 MLYwhere doses ≥ 1258 μg/mL of DA proved to be highly toxic following 24-hour treatment.
Secondly, while the study design and approach would likely comply with the OECD 473 guideline and GLP, the procedure deviates somewhat from the current protocol by collecting blood from two [supposedly] healthy subjects rather than one. Of concern is that the male subject's lymphocytes reacted (aberrations reported); yet those of the female didn't. While there are any number of biological reasons for this discrepancy (unhealthy male donor, currently undergoing therapy, viral exposure within the 6 months prior to having his blood drawn, etc). because the report only states that the donors were healthy, we would have requested the test to be repeated using [probably] the same female and replace the man with another individual. Confirmation that both were healthy for at least 6 months prior to conducting the study by asking questions, etc. would have been a documented criterion for selection. Our opinion is that the results most likely would have been negative in both individuals of the repeated test.
For these reasons, we deem this study to have limited value.
The following read-across in vitro gene mutation tests have been conducted with similar substances:
An in vitro mammalian cell gene mutation assay was performed with diisononyl adipate (CAS 33703-08-1) according to OECD guideline 476 and under conditions of GLP (McKee et al., 1986). Mutations at the TK locus of mouse-lymphoma L5178Y cells were investigated at test substance concentrations ranging from 7.5-100 µL/mL in the absence of metabolic activation (S9 mix) or 5.6-100 µL/mL in the presence of metabolic activation. Assuming a test substance density of 0.92 g/mL, these concentrations approximately corresponded to mass concentrations of 6.8-92 mg/mL and 5.1-92 mg/mL, respectively. After exposure to the test substance, cytotoxicity was observed at concentrations ≥ 10 µL/mL with relative total growth values ≤ 37%. After a 2-day expression period of the cultures, the resistance to 5-trifluorothymidine (TFT) was determined in all experiments. The test substance did not induce a significant increase in the mutant frequency at any dose concentration. The positive controls significantly increased mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system. In conclusion, Diisononyl adipate did not induce the mutant frequency in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.
An in vitro mammalian cell gene mutation assay with Bis(2-ethylhexyl) adipate (CAS 103-23-1) was performed similar to OECD guideline 476 (McGregor et al., 1988). Mutations at the TK locus of mouse-lymphoma L5178Y cells in the presence of metabolic activation (S9 mix) were investigated in two independent experiments at test substance concentrations ranging from 312.5 to 5000 µg/mL. In two further experiments, cells were exposed to the test substance at concentrations ranging from 1000 to 5000 µg/mL in the absence of metabolic activation. The treatment of cells in all experiments included an exposure period of 4 h, followed by an expression period of 48 h and a selection period of 11-14 days in the presence of 5-trifluorothymidine (TFT). The test substance did not induce a significant increase in the mutant frequency at any test substance concentration in the absence of S9 mix. No significant increase in mutant frequency was observed in the first experiment in the presence of S9 mix. In contrast, a statistically significant increase in the mutant frequency was observed in cells treated at concentrations ≥ 2000 µg/mL in the second experiment with S9 mix. However, since no dose-response relationship was observed and precipitation was evident at concentrations of 1000 µg/mL, the slight significant increase in mutant frequencies in the presence of S9 mix was not considered to be of biological relevance. In addition, significant cytotoxicity, as indicated by a reduction in relative total growth, was noted in all experiments at concentrations ≥ 1000 µg/mL, both with and without S9 mix. The positive controls significantly increased mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system. In conclusion, the test substance did not induce the mutant frequency in mouse-lymphoma L5178Y cells in the presence and absence of metabolic activation.
In addition, an in vitro mammalian cell micronucleus test (OECD guideline 487) has been conducted with the read-across substance, diisononyl adipate. Under the experimental conditions described, the substance was 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.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Justification for type of information:
- Please see Section 13 below.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- 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
- 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:
- March 26 - April 18, 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study has been performed according to OECD and EC guidelines and according to GLP principles.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine gene
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9-mix induced with Aroclor 1254
- Test concentrations with justification for top dose:
- Preliminary toxicity test: the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
The selected treatment-levels for the main test were: 312.5, 625, 1250, 2500 and 5000 μg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item DIISODECYL ADIPATE was dissolved in ethanol and ethanol is accepted and approved by
authorities and international guidelines. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- without S9
Migrated to IUCLID6: (1 μg/plate for TA1535 and TA100) - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9
Migrated to IUCLID6: (50 μg/plate for TA1537) - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- without S9
Migrated to IUCLID6: (0.5 μg/plate for TA98) - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9
Migrated to IUCLID6: (0.5 μg/plate for TA102) - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-Anthramine (2 μg/plate for TA1535, TA1537, TA98 and TA100)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-Anthramine (10 μg/plate for TA102)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (second experiment with S9-mix only)
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours
NUMBER OF REPLICATIONS:
- Preliminary toxicity test: one plate/dose-level in strain TA98, TA100 and TA102
- Mutagenicity experiments: three plates/dose-level in strain TA1535, TA1537, TA98, TA100 and TA102
DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined. - Evaluation criteria:
- Acceptance criteria:
This study is considered valid if the following criteria are fully met:
. the number of revertants in the vehicle controls is consistent with our historical data,
. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the numberof revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a
positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained. - Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Preliminary toxicity test: A moderate emulsion was noted in the Petri plates when scoring the revertants at dose-levels ≥ 1000 μg/plate.
Mutagenicity experiments: A moderate emulsion was noted in the Petri plates when scoring the revertants at dose-levels ≥ 1250 μg/plate.
RANGE-FINDING/SCREENING STUDIES:
No noteworthy toxicity was noted towards the three strains used, with and without S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria, see section evaluation cirteria.
The study was therefore considered valid.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No noteworthy toxicity was noted towards all the strains used, both with and without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. - Conclusions:
- Interpretation of results (migrated information):
negative
Under the experimental conditions, the test item DIISODECYL ADIPATE does not show mutagenic activity in the bacterial reverse mutation test with
Salmonella typhimurium. - 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:
- March 20 - May 23, 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study has been performed according to OECD and EC guidelines and according to GLP principles.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: human
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9 mix
- Test concentrations with justification for top dose:
- No preliminary cytotoxicity test was performed. The highest dose-level for treatment in the first experiment was selected on the basis of pH,
osmolality and solubility.
First cytogenetic experiment: 19.49, 38.99, 77.98, 155.9, 311.9, 623.9, 1248 and 2495 μg/mL with and without S9 mix.
Second cytogenetic experiment: 77.98, 155.9, 311.9, 623.9, 1248 and 2495 μg/mL with and without S9 mix. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test item being almost insoluble in water (information supplied by the Sponsor) and in
dimethylsulfoxide, it was dissolved in ethanol. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9-mix
Migrated to IUCLID6: (3 μg/mL, 3 hours of treatment) - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9-mix
Migrated to IUCLID6: (0.2 μg/mL, continuous treatment) - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9-mix
Migrated to IUCLID6: (25 μg/mL and 50 μg/mL, 3 hours of treatment) - Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours (with and without S9-mix), 20 and 44 hours (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 and 44 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid solution (10 μg/mL)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: Duplicate cultures
NUMBER OF CELLS EVALUATED: Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture
whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberrations were observed.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (number of cells in mitosis/1000 cells examined)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- Acceptance criteria:
This study was considered valid since the following criteria were met:
. the frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with our historical data,
. the frequency of cells with structural chromosome aberrations in the positive controls was significantly higher than that of the controls and
consistent with our historical data.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the
dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of
biological relevance, was also taken into account in the evaluation of the findings. - Statistics:
- Statistical analysis:
For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was
compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ2 test, in which p = 0.05 was used as the
lowest level of significance. - Species / strain:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (3 hour treatment)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (3 hour treatment)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- (20 hour and 44 hour treatment)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (20 hour and 44 hour treatment)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- (3 hour treatment)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (3 hour treatment)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No variation of pH related to the presence of the test item was noted.
- Effects of osmolality: No effects (at the dose-level of 2495 μg/mL, the osmolality value was equal to 281 mOsm/kg H2O (330 for the vehicle control)
- Precipitation: In the culture medium, the dose-level of 2495 μg/mL (obtained with treatment of the supplied test item at the volume of 15 μL/5.5 mL culture medium) showed a slight to moderate emulsion.
RANGE-FINDING/SCREENING STUDIES: No preliminary cytotoxicity test was performed.
COMPARISON WITH HISTORICAL CONTROL DATA: The frequencies of cells with structural chromosome aberrations of the vehicle and positive
controls were as specified in acceptance criteria (see section evaluation criteria). - Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation (3 hour treatment)
negative without metabolic activation (3 hour treatment)
positive without metabolic activation (20 hour and 44 hour treatment)
Under the experimental conditions, the test item DIISODECYL ADIPATE induced chromosome aberrations in cultured human lymphocytes, following a long-treatment without S9 mix and at toxic dose-level. - 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:
- 6 March 2002 until 13 June 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: According to GLP and OECD 476
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- The mutation rate of control and treated cultures were compared using the fluctuation assay approach.
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine Kinase (TK) locus
- Test concentrations with justification for top dose:
- 78.63, 157.3, 314.5, 629, 1258 and 2516 µg/mL (1st and 2nd experiment, without S9)
250, 375, 500, 750, 75 and 1000 µg/mL (3rd experiment, without S9)
78.63, 157.3, 314.5, 629, 1258 and 2516 µg/mL (1st and 2nd experiment, with S9) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test material was not soluble in water and DMSO. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- pos. control cyclophosphamide, 3 µg/mL, with S9 Migrated to IUCLID6: without S9, 25 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not relevant
- Exposure duration: 3 hours (without S9, 1st experiment and with S9, both experiments); 24 hours (without S9, 2nd and 3rd experiment)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days
SELECTION AGENT (mutation assays): 5-trifluorothymidine
NUMBER OF REPLICATIONS: 2 tubes per concentration containing each 10 x10^6 (3-h treatment) or 3x10^6 (24-h treatment) cells were treated
NUMBER OF CELLS EVALUATED: 2000 cells/well (one 96-well plate per culture = two plates per dose level, except for the vehicle control, where two 96-well plates per culture were used = total of four plates).
DETERMINATION OF CYTOTOXICITY
- Method: relative survival after treatment and relative total growth during 2-day expresion.
OTHER EXAMINATIONS:
- Other: colony size was determined (differentiation between small colonies (<25% of diameter of well) and large colonies (>25% of diameter of well).
OTHER: - - Evaluation criteria:
- Reported criteria for assay acceptance were: (1) The cloning efficieny of the vehicle controls should be average absolute cloning efficiency should be between 0.6-1.4 for CE0 and between 0.7-1.3 for CE2 (CE0 = cloning efficiency after treatment; CE2 = cloning efficiency at the end of the expression period); (2) The mutation frequency of the vehicle controls should be between 60-250x10^6; (3) The mutation frequency of the positive controls should be more than 2-fold that of the vehicle controls, and consistent with historical control data.
The evaluation criteria were: a reproducible two-fold increase in the mutant frequency when compared with the vehicle controls, at any dose level and/or evidence of a dose-relationship were considered as a positive result. Reference to historical data (presented in the report), or other considerations of biological relevance were also taken into account in the evalaution. Postive response observed only at high levels of cytotoxicity (survival lower than 10%) were not considered. - Statistics:
- None were applied.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Summary tables of the rsults are shown in the tables below. For evaluation see "Overall remarks, attachments".
- Conclusions:
- Interpretation of results (migrated information):
ambiguous without metabolic activation
Without metabolic activation an ambigous increase in mutant frequency was observed at concentrations ≥500 µg/mL. At these concentrations increases in the number of small colonies were observed, which may indicate the occurrence of gross chromosomal aberrations. - 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
- 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
- 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; 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 - 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).
- 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'.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Justification for type of information:
- Please see Section 13 below.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥ 10 µL/mL with and without S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Justification for type of information:
- Please see Section 13 below.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- only in experiment 4, an increase in mutant fraction was observed from 2000 µg/mL
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from approx. 1000 ug/mL in all experiments
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions (no data on test substance purity, limited documentation)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- (no data on colony sizing, very high concentrations tested, limited documentation; no data on analytical purity of the test substance)
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Fischer´s medium (F0) was supplemented with 2 M L-glutamine, sodium pyruvate (110 µg/mL) 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (F10P).
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9-mix), prepared with the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment 1: 312.5, 625, 1250, 2500, and 5000 µg/mL
Experiment 2: 1800, 2600, 3400, 4200, and 5000 µg/mL
Experiment 3 and 4: 1000, 2000, 3000, 4000, and 5000 µg/mL - Vehicle / solvent:
- acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: (+S9): 3-Methylcholanthrene (MCA; 2.5 µg/mL); (-S9): mehtyl metahnesulphonate (MMS; 15 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
If no clear positive response was observed in two experiments in the absence of S9 mix, 2 experiments were performed in the presence of S9-mix. Insignificant responses were investigated by further tests involving RLN (uninduced S9) activation.
DURATION
- Preincubation period: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 11-14 days at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): 14-17 days
SELECTION AGENT (mutation assays): trifluorothymidine (3 µg/mL)
STAIN (for cytogenetic assays): no data
NUMBER OF REPLICATIONS: duplicates per concentration per experiment (negative control: 3)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth - Evaluation criteria:
- Increase in mutant frequency compared to negative control.
Mutant fraction (MF) was calculated as follows: 200 x (mutant clones per plate)/(total clones per plate) = mutants/10E+06 clonable cells
Quality control criteria:
- The cloning efficiency of the solvent control has to be between 50% and 60%.
- Average mutant fraction of the solvent controls has to be > 15 and 110 mutants per 10E+06 surviving cells.
- At least two solvent control cultures have to be accepted.
- A positive control culture is rejected if a chi-square test for consistency of the acceptable mutant fractions shows P<5%.
- A positive control culture is rejected if the relative total growth (RTG) is < 1%. - Statistics:
- The statistical analysis was based upon the mathematical model proposed for this system and consisted of a dose-trend test and a variance anaysis of pair-wise comparisons of each dose against the vehicle control.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- only in experiment 4, an increase in mutant fraction was observed from 2000 µg/mL
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from approx. 1000 µg/mL in all experiments
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at 1000 µg/mL, but testing was continued up to 5000 µg/mL
RANGE-FINDING/SCREENING STUDIES: a screening test was performed to find the highest applicable concentration
The first experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/mL .
ADDITIONAL INFORMATION ON CYTOTOXICITY: significant toxicity (RTG < 50%) was observed in all experiments (exp 1: from 1250 µg/mL, exp 2: from 1800 µg/mL, exp 3: from 1000 µg/mL, and exp 4: from 2000 µg/mL)
In experiment 4, the LOED (lowest observed effective dose) was 2000 µg/mL. However, the test substance was considered as negative in this chromosome aberration test, because:
- precipitation occurred from 1000 µg/mL
- no clear dose-relationship in mutant frequency increase was observed,
- cytotoxicity occurred from 1000 µg/mL
- the positive result was not reproducible in the other experiments, although the same conditions (with S9, same concentration) were applied and the relative total growth was comparable, no increase in the average mutation factor was observed in experiment 3. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative - 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
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions (limited information on method, e.g. no exposure duration reported)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- limited information on method, no data on exposure duration, selection and fixation time, exposure concentrations do not cover a range from maximum to no cytotoxicity
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Fisher medium for leukemic mouse cells supplemented with 0.1% pluronic and 10% heat-inactivated horse serum (F10P)
- Metabolic activation:
- with and without
- Metabolic activation system:
- post-mitochondrial fraction (S9 mix), prepared from the livers of rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- Without S9 mix: 7.5, 10, 13, 18, 24, 56, 75 and 100 µL/mL, corresponding to approx. 6.8-92 mg/mL assuming a density of 0.92 g/mL
With S9 mix: 5.6, 10, 13, 18, 24, 32, 42, 56, 75 and 100 µL/mL, corresponding to approx. 5.1-92 mg/mL assuming a density of 0.92 g/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- 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
- Remarks:
- ethylmethanesulphonate: 0.5 and 1.0 µL/mL (without S9 mix); 7,12-dimethylbenzanthracene: 5.0 and 7.5 µL/mL (with S9 mix)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Expression time (cells in growth medium): 2 days
SELECTION AGENT (mutation assays): 3 µg/mL triflourothymidine (TFT)
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth, cloning efficiency - Evaluation criteria:
- The test substance was considered to be mutagenic, if the increase in the mutant frequency was greater than two-fold over the concurrent vehicle control value.
- Statistics:
- Mean values and standard deviations were calculated.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥ 10 µL/mL with and without S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity, as determined by a relative suspension growth ≤ 37%, was evident at test substance concentrations ≥ 10 µL/mL in the absence and presence of S9 mix.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Referenceopen allclose all
Tables Mitotic index and chromosome aberrations, see attachment Tables in vitro mamm chrom
| without S9 | without S9 | |||||||
| 1st experiment | 2nd experiment | |||||||
| 3-h treatment | 24-h treatment | |||||||
| dose (ug/mL) | RTG % | RS % | MF*10^-6 | R | RTG % | RS % | MF*10^-6 | R |
| 0 | 100 | 100 | 92 | 1.0 | 100 | 100 | 100 | 1.0 |
| 78.63 | 78 | 91 | 127 | 1.4 | 83 | 86 | 156 | 1.6 |
| 157.3 | 77 | 79 | 115 | 1.2 | 87 | 91 | 121 | 1.2 |
| 314.5 | 74 | 75 | 115 | 1.2 | 92 | 82 | 120 | 1.2 |
| 629 | 81 | 75 | 119 | 1.3 | 71 | 39 | 215 | 2.1 |
| 1258 | 65 | 102 | 157 | 1.7 | 10 | 2 | 600 | 6.0 |
| 2516 | 71 | 71 | 156 | 1.7 | - | - | - | - |
| MMS | 41 | 71 | 434 | 4.7 | 72 | 68 | 525 | 5.2 |
| MMS = methylmethanesulphonate |
| RTG = relative total growth |
| RS = relative survival |
| MF = mutation frequency |
| R = ratio between mutation frequency of treated cells / mutation frequency of control cells |
| without S9 | ||||
| 3rd experiment | ||||
| 24-h treatment | ||||
| dose (ug/mL) | RTG % | RS % | MF*10^-6 | R |
| 0 | 100 | 100 | 143 | 1.0 |
| 250 | 194 | 95 | 78 | 0.5 |
| 375 | 213 | 50 | 114 | 0.8 |
| 500 | 248 | 42 | 92 | 0.6 |
| 750 | 130 | 13 | 221 | 1.5 |
| 875 | 118 | 8 | 233 | 1.6 |
| 100 | 43 | 4 | 412 | 2.9 |
| MMS | 206 | 85 | 334 | 2.3 |
| with S9 | with S9 | |||||||
| 1st experiment | 2nd experiment | |||||||
| 3-h treatment | 3-h treatment | |||||||
| dose (ug/mL) | RTG % | RS % | MF*10^-6 | R | RTG % | RS % | MF*10^-6 | R |
| 0 | 100 | 100 | 123 | 1.0 | 100 | 100 | 124 | 1.0 |
| 78.63 | 73 | 93 | 100 | 0.8 | 189 | 95 | 54 | 0.4 |
| 157.3 | 82 | 85 | 111 | 0.9 | 108 | 85 | 117 | 0.9 |
| 314.5 | 99 | 105 | 108 | 0.9 | 111 | 79 | 116 | 0.9 |
| 629 | 97 | 98 | 116 | 0.9 | 115 | 94 | 115 | 0.9 |
| 1258 | 81 | 89 | 120 | 1.0 | 117 | 80 | 121 | 1.0 |
| 2516 | 85 | 84 | 94 | 0.8 | 130 | 81 | 84 | 0.7 |
| CPA | 40 | 79 | 617 | 5.0 | 38 | 47 | 1010 | 8.2 |
| CPA = cyclophosphamide |
| RTG = relative total growth |
| RS = relative survival |
| MF = mutation frequency |
| R = ratio between mutation frequency of treated cells / mutation frequency of control cells |
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. |
3.6S |
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. |
5.9S |
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. |
3.3S |
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. |
4.2S |
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
Table 1. Results of the Mouse Lymphoma Cell Assay
Experiment 1 - without S9 |
Experiment 2 - without S9 |
||||||||||
Conc. (µg/mL) |
CE (%) |
RTG (%) |
MC |
MF |
AVG MF |
Conc. (µg/mL) |
CE (%) |
RTG (%) |
MC |
MF |
AVG MF |
Acetone |
78 |
96 |
60 |
26 |
|
Acetone |
92 |
100 |
75 |
27 |
|
0 |
84 |
85 |
49 |
19 |
24 |
0 |
63 |
97 |
31 |
16 |
22 |
91 |
105 |
65 |
24 |
103 |
104 |
61 |
20 |
||||
85 |
114 |
67 |
26 |
84 |
98 |
63 |
25 |
||||
312.5 |
75 |
74 |
64 |
28 |
26 |
1800 |
88 |
26 |
41 |
16 |
18 |
80 |
80 |
57 |
24 |
74 |
25 |
47 |
21 |
||||
625.0 |
88 |
75 |
35 |
13 |
20 |
2600 |
73 |
24 |
51 |
23 |
18 |
80 |
70 |
64 |
27 |
88 |
23 |
36 |
14 |
||||
1250 |
69 |
25 |
58 |
28 |
23 |
3400 |
97 |
20 |
48 |
17 |
16 |
75 |
38 |
39 |
17 |
93 |
15 |
44 |
16 |
||||
2500 |
79 |
13 |
50 |
21 |
27 |
4200 |
78 |
15 |
35 |
15 |
20 |
70 |
16 |
68 |
32 |
69 |
13 |
50 |
24 |
||||
5000 |
87 |
13 |
48 |
18 |
|
5000 |
74 |
12 |
37 |
17 |
17 |
lethal |
|
|
|
72 |
13 |
36 |
17 |
||||
(MMS) 15 |
32 |
25 |
112 |
119 |
118* |
(MMS) 15 |
44 |
29 |
110 |
83 |
83* |
44 |
24 |
156 |
118 |
39 |
34 |
97 |
84 |
||||
Experiment 3 - with S9 |
Experiment 4 – with S9 |
|||||||||
Conc. (µg/mL) |
CE (%) |
RTG (%) |
MC |
MF |
AVG |
CE (%) |
RTG (%) |
MC |
MF |
AVG MF |
Acetone |
80 |
98 |
72 |
30 |
|
102 |
116 |
82 |
27 |
|
0 |
76 |
109 |
55 |
24 |
33 |
109 |
101 |
114 |
35 |
37 |
70 |
98 |
88 |
42 |
100 |
101 |
111 |
37 |
|||
73 |
94 |
78 |
36 |
68 |
82 |
102 |
50 |
|||
1000 |
65 |
39 |
47 |
24 |
32 |
93 |
70 |
116 |
42 |
40 |
75 |
38 |
90 |
40 |
100 |
67 |
115 |
38 |
|||
2000 |
50 |
27 |
53 |
36 |
33 |
111 |
28 |
294 |
88 |
73* |
48 |
22 |
43 |
30 |
101 |
32 |
176 |
58 |
|||
3000 |
59 |
18 |
55 |
31 |
34 |
93 |
23 |
226 |
81 |
85* |
59 |
19 |
65 |
37 |
91 |
21 |
242 |
89 |
|||
4000 |
56 |
16 |
61 |
37 |
39 |
78 |
20 |
187 |
80 |
80* |
53 |
22 |
66 |
41 |
100 |
26 |
238 |
79 |
|||
5000 |
40 |
16 |
37 |
31 |
34 |
94 |
28 |
240 |
85 |
81* |
48 |
13 |
53 |
37 |
77 |
27 |
177 |
77 |
|||
(MCA) 2.5 |
29 |
16 |
187 |
214 |
222* |
55 |
11 |
603 |
364 |
363* |
32 |
21 |
223 |
231 |
55 |
11 |
592 |
361 |
|||
CE = cloning efficiency (%)
RTG = relative total growth (%)
MC = mutant colony count
MF = mutant fraction (mutant colony per 10E+06 clonable cells)
AVG MF = group average mutant fraction
* Statistically significant different from control (P < 5%)
MMS = methyl metahnesulphonate
MCA = 3-methylcholanthrene
Table 1. Results of mouse lymphoma assay without metabolic activation
Concentration [µL/mL] |
Relative cloning [%] |
Relative Total Growth [%] |
Mutants per 1E+06 surviving cells |
0 (Acetone) |
100 |
100 |
60 |
0 (Acetone) |
100 |
100 |
30 |
7.5 |
96 |
92 |
30 |
10 |
165 |
36 |
40 |
13 |
116 |
33 |
40 |
18 |
160 |
23 |
40 |
24 |
153 |
21 |
40 |
56 |
123 |
13 |
40 |
75 |
88 |
11 |
30 |
100 |
99 |
15 |
50 |
EMS, 0.5 |
42 |
55 |
2570* |
EMS, 1.0 |
8 |
29 |
1230* |
EMS = ethylmethane sulfonate
*denotes a result equal or greater than twice the concurrent vehicle control
Table 2. Results of mouse lymphoma assay with metabolic activation
Concentration [µL/mL] |
Relative cloning [%] |
Relative Total Growth [%] |
Mutants per 1E+06 surviving cells |
0 (Acetone) |
100 |
100 |
50 |
5.6 |
88 |
75 |
70 |
10 |
68 |
37 |
30 |
13 |
53 |
26 |
50 |
18 |
87 |
22 |
40 |
24 |
76 |
25 |
50 |
32 |
77 |
28 |
40 |
42 |
103 |
20 |
30 |
56 |
91 |
21 |
30 |
75 |
96 |
18 |
50 |
100 |
128 |
27 |
40 |
DMBA, 7.5 |
24 |
22 |
1130* |
DMBA, 5.0 |
71 |
60 |
370* |
DBMA = 7,12-Dimethylbenzanthracene
*denotes a result equal or greater than twice the concurrent vehicle control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The clastogenic potential of read-across substance, bis(tridecyl) adipate (CAS 16958-92-2), was investigated in an in vivo Mammalian Erythrocyte Micronucleus Test in Sprague-Dawley rats similar to OECD Guideline 474 (Skinner, 1985). The test substance was applied to the clipped dorsal skin of 10 animals per sex and group at dose levels of 800 and 2000 mg/kg bw/day. Treatment of the animals was performed daily for 5 days/week over a period of 13 weeks. A similar constituted control group remained untreated and served as controls. At the end of exposure, the femoral bone marrow and peripheral blood smears of 5 animals per sex and dose were prepared and the incidence of micronucleated cells per 1000 polychromatic or normochromatic erythrocytes were scored in the respective tissues. In addition, the number of polychromatic and normochromatic erythrocytes was determined and a ratio of both was calculated in order to determine cytotoxic effects of the test substance. The obtained ratios of poly- to normochromatic cells did not indicate cytotoxicity in bone barrow and peripheral blood of treated animals. The frequency of micronuclei in polychromatic and normochromatic erythrocytes of the femoral bone marrow and peripheral blood of treated animals was not increased at any dose level compared to controls after 13-week dermal exposure.
Under the conditions of this experiment, Bis(tridecyl) adipate did not induce clastogenicity in male and female Sprague Dawley rats in the micronucleus assay.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Justification for type of information:
- Please see Section 13 below.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- not applicable
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Well documented study report, comparable to guideline study with acceptable restrictions (no data on analytical purity, non-GLP, no positive control)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- no positive control
- Principles of method if other than guideline:
- The present study (Study ID 40553) was part of a multiple endpoint study (Study ID 40552). Details on methods for dosing, animal care etc. were derived from the 13-weeks-repeated dose dermal study.
- GLP compliance:
- no
- Remarks:
- The study was conducted according to GLP regulations, but the report was not reviewed by the quality assurance unit.
- Type of assay:
- micronucleus assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Details on the strain: Rat/crl COBS CD[SD] BR/Charles River, Lakeview, New Jersey
- Source: Charles River, Lakeview, New Jersey
- Age at study initiation: 49 days
- Weight at study initiation: males: 161.9 - 166.8 g; females: 149.2 - 150.5 g
- Housing: individually, in suspended, stainless steel cages, with wire mesh bottoms and fronts
- Diet: Purina Certified Lab Chow #5002 in pellet form; ad libitum
- Water: tap water, delivered by an automatic watering system; ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- dermal
- Vehicle:
- - Vehicle(s)/solvent(s) used: none
- Details on exposure:
- TEST SITE
- Area of exposure: on the backs of the animals, beginning at the scapula and continuing laterally and posteriorly
- Type of wrap if used: no wrap. Animals were fitted with cardboard "Elisabethan" collars to minimize ingestion of test material.
- Time intervals for shavings or clipplings: approx. 24 h before the start of dosing, hair was then reclipped as necessary, but at least once per week
REMOVAL OF TEST SUBSTANCE
- Washing: approx. 24 h after the last dose each week, as much residual test substance as practical was wiped off with gauze pads.
TEST MATERIAL
- Constant volume or concentration used: yes - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 24 h, 5 days/week
- Post exposure period:
- none
- Remarks:
- Doses / Concentrations:
800 and 2000 mg/kg bw/day
Basis:
nominal conc. - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- None.
Since the study was part of a 13-weeks repeated dose dermal study, no positive control was run in parallel. - Tissues and cell types examined:
- Peripheral blood smears and bone marrow were taken from the femurs of 5 animals/sex/dose.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
The doses were the same as chosen for the 13-weeks repeated dermal dose study.
TREATMENT AND SAMPLING TIMES:
To ascertain if the bone marrow and peripheral blood were equally sensitive for micronuclei detection, each target tissue was scored as an independent phase of the study and the results were compared. Three peripheral blood slides and four bone marrow slides were made for each animal.
DETAILS OF SLIDE PREPARATION:
Slides were air dried and fixed in absolute methanol for 15 min. After the slides had dried, they were placed in an acridine orange (0.125 mg/mL in Giordano's buffer, pH 5.4-6.5) solution for 7 to 10 min, rinsed in Giordano's buffer and washed in fresh Giordano's buffer for an additional 5-10 min.
METHOD OF ANALYSIS:
1000 polychromatic erythrocytes and 1000 normochromatic erythrocytes were scored in each target tissue to determine the incidence of micronuclei. - Statistics:
- Several statistical methods, including ANOVA (analysis of variance) and SAS GLM (general linear model) models, were applied to the data. The ANOVA F test was used to determine significant differences between mean values. The Tukey's Studentized Range Test and the Scheffe's Test were performed to determine which means were different. SAS GLM is a studentized linear regression analysis that can be used to determine dose responsiveness.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- not applicable
- Conclusions:
- Interpretation of results (migrated information): negative
Referenceopen allclose all
Table 1: Micronucleus assay of bone marrow from rats treated dermally with the substance for 13 weeks (averaged data)
Dose [mg/kg bw/day] |
Sex |
No. of animals |
PCE/NCE |
% PCE with MN |
% NCE with MN |
0 |
F |
5 |
0.38 (0.19) |
0.12 (0.11) |
0.04 (0.05) |
0 |
M |
5 |
0.44 (0.17) |
0.04 (0.05) |
0.02 (0.04) |
0 |
M+F |
10 |
0.41 (0.17) |
0.08 (0.09) |
0.03 (0.05) |
800 |
F |
5 |
0.40 (0.12) |
0.14 (0.11) |
0.04 (0.05) |
800 |
M |
5 |
0.40 (0.08) |
0.08 (0.13) |
0.00 (0.00) |
800 |
M+F |
10 |
0.40 (0.10) |
0.11 (0.12) |
0.02 (0.04) |
2000 |
F |
5 |
0.46 (0.16) |
0.06 (0.05) |
0.02 (0.04) |
2000 |
M |
5 |
0.41 (0.09) |
0.02 (0.04) |
0.08 (0.08) |
2000 |
M+F |
10 |
0.44 (0.12) |
0.04 (0.05) |
0.05 (0.07) |
Averaged data: means (standard deviation)
PCE: polychromatic erythrocytes
NCE: normochromatic erythrocytes
MN: micronuclei
Table 2: Micronucleus assay of peripheral red blood cells from rats treated dermally with the substance for 13 weeks (averaged data)
Dose [mg/kg bw/day] |
Sex |
No. of animals |
PCE/NCE |
% PCE with MN |
% NCE with MN |
0 |
F |
5 |
0.02 (0.00) |
0.02 (0.04) |
0.02 (0.04) |
0 |
M |
5 |
0.02 (0.00) |
0.14 (0.11) |
0.02 (0.04) |
0 |
M+F |
10 |
0.02 (0.00) |
0.08 (0.10) |
0.02 (0.04) |
800 |
F |
5 |
0.02 (0.00) |
0.10 (0.12) |
0.04 (0.05) |
800 |
M |
5 |
0.03 (0.01) |
0.08 (0.18) |
0.00 (0.00) |
800 |
M+F |
10 |
0.02 (0.01) |
0.09 (0.14) |
0.02 (0.04) |
2000 |
F |
5 |
0.02 (0.01) |
0.08 (0.08) |
0.02 (0.04) |
2000 |
M |
5 |
0.03 (0.01) |
0.06 (0.09) |
0.00 (0.00) |
2000 |
M+F |
10 |
0.02 (0.00) |
0.07 (0.08) |
0.01 (0.03) |
Averaged data: means (standard deviation)
PCE: polychromatic erythrocytes
NCE: normochromatic erythrocytes
MN: micronuclei
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Based on the above considerations, diisodecyl adipate does not need to be classified according to the CLP Regulation (EC) 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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