Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Based on the available weight of evidence from in vitro genotoxicity assays with the test substance or substances representative of its main constituents, the test substanceis considered to be non-genotoxic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- (Q)SAR
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- QSAR prediction from an well known and acknowledged tool. See below under 'Overall remarks, attachments' for applicability domain and 'attached background material section' for methodology.
- Qualifier:
- according to guideline
- Guideline:
- other: REACH guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals
- Principles of method if other than guideline:
- Since the test substance is a UVCB, the mutagenicity potential was predicted for the all the constituents.
- GLP compliance:
- no
- Type of assay:
- other: QSAR prediction
- Key result
- Species / strain:
- other: QSAR prediction from TEST v4.2.1
- Remarks:
- Ames Mutagenicity Test
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Remarks:
- predicted for all the constituents
- Species / strain:
- S. typhimurium TA 1535
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 1537
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 97
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 100
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 102
- Genotoxicity:
- not specified
- Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- Based on the negative mutagenicity predictions for all the constituents using the Consensus method of the T.E.S.T. v4.2.1 program, the test substance is overall considered to be non-mutagenic
- Executive summary:
The mutagenicity potential of the test substance 'Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide' (UVCB) was predicted using the Consensus method of the T.E.S.T. v4.2.1 program. Since the test substance is a UVCB, the mutagenicity potential was predicted for all the individual constituents, using SMILES as the input parameter. All the constituents were predicted to be negative for mutagenicity (US EPA, 2019), indicating that the test substance can be overall considered to be non-mutagenic. Applicability domain evaluation was performed by checking the descriptor and structural fragment domains of the individual QSAR methods (i.e., FDA, hierarchical clustering and nearest neighbour methods) underlying Consensus model predictions. Since an experimental value could be identified for cetyl stearate, no domain evaluation was necessary for it. The domain evaluation of the remaining all constituents indicated that they were within both descriptor and structural fragment domains of the FDA and hierarchical clustering methods, but not completely within domain for the structural fragments identified for the three nearest neighbours. Further, the prediction accuracy of the binary toxicity endpoints, can be evaluated in terms of the fraction of compounds that are predicted accurately and are described based on three statistics parameters: concordance, sensitivity, and specificity. In general, if the concordance is greater than or equal to 0.8, the model is considered to be valid. And both the leave-one-out sensitivity and specificity must be at least 0.5 to avoid using models which are heavily biased to predict either active or inactive scores. Out of three statistical parameters, the concordance and specificity fractions of all the constituents are above the required cut-offs. For sensitivity, effectively only one chemical was identified to be experimentally active but not predicted correctly for constituent 1. Given the structural differences of this chemical compared to the target, the overall accuracy of the model and its prediction is not expected to be compromised. Therefore, the predictions can be overall considered to be accurate with medium to high confidence.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- (Q)SAR
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- QSAR prediction from a well-known and acknowledged tool. See below 'attached background material section' for detailed prediction results and applicability domain evaluation.
- Qualifier:
- according to guideline
- Guideline:
- other: REACH guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals
- Principles of method if other than guideline:
- Since the test substance is a UVCB, the mutagenicity potential was predicted for all the individual constituents, using SMILES as the input parameter.
- GLP compliance:
- no
- Type of assay:
- other: QSAR prediction
- Key result
- Species / strain:
- other: QSAR prediction from Mutagenicity (Ames test) model (CAESAR) 2.1.13 of the VEGA v1.2.4 program
- Remarks:
- Ames Mutagenicity Test
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Remarks:
- predicted for all the constituents
- Species / strain:
- S. typhimurium TA 1535
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 1537
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 98
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 100
- Genotoxicity:
- not specified
- Species / strain:
- S. typhimurium TA 102
- Genotoxicity:
- not specified
- Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- Based on the negative mutagenicity predictions for all the constituents using the Mutagenicity (Ames test) model (CAESAR) 2.1.13 of the VEGA v1.2.4 program, the test substance is overall considered to be non-mutagenic.
- Executive summary:
The mutagenicity potential of the test substance 'Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide' (UVCB) was predicted using the Mutagenicity (Ames test) model (CAESAR) 2.1.13 of the VEGA v1.2.4 program. Since the test substance is a UVCB, the mutagenicity potential was predicted for all the individual constituents, using SMILES as the input parameter. All the constituents were predicted to be negative for mutagenicity (VEGA, 2019), indicating that the test substance can be overall considered to be non-mutagenic.The applicability domain of predictions is assessed using an Applicability Domain Index (ADI) that has values from 0 (worst case) to 1 (best case). A global ADI is calculated by grouping several other indices such as similar molecules with known experimental value, accuracy of prediction for similar molecules, concordance for similar molecules, model's descriptors range check and atom centered fragments similarity checks. Usually values lower than 0.75 indicate that the similar compound has important differences compared to the target. Considering that not all constituents were in domain, the overall predictions can be considered to be reliable with restrictions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From November 09, 2020 to March 01, 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 29 July 2016
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Qualifier:
- according to guideline
- Guideline:
- other: Method B67 of Commission Regulation (EC) No. 440/2008
- Version / remarks:
- 26 September 2019
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine kinase, TK +/-, locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Metabolic activation:
- with and without
- Metabolic activation system:
- The S9 Microsomal Enzyme Fractions were purchased from Moltox, Lot no 4272 with the expiry date of 16 July 2022, and Lot no 4217 with the expiry date of 05 March 2022, were used in this study. The protein content was adjusted to approximately 20 mg/ml prior to use. The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.
- Test concentrations with justification for top dose:
- Preliminary test: 0, 0.63, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μg/mL with and without metabolic activation (S9)
Main experiment: 0, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL with and without metabolic activation (S9)
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose levels in the Mutagenicity Test were limited by the onset of test item precipitate in both the absence and presence of metabolic activation, as recommended by the OECD 490 guideline. - Vehicle / solvent:
- Acetone
Supplier: Acros
Batch number: 1911510
Purity: 99.9%
Expiry date: 29 July 2021 - Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Test System
Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier. Master stocks of cells were tested and found to be free of mycoplasma.
Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Test Item Preparation
The test item was a UVCB*, therefore, the maximum proposed dose level in the solubility test was set at 5000 μg/mL, the maximum recommended dose level and no correction for the purity of the test item was applied to the formulations. The test item was found to be immiscible in RO medium at 50 mg/mL, and DMSO and acetone at 500 and 250 mg/mL. The test item was miscible in acetone at 125 mg/ml. Acetone was therefore selected as the solvent. However, acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 125 mg/mL and dosed at 0.5% to give a maximum achievable dose level of 625 μg/mL There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Test Procedure
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 1x10E7 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9). Due to the precipitate observed in the solubility check, the maximum dose concentration was limited to 160 μg/mL for both exposure groups and the dose range used in the preliminary toxicity test was 0, 0.63, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μg/mL for both of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2x10E5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2x10E5 cells/mL in R20 medium. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB*) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 %. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).
Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 107 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (0, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL in both the absence and presence of metabolic activation), solvent and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL. The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood. - Evaluation criteria:
- Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20 % survival, but no less than 10% survival (90% toxicity). Relative Total Growth values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, i.e. the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories.
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. 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. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- It was concluded that did not induce biologically relevant increases in mutation at the TK +/- locus of L5178Y mouse lymphoma cells that exceeded the Global Evaluation Factor (GEF) when tested for 4 h in the absence and presence of a rat liver metabolic activation system (S-9). Solvent, vehicle and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion.
- Remarks on result:
- other: no mutagenic potential
- Conclusions:
- Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y mouse lymphoma cells at the TK +/- locus, in the absence and presence of metabolic activation.
- Executive summary:
A study was conducted to determine the mutagenic potential of the test substance on thymidine kinase, TK +/-, locus of L5178Y mouse lymphoma cells, according to the OECD Test Guideline 490, in compliance with GLP. L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) cells were counted and processed to give 1 x 10E7 cells/mL in 10 mL aliquots in test medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate, both with and without metabolic activation (2% S9 final concentration) at eight dose levels (0, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL) in duplicate, both the absence and presence of metabolic activation (2% S9), solvent and positive controls for 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9). The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood. There was no evidence of any toxicity, in either the absence or presence of metabolic activation, following exposure to the test substance, as indicated by the %RSG and RTG values. There was also no evidence of any marked reductions in cloning efficiency in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Test substance precipitate was observed at 80 μg/mL in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances. The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline. Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y mouse lymphoma cells at the TK +/- locus, in the absence and presence of metabolic activation (Willey, 2022).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Study period:
- From August 01, 2017 to October 30, 2017
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 h. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used are:
Preliminary toxicity test: male, aged 28 years
Main Experiment: male, aged 26 years - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver homogenate metabolizing system S9 fraction (20% (v/v))
- Test concentrations with justification for top dose:
- Preliminary test: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL
Main experiment: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/mL
The test substance was insoluble in Dimethyl sulphoxide, Acetone and Tetrahydrofuran at 200, 100 and 50 mg/mL. The test substance was insoluble in Minimal Essential Medium (MEM) at 20 mg/mL but was partially soluble/ suspendable in MEM at 10 mg/mL in solubility checks performed in-house. Therefore 1000 µg/mL was considered to be the maximum achievable dose level due to formulation difficulties. The selection of the maximum dose level for the main experiment was based on the lowest precipitating dose level and was 125 µg/mL for the 4(20)-h exposure groups and for the continuous exposure group. - Vehicle / solvent:
- The test substance was insoluble in Dimethyl sulphoxide, Acetone and Tetrahydrofuran at 200, 100 and 50 mg/mL. The test substance was insoluble in Minimal Essential Medium (MEM) at 20 mg/mL but was partially soluble/ suspendable in MEM at 10 mg/mL in solubility checks performed in-house. Therefore MEM was used as vehicle.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Identity: Minimal Essential Medium, Supplier: Sigma, Batch number: RNBF9655, Expiry Date: September 2018, Purity: Treated as 100%
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 h. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used are:
Preliminary toxicity test: male, aged 28 years
Main experiment: male, aged 26 years
Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Batch Nos. PB/NF S9 30/6/17 and 20/8/17 were used in this study. Prior to use each batch of S9 is tested for its capability to activate known mutagens in the Ames test. The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%. - Evaluation criteria:
- Data Evaluation
The following criteria were used to determine a valid assay:
1) The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
2) All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
3) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
4) The required number of cells and concentrations were analyzed. - Statistics:
- Statistical Analysis
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989). A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis. - Key result
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Tested concentrations: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: no mutagenic potential
- Conclusions:
- Under the conditions of the study, the read across substance was considered to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation. Based on the results of the read across study, similar absence of clastogenicity can be considered for the phosphate ester constituent of the test substance.
- Executive summary:
An in vitro study was conducted to determine the clastogenicity of the read across substance, 'mono- and di- C16 PSE, K+ and H3PO4', using human lymphocytes, according to OECD Guideline 473 (Chromosome Aberration test), in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the read across substance, were evaluated for chromosome aberrations at doses 0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL, together with vehicle and positive controls. In this study, three exposure conditions were investigated: 4 h exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-h expression period, 4 h exposure in the absence of metabolic activation (S9) with a 20-h expression period and a 24-h exposure in the absence of metabolic activation. The dose levels used in the main experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances (Mitomycin C and Cyclophosphamide) induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The read across substance was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. Under the conditions of the study, the read across substance was considered to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation. (Envigo, 2017). Based on the results of the read across study, similar absence of clastogenicity can be considered for the phosphate ester constituent of the test substance.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Study period:
- From February 11, 1998 to April 20, 1998
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification.
- Reason / purpose for cross-reference:
- read-across source
- 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
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured human peripheral lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: F10 complete culture medium, containing Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively), sodium bicarbonate (1.2 g/L) and 30 U/mL heparin.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment 1:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
48 h treatment, 48 h harvest time, without metabolic activation: 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 48 h harvest time, with metabolic activation: 1000 µg/mL
Experiment 2:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.9% DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: mitomycine C (0.2 µg/mL, 24 h treatment time, -S9; 0.1 µg/mL, 48 h treatment time, -S9) and cyclophosphamide (15 µg/mL in nutrient medium, 3 h treatment time, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h with metabolic activation, 24 and 48 h without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h fixation time with and without metabolic activation
SPINDLE INHIBITOR (cytogenetic assays): 0.5 µg/mL colcemid, added 3 hours before harvest time (21 h with metabolic activation, 21 and 45 h without metabolic activation)
STAIN (for cytogenetic assays): 5% (v/v) Gimsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 (100 per culture, duplicate cultures)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
OTHER:
Blood samples were taken from healthy, adult male volunteers by venapuncture, using a sterile vessel containing sodium heparine. The blood samples were stored at 4-25 ºC. Cultures were started within 4 h after blood collection. The average generation time of the cells is 13.6-14.1 h. Cultures were incubated at 37 ºC for 48 h prior to exposure to the test substance. - Evaluation criteria:
- A chromosome aberration test was considered acceptable if it met the following criteria:
a) The numbers of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range (min = 0, max = 5 (mean = 0.9, standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min = 0, max = 5 (mean = 0.7, standard deviation = 0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded)
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
c) A homogeneous response between the replicate cultures is observed.
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
b) A statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. - Statistics:
- The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.
- Key result
- Species / strain:
- lymphocytes: cultured human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the culture medium at 1000 µg/mL.
RANGE-FINDING/SCREENING STUDIES:
A range-finding test was performed to select the dose levels for the main experiment, using 10, 33, 100, 333 and 1000 µg/mL, with and without metabolic activation. The test substance precipitated in the culture medium at 1000 µg/mL (see Table 1).
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed at any concentration, with or without metabolic activation (see Table 2 and 3). - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- no mutagenic potential
- Conclusions:
- Under the read across study results, the ester constituent of the test substance is considered to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation.
- Executive summary:
A study was conducted to determine the clastogenicity of the read across substance, ‘Octyldodecyl isooctadecanoate' (purity: 100%), using Chromosome Aberration test in human lymphocytes , according to the OECD Guideline 473, in compliance with GLP. Cultures of human lymphocytes were treated with the read across substance and were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Two experiments under different exposure conditions were investigated in the study: Experiment 1 (a) 24 h exposure in the absence of an induced rat liver homogenate metabolizing system (S9), with cell harvest after a 24 h expression period in absence of metabolic activation (at test concentrations of 100, 333 and 100 μg/mL), (b) 48 h exposure in the absence of metabolic activation (S9), with a 48 h expression period in absence of metabolic activation (at test concentrations of 1000 μg/mL), (c) a 3 h exposure in the presence of metabolic activation, with 24 h harvest period in presence of metabolic activation (at test concentrations of 100, 333 and 1000 μg/mL) and (d) a 3 h exposure in the presence of metabolic activation with 48 h harvest period in presence of metabolic activation (at test concertation of 1000 μg/mL). Experiment 2 (a) a 24 h treatment in absence of metabolic activation with 24 h harvest period in absence of metabolic activation (at test concentration of 100, 333 and 1000 μg/mL) and (b) 3 h exposure in presence of metabolic activation with 24 h harvest period in presence of metabolic activation (at test concentration 100, 333 and 1000 μg/mL). The dose levels used in the main experiment (mentioned above) were selected using data from the preliminary toxicity test (using 10, 33, 100, 333 and 1000 µg/mL, with and without metabolic activation), where the results indicated that the maximum concentration should be limited to 1000 µg/mL, due to precipitation. The incidences of aberrant cells in the vehicle (dimethyl sulphoxide (DMSO)) control group were within the range expected for normal human lymphocytes. All the positive control substances (i.e., Mitomycin C and Cyclophosphamide) induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The read across substance was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations. Under study conditions, the read across substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (Notox, 1998). Based on the results of the read across study, a similar absence of genotoxicity in the chromosomal aberration assay can be expected for the ester constituent of the test substance
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Study period:
- no informaton available
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: minimum essential medium
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver microsomal fraction from male rats prepared according to Ames et al., 1977
- Test concentrations with justification for top dose:
- 0.6, 10.0 and 20.0 µg/mL
- Vehicle / solvent:
- Ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 7 and 24 (or 28) hours at 20 µg/ml, 18 hours at 0.6, 10 and 20 µg/ml
SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.2 µg/ml
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2 cultures per concentration
NUMBER OF CELLS EVALUATED: 100 per slide, 200 per concentration
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Evaluation criteria:
- To be considered positive, either a statistically significant, concentration-related increase in the number of structural chromosome aberrations, or a statistically significant positive response at one of the concentrations
- Statistics:
- Chi-squared test performed for cells with aberration (excluding gaps)
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: presumably >20 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: insoluble
- Precipitation: no data
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: yes, but no data presented
COMPARISON WITH HISTORICAL CONTROL DATA: no data
ADDITIONAL INFORMATION ON CYTOTOXICITY: at 20 µg/ml, mitotic index not reduced, plating efficiency not reduced - Remarks on result:
- other: no mutagenic potential
- Conclusions:
- Based on the results of the read across study, the test substance, hexadecanol is considered to be non-genotoxic with and without metabolic activation in chromosome aberration test
- Executive summary:
A study was conducted to determine the genotoxicity of the read across substance, behenyl alcohol (C22), according to a method similar to the OECD Guideline 473, using in vitro chromosome aberration study in Chinese hamster lung fibroblasts (V79). Chinese hamster lung fibroblasts (V79) in duplicates were incubated with 0.6, 10.0 and 20.0 µg/mL of read across substance dissolved in ethanol for 4 h, with and without metabolic activation. There was no evidence of cytotoxicity at this dose level. At 20 µg/mL, mitotic index as well as plating efficiency were not reduced. The read across substance did not increase the incidence of chromosome aberrations in Chinese hamster V79 cells in the presence or absence of metabolising fraction at concentrations up to 20 µg/mL. The negative and positive controls were considered to be valid. Under the study conditions, the read across substance was determined to be non-genotoxic with and without metabolic activation (Iglesias, 2002). Based on the results of the read across study, similar absence of clastogenic potential can be considered for the constituent alcohol.
Referenceopen allclose all
Results
Constituents |
Name |
SMILES |
TEST - Predicted Ames Mutagenicity value from Consensus method |
TEST - Predicted Ames Mutagenicity result from Consensus method |
Prediction statistics for similar chemicals - External and Training Dataset |
||
Concordance |
Sensitivity |
Specificity |
|||||
1 |
mono- C16 PSE, K+ and mono-C16 PSE |
CCCCCCCCCCCCCCCCOP(O)(O)=O |
0.14 |
Negative |
0.89 and 0.90 |
0 and 0 |
1 and 1 |
2 |
di- C16 PSE, K+ and di- C16 PSE |
CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC |
0.07 |
Negative |
1 and 0.90 |
1 and 0 |
1 and 1 |
3 |
Cetyl alcohol |
CCCCCCCCCCCCCCCCO |
-0.06 |
Negative |
1 and 1 |
N/A and NA |
1 and 1 |
4 |
Isostearyl alcohol |
CC(C)CCCCCCCCCCCCCCCO |
-0.09 |
Negative |
1 and 1 |
N/A and NA |
1 and 1 |
5 |
Stearic acid/potassium stearate |
CCCCCCCCCCCCCCCCCC(=O)O |
-0.06 |
Negative |
1 and 1 |
N/A and NA |
1 and 1 |
6 |
Cetyl stearate |
CCCCCCCCCCCCCCCCCC(=O)O(CCCCCCCCCCCCCCCC) |
0 |
Negative* |
- |
- |
- |
7 |
Cetyl Isostearate |
CC(C)CCCCCCCCCCCCCCC(=O)O(CCCCCCCCCCCCCCCC) |
0.03 |
Negative |
1 and 0.90 |
1 and 0 |
1 and 1 |
8 |
Isostearyl Isostearate** |
CC(C)CCCCCCCCCCCCCCC(=O)O(CCCCCCCCCCCCCCCC(C)C) |
0.06 |
Negative |
1 and 0.90 |
1 and 0 |
1 and 1 |
9 |
Isostearyl stearate |
CC(C)CCCCCCCCCCCCCCCOC(=O)CCCCCCCCCCCCCCCCC |
0.06 |
Negative |
1 and 0.90 |
1 and 0 |
1 and 1 |
N/A: Not available
*Experimental result available. So no need to perfprm domain evaluation.
In general, if the concordance is greater than or equal to 0.8, the model is considered to be valid. In addition both the leave-one-out sensitivity and specificity must be at least 0.5 to avoid using models which are heavily biased to predict either active or inactive scores.
**representative component of the 'esters of C18 (branched and linear) fatty acids with C18 (branched and linear alcohols)'
For more details on results, kindly refer the attached background material section of the IUCLID.
Results
| Constituents | Name | SMILES | VEGA - Mutagenicity (Ames test) model (CAESAR) 2.1.13 | Global Applicability domain Index | ID/OD |
| 1 | mono- C16 PSE, K+ and mono-C16 PSE | CCCCCCCCCCCCCCCCOP(O)(O)=O | Non-mutagenic | 0.93 | ID |
| 2 | di- C16 PSE, K+ and di- C16 PSE | CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC | Non-mutagenic | 0.935 | ID |
| 3 | Cetyl alcohol | CCCCCCCCCCCCCCCCO | Non-mutagenic | 0.882 | Could be OD |
| 4 | Isostearyl alcohol | CC(C)CCCCCCCCCCCCCCCO | Non-mutagenic | 0.877 | Could be OD |
| 5 | Stearic acid/potassium stearate | CCCCCCCCCCCCCCCCCC(=O)O | Non-mutagenic* | 1 | ID |
| 6 | Cetyl stearate | CCCCCCCCCCCCCCCCCC(=O)O(CCCCCCCCCCCCCCCC) | Non-mutagenic | 0 | OD |
| 7 | Cetyl Isostearate | CC(C)CCCCCCCCCCCCCCC(=O)O(CCCCCCCCCCCCCCCC) | Non-mutagenic | 0 | OD |
| 8 | Isostearyl Isostearate* | CC(C)CCCCCCCCCCCCCCC(=O)O(CCCCCCCCCCCCCCCC(C)C) | Non-mutagenic | 0 | OD |
| 9 | Isostearyl stearate | CC(C)CCCCCCCCCCCCCCCOC(=O)CCCCCCCCCCCCCCCCC | Non-mutagenic | 0 | OD |
| ID = In domain | OD = Out of domain | *Experimental result |
*representative component of the 'esters of C18 (branched and linear) fatty acids with C18 (branched and linear alcohols)'
For more detailed prediction results and applicability domain analysis of models, kindly refer the attached background material section of the IUCLID.
Result
Preliminary Cytotoxicity Test
The dose range of the test item used in the preliminary toxicity test was 0.63 to 160 μg/mL. There was evidence of very modest dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in both of the exposure groups. Precipitate of the test item was observed at and above 40 μg/mL in both the absence and presence of metabolic activation at the end of the exposure period. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose levels in the subsequent mutagenicity test were limited by the onset of test item precipitate in both the absence and presence of metabolic activation.
Mutagenicity Test
There was no evidence of any toxicity, in either the absence or presence of metabolic activation, following exposure to the test item, as indicated by the %RSG and RTG values. There was also no evidence of any marked reductions in cloning efficiency (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Test item precipitate was observed at 80 μg/mL in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances. The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test item did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.
Conclusion
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.
Results
Preliminary toxicity test
The dose range for the preliminary toxicity test was 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL.The maximum dose was the maximum achievable dose level due to formulation difficulties. A precipitate of the test substance was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 µg/mL in the 4(20)-h exposure group in the absence of S9, at and above 125 µg/mL in the presence of S9, and at and above 62.5 µg/mL in the continuous exposure group. Precipitate from the test substance was also noted on the slides at 1000 µg/mL in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1000 µg/mL in all three exposure groups. The maximum dose selected for mitotic index analysis was limited to 125 µg/mL based on the precipitate observations.The test substance induced no evidence of toxicity in any of the exposure groups. The selection of the maximum dose level for the main experiment was based on the lowest precipitating dose level and was 125 µg/mL for the 4(20)-h exposure groups and for the continuous exposure group.
Table 1: Mitotic Index - Preliminary toxicity test
Dose Level (µg/mL) |
4(20)-h Without S9 |
4(20)-h With S9 |
24-h Without S9 |
|||
Mitotic Index |
% of Control |
Mitotic Index |
% of Control |
Mitotic Index |
% of Control |
|
0 |
8.35 |
100 |
10.55 |
100 |
16.25 |
100 |
3.91 |
- |
- |
- |
- |
- |
- |
7.81 |
6.65 |
80 |
- |
- |
- |
- |
15.63 |
8.10 |
97 |
7.55 |
72 |
17.55 |
108 |
31.25 |
17.05 P |
204 |
6.95 |
66 |
14.35 |
88 |
62.5 |
- P |
- |
8.80 |
83 |
13.30 P |
82 |
125 |
8.35 P |
100 |
9.90 P |
94 |
13.95 P |
86 |
250 |
- P |
- |
- P |
- |
- P |
- |
500 |
- P |
- |
- P |
- |
- P |
- |
1000 |
- P† |
- |
- P† |
- |
- P† |
- |
- = Not assessed for mitotic index
P = Precipitate observed at end of exposure period in blood-free cultures
† = Precipitate observed on the slides
Chromosome aberration test – main experiment
The qualitative assessment of the slides determined that there was no marked toxicity as in the preliminary toxicity test and that there were metaphases suitable for scoring present up to 125 µg/mL in all three exposure groups. Precipitate observations were made at the end of exposure in blood-free cultures and precipitate was noted at and above 62.5 µg/mL in the absence of S9 and at and above 31.25 µg/mL in the presence of S9. The mitotic index data for the Main Experiment confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the three exposure groups. The maximum dose level selected for metaphase analysis was the lowest precipitating dose level in each of the exposure groups and was 62.5 µg/mL in the absence of S9 and 31.25 µg/mL in the presence of S9.
Table 2: Mitotic Index – Main experiment(4(20)-h Exposure Groups)
Dose Level (mg/mL) |
4(20)-h Without S9 |
4(20)-h With S9 |
||||||
A |
B |
Mean |
% of Control |
A |
B |
Mean |
% of Control |
|
0 |
9.75 |
11.70 |
10.73 |
100 |
5.95 |
5.05 |
5.50 |
100 |
3.91 |
- |
- |
- |
- |
- |
- |
- |
- |
7.81 |
- |
- |
- |
- |
5.25 |
6.20 |
5.73 |
104 |
15.63 |
9.75 |
10.30 |
10.03 |
93 |
7.25 |
4.35 |
5.80 |
105 |
31.25 |
11.15 |
9.75 |
10.45 |
97 |
4.95 P |
5.20 P |
5.08 |
92 |
62.5 |
12.05 P |
11.05 P |
11.55 |
108 |
- P |
- P |
- |
- |
125 |
- P |
- P |
- |
- |
- P |
- P |
- |
- |
MMC 0.4 |
- P |
- P |
- |
- |
NA |
NA |
NA |
NA |
CP 2 |
NA |
NA |
NA |
NA |
4.25 |
3.35 |
3.80 |
69 |
MMC = Mitomycin C
CP = Cyclophosphamide
P = Precipitate observed at end of exposure period in blood-free cultures
NA = Not applicable
- = Not assessed for mitotic index
Table 3: Mitotic Index – Main experiment (24-h Exposure Group)
Dose Level (µg/mL) |
24-h Without S9 |
|||
A |
B |
Mean |
% of Control |
|
0 |
14.20 |
13.65 |
13.93 |
100 |
3.91 |
- |
- |
- |
- |
7.81 |
- |
- |
- |
- |
15.63 |
10.90 |
12.45 |
11.68 |
84 |
31.25 |
9.95 |
10.30 |
10.13 |
73 |
62.5 |
14.35 P |
18.10 P |
16.23 |
117 |
125 |
- P |
- P |
- |
- |
MMC 0.1 |
7.75 |
5.70 |
6.73 |
48 |
Table 4: Main experiment (4-h treatment, 24 h Harvest) without metabolic treatment
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of Cells Scored |
Number of Aberrations |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
|||||||
Gaps |
Chromatid |
Chromosome |
Others |
||||||||||
Breaks |
Exchanges |
Breaks |
Exchanges |
X |
(+ Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
|||||
Vehicle Control (MEM) |
A |
9.75 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
11.70 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
21.45 |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
(100) |
(0.0) |
(0.0) |
|||||||||||
A |
9.75 |
150 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
|
15.63 |
B |
10.30 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
20.05 |
300 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
(93) |
(0.3) |
(0.0) |
|||||||||||
A |
11.15 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
31.25 |
B |
9.75 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
20.90 |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
(97) |
(0.0) |
(0.0) |
|||||||||||
A |
12.05 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
62.5 |
B |
11.05 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
23.10 |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
(108) |
(0.0) |
(0.0) |
|||||||||||
Positive Control |
A |
5.90 |
96a |
4 |
8 |
9 |
2 |
0 |
0 |
23 |
19 |
22 |
18 |
MMC 0.4 |
B |
7.60 |
46a |
0 |
10 |
11 |
1 |
0 |
0 |
22 |
22 |
15 |
15 |
µg/mL |
Total |
13.50 |
142 |
4 |
18 |
20 |
3 |
0 |
0 |
45 |
41 |
37 |
33*** |
(63) |
(26.1) |
(23.2) |
|||||||||||
MMC - Mitomycin C
a - Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed
*** - P < 0.001
MEM - Minimal Essential Medium
Table 5: Main experiment (4-h treatment, 24 h Harvest) with metabolic treatment
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of Cells Scored |
Number of Aberrations |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
|||||||
Gaps |
Chromatid |
Chromosome |
Others |
||||||||||
Breaks |
Exchanges |
Breaks |
Exchanges |
X |
(+ Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
|||||
Vehicle Control (MEM) |
A |
5.95 |
150 |
0 |
5 |
0 |
0 |
0 |
0 |
5 |
5 |
4 |
4 |
B |
5.05 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
11.00 |
300 |
0 |
5 |
0 |
0 |
0 |
0 |
5 |
5 |
4 |
4 |
|
(100) |
(1.3) |
(1.3) |
|||||||||||
A |
5.25 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
7.81 |
B |
6.20 |
150 |
0 |
3 |
0 |
1 |
0 |
0 |
4 |
4 |
4 |
4 |
µg/mL |
Total |
11.45 |
300 |
0 |
3 |
0 |
1 |
0 |
0 |
4 |
4 |
4 |
4 |
(104) |
(1.3) |
(1.3) |
|||||||||||
A |
7.25 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
15.63 |
B |
4.35 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
11.60 |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
(105) |
(0.0) |
(0.0) |
|||||||||||
A |
4.95 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
31.25 |
B |
5.20 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
10.15 |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
(92) |
(0.0) |
(0.0) |
|||||||||||
Positive Control |
A |
4.25 |
104a |
0 |
17 |
2 |
0 |
0 |
0 |
19 |
19 |
15 |
15 |
CP 2 |
B |
3.35 |
109a |
1 |
11 |
5 |
1 |
0 |
0 |
18 |
17 |
16 |
15 |
µg/mL |
Total |
7.60 |
213 |
1 |
28 |
7 |
1 |
0 |
0 |
37 |
36 |
31 |
30*** |
(69) |
(14.6) |
(14.1) |
|||||||||||
Table 6: Main experiment (24-h treatment) without metabolic treatment
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of Cells Scored |
Number of Aberrations |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
|||||||
Gaps |
Chromatid |
Chromosome |
Others |
||||||||||
Breaks |
Exchanges |
Breaks |
Exchanges |
X |
(+ Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
|||||
Vehicle Control (MEM) |
A |
14.20 |
150 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
2 |
1 |
B |
13.65 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
27.85 |
300 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
2 |
1 |
|
(100) |
(0.7) |
(0.3) |
|||||||||||
A |
10.90 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
15.63 |
B |
12.45 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
23.35 |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
(84) |
(0.0) |
(0.0) |
|||||||||||
A |
9.95 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
31.25 |
B |
10.30 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
20.25 |
300 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
(73) |
(0.3) |
(0.3) |
|||||||||||
A |
14.35 |
150 |
0 |
0 |
0 |
2 |
0 |
0 |
2 |
2 |
2 |
2 |
|
62.5 |
B |
18.10 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
µg/mL |
Total |
32.45 |
300 |
0 |
0 |
0 |
2 |
0 |
0 |
2 |
2 |
2 |
2 |
(117) |
(0.7) |
(0.7) |
|||||||||||
Positive Control |
A |
7.75 |
45a |
4 |
24 |
1 |
3 |
0 |
0 |
32 |
28 |
19 |
18 |
MMC 0.1 |
B |
5.70 |
93a |
2 |
14 |
1 |
2 |
0 |
0 |
19 |
17 |
15 |
15 |
µg/mL |
Total |
13.45 |
138 |
6 |
38 |
2 |
5 |
0 |
0 |
51 |
45 |
34 |
33*** |
(48) |
(24.6) |
(23.9) |
|||||||||||
Table 7: Mean Frequency of Polyploid Cells (%)
Dose Level (µg/mL) |
Exposure Group |
||
4(20)-h Without S9 |
4(20)-h With S9 |
24-h Without S9 |
|
0 |
0 |
0 |
0 |
7.81 |
- |
0 |
0 |
15.63 |
0 |
0 |
0 |
31.25 |
0 |
0 |
0 |
62.5 |
0 |
0 |
- |
MMC 0.4 |
0 |
NA |
NA |
MMC 0.1 |
NA |
NA |
0 |
CP 5 |
NA |
0 |
NA |
Validity of assay
The assay was considered valid as it met all of the following criteria:
1) The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
2) All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
3) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
4) The required number of cells and concentrations were analyzed.
The test substance did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. Thetest substancedid not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.
Table 1: results of range-finding assay
Metabolic activation |
Test substance concentration (μg/mL) |
Mitotic index (%) |
|
|
|
24 h harvesting time |
48 h harvesting time |
- |
Vehicle control (DMSO) |
100 |
100 |
- |
10 |
91 |
98 |
- |
33 |
86 |
98 |
- |
100 |
91 |
105 |
- |
333 |
93 |
105 |
- |
1000 |
100 |
91 |
+ |
Vehicle control (DMSO) |
100 |
|
+ |
10 |
92 |
- |
+ |
33 |
90 |
- |
+ |
100 |
94 |
- |
+ |
333 |
98 |
- |
+ |
1000 |
94 |
- |
Table 2: Chromosome aberrations, summarised data, experiment 1
Metabolic activation |
Test substance concentration (μg/mL) |
Total number of metaphases analysed |
Total number of aberrations1 |
Number of cells with aberrations1 |
Mitotic index (%)2
|
||
|
|
|
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
24 h treatment, 24 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
1 |
1 |
1 |
1 |
100 |
- |
100 |
200 |
7 |
6 |
6 |
5 |
98 |
- |
333 |
200 |
1 |
1 |
1 |
1 |
106 |
- |
1000 |
200 |
3 |
2 |
3 |
2 |
106 |
- |
mitomycin C |
200 |
41 |
37 |
38*** |
34*** |
61 |
48 h treatment, 48 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
1 |
1 |
1 |
1 |
100 |
- |
1000 |
200 |
1 |
1 |
1 |
1 |
81 |
- |
mitomycin C |
150 |
72 |
65 |
59*** |
55*** |
49 |
3 h treatment, 24 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
4 |
2 |
4 |
2 |
100 |
+ |
100 |
200 |
4 |
4 |
4 |
4 |
68 |
+ |
333 |
200 |
0 |
0 |
0 |
0 |
81 |
+ |
1000 |
200 |
1 |
1 |
1 |
1 |
73 |
+ |
cyclophosphamide |
200 |
89 |
75 |
71*** |
63*** |
33 |
3 h treatment, 48 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
9 |
9 |
5 |
5 |
100 |
+ |
1000 |
200 |
1 |
1 |
1 |
1 |
81 |
1gaps include chromatid and isochromatid (chromosome) gaps
2percentage of metaphases per 1000 cells, compared to control
* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)
Table 3: Chromosome aberrations, summarised data, experiment 2
Metabolic activation |
Test substance concentration (μg/mL) |
Total number of metaphases analysed |
Total number of aberrations1 |
Number of cells with aberrations1 |
Mitotic index (%)2
|
||
|
|
|
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
24 h treatment, 24 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
0 |
0 |
0 |
0 |
100 |
- |
100 |
200 |
2 |
2 |
2 |
2 |
99 |
- |
333 |
200 |
4 |
4 |
4 |
4 |
113 |
- |
1000 |
200 |
1 |
1 |
1 |
1 |
97 |
- |
mitomycin C |
150 |
63 |
63 |
53*** |
53*** |
39 |
3 h treatment, 24 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
3 |
3 |
3 |
3 |
100 |
+ |
100 |
200 |
2 |
2 |
2 |
2 |
108 |
+ |
333 |
200 |
2 |
2 |
2 |
2 |
101 |
+ |
1000 |
200 |
2 |
2 |
2 |
2 |
104 |
+ |
Cyclophosphamide |
200 |
111 |
109 |
80*** |
79*** |
32 |
1gaps include chromatid and isochromatid (chromosome) gaps
2percentage of metaphases per 1000 cells, compared to control
* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)
Table 1 Cytogenicity: 7 hour fixation. Aberrations in 200 cells
Activation |
Concentration µg/mL |
Percent aberrant cells |
||
incl gaps |
excl gaps |
exchanges |
||
Without |
0* |
4.0 |
1.5 |
0 |
20 |
2.5 |
0.5 |
0 |
|
With |
0* |
4.0 |
1.5 |
0 |
20 |
7.0 |
2.5 |
0 |
|
* Solvent control with ethanol
** Only 100 cells counted for positive controls
Table 2 Cytogenicity: 18 hour fixation. Aberrations in 200 cells
Activation |
Concentration µg/mL |
Percent aberrant cells |
||
incl gaps |
excl gaps |
exchanges |
||
Without |
Negative control |
5.5 |
1.5 |
0 |
0* |
4.0 |
1.5 |
0.5 |
|
0.6 |
4.5 |
2.0 |
0 |
|
10 |
4.0 |
1.0 |
0.5 |
|
20 |
3.0 |
0.5 |
0 |
|
Positive control** |
12.0 |
9.0 |
4.0 |
|
With |
Negative control |
2.5 |
1.5 |
0 |
0* |
2.5 |
1.5 |
0.5 |
|
0.6 |
5.5 |
3.0 |
0.5 |
|
10 |
4.0 |
2.5 |
0 |
|
20 |
4.0 |
2.5 |
0.5 |
|
Positive control** |
16.0 |
13.0 |
5.5 |
|
* Solvent control with ethanol
** Only 100 cells counted for positive controls
Table 3 Cytogenicity: 18 hour fixation. Aberrations in 200 cells
Activation |
Concentration µg/mL |
Percent aberrant cells |
||
incl gaps |
excl gaps |
exchanges |
||
Without |
0* |
6.0 |
2.5 |
0.5 |
20 |
3.5 |
2.0 |
0 |
|
With |
0* |
1.0 |
0.5 |
0 |
20 |
4.0 |
2.5 |
0.5 |
|
* Solvent control with ethanol
** Only 100 cells counted for positive controls
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test:
Study 1: The mutagenicity potential of the test substance 'Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide' (UVCB) was predicted using the Mutagenicity (Ames test) model (CAESAR) 2.1.13 of the VEGA v1.2.4 program. Since the test substance is a UVCB, the mutagenicity potential was predicted for all the individual constituents, using SMILES as the input parameter. All the constituents were predicted to be negative for mutagenicity (VEGA, 2019), indicating that the test substance can be overall considered to be non-mutagenic.The applicability domain of predictions is assessed using an Applicability Domain Index (ADI) that has values from 0 (worst case) to 1 (best case). A global ADI is calculated by grouping several other indices such as similar molecules with known experimental value, accuracy of prediction for similar molecules, concordance for similar molecules, model's descriptors range check and atom centered fragments similarity checks. Usually values lower than 0.75 indicate that the similar compound has important differences compared to the target. Considering that not all constituents were in domain, the overall predictions can be considered to be reliable with restrictions.
Study 2: The mutagenicity potential of the test substance 'Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide' (UVCB) was predicted using the Consensus method of the T.E.S.T. v4.2.1 program. Since the test substance is a UVCB, the mutagenicity potential was predicted for all the individual constituents, using SMILES as the input parameter. All the constituents were predicted to be negative for mutagenicity (US EPA, 2019), indicating that the test substance can be overall considered to be non-mutagenic. Applicability domain evaluation was performed by checking the descriptor and structural fragment domains of the individual QSAR methods (i.e., FDA, hierarchical clustering and nearest neighbour methods) underlying Consensus model predictions. Since an experimental value could be identified for cetyl stearate, no domain evaluation was necessary for it. The domain evaluation of the remaining all constituents indicated that they were within both descriptor and structural fragment domains of the FDA and hierarchical clustering methods, but not completely within domain for the structural fragments identified for the three nearest neighbours. Further, the prediction accuracy of the binary toxicity endpoints, can be evaluated in terms of the fraction of compounds that are predicted accurately and are described based on three statistics parameters: concordance, sensitivity, and specificity. In general, if the concordance is greater than or equal to 0.8, the model is considered to be valid. And both the leave-one-out sensitivity and specificity must be at least 0.5 to avoid using models which are heavily biased to predict either active or inactive scores. Out of three statistical parameters, the concordance and specificity fractions of all the constituents are above the required cut-offs. For sensitivity, effectively only one chemical was identified to be experimentally active but not predicted correctly for constituent 1. Given the structural differences of this chemical compared to the target, the overall accuracy of the model and its prediction is not expected to be compromised. Therefore, the predictions can be overall considered to be accurate with medium to high confidence.
Based on the negative mutagenicity predictions for the constituents using the QSAR models, the test substance is overall considered to be non-mutagenic.
Mammalian cell gene mutation assay:
An in vitro study was conducted to determine the mutagenic potential of the test substance on thymidine kinase, TK +/-, locus of L5178Y mouse lymphoma cells, according to the OECD Test Guideline 490, in compliance with GLP. L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) cells were counted and processed to give 1 x 10E7 cells/mL in 10 mL aliquots in test medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate, both with and without metabolic activation (2% S9 final concentration) at eight dose levels (0, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL) in duplicate, both the absence and presence of metabolic activation (2% S9), solvent and positive controls for 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9). The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood. There was no evidence of any toxicity, in either the absence or presence of metabolic activation, following exposure to the test substance, as indicated by the %RSG and RTG values. There was also no evidence of any marked reductions in cloning efficiency in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Test substance precipitate was observed at 80 μg/mL in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances. The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline. Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y mouse lymphoma cells at the TK +/- locus, in the absence and presence of metabolic activation (Willey, 2022).
Chromosomal aberration assay:
In absence of clastogenicity assay with the test substance, the endpoint has been assessed based on studies for read across substances representative of the main constituents, which can be categorised as phosphate esters (PSE i.e., mono- and di C16 PSE, K+: 10 -40%), alkyl esters (i.e., C16-18 linear and branched fatty acid esters: 39 -87%) and alcohols (i.e., C16 -18 linear or branched alcohol: 10 -30%). The results are presented below:
Constituent: PSE - read across study:
An in vitro study was conducted to determine the clastogenicity of the read across substance, 'mono- and di- C16 PSE, K+ and H3PO4', using human lymphocytes, according to OECD Guideline 473 (Chromosome Aberration test), in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the read across substance, were evaluated for chromosome aberrations at doses 0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL, together with vehicle and positive controls. In this study, three exposure conditions were investigated: 4 h exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-h expression period, 4 h exposure in the absence of metabolic activation (S9) with a 20-h expression period and a 24-h exposure in the absence of metabolic activation. The dose levels used in the main experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances (Mitomycin C and Cyclophosphamide) induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The read across substance was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. Under the conditions of the study, the read across substance was considered to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation. (Envigo, 2017). Based on the results of the read across study, similar absence of clastogenicity can be considered for the phosphate ester constituent of the test substance.
Constituent: Alkyl ester - read across study:
An in vitro study was conducted to determine the clastogenicity of the read across substance, ‘Octyldodecyl isooctadecanoate' (purity: 100%), using human lymphocytes , according to the OECD Guideline 473, in compliance with GLP. Cultures of human lymphocytes were treated with the read across substance and were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Two experiments under different exposure conditions were investigated in the study: Experiment 1 (a) 24 h exposure in the absence of an induced rat liver homogenate metabolizing system (S9), with cell harvest after a 24 h expression period in absence of metabolic activation (at test concentrations of 100, 333 and 100 μg/mL), (b) 48 h exposure in the absence of metabolic activation (S9), with a 48 h expression period in absence of metabolic activation (at test concentrations of 1000 μg/mL), (c) a 3 h exposure in the presence of metabolic activation, with 24 h harvest period in presence of metabolic activation (at test concentrations of 100, 333 and 1000 μg/mL) and (d) a 3 h exposure in the presence of metabolic activation with 48 h harvest period in presence of metabolic activation (at test concertation of 1000 μg/mL). Experiment 2 (a) a 24 h treatment in absence of metabolic activation with 24 h harvest period in absence of metabolic activation (at test concentration of 100, 333 and 1000 μg/mL) and (b) 3 h exposure in presence of metabolic activation with 24 h harvest period in presence of metabolic activation (at test concentration 100, 333 and 1000 μg/mL). The dose levels used in the main experiment (mentioned above) were selected using data from the preliminary toxicity test (using 10, 33, 100, 333 and 1000 µg/mL, with and without metabolic activation), where the results indicated that the maximum concentration should be limited to 1000 µg/mL, due to precipitation. The incidences of aberrant cells in the vehicle (dimethyl sulphoxide (DMSO)) control group were within the range expected for normal human lymphocytes. All the positive control substances (i.e., Mitomycin C and Cyclophosphamide) induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The read across substance was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations. Under study conditions, the read across substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (Bertens, 1998).Based on the results of the read across study, a similar absence of genotoxicity in the chromosomal aberration assay can be expected for the ester constituent of the test substance.
Constituent: Alcohol - read across study:
An in vitro study was conducted to determine the genotoxicity of the read across substance, behenyl alcohol (C22) using Chinese hamster lung fibroblasts (V79), according to a method similar to the OECD Guideline 473. Chinese hamster lung fibroblasts (V79) in duplicates were incubated with 0.6, 10.0 and 20.0 µg/mL of read across substance dissolved in ethanol for 4 h, with and without metabolic activation. There was no evidence of cytotoxicity at this dose level. At 20 µg/mL, mitotic index as well as plating efficiency were not reduced. The read across substance did not increase the incidence of chromosome aberrations in Chinese hamster V79 cells in the presence or absence of metabolising fraction at concentrations up to 20 µg/mL. The negative and positive controls were considered to be valid. Under the study conditions, the read across substance was determined to be non-genotoxic with and without metabolic activation (Iglesias, 2002). Based on the results of the read across study, similar absence of clastogenic potential can be considered for the alcohol constituent of the test substance.
Overall based on the available weight of evidence from in vitro genotoxicity assays with the test substance or substances representative of its main constituents, the test substance, ‘Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide’ is considered to be non-genotoxic.
Justification for classification or non-classification
Based on the available weight of evidence from in vitro genotoxicity assays with the test substance or substances representative of its main constituents, the test substance does not warrant a classification for mutagenicity according to the EU CLP criteria (1272/2008/EC).
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