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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1997

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
Reaction products of coconut oil with polyethyleneglycol and 2-ethyl-2-(hydroxymethyl)propane-1,3-diol
EC Number:
640-964-5
Cas Number:
218451-68-4
Molecular formula:
not applicable because UVCB
IUPAC Name:
Reaction products of coconut oil with polyethyleneglycol and 2-ethyl-2-(hydroxymethyl)propane-1,3-diol
Details on test material:
- Name of test material (as cited in study report): Marlowet 4753
- Physical state: slightly yellow liquid, containing flakes and crystals
- Lot/batch No.: 01/96
- Expiration date of the lot/batch: no data
- Other:
Date of production: 14th week 1996
Stability: > 1 year

Method

Species / strain
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
Controlsopen allclose all
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

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

Table #1a: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

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
Solvent: Acetone

Applicant's summary and conclusion

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.