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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered substance was tested under OECD 471 guidelines and was found not to induce point mutations for frame-shift mutations with and without metabolic activation in TA 97, TA 98, TA 100, TA 102 or TA 1535 stains of E coli. Sasol (LAUS 2016). The results of this study it is concluded that 2,2 -bis[[(2 -hexyl-1 -oxodecyl)oxy]methyl]-1,3 -propanediyl bis(2 -hexyldecanoate), the registered substance, is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

A mouse lymphoma assay for 85186 -89 -6 (now 85566 -29 -6) under OECD 476 guidelines and a chromosal aberration test to human lymphocytes for 403507 -18 -6 under OECD 473 guidelines were negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 31 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Production plant, Batch no. 04549/MA
- Expiration date of the lot/batch: 15 May 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Species / strain / cell type:
S. typhimurium, other: TA97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S9 enzymes from the livers male Sprague-Dawley rats, treated with 500 mg Aroclor 1254/kg bw intraperitoneally
Test concentrations with justification for top dose:
Experiment using the plate incorporation method: 5000 / 1500 / 500 / 150 / 50 µg/plate
Repetition experiment using the preincubation method: 5000 / 2500 / 1250 / 625 / 313 / 156 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetra hydrofurane (THF)
- Justification for choice of solvent/vehicle: In a preliminary test, the solubility of the test item was determined in a concentration of 50 g/L in demineralised water, DMSO, Ethanol; and in 200 g/L in THF. THF was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viabilty of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene, 20 µg/plate in DMSO with strains TA97a, TA98 and TA102; Sodium Azide, 1 µg/plate in H2O with strains TA100 and TA1535
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Amino-anthracene, 1 µg/plate in DMSO with strains TA97a, TA100, TA102 and TA1535; Benzo-a-pyrene, 20 µg/plate in DMSO with strain TA98
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in the first experiment; pre-incubation in the second experiment

DURATION
- Preincubation period: 20 min (only in the second experiment)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
- Method: In the pre-experiment: determination of titre (the test item was considered non-toxic, if the quotient titre/toxicity is below 2), in the main experiments: evaluation of background lawn, reduction in number of revertants in comparison to negativ/solvents control

OTHER EXAMINATIONS:
- Visual counting of mutant colonies, a spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations.
- Quality control of bacterial strains: genotype confirmation for each batch of bacteria before stock culture preparation: All bacterial strains were tested for histidine requirement, ampicillin resistence, crystal violet sensitivity, UV sensitivity and spontaneous revertants, furthermore the following examinations were performed: determination of titre, toxicity control, sterility control and positive control.
- Visual inspection of precipitation: Plates were checked for precipitation of test item at the end of the incubation

Evaluation criteria:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (revertants less mean spontaneous revertants) were also calculated.

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate in at least one strain exceeding an increase factor of 2 (in tester strains TA 97a, TA98, TA100 and TA102) and an increase factor of 3 (in tester strain TA1535) as compared to the reversion rate of the solvent control can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
not performed
Key result
Species / strain:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not soluble in water
- Precipitation: Precipitated/undissolved test item was not observed at any of the concentrations tested.
- Other confounding effects: nothing mentioned


COMPARISON WITH HISTORICAL CONTROL DATA: Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory, differences were only marginal and no critical impact on the outcome of the study was expected. All positive control showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.
For the solvent control THF, no historical data are available. This can be seen as uncritical, because the values of the spontaneous revertants are in normal range of the data for the other solvent controls (demin. water and DMSO) in the test facility.
Table #1: Salmonella typhimurium reverse mutation assay without metabolic activation, plate incorporation test
Concentration [µg/plate] Revertant colonies / plate
TA 97a TA98 TA100 TA102 TA1535
mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l)
Negative control (H2O) 73 ± 8.5 - 10 ± 3.1 - 62 ± 2.0 - 263 ± 25.5 - 20 ± 2.1 -
Solvent control (DMSO) 82 ± 3.2 - 9 ± 2.5 - 64 ± 9.3 - 235 ± 68.5 - 17 ± 2.6 -
Solvent control (THF) 69 ± 11.9 - 12 ± 3.5 - 61 ± 1.0 - 352 ± 13.9 - 21 ± 2.6 -
5000 59 ± 9.3 0.86 13 ± 3.5 1.08 61 ± 5.1 1.00 379 ± 8.3 1.08 16 ± 2.5 0.76
1500 68 ± 6.5 0.99 10 ± 2.1 0.83 61 ± 4.4 1.00 359 ± 24.1 1.02 17 ± 4.5 0.81
500 76 ± 4.7 1.10 10 ± 1.5 0.83 65 ± 8.4 1.07 291 ± 34.9 0.83 16 ± 0.6 0.76
150 68 ± 8.7 0.99 15 ± 1.2 1.42 61 ± 3.5 1.00 363 ± 20.1 1.03 17 ± 1.2 0.81
50 64 ± 4.6 0.93 15 ± 2.1 1.25 67 ± 4.9 1.10 311 ± 2.3 0.88 19 ± 0.6 0.90
Positive controls 740 ± 170.9 9.02 311 ± 49.7 34.56 315 ± 26.1 5.08 725 ± 63.3 3.09 341 ± 81.0 17.05
SD = Standard deviation   f(l) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
*mean from 3 replicates
Table #2: Salmonella typhimurium reverse mutation assay with metabolic activation, plate incorporation test
Concentration [µg/plate] Revertant colonies / plate
TA 97a TA98 TA100 TA102 TA1535
mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l)
Negative control (H2O) 114 ± 7.9 - 14 ± 3.8 - 66 ± 4.0 - 291 ± 26.0 - 20 ± 2.1 -
Solvent control (DMSO) 128 ± 8.3 - 13 ± 1.2 - 63 ± 2.0 - 309 ± 28.4 - 17 ± 2.6 -
Solvent control (THF) 82 ± 8.3 - 17 ± 0.6 - 73 ± 1.5 - 359 ± 23.4 - 21 ± 2.6 -
5000 116 ± 18.9 1.41 11 ± 3.1 0.65 71 ± 16.9 0.97 369 ± 19.7 1.03 16 ± 2.5 0.95
1500 112 ± 10.5 1.37 16 ± 1.0 0.94 66 ± 11.0 0.90 355 ± 9.2 0.99 17 ± 4.5 0.74
500 104 ± 8.7 1.27 15 ± 2.0 0.88 69 ± 8.1 0.95 267 ± 66.0 0.74 16 ± 0.6 0.79
150 89 ± 22.2 1.09 11 ± 2.0 0.65 69 ± 10.0 0.95 330 ± 44.2 0.92 17 ± 1.2 1.00
50 75 ± 6.0 0.91 16 ± 2.5 0.94 75 ± 3.5 1.03 299 ± 52.8 0.83 19 ± 0.6 0.79
Positive controls 567 ± 84.5 4.43 59 ± 3.5 5.54 627 ± 41.1 9.95 671 ± 151.4 2.17 341 ± 81.0 6.84
SD = Standard deviation   f(l) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
*mean from 3 replicates
Table #3: Salmonella typhimurium reverse mutation assay without metabolic activation, pre-incubation test
Concentration [µg/plate] Revertant colonies / plate
TA 97a TA98 TA100 TA102 TA1535
mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l)
Negative control (H2O) 86 ± 13.7 - 12 ± 3.1 - 90 ± 17.3 - 428 ± 64.4 - 21 ± 2.5 -
Solvent control (DMSO) 88 ± 4.7 - 12 ± 1.2 - 94 ± 5.3 - 332 ± 43.3 - 21 ± 3.5 -
Solvent control (THF) 86 ± 2.1 - 15 ± 2.0 - 99 ± 12.0 - 351 ± 28.1 - 28 ± 3.0 -
5000 94 ± 4.0 1.09 10 ± 0.6 0.67 92 ± 16.8 0.93 398 ± 45.3 1.13 26 ± 5.2 0.93
2500 101 ± 5.0 1.17 9 ± 1.7 0.60 104 ± 11.0 1.05 413 ± 26.6 1.18 24 ± 6.7 0.86
1250 97 ± 15.3 1.13 13 ± 1.2 0.87 117 ± 1.0 1.18 388 ± 56.0 1.11 24 ± 3.6 0.86
625 94 ± 18.2 1.09 15 ± 1.0 1.00 106 ± 8.3 1.07 383 ± 53.7 1.09 25 ± 1.2 0.8
312 111 ± 17.4 1.29 19 ± 2.3 1.27 104 ± 7.2 1.05 292 ± 13.9 0.83 24 ± 7.2 0.86
156 108 ± 9.0 1.29 16 ± 6.1 1.07 106 ± 13.9 1.07 280 ± 6.9 0.80 21 ± 6.1 0.75
Positive controls 565 ± 196.3 6.42 467 ± 32.2 38.92 525 ± 54.0 5.83 1296 ± 57.7 3.90 284 ± 32.7 13.52
SD = Standard deviation   f(l) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
*mean from 3 replicates
Table #4: Salmonella typhimurium reverse mutation assay with metabolic activation, pre-incubation test
Concentration [µg/plate] Revertant colonies / plate
TA 97a TA98 TA100 TA102 TA1535
mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l) mean*±SD f(l)
Negative control (H2O) 90 ± 4.2 - 15 ± 3.5 - 101 ± 11.0 - 336 ± 64.0 - 22 ± 3.0 -
Solvent control (DMSO) 87 ± 6.7 - 12 ± 1.5 - 89 ± 7.8 - 407 ± 67.7 - 23 ± 3.1 -
Solvent control (THF) 78 ± 14.4 - 17 ± 1.5 - 95 ± 6.2 - 405 ± 70.5 - 18 ± 3.0 -
5000 116 ± 16.0 1.49 16 ± 2.5 0.94 127 ± 22.1 1.32 417 ± 77.4 1.03 24 ± 4.7 1.33
2500 100 ± 12.5 1.28 10 ± 2.3 0.59 124 ± 11.5 1.29 391 ± 63.8 0.97 24 ± 1.5 1.33
1250 116 ± 16.5 1.49 19 v 8.9 1.12 112 ± 18.2 1.17 421 ± 54.5 1.04 23 ± 4.5 1.28
625 98 ± 21.2 1.26 23 ± 2.6 1.35 129 ± 7.5 1.34 368 ± 26.2 0.91 21 ± 4.0 1.17
312 95 ± 9.6 1.22 16 ± 3.0 0.94 116 ± 12.2 1.21 319 ± 28.9 0.79 22 ± 1.7 1.22
156 107 ± 3.6 1.37 15 ± 4.4 0.88 93 ± 2.3 0.97 297 ± 12.9 0.73 23 ± 6.0 1.28
Positive controls 672 ± 184.7 7.72 77 ± 18.2 6.42 507 ± 67.4 5.70 931 ± 30.3 2.29 106 ± 33.0 4.61
SD = Standard deviation   f(l) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
*mean from 3 replicates
Conclusions:
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

The study procedures described in this report were based on the most recent OECD and EC guidelines and performed under the principles of GLP. The test item 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). In the first experiment, 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) (dissolved in THF) was tested up to nominal concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. On the base of the first experiment, 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) was tested up to nominal concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strain using the pre-incubation method. 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

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:
26 Mar - 08 June 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. 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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from rats pretreated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (12%, v/v))
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
+ S9: cyclophosphamide, 15 and 5 µg/mL for 3 and 24 h treatment, respectively; - S9: methylmethanesulfonate, 7.5 µg/mL
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: No
- Exposure duration: cells were exposed to the test material for 3 h and 24 h in the presence and absence of S9-mix, respectively.
- Expression time (cells in growth medium): For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. For determination of the mutation frequency cells were plated and incubated for 11-12 days. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
Several criteria including a concentration-related, or a reproducible increase in mutation frequencies determined a positive result.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 333 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 333 µg/mL

RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h respectively with a number of increasing test substance concentrations. The highest concentration tested was 750 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h or 24 h incubation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls
Remarks on result:
other: all strains/cell types tested

Table 1: Results of experiment 1

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

104

100

100

74

SC2

85

97

0.3

99

98

104

102

74

1

101

102

108

109

71

3

100

101

107

107

94

10

93

98

104

97

67

33

120

94

100

120

63

100

113

101

107

121

61

333*

104

113

120

124

64

750*

405

101

107

112

74

MMS

71

68

72

51

835

With 8% (v/v) metabolic activation, 3 h treatment

SC1

100

70

100

100

65

SC2

69

64

0.3

96

60

86

83

74

1

115

68

98

113

60

3

109

40

57

62

84

10

127

72

104

132

52

33

114

46

66

75

84

100

122

76

108

133

63

333*

115

62

89

102

72

750*

104

58

84

87

53

CP

50

32

45

22

1617

 

Table 2: Results of experiment 2

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

66

100

100

90

SC2

79

75

0.3

112

77

106

119

88

1

116

80

110

128

82

3

117

72

100

117

79

10

120

85

117

140

66

33

114

74

101

116

83

100

121

69

95

115

83

333*

116

70

97

112

70

750*

116

66

91

106

71

MMS

101

49

67

68

1502

With 12% (v/v) metabolic activation, 3 h treatment

SC1

100

93

100

100

80

SC2

93

76

0.3

103

84

90

93

74

1

113

83

89

101

81

3

107

97

104

112

60

10

105

94

101

107

80

33

103

93

100

103

67

100

102

105

114

116

57

333*

106

91

99

104

74

750*

103

93

100

103

73

CP

72

75

81

58

1082

 

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06. Oct. - 16. Feb. 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD). (Purity of test substance not given, evalation criteria not given.)
Justification for type of information:
see chapter 13 read-across justification
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
purity of test substance is not given (responsibility of the sponsor)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagles essential medium with HEPES buffer (MEM), supplemented with:
L-glutamine, penicillin/streptomycin, amphotericin B, 15% foetel calf serum
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats pretreated with phenobarbitone (80 mg/kg) and ß-naphtoflavone (100 mg/kg)
Test concentrations with justification for top dose:
Experiment I:
4 hour (with and without): 40, 80, 160, 240*, 320*, 400* µg/mL

Experiment II
4 hour (with): 40, 80, 160, 240*, 320*, 400* µg/mL
24 hour (without): 40, 80, 160, 240*, 320*, 400* µg/mL

* Dose levels (plus control dose) selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC; 0.2 and 0.4 µg/mL; -S9), cyclophosphamide (CP; 10 µg/mL; +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24 h (without)
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20 h; 24 h treatment: 0 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL (demecolcine)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 minutes

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
no data
Statistics:
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.
Species / strain:
lymphocytes: cultured peripheral human 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: cloudy precipitates were observed at and above 40 and 80 µg/mL in the 24-hour continuous and 4-hour pulse treatment groups, respectively

RANGE-FINDING/SCREENING STUDIES:
The dose range tested was 10-320 µg/mL. The test material produced some weak toxicity in the 4-hour treatment group but not the 24-hour treatment group. Toxicity could not be reproduced in the main experiment (scorable metaphases at every dose level).
COMPARISON WITH HISTORICAL CONTROL DATA:
The results are in range with historical control data.

Remarks on result:
other: all strains/cell types tested

Table 3 + 4: Test results of experiment I.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 Experiment I

in µg/mL

in %

with gaps

without gaps

Exposure period 4h, fixation time 20h, without S9 mix

control

0

100

1

0

MMC

0.4

35

53

37

Test substance

240

98P

0.5

0

320

110P

0

0

400

91P

0.5

0.5

Exposure period 4h, fixation time 20h, with S9 mix

control

0

100

1

0.5

CP

10

20

35.5

28.5

Test substance

240

89P

1.5

0

320

89P

0.5

0

400

108P

3.5

1

Test item

Concentration

Mitotic Index

Aberrant cells in %

 Experiment II

in µg/mL

in %

with gaps

without gaps

Exposure period + fixation time 24h, without S9 mix

control

0

100

1.5

0

MMC

0.4

41

70

67

Test substance

240

61

0.5

0.5

320

53

0.5

0

400

83

0

0

Exposure period 4h, fixation time 20h, with S9 mix

control

0

100

0.5

0

CP

10

30

67

56

Test substance

240

103

1

0

320

76

1

0

400

110

1.5

0.5

Table5 +6: Mean Frequency of Polyploid Cells (%)

Experiment I

dose level µg/mL

harvest time 24 hours

4 hours without S9

4 hours with S9

0

0.0

0.0

240

0.0

0.0

320

0.0

0.0

400

0.0

0.0

MMC 0.4

0.0

NA

CP 10

NA

0.0

dose level µg/mL

harvest time 24 hours

24 hours without S9

4 hours with S9

0

0.5

0.0

240

0.0

0.0

320

0.0

0.0

400

0.0

0.5

MMC 0.4

0.0

NA

CP 10

NA

0.0

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The registered substance is substantially similar to CAS 85566 -29 -6 (formerly 85186 -89 -6), and CAS 403507 -18-6 which are part of the polyol ester category. In summary, several studies are available to assess the mutagenic potential in bacteria within the polyol esters category all providing negative results. Furthermore, no cytogenicity in mammalian cells in-vitro and no mutagenicity in mammalian cells in-vitro were observed with other members of the polyol esters category.

In conclusion, all available and reliable in vitro and in vivo studies conducted with polyol esters category members revealed no effects on genetic toxicity. 


Short description of key category information:
In none of these studies mutagenicity in bacteria could be observed.
In none of these studies clastogenic effects in mammalian cells could be observed.
In none of these studies mutagenicity in mammalian cells could be observed.

Endpoint Conclusion: No adverse effect observed (negative)

The category justification is in IUCLID section 13.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint".Since the category concept is applied to the polyol esters, data gaps will be filled by interpolation, as part of a read across approach from a representative category member(s) to avoid unnecessary animal testing. Additionally, once the category concept is applied, substances will be classified and labelled on this basis. Therefore, based on the group concept, all available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.