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Registration Dossier
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EC number: 640-964-5 | CAS number: 218451-68-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Coconut oil, reaction products with polyethylene glycol and trimethylolpropane has been assessed for genetic toxicity in several in vitro systems, all studies were conducted according to OECD test guidelines and GLP. In vitro studies in bacteria (Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537) and mouse lymphoma L5178Y cells have demonstrated no genetic toxicity of Coconut oil, reaction products with polyethylene glycol and trimethylolpropane when tested in the presence or absence of exogenous metabolic activation. A Chromosomal aberration study in Chinese Hamster Ovary cells demonstrated weak effects only in the presence of exogenous metabolic activation at a concentration deemed toxic to the cells.
Link to relevant study records
- 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:
- October - November 1996
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 or Phenobarbital/beta-Naphthoflavone induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Plate incorporation test:
50 / 160 / 500 / 1600 / 5000 µg/plate with all strains with and without S9 mix
30 / 100 / 300 / 100 / 3000 µg/plate, repetition with strain TA 98 without S9 mix (due to failure of positive control and toxicity)
10 / 30 / 100 / 300 / 100 µg/plate, repetition with strain TA 1537 with S9 mix (due to toxicity)
1 / 3 / 10 / 30 / 100 µg/plate, repetition with strain 1537 without S9 mix (due to failure of positive control and toxicty)
Preincubation test:
10 / 30 / 100 / 300 / 1000 µg/plate with strain TA 98 with and without S9 mix
30 / 100 / 300 / 100 / 3000 µg/plate with strain TA 100 with and without S9 mix
16 / 50 / 160 / 500 / 1600 µg/plate with strain TA 1535 with and without S9 mix
10 / 30 / 100 / 300 / 1000 µg/pklate with strain TA 1537 with S9 mix
0.1 / 0.3 / 1 / 3 / 10 µg/plate with strain TA 1537 without S9 mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: not mentioned - Untreated negative controls:
- yes
- Remarks:
- water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone (25 µL/plate)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Nitrofluorene (2.5 µg in DMSO/plate) for the strain TA 98; Sodium azide (5.0 µg in aqua bidest./plate) for strain TA 100; Sodium azide (2.5 µg in aqua bidest./plate) for strain TA 1535; 9-Aminoacredine (25 µg in DMSO/plate) for strain TA 1537
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone (25 µL/plate)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Aminoanthracene (2.5 µg in DMSO/plate) with all strains
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and preincubation, performed in two independent tests
DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 per concentration
DETERMINATION OF CYTOTOXICITY
- Method: examination of bacterial background lawn (reduced or absent), a reduction in the number of spontaneous revertants was jugded as also indicative of toxicity
OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (acetone), test substance concentrations and positive controls; determination of the titers of overnight cultures
OTHER: none - Evaluation criteria:
- Criteria for determination of a valid test:
- in the the solvent control, each tester strain culture must exhibit a characteristic meean number of spontanious revertants
- to ensure that appropriate numbers of bacteria are plated, overnight culture titers must be in excess of 10E8 bacteria/mL - Statistics:
- no statistics performed
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- MA, resp. at 5000 µg/plate with S9 in the plate incorporation test
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate with and without S9 in the plate incorporation test
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate with and without S9 in the plate incorporation test
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1600 µg/plate and above with S9, resp. 100 µg/plate and above without S9 in the plate incorporation test
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not mentioned
- Precipitation: no
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES: not performed
COMPARISON WITH HISTORICAL CONTROL DATA: not performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the plate incorporation test, treatment with 50 - 5000 µg/plate was associated with excess toxicity to strain TA 1537. This part of the test was repeated with lower test subtance concentrations. In the preincubation test,all strains were exposed to lower, toxicity adjusted test substance concentrations. - Remarks on result:
- other: strain/cell type: Salmonella typhimurium
- Conclusions:
- In the plate incorporation test without metabolic activation with strain TA 1535 treatment with 5000 µg/plate Coconut oil, reaction product with polyethylene glycol and trimethylolpropane resulted in an increased revertant frequency (factor 4.2) but accompanied with toxicity. Therefore, this mutagenic response was jugded by the author of the study to be of no relevance. In the preincubation test, all tester strains were exposed to lower, toxicity adjusted test compound concentratons. With the above mentioned exception, treatment with the test compound did not result in a significant increase in the revertant frequency of the tester strains. All four tester strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activaton by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable.
Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was jugded not to be a bacterial mutagen. - Executive summary:
Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was tested for its ability to induce reverse mutation in an in vitro bacterial system.
Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were treated with the test compound by the Ames test plate incorporation as well as the preincubation method. Five dose levels covering a total range between 0.1 and 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Aroclor 1254 or Phenobarbital/beta-Naphthoflavone induced rat liver S9 mix) were employed.
All four bacterial strains exhibited mutagenic responses to the appropriate positive control substance. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range.
A reproducible mutagenic activity of the test compound to any of the tester strains TA 98, TA 100, TA 1535 or TA 1537 was not observed with and without metabolic activation.
It is therefore concluded, that Coconut oil, reaction products with polyethylene glycol and trimethylolpropane is not a bacterial mutagen.
- 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:
- September - October 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Well-documented guideline study according to GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Ham's F-10 containing HEPES buffer and supplemented with minocycline (basic medium, medium for treatment in the presence of S9 mix and for washing cultures before or after treatment), basic medium with 10% (v/v) foetal bovine serum (medium for cell growth and treatment in the absence of S9 mix)
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- other: Modal chromosome number: 21, generation time ca 12 - 14 h
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from S9 fraction obtained from male rats dosed with Arochlor 1254
- Test concentrations with justification for top dose:
- 20, 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/ml
- Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: with S9 mix: cyclophosphamid (CP); without S9 mix: methyl methanesulphonate (MMS)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: -S9/+S9: 6 hours
- Fixation time (start of exposure up to fixation or harvest of cells): -S9/+S9: 24 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: two cultures per dose level, controls and vehicle control
NUMBER OF CELLS EVALUATED: 200 metaphases per dose level (100 per replicate)
DETERMINATION OF CYTOTOXICITY
- Method: Slides were examined for evidence of metaphase cells and signs of cellular necrosis. From the cell counts, the number if cells recovered per culture, was calculated. This was then compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Other: The osmotic pressure of selected concentrations of the test substance was measured; observations of precipitation were made at the end of the treatment period. - Evaluation criteria:
- The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historical negative control data.
The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99% confidence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate.
A test was positive if the response in at least one acceptable dose level was significant by the criterion described above.
A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence limits).
Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive. - Statistics:
- No statistical analysis was performed.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- for details see table 2 below
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- for details see table 1 below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- for details see table 2 below
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- for details see table 1 below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the colour of the medium was not changed by the test substance indicating no change of the pH
- Effects of osmolality: effects on osmotic pressure only at toxic concentrations (at 5000 µg/mL, both in the presence and absence of S9 mix)
- Precipitation: precipitation was noted at dose levels of 156-5000 µg/ml in both the presence and absence of S9 mix
RANGE-FINDING/SCREENING STUDIES: In the presence of S9 mix, toxicity was observed in the cultures treated with 156-5000 µg/mL. In the cultures treated with 625 µg/mL and above, there were no metaphase cells for assessment. There were reduced cell counts (below 50% of the vehicle control cultures) in the cultures treated with 313 µg/mL (mean cell count of 36% compared to the vehicle control cultures). The concentration of 156 µg/mL was deemed toxic to the cells from culture and slide observation.
In the absence of S9 mix, toxicity was observed in the cultures treated with 313-5000 µg/mL. In the cultures treated with 625 µg/mL and above, there were no metaphase cells for assessment. There were reduced cell counts (below 50% of the vehicle control cultures) in the cultures treated with 313 µg/mL (mean cell couant of 28% compared to the vehicle control cultures).
COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures had levels of structural and numerical aberration within the 95% confidence limits of the historical negative control data.
OTHER: Due to the positive response observed in the first test, it was not deemed necessary to conduct a second test. - Conclusions:
- It was concluded that Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was a weak clastogen when tested with Chinese hamster ovary cells in vitro in the presence of S9 mix at a concentration deemed toxic to the cells (313 µg/mL)
- Executive summary:
A chromosomal aberrations assay was performed with the test item Coconut oil, reaction products with polyethylene glycol and trimethylolpropane in duplicate. Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls.
The test was conducted in the presence and absence of S9 mix (exogenous source of metabolic activation). S9 is a post-mitochondrial supernatant fraction prepared from the livers of adult, male rats treated with Aroclor 1254. S9 mix is S9 and a NADPH-generating system.
Cultures, established approx. 20 h before testing, were treated for 6 h in the presence and absence of S9 mix. Cultures were harvested at 24 h post treatment.
Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix. It was tested up to the maximum permitted concentration of 5000 µg/mL. Toxicity was noted at 156 -5000 µg/mL in the presence of S9 mix and at 313 -5000 µg/mL in the absence of S9 mix.
Coconut oil, reaction products with polyethylene glycol and trimethylolpropane induced structural chromosomal aberrations in the presence of S9 mix and was non clastogenic in the absence of S9 mix.
In conclusion, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was a weak clastogen when tested with Chinese hamster ovary cells in vitro in the presence of S9 mix at a concentration deemed toxic to the cells (313 µg/mL).
- 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:
- September 10 to November 10, 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: well documented study performed according to OECD Guideline and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 medium supplemented with penicillin, streptomycin, sodium bicarbonate and pluronic acid (basic culture medium), R0P supplemented with 5% v/v heat-inactivated horse serum (R5P) (medium used during treatment for 4 h), R0P supplemented with 10% v/v heat-inactivated horse serum (R10P) (medium used during treatment for 24h), R0P supplemented with heat-inactivated horse serum (20% v/v), sodium pyruvate, and amphotericin B (fungizone) (cloning medium), cloning medium supplemented with trifluorothymidine (FT) (medium for selection of tk-tk- cells)
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: Generation time of about 11 h, stable near diploid chromosome number
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 mix of Arochlor 1254-induced male rats
- Test concentrations with justification for top dose:
- Toxicity test (range finding): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL both in the absence and presence of S9 mix
Assay 1 (in the absence of S9 mix, 4h exposure period): 45, 60, 90, 135, 195, 270, 360 and 465 µg/ml
Assay 2 (in the presence of S9 mix, 4h exposure period): 45, 60, 90, 135, 195, 270, 360 and 465 µg/ml
Assay 3 (in the absence of S9 mix, 24h exposure period): 30, 60, 90, 120, 150, 180, 210 and 240 µg/mL
Assay 4 (in the presence of S9 mix, 4h exposure period): 120, 150, 180, 210, 240, 270, 300 and 330 µg/mL
Each dose range selection was based on all available results at the time the selection was made. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not mentioned - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: in the absence of metabolic activation (4 h/24h exposure): ethyl methanesulphonate (EMS, 250/150 µg/ml) and methyl methanesulphonate (MMS, 10/5 µg/ml); in the presence of metabolic activation: 3-methylcholanthrene (3-MC, 2.5 and 10 µg/ml)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Assay 1 (without S9 mix): 4 h
Assay 2 (with S9 mix): 4 h
Assay 3 (without S9 mix): 24 h
Assay 4 (with S9 mix): 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): at least 12 days (mutation selection assay), at least 9 days (cloning efficienty assay)
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: 2 (test item and positive control, 4 (vehicle control)
NUMBER OF CELLS EVALUATED: mutation selection assay: 2000 per well (1x10E4 cells/mL); cloning efficiency assay: 1.6 cells per well (8 cells/mL)
DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth, recorded over 2 days following treatment using a haemocytometer
OTHER EXAMINTATIONS
- Colony sizing: ratio of small to large type mutants
- Observations on precipitation of the test substance: after dosing and at the end of the exposure period
- Observations of pH: colour change in indicator in medium - Evaluation criteria:
- Criteria for a positive result:
-one or more concentrations were biologically significant and there was a significant linear trend
-in the absence of a linear trend if there was mitigating evidence ( e.g. presence of similar level of toxicity at all concentrations; in such a case, the confirmatory experiment would have been expected to assess concentrations covering different levels of toxicity, to establish a linear trend.
Additional comparisons that can aid interpretation of results include:
-comparison of the induced mutant fraction with the historical maximum for difference between vehicle controls (No Effect Maximum)
-comparison of the mutant fraction of a treated group with the historical range of vehicle control values
A test item was positive if 2 positive experiments out of 2 were recorded within the same activation condition. Test items that gave a negative response in the standard exposure in the absence of S9 mix, but gave a positive response in the extended exposure, were liable to a confirmatory experiment with the extended exposure.
Criteria for a negative result:
In the absence of any significant findings or other criteria for a positive response, a test item was defined as non-mutagenic, provided data were obtained in both the absence and the presence of S9 mix that accompanied one or more of the following:
- the predetermined maximum concentration of 5000 µg/mL or 10 mM, whichever is lower
- the highest practical concentration limited by the solubility or pH of the test item
- RTG in the range 10-20%
A chemical may be determined to be non-mutagenic when there was no treatment showing an RTG value between 10 and 20%. These situations are as follows:
- no evidence of mutagenic activity in a series of data points within 100% to 20% RTG and there was at least one data point between 20% and 25% RTG
- no evidence of mutagenic activity in a series of data points between 100% to 25% RTG and there was also a data point between 10 and 1% RTG - Statistics:
- Data were analysed using methods outlined in Robinson et al (1989).
- Determination of the heterogeneity factor for each dose level
- Comparison of the heterogeneity factor with the historical control for consistency using a one-sided F-test (Pearson and Hartley (1989)) at P>0.01. Any dose levels where either the mutant or the survival heterogeneity factors were significantly higher than their respective historical controls were excluded from all statistical analysis.
- Determination of the heterogeneity factor for the experiment and comparison with the historical controls for consistency using a one-sided F-test at P<0.01. If either the mutant or the survival heterogeneity factors were significantly higher than their respective historical controls the assay was discarded and the old historical controls remained in place. Otherwise, new historical control heterogeneity factors were calculated.
- Calculation of the log mutant fraction for each dose level and comparison to the control at P<0.05 using a one-sided Dunnett's test (Dunnett (1955)) for an increase in mutant fraction, if the treatment dose mutant fraction was greater than the control.
- Test for linear increasing trend of mutant fraction with increasing dose of test item with a one sided Chi2 test (Pearson and Hartley (1989)) (at P<0.05), if the direction of the slope parameter was positive. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- for details see tables 2a,b,c,d below
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- for details see table 1 below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no data
- Water solubility: not soluble
- Precipitation: yes
Assays 1 and 2: the test item precipitated at the 3 highest dose levels of 270, 360 and 465 µg/mL in both the absence and the presence of S9 mix, though it should be noted that only the 270 µg/mL treatment in the presence of S9 mix was included in the final assessment.
Assays 3 and 4: the test item precipitated at the highest 2 concentrations of 210 and 240 µg/mL in the absence of S9 mix, though neither concentration was assessed. In the presence of S9 mix, the test item precipitated at the highest 4 concentrations of 240, 270, 300 and 330 µg/mL. Only the 240 µg/mL was assessed and it was ultimately excluded from the analysis due to excessive toxicity.
- Other confounding effects: An assumed handling error occurred in one of the replicate cultures at the treatment in the presence of S9 mix in the 2nd Assay at a dose level of 195 µg/mL. The group was therefore excluded from the statistical analysis. The results for the 195 µg/mL group are represented by a single culture.
RANGE-FINDING/SCREENING STUDIES:
Absence of S9 mix, 4h exposure period: 150 µg/mL reduced suspension growth to 64.5% of the vehicle control value, all higher concentrations were lethal.
Presence of S9 mix, 4h exposure: 150 µg/mL resulted in a relative suspension growth of 45.5%, all higher concentrations were lethal.
Absence of S9 mix, 24h exposure period: concentrations of 50 and 150 µg/mL resulted in relative suspension growth values of 60.5% and 26,5%, respectively, all higher concentrations were lethal.
COMPARISON WITH HISTORICAL CONTROL DATA: The solvent control mean mutant fractions were within the normal ranges experienced in the testing laboratory and reported in the literature with the used cell line (Mitchell et al (1997)). - Conclusions:
- Coconut oil, reaction products with polyethylene glycol and trimethylolpropane is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in dimethylsulphoxide at concentrations extending into the toxic range and close to or exceeding its limit of solubility in the test system.
- Executive summary:
The test item, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane, was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine kinase locus: tk+tk- to tk-tk-. Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was formulated in dimethylsulphoxide. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254 -induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). The study was designed to be consistent with ICH Guidelines, OECD Guideline No. 476 and EC Directive 2000/32/EC B.17.
In preliminary cytotoxicity tests, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was shown to be of a moderate order of toxicity, causing reduced rates of cell growth at concentrations above 50 µg/mL and complete cell death at 500 µg/mL. Concentrations of 500 µg/mL and higher exceeded the limit of solubility of Coconut oil, reaction products with polyethylene glycol and trimethylolpropane in the test system.
Four independent mutation assay were conducted, as follows:
Assay No. Presence of absence of S9 Treatment time (hours) Final concentrations (µg/mL) 1 Absence 4 60, 90, 135, 195 2 Presence 4 60, 90, 135, 195, 270 3 Absence 24 60, 90, 120, 150, 180 4 Presence 4 120, 150, 180, 210, (240: too toxic to include in final assessment) Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of 'small' type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate.
Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value. In addition, the results were analysed for comparison of the log mutant fraction between the vehicle controls and each concentration of Coconut oil, reaction products with polyethylene glycol and trimethylolpropane. In addition all the experiments were tested for dose-related trends in mutant fraction.
No evidence of mutagenic activity was obtained with Coconut oil, reaction products with polyethylene glycol and trimethylolpropane in any of the 4 assays.
Results were obtained at concentrations resulting in a critical level of toxicity in both assays in the presence of S9 mix and in the assay in the absence of S9 mix with a 24 h exposure period. In addition, all assays included results from concentrations of Coconut oil, reaction products with polyethylene glycol and trimethylolpropane that were close to, or exceeded its limit of solubitliy in the test system.
Although results were not obtained at a critical level of toxicity in the assay in the absence of S9 mix with a 4 h exposure period, no increase in mutant fraction was seen at a concentration of 195 µg/mL, while a concentration of 270 µg/mL (being well below a 2 -fold increase in concentration) was too toxic for assessment. It was therefore considered that an unequivocal conclusion of not mutagenic could be made for Coconut oil, reaction products with polyethylene glycol and trimethylolpropane.
In conclusion, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was not mutagenic in mouse lymphoma L5178Y cells, in either the absence of the presence of S9 mix, when tested in dimethylsulphoxide at concentrations extending into the toxic range and close to or exceeding its limit of solubility in the test system.
Referenceopen allclose all
Table #1a: Plate incorporation test: Number of revertants per plate (mean of 3 plates) |
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| Concentration µg/plate |
Strain TA 98 | Strain TA 100 | Strain TA 1535 | Strain TA 1537 | |||||||||||
| + S9 mix | - S9 mix | Cytotoxicity | + S9 mix | - S9 mix | Cytotoxicity | + S9 mix | - S9 mix | Cytotoxicity | + S9 mix | - S9 mix | Cytotoxicity | ||||
| Neg. control | 40 ± 13 | 26 ± 3 | no | 155 ± 35 | 145 ± 11 | no | 11 ±1 | 9 ± 2 | no | 17 ± 2 | 22 ± 4 | no | |||
| Solvent control | 41 ± 5 | 24 ± 5 | no | 135 ± 31 | 156 ± 11 | no | 10 ± 1 | 7 ± 2 | no | 16 ± 10 | 16 ± 3 | no | |||
| 50 | 34 ± 10 | 19 ± 2 | no | 128 ± 6 | 108 ± 21 | no | 12 ± 3 | 7 ± 4 | no | 20 ± 1 | 16 ± 3 | no | |||
| 160 | 31 ± 3 | 22 ± 5 | no | 143 ± 41 | 125 ± 14 | no | 15 ± 6 | 8 ± 1 | no | 13 ± 3 | 5 ± 1 | +S9: no, -S9: yes | |||
| 500 | 47 ± 8 | 16 ± 7 | no | 137 ± 5 | 124 ± 12 | no | 13 ± 3 | 7 ± 2 | no | 13 ± 2 | 5 ± 3 | +S9: no, -S9: yes | |||
| 1600 | 39 ± 9 | 13 ± 6 | no | 148 ± 18 | 113 ± 6 | no | 7 ± 2 | 4 ± 0 | no | 5 ± 2 | 2 ± 0 | yes | |||
| 5000 | 36 ± 5 | 7 ± 5 | yes | 118 ± 6 | 107 ± 11 | yes | 3 ± 1 | 28 ± 7 | yes | 5 ± 2 | 3 ± 2 | yes | |||
| Pos. control | 623 ± 37 | 67 ± 12 | no | 947 ± 150 | 586 ± 19 | no | 312 ± 52 | no | 52 ± 10 | 32 ± 14 | no | ||||
| Solvent: Acetone | |||||||||||||||
Table #1b: Repetition of Plate incorporation test: Number of revertants per plate (mean of 3 plates) |
|||||||||||||||
| Concentration µg/plate |
Strain TA 98 | Strain TA 1537 | |||||||||||||
| + S9 mix | - S9 mix | Cytotoxicity | + S9 mix | - S9 mix | Cytotoxicity | ||||||||||
| Neg. control | not tested | 15 ± 3 | no | 16 ± 4 | 19 ± 4 | no | |||||||||
| Solvent control | 14 ± 4 | no | 14 ± 4 | 14 ± 6 | no | ||||||||||
| 1 | not tested | not tested | 13 ± 8 | -S9: no | |||||||||||
| 3 | 8 ± 5 | -S9: no | |||||||||||||
| 10 | 19 ± 6 | 7 ± 2 | no | ||||||||||||
| 30 | 16 ± 9 | no | 15 ± 2 | 6 ± 3 | no | ||||||||||
| 100 | 12 ± 3 | no | 21 ± 7 | 4 ± 2 | + S9: no, -S9: yes | ||||||||||
| 300 | 16 ± 4 | no | 15 ± 5 | not tested | +S9: no | ||||||||||
| 1000 | 10 ± 2 | no | 9 ± 3 | +S9: no | |||||||||||
| 3000 | 8 ± 3 | yes | not tested | ||||||||||||
| Pos. control | 147 ± 5 | no | 62 ± 5 | 70 ± 18 | no | ||||||||||
| Solvent: Acetone | |||||||||||||||
Table #2a: Preincubation test: Number of revertants per plate (mean of 3 plates) |
|||||||||||||||
| Strain TA 98 | Strain TA 100 | Strain TA 1535 | Strain TA 1537 | ||||||||||||
| Concentration µg/plate |
+ S9 mix | - S9 mix | Cytotoxicty | Concentration µg/plate |
+ S9 mix | - S9 mix | Cytotoxicity | Concentration µg/plate |
+ S9 mix | - S9 mix | Cytotoxicity | Concentration µg/plate |
+ S9 mix | - S9 mix | Cytotoxicity |
| Neg. control | 26 ± 6 | 16 ± 5 | no | Neg. control | 204 ± 20 | 158 ± 14 | no | Neg. control | 19 ± 7 | 14 ± 4 | no | Neg. control | 45 ± 5 | 22 ± 2 | no |
| Solvent control | 25 ± 3 | 22 ± 6 | no | Solvent control | 194 ± 27 | 155 ± 11 | no | Solvent control | 15 ± 4 | 9 ± 2 | no | Solvent control | 44 ± 3 | 18 ± 1 | no |
| 10 | 31 ± 6 | 22 ± 5 | no | 30 | 177 ± 7 | 135 ± 16 | no | 16 | 16 ± 4 | 17 ± 3 | no | 0.1 | not tested | 20 ± 2 | S9: no |
| 30 | 31 ± 7 | 19 ± 3 | no | 100 | 187 ± 6 | 163 ± 19 | no | 50 | 21 ± 3 | 12 ± 3 | no | 0.3 | 13 ± 3 | S9: no | |
| 100 | 35 ± 5 | 17 ± 3 | no | 300 | 185 ± 22 | 170 ± 12 | no | 160 | 17 ± 4 | 10 ± 2 | no | 1 | 24 ± 5 | S9: no | |
| 300 | 39 ± 3 | 19 ± 6 | no | 1000 | 215 ± 8 | 160 ± 7 | no | 500 | 17 ± 8 | 11 ± 4 | no | 3 | 24 ± 3 | S9: no | |
| 1000 | 30 ± 7 | 15 ± 2 | no | 3000 | 200 ± 16 | 148 ± 12 | no | 1600 | 12 ± 1 | 10 ± 3 | no | 10 | 52 ± 5 | 26 ± 6 | no |
| Pos.control | 1523 ± 75 | 149 ± 9 | no | Pos. control | 1877 ± 44 | 599 ± 18 | no | Pos.control | 225 ± 20 | 352 ± 15 | no | 30 | 47 ± 7 | not tested | +S9 : no |
| 100 | 48 ± 8 | +S9 : no | |||||||||||||
| 300 | 49 ± 2 | +S9 : no | |||||||||||||
| 1000 | 48 ± 12 | +S9 : no | |||||||||||||
| Pos. Control | 179 ± 8 | 52 ± 3 | no | ||||||||||||
| Table 1: Toxicity data | ||||||||
| Concentration µg/mL |
With S9 Mix | Without S9 Mix | ||||||
| Observations | Toxic Judge | Observations | Toxic Judge | |||||
| Index | Culture | Slide | Index | Culture | Slide | |||
| Neg. Control 1% DMSO |
107 | Nil toxicity | Nil toxicity | - | 100 | Nil toxicity | Nil toxicity | - |
| 93 | - | 100 | - | |||||
| 20 | 99 | - | 108 | - | ||||
| 95 | - | 109 | - | |||||
| 39 | 101 | - | 96 | - | ||||
| 97 | - | 106 | - | |||||
| 78 | 101 | - | 100 | - | ||||
| 93 | - | 88 | - | |||||
| 156 | 63 | Slightly rounded cells | Slightly sparse metaphase cells | t | 73 | - | ||
| 63 | t | 70 | - | |||||
| 313 | 30 | Sparse metaphase cells | tt | 26 | Slightly sparse metaphase cells | tt | ||
| 42 | tt | 30 | tt | |||||
| 625 | 0 | Rounded cells | No metaphase cells | ttt | 5 | Cells look fixed | Very sparse metaphase cells | ttt |
| 0 | ttt | 2 | ttt | |||||
| 1250 | 0 | Rounded cells, looked fixed | ttt | 0 | No metaphase cells | ttt | ||
| 0 | ttt | 0 | ttt | |||||
| 2500 | 0 | ttt | 0 | ttt | ||||
| 0 | ttt | 0 | ttt | |||||
| 5000 | 0 | ttt | 0 | ttt | ||||
| 0 | ttt | 0 | ttt | |||||
| t = Toxicity evident from morphological changes | ||||||||
| tt = Toxicity evident from reduced cell count (< 50% of vehicle) | ||||||||
| ttt = Too toxic for metaphase assessment | ||||||||
| Table 2. Aberration Data | ||||||||
| Exposure duration / Harvest Time | S9 mix | Concentration (µg/ml) | Aberration frequency | Aberrant Cell Frequency (%) | ||||
| (Lesions/ Cell) | Including Gaps | Excluding Gaps | ||||||
| 6 h / 24 h | + | 0 (DMSO) | 0.00 | 0 | 0 | |||
| 0.01 | 1 | 0 | ||||||
| 78 | 0.02 | 2 | 0 | |||||
| 0.00 | 0 | 0 | ||||||
| 156 | 0.04 | 2 | 2 | |||||
| 0.04 | 4 | 2 | ||||||
| 313 | 0.21+ | 9+ | 8+ | |||||
| 0.16+ | 7+ | 5+ | ||||||
| 40 µg/ml CP | 0.20+ | 11+ | 9+ | |||||
| 50 µg/ml CP | 0.24+ | 16+ | 16+ | |||||
| 6 h / 24 h | - | 0 (DMSO) | 0.01 | 1 | 0 | |||
| 0.00 | 0 | 0 | ||||||
| 78 | 0.00 | 0 | 0 | |||||
| 0.00 | 0 | 0 | ||||||
| 156 | 0.00 | 0 | 0 | |||||
| 0.01 | 1 | 0 | ||||||
| 313 | 0.00 | 0 | 0 | |||||
| 0.01 | 1 | 0 | ||||||
| 20 µg/ml MMS | 0.08+ | 6+/- | 6+ | |||||
| 30 µg/ml MMS | 0.13+ | 10+ | 8+ | |||||
| + = Positive aberration frequency | ||||||||
| +/- = Suspicious aberration frequency | ||||||||
| Table 1: Toxicity test | |||||||
| Concentration | Absence of S9 mix, 4h exposure | Presence of S9 mix, 4h exposure | Absence of S9 mix, 24h exposure | ||||
| (µg/mL) | Relative Suspension Growth (%) | Relative Suspension Growth (%) | Relative Suspension Growth (%) | ||||
| 0 (Vehicle control) | 100 | 100 | 100 | ||||
| 0.5 | 95.1 | 97.5 | 76.6 | ||||
| 1.5 | 105.3 | 84.1 | 95.6 | ||||
| 5 | 75.9 | 100.6 | 73.1 | ||||
| 15 | 89.4 | 93.1 | 89.0 | ||||
| 50 | 92.8 | 90.8 | 60.5 | ||||
| 150 | 64.5 | 45.5 | 26.3 | ||||
| 500 pptn | 0.0 | 0.0 | 0.0 | ||||
| 1500 pptn | 0.0 | 0.0 | 0.0 | ||||
| 5000 pptn | 0.0 | 0.0 | 0.0 | ||||
| pptn = precipitation | |||||||
| Table 2a: Mutation test in the absence of S9 Mix (4h exposure), Summary of means of data (Assay 1) | |||||||
| Concentration | Relative total growth (%) | Mutant fraction | Induced mutant fraction (x 10-6) | ||||
| (µg/mL) | (x 10-6) | ||||||
| 0 (Vehicle control) | 100 | 62 | n.a. | ||||
| 45 | NPS | NPS | NPS | ||||
| 60 | 93 | 49 | - | ||||
| 90 | 95 | 79 | 17 | ||||
| 135 | 64 | 87 | 16 | ||||
| 195 | 51 | 57 | - | ||||
| 270 pptn | NPT | NPT | NPT | ||||
| 360 pptn | NPT | NPT | NPT | ||||
| 465 pptn | NPT | NPT | NPT | ||||
| positive control EMS (250 µg/mL) | 53 | 934 | 872* | ||||
| positive control MMS (10 µg/mL) | 31 | 1168 | 1106* | ||||
| NPS: not plated - Surplus | |||||||
| NPT: not plated - Toxic | |||||||
| n.a.: not applicable | |||||||
| * significant difference in log mutant fraction compared with vehicle control (P<0.05) | |||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of vehicle group | |||||||
| - : IMF <= 0 | |||||||
| Table 2b: Mutation test in the presence of S9 Mix (4h exposure), Summary of means of data (Assay 2) | |||||||
| Concentration | Relative total growth (%) | Mutant fraction | Induced mutant fraction (x 10-6) | ||||
| (µg/ml) | (x 10-6) | ||||||
| 0 (Vehicle control) | 100 | 72 | n.a. | ||||
| 45 | NPS | NPS | NPS | ||||
| 60 | 105 | 90 | 18 | ||||
| 90 | 98 | 88 | 16 | ||||
| 135 | 73 | 86 | 14 | ||||
| 195 | 32 | 67 | (-) | ||||
| 270 pptn | 11 | 83 | 11 | ||||
| 360 pptn | NPT | NPT | NPT | ||||
| 465 pptn | NPT | NPT | NPT | ||||
| positive control 3-MC (2.5 µg/mL) | 79 | 446 | 374* | ||||
| positive control 3-MC (10 µg/mL) | 50 | 824 | 752* | ||||
| NPS: not plated - Surplus | |||||||
| NPT: not plated - Toxic | |||||||
| n.a.: not applicable | |||||||
| (): Results presented for a single culture only and therefore excluded from statistical analysis | |||||||
| * significant difference in log mutant fraction compared with vehicle control (P<0.05) | |||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of vehicle group | |||||||
| - : IMF <= 0 | |||||||
| Table 2c: Mutation test in the absence of S9 Mix (24h exposure), Summary of means of data (Assay 3) | |||||||
| Concentration | Relative total growth (%) | Mutant fraction | Induced mutant fraction (x 10-6) | ||||
| (µg/ml) | (x 10-6) | ||||||
| 0 (Vehicle control) | 100 | 66 | n.a. | ||||
| 30 | NPS | NPS | NPS | ||||
| 60 | 81 | 52 | - | ||||
| 90 | 62 | 59 | - | ||||
| 120 | 34 | 70 | 5 | ||||
| 150 | 32 | 72 | 6 | ||||
| 180 | 15 | 71 | 5 | ||||
| 210 pptn | NPT | NPT | NPT | ||||
| 240 pptn | NPT | NPT | NPT | ||||
| positive control EMS (150 µg/mL) | 28 | 2469 | 2403* | ||||
| positive control MMS (5 µg/mL) | 30 | 1746 | 1680* | ||||
| NPS: not plated - Surplus | |||||||
| NPT: not plated - Toxic | |||||||
| n.a.: not applicable | |||||||
| * significant difference in log mutant fraction compared with vehicle control (P<0.05) | |||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutantt fraction of vehicle group | |||||||
| - : IMF <= 0 | |||||||
| Table 2d: Mutation test in the presence of S9 Mix (4h exposure), Summary of means of data (Assay 4) | |||||||
| Concentration | Relative total growth (%) | Mutant fraction | Induced mutant fraction (x 10-6) | ||||
| (µg/ml) | (x 10-6) | ||||||
| 0 (Vehicle control) | 100 | 94 | n.a. | ||||
| 120 | 71 | 109 | 15 | ||||
| 150 | 56 | 104 | 10 | ||||
| 180 | 31 | 110 | 17 | ||||
| 210 | 20 | 117 | 23 | ||||
| 240 pptn | 9 | (156) | (62) | ||||
| 270 pptn | NPT | NPT | NPT | ||||
| 300 pptn | NPT | NPT | NPT | ||||
| 330 pptn | NPT | NPT | NPT | ||||
| positive control 3-MC (2.5 µg/mL) | 65 | 719 | 625* | ||||
| positive control 3-MC (10 µg/mL) | 50 | 916 | 823* | ||||
| NPT: not plated - Toxic | |||||||
| n.a.: not applicable | |||||||
| * significant difference in log mutant fraction compared with vehicle control (P<0.05) | |||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutantt fraction of vehicle group | |||||||
| (): Treatment giving an RTG value below the minimum acceptable level of 10% and therefore excluded from further analysis | |||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In the in vivo Micronucleus test in mice,
Coconut oil, reaction products with polyethylene glycol and
trimethyolpropane did not induce any chromosomal damage.
Link to relevant study records
- 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
- Study period:
- October - December 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Initial age at start of acclimatisation: 6-12 weeks
- Age at start of treatment: minimum 7 weeks
- Weight at study initiation: 28.4 g ± 1.5 g (males), 23.4 g ± 1.3 g (females); weight variation < 10 % of mean weight for each sex
- Assigned to test groups randomly: yes
- Fasting period before study: 4hrs
- Housing: 5 animals of identical sex per IVC cage (Polysulphone), Type II L
- Bedding: Altromin saw fiber bedding (batch: 160812)
- Diet (e.g. ad libitum): ad libitum (Altromin 1324, Batch: 0702)
- Water (e.g. ad libitum): ad libitum (tap water, sulphur acidified)
- Acclimation period: adequate duration, not specified
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10x
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: cotton seed oil
- Justification for choice of solvent/vehicle: relatively non-toxic to the animals
- Concentration of test material in vehicle: 200 mg/mL (based on 98.5 % active component content)
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no. (if required): MKBJ0602V - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
not specified - Duration of treatment / exposure:
- 44 hours, negative control and dose group additional 68 hours
- Frequency of treatment:
- single treatment
- Post exposure period:
- none
- Remarks:
- Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc. - No. of animals per sex per dose:
- 5 (3 in pre-experiment)
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide (CPA)
- Justification for choice of positive control(s): not mentioned
- Supplier: Sigma
- Batch no.: 120M1253V
- Route of administration: intraperitoneal (ip)
- Doses / concentrations: 10 mg/kg bw, single dose
- Volume administered: 10 mL/kg/ bw, CPA dissolved in physiological saline - Tissues and cell types examined:
- number of immature erythrocytes, number of micronucleated immature erythrocytes, percentage of micronucleated cells, percentage of immature among total erythrocytes
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Prior to the micronucleus test, the maximum tolerable dose (MTD) was determined in a pre-experiment. The MTD is defined as the dose producing signs of toxicity such as lethargy, palpebral closure, prone position etc..
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of peripheral blood was carried out on all animals (5 per sex and dose group) 44 and 68 hours after single treatment.
DETAILS OF SLIDE PREPARATION:
No slides were prepared.
METHOD OF ANALYSIS:
Samples, including positive and negative controls, were evaluated using a flow cytometer (FACScan, BD Biosciences). Anti-CD71 antibodies were labelled with Fluorescein-isothiocyanate (FITC), anti-CD61 antibodies were labelled with Phycoerythrin (PE). Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensities were recorded on the FL1, FL2, and FL3 channels for FITC, PE and PI, respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect occurring possible cytotoxic effects of the test item, the ratio between immature and mature erythrocytes was determined. The results were expressed as relative PCE (rel. PCE=proportion of polychromatic [immature] erythrocytes among total erythrocytes). - Evaluation criteria:
- The mean relative PCE and the mean and standard deviation of the relative ratio of micronucleated polychromatic erythrocytes to total PCE were calculated for each group (per sex). A dose-related increase in the number of micronucleated cells and/or a biologically relevant increase in the number of micronucleated cells for at least one of the dose groups were considered as criteria for a positive result. The biological relevance as well as the statistical significance of the results are the criterion for interpretation.
- Statistics:
- The nonparametric Mann-Whitney Test was used to analyze differences between the groups.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- other: not performed
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: none observed
- Evidence of cytotoxicity in tissue analyzed: no tissue analyzed
- Rationale for exposure: 2000 mg/kg was chosen as MTD
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically relevant increase of micronuclei was found, nor were any significant differences between the negative control groups, dose groups and the historical control data found.
- Ratio of PCE/NCE (for Micronucleus assay): No significant difference between the negative control groups, dose groups and the historical control data found.
- Appropriateness of dose levels and route: Both, MTD and oral administration, were appropriate to investigate the genotoxic potential of teh test item. The positive control groups showed a significantly increased micronucleus frequency.
- Statistical evaluation: No statistically significant differences between the negative control groups and the dose group were found. - Conclusions:
- During the study an under the experimental conditions reported, the test item Coconut oil, reaction products with polyethylene glycol and trimethylolpropane did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.
- Executive summary:
This study was performed to investigate the potential of Coconut oil, reaction products with polyethylene glycol and trimethylpropane to induce micronuclei in polychromatic erythrocytes (PCE) in the mouse, which is the endpoint of this test to assess genotoxicity.
The test item was diluted in Cottonseed Oil. The volume administered orally was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after a single application of the test item.
A pre-experiment was performed as range finding study based on the OECD guideline 474 and other relevant documents. A dose of 2000 mg/kg bw was selected as maximum tolerable dose (MTD). No signs of toxicity were noted.
In the main experiment 2000 mg/kg bw was tested as the maximum tolerable dose (1 MTD). The volume administered was 10 mL/kg bw orally. No toxicity was observed during the treatment.
For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (44 h, 68 h) were within the range of the laboratory control data. The mean values noted for the dose group treated with the test item were within the negative control data range.
No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.
The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p<0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated. Based on this data this increase was regarded as not biological relevant.
Cyclophosphamide was used as positive control. The intraperitoneal administration of 40 mg CPA/kg bw induced a significant increase in micronucleus frequency. This demonstrates the validity of the assay.
Reference
| Dose [mg/kg bw] | Sex | Time [h] | bw mean [g] ± SD | bw variation [%] | MN total | MN [%] mean±SD | rel. PCE mean |
| 40 (positive control) | male | 44 | 28.1 ± 1.2 | ± 5.5 | 15.8 | 3.16 * ± 0.97 | 0.94 |
| 40 (positive control) | female | 44 | 23.5 ± 1.7 | ± 7.7 | 13.86 | 3.47 * ± 1.07 | 1.11 |
| 0 (negative control) | male | 44 | 28.1±2.4 | ± 9.8 | 1.16 | 0.23 ± 0.07 | 2.48 |
| 0 (negative control) | female | 44 | 23.7±0.4 | ± 1.9 | 1.18 | 0.24 ± 0.05 | 2.04 |
| 0 (negative control) | male | 68 | 28.1±2.4 | ± 9.8 | 0.89 | 0.22 ± 0.09 | 2.64 |
| 0 (negative control) | female | 68 | 23.7±0.4 | ± 1.9 | 1.13 | 0.23 ± 0.09 | 2.96 |
| 2000 | male | 44 | 29.1 ± 0.3 | ± 1.2 | 0.75 | 0.19 ± 0.04 | 3.28 |
| 2000 | female | 44 | 23.0 ± 1.6 | ± 8.5 | 1.41 | 0.28 ± 0.09 | 1.64 |
| 2000 | male | 68 | 29.1 ± 0.3 | ± 1.2 | 1.32 | 0.26 ± 0.06 | 3.69 |
| 2000 | female | 68 | 23.0 ± 1.6 | ± 8.5 | 1.03 | 0.26 ± 0.09 | 1.86 |
| * - p < 0.05 | SD - standard deviation | bw - initial body | |||||
| MN = (micronucleated PCE/total PCE) x100 | rel. PCE = (total PCE/total erythrocytes)x100 | ||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In the Ames test, Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were treated with the test compound by the plate incorporation as well as the preincubation method. Five dose levels covering a total range between 0.1 and 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Aroclor 1254 or Phenobarbital/beta-Naphthoflavone induced rat liver S9 mix) were employed. In the plate incorporation test without metabolic activation with strain TA 1535 treatment with 5000 µg/plate Coconut oil, reaction product with polyethylene glycol and trimethylolpropane resulted in an increased revertant frequency (factor 4.2) but accompanied with toxicity. Therefore, this mutagenic response was jugded to be of no relevance. In the preincubation test, all tester strains were exposed to lower, toxicity adjusted test compound concentrations. With the above mentioned exception, treatment with the test compound did not result in a significant increase in the revertant frequency of the tester strains. Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was jugded not to be a bacterial mutagen.
The chromosomal aberrations assay was performed with the test item Coconut oil, reaction products with polyethylene glycol and trimethylolpropane in duplicate. The test was conducted in the presence and absence of an exogenous source of metabolic activation (S9 mix, prepared from the livers of adult, male rats treated with Aroclor 1254). Cultures, established approx. 20 h before testing, were treated for 6 h in the presence and absence of S9 mix. Cultures were harvested at 24 h post treatment.
Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix. It was tested up to the maximum permitted concentration of 5000 µg/mL. Toxicity was noted at 156 -5000 µg/mL in the presence of S9 mix and at 313 -5000 µg/mL in the absence of S9 mix.
Coconut oil, reaction products with polyethylene glycol and trimethylolpropane induced structural chromosomal aberrations in the presence of S9 mix (only a weak effect at 313 µg/mL, a concentration deemed toxic to the cells) and was non clastogenic in the absence of S9 mix.
In the 3rd genetic toxicity study, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane, was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine kinase locus: tk+tk- to tk-tk-. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254 -induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix).
In preliminary cytotoxicity tests, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was shown to be of a moderate order of toxicity, causing reduced rates of cell growth at concentrations above 50 µg/mL and complete cell death at 500 µg/mL. Concentrations of 500 µg/mL and higher exceeded the limit of solubility of Coconut oil, reaction products with polyethylene glycol and trimethylolpropane in the test system. Coconut oil, reaction products with polyethylene glycol and trimethylolpropane was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in dimethylsulphoxide at concentrations extending into the toxic range and close to or exceeding its limit of solubility in the test system.
The aim of the in vivo study was to investigate the genotoxicity of Coconut oil, reaction products with polyethylene glycol and trimethylolpropane and its potential to induce micronuclei in polychromatic erythrocytes in the mouse. The test item was diluted in Cottonseed Oil and a volume of 10 mL/kg bw was administered to the animals, corresponding to a dose of 2000 mg/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after single application of the test item. No toxicity was observed during the treatment and no significant increase of micronuclei was found after treatment with Coconut oil, reaction products with polyethylene glycol and trimethylolpropane. Hence, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane can be considered as non-mutagenic with respect to clastogenicity and/or aneugenicity.
In conclusion the weakly positive result in the in vitro chromosome aberration study using a rodent cell line (CHO cells) derived from cancer tissues that is lacking proper cell cycle control has to be seen in the context of the negative result obtained in the in vivo assay. In vivo studies do assess genotoxicity under more realistic conditions, therefore the negative result of the Mammalian Micronucleus Test shows that Coconut oil, reaction products with polyethylene glycol and trimethylolpropane does not induce any genetic toxicity in mouse erythrocytes.
Short description of key information:
Coconut oil, reaction products with polyethylene glycol and
trimethylolpropane has been assessed for genetic toxicity in several in
vitro systems and one in vivo study, all studies were conducted
according to OECD test guidelines and GLP. In vitro studies in bacteria
(Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537) and
mouse lymphoma L5178Y cells have demonstrated no genetic toxicity of
Coconut oil, reaction products with polyethylene glycol and
trimethylolpropane when tested in the presence or absence of exogenous
metabolic activation. A Chromosomal aberration study in Chinese Hamster
Ovary cells demonstrated weak effects only in the presence of exogenous
metabolic activation at a concentration deemed toxic to the cells. In
the in vivo study in mice, Coconut oil, reaction products with
polyethylene glycol and trimethyolpropane did not induce any chromosomal
damage.
Endpoint Conclusion:
Justification for classification or non-classification
Based on the negative results of the Mammalian in vivo Micronucleus Test and supported by the outcome of the numerous available in vitro studies, Coconut oil, reaction products with polyethylene glycol and trimethylolpropane is not classified as genotoxic.
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