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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
19 Dec 2006 - 16 Jan 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance Hexadecyl 2-ethylhexanoate. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Groupe Interministéririel des Produits Chemiques (GIPC), 12 rue Villiot, Paris cedex 12, France
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexadecyl 2-ethylhexanoate
EC Number:
261-619-1
EC Name:
Hexadecyl 2-ethylhexanoate
Cas Number:
59130-69-7
Molecular formula:
C24H48O2
IUPAC Name:
hexadecyl 2-ethylhexanoate
Details on test material:
- Name of test material (as cited in study report): Ethyl 2 Hexanoate de Cetyle
- Identification No: 06-901
- Lot/batch No.: 06090084
- Physical state: thick colouless liquid
- Analytical purity: 99.9%
- Expiration date of the lot/batch: 04 Sep 2008
- Storage condition of test material: at room temperature

Method

Target gene:
his
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
Pretest: 10, 100, 500, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Main test: 312.5, 625, 1250, 2500, 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (batch: V6I963106L, CARLO ERBA, Val de Reuil, France)
- Justification for choice of solvent/vehicle: as test material is not soluble in water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine (2AM, 2 µg/plate for TA1535, TA1537, TA98 and TA100, 10 µg/plate for TA102)
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide (NAN3, 1 µg/plate for TA1535 and TA100); 9-Aminoacridine (9AA, 50 µg/plate for TA1537); 2-Nitrofluorene (2NF, 0.5 µg/plate for TA98); Mitomycine C (MMC, 0.5 µg/plate for TA102)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation): pretest, experiment 1, experiment 2 without S9 mix
Preincubation: experiment 2 with S9 mix

DURATION
- Preincubation period: 60 min, 37°C
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
An reproducible 2-fold increase (for the TA98, TA100 and TA102 strains) or a 3-fold increase (fort the TA1535 and TA1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered a positive results. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
The mean number of revertants with the corresponding standard deviation and ratio were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: a moderate emulsion was observed in the plates at dose levels of ≥625 µg/plate and ≥2500 µg/plate, with and without S9 mix, respectively.

RANGE-FINDING/SCREENING STUDIES:
A moderate emulsion was observed in the plates at dose levels ≥ 500 µg/plate. No noteworthy toxicity was noted towards the three strains used, either with or without S9 mix

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in any strains at any dose-level tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Pre test:

Dose Level (µg/plate) Liver S9 mix TA 98 TA100 TA102
Ethanol - 25 110 428
10 - 24 105 405
100 - 18 120 410
500 - 23* 131* 481*
1000 - 30* 111* 449*
2500 - 28* 116* 487*
5000 - 29* 137* 499*
Ethanol + 36 89 526
10 + 42 89 489
100 + 36 103 619
500 + 40* 99* 601*
1000 + 46* 101* 565*
2500 + 32* 99* 550*
5000 + 35* 86* 590*

*: moderate emulsion

Main test experiment 1:

Dose Level (µg/plate) Liver S-9 mix TA1535 TA1537 TA98 TA100 TA102
Individual revertant colony counts mean Individual revertant colony counts mean Individual revertant colony counts mean Individual revertant colony counts mean Individual revertant colony counts mean
Ethanol - 17 16 19 17 6 6 6 6 23 36 25 28 104 108 104 105 372 362 388 374
312.5 - 24 23 25 24 5 12 4 7 28 26 18 24 129 140 110 126 357 384 296 346
625 - 19 12 24 18 4 ap 8 6 14 18 19 17 111 89 93 98 332 398 406 379
1250 - 16 20 16 17 5 5 11 7 24 25 12 20 101 93 104 99 394 390 408 397
2500 - 23* 13* 13* 16 8* 10* 12* 10 10* 20* 18* 16 117* 96* 104* 106 286* 347* 338* 324
5000 - 25* 19* 18* 21 5* 6* 6* 6 34* 24* 17* 25 113* 122* 1216* 117 358* 316* 359* 344
NAN3 (1 µg/plate) - 468* 60* 582* 552                 672 647 600 640        
9AA (50 µg/plate) -         105 143 117 122                        
2NF (0.5 µg/plate) -                 160 153 134 149                
MMC (0.5 µg/plate -                                 2442 2024 2302 2256
                                           
Ethanol + 16 14 16 25 12 10 14 12 26 19 40 28 98 93 101 97 382 248 230 287
312.5 + 13 14 22 16 10 4 11 8 26 26 16 23 104 90 77 90 283 216 216 238
625 + 14* 14* 22* 17 2* 13* 4* 6 19* 25* 18* 21 84* 97* 99* 93 425* 505* 287* 406
1250 + 8* 14* 14* 12 4* 6 5* 5 29* 44* 25* 33 109* 84* 90* 94 359* 356* 353* 356
2500 + 14* 12* 14* 13 2* 10* 6* 6 29* 23* 20* 24 91* 80* 99* 90 443* 190* 396* 343
5000 + 16* 35* 13* 21 11* 11* 12* 11 19* 18* 41* 26 93* 78* 114* 95 295* 490* 446* 410
2AM (2 µg/plate) + 116 126 122 121 85 69 104 86 959 1054 1035 1016 679 703 701 700        
2AM (10 µg/plate) +                                 2177 3035 3289 2834

*:moderate emulsion

ap: aberrant plate

Main test experiment 2:

Dose Level (µg/plate) Liver S-9 mix TA1535 TA1537 TA98 TA100 TA102
Individual revertant colony counts mean  Individual revertant colony counts mean  Individual revertant colony counts mean  Individual revertant colony counts mean  Individual revertant colony counts mean 
Ethanol - 26 26 25 26 4 4 6 5 11 20 29 20 95 92 96 94 279 315 281 292
312.5 - 31 18 26 25 7 2 1 3 17 17 19 18 113 102 95 103 333 320 320 324
625 - 34 23 32 30 7 7 1 5 19 12 20 17 107 99 120 109 296 378 254 309
1250 - 26 14 26 22 6 5 6 6 11 22 19 17 92 80 113 95 365 338 347 350
2500 - 22* 23* 25* 23 1* 6* 4* 4 19* 31* 23* 24 99* 116* 111* 109 332* 337* 296* 322
5000 - 29* 17* 23* 23 1* 10* 5* 5 22* 42* 19* 28 96* 102* 140* 113 392* 308* 265* 322
NAN3 (1 µg/plate) - 484 576 493 518         593 565 543 567        
9AA (50 µg/plate) -         171 87 ap 129                        
2NF (0.5 µg/plate) -                 95 104 137 112                
MMC (0.5 µg/plate -                                 1871 1811 2003 1895
Ethanol + 19 18 18 18 4 7 2 4 22 31 28 27 72 91 77 80 429 441 353 408
312.5 + 26 17 16 20 8 12 5 8 42 28 28 33 97 114 107 106 446 455 341 414
625 + 15* 14* 18* 16 10* 12* 10* 11 32* 34* 32* 33 97* 78* 96* 90 531* 441* 428* 467
1250 + 17* 19* 20* 19 5* 5* 16* 9 22* 25* 32* 26 121* 109* 90* 107 493* 410* 382* 428
2500 + 20* 22* 20* 21 14* 10* 7* 10 71* 32* 52* 52 79* 96* 119* 98 462* 426* 432* 440
5000 + 24* 11* 20* 18 11* 12* 8* 10 44* 31* 35* 37 104* 125* 99* 109 510* 366* 416* 464
2AM (2 µg/plate) + 113 96 105 105 105 101 104 103 1212 1315 1238 1255 770 696 685 717        
2AM (10 µg/plate) +                                 1812 2061 1759 1877

*:moderate emulsion

ap: aberrant plate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative