Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies of bacterial reverse mutation (Ames test), micronucleus formation in cultured human lymphocytes and mammalian cell forward mutation (HPRT Assay) are available for the submission substance [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol]. All thre studies report negative results.

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:
weight of evidence
Study period:
06 June 2017 to 11 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Lot number: CVR83297
Purity: 97.2%
Molecular weight: 1281.67 g/mol
Appearance: Clear yellow-orange resin
Storage: Room temperature; protected from light
Expiry date: 27 December 2018
Target gene:
The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO) for the test substance; all positive controls were diluted in DMSO except for sodium azide, which was diluted in sterile water - Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (pre-incubation: incubated with shaking for 60±2 minutes at 37±2°C)
DURATION: Exposure duration: 48 to 72 hours
NUMBER OF REPLICATIONS: 2 in the initial-toxicity mutation assay; 3 in the confirmatory mutagenicity assay
NUMBER OF CELLS EVALUATED: >/= 0.3 x 10^8 cells/plate
DETERMINATION OF CYTOTOXICITY- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations. Strains TA1535 and TA1537Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range. Strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: Precipitate was observed beginning at 1500 or at 5000 µg per plate with all conditions in intial-toxicity mutation assay and confirmatory mutagenicity assay.
Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the study indicate that, under the conditions of this study, Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.
Executive summary:

The test substance [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was tested in an Ames test to evaluate its mutagenic potential by measuring its ability to induce reverse mutations Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia coli strain WP2uvrA, in the presence and absence of an exogenous metabolic activation system (induced rat liver S9 fraction).  Dimethyl sulphoxide (DMSO) was used as the vehicle. In an initial assay, concentrations of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 ¿g/plate were tested. No toxicity was observed. Precipitation of the test material was observed at concentrations of 1500 and 5000 ¿g/plate. No positive responses were observed with any of the tester strains in either the presence or absence of S9 activation. In a confirmatory mutagenicity assay, the concentrations tested were 15.0, 50.0, 150, 500, 1500 and 5000 ¿g/plate. No toxicity was observed. Precipitate was observed beginning at 1500 and 5000 ¿g/plate. No positive responses were observed with any of the tester strains in either the presence or absence of S9 activation. The results of this study indicate that the test material [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] is not mutagenic under the conditions of the assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 May 2017 to 28 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Source and lot/batch No.of test material: Sponsor and CVR83297
Expiration date of the lot/batch: 27 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage condition of test material: Room temperature
Stability under test conditions: Based on the expiration date provided in the Certificate of Analysis, the test substance was considered stable through 27 December 2018
Solubility of the test substance in the solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL with sonication at 26.7ºC for 10 minutes in the solubility test conducted at BioReliance
Target gene:
The purpose of this study was to evaluate a test article for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 2.00, 4.00, 8.00, 16.0 and 32.0 µg/mL with and without S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO as vehicle control and to dilute positive controls
Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
Exposure duration: 5 hours
Expression time (cells in growth medium): 7 days
Selection time (if incubation with a selection agent): 7 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the mutagenicity assay
NUMBER OF CELLS EVALUATED: 2.4 x 10e6 cells per culture
DETERMINATION OF CYTOTOXICITY- Method: adjusted relative survival
Evaluation criteria:
The test substance was considered to have produced a positive response if it induced a dose-related increase in mutation frequency and an increase exceedeing 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). . If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result was considered equivocal. If none of these criteria were met, the results were considered to be negative. Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Statistics:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preliminary toxicity assay: Adjusted relative survival (ARS) was 11.79% at 1000 µg/mL with S9 and 18.94% at 250 µg/mL without S9. Definitive mutagenicity assay: The average ARS was 101.86 and 82.82% at 32.0 µg/mL with and without S9, respectively
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Effects of pH: Preliminary Toxicity Assay: The pH of the solvent and cultures at concentrations ¿62.5 µg/mL with and without S9 was adjusted to maintain neutral pH.
Definitive Mutagenicity Assay: The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
Effects of osmolality: Preliminary Toxicity Assay: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. - Precipitation: Preliminary Toxicity Assay: Visible precipitate was observed at concentrations ¿125 µg/mL at the beginning of treatment and at concentrations ¿31.3 µg/mL by the end of treatment.
Definitive Mutagenicity Assay: Visible precipitate was observed at a concentration of 32.0 µg/mL at the beginning of treatment and end of treatment.

Summary of findings

 

-S9

+S9

Relative survival

Cloning efficiency

MF (x10-6)

Relative survival

Cloning efficiency

MF (x10-6)

DMSO

-

62.0%

3.63

-

88.3%

3.31

2 µg/mL

91.1%

67.0%

3.73

98.3%

93.5%

1.16

4 µg/mL

99.2%

56.5%

9.21

101.0%

84.8%

5.64

8 µg/mL

91.1%

68.9%

1.80

95.6%

95.2%

5.39

16 µg/mL

89.7%

66.1%

4.48

93.3%

77.8%

9.74

32 µg/mLp

82.8%

57.7%

7.86

101.9%

78.5%

2.24

EMS

62.7%

54.3%

391.31**

 

 

 

B(a)P

 

 

 

46.7%

86.5%

164.22**

**significantly different to controls (p<0.01)

pvisible precipitate

Conclusions:
Under the conditions of the assay described in this report, Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.
Executive summary:

The test substance [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine. Dimethyl sulphoxide (DMSO) was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.  The highest concentration evaluated approximated the limit concentration for this assay.  Visible precipitate was observed at concentrations ¿125 µg/mL at the beginning of treatment and at concentrations ¿31.3 µg/mL by the end of treatment.  Adjusted relative survival was 11.79% at a concentration of 1000 µg/mL with S9 and 18.94% at a concentration of 250 µg/mL without S9.  Adjusted relative survival approximated to 0% at all higher concentrations with and without S9.  Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 2.00, 4.00, 8.00, 16.0 and 32.0 µg/mL with and without S9. In the definitive mutagenicity assay, visible precipitate was observed at a concentration of 32.0 µg/mL at the beginning and end of treatment.  The average adjusted relative survival was 101.86 and 82.82% at a concentration of 32.0 µg/mL with and without S9, respectively.  Cultures treated at all concentrations with and without S9 were chosen for mutant selection.  No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p >0.01).  The positive controls induced significant increases in mutant frequency (p <0.01). These results indicate that the test material [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was negative for the ability to induce forward mutations at the HPRT locus of CHO cells in the presence and absence of an exogenous metabolic activation system.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 May 2017 to 27 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Lot number: CVR 83297
CAS number: 154565-28-3
Purity: 97.2%
Molecular weight: 1281.67 g/mol
Appearance: Clear yellow-orange resin
Storage: Room temperature; protected from light
Expiry date: 27 December 2018
Target gene:
Not applicable (micronucleus induction)
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Peripheral blood lymphocytes were obtained from a healthy non-smoking individuals. The donors had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals (Clare et al., 2006).
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 µg/mL concentration to block cytokinesis.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 0.2, 0.6, 2, 6, 20, 60, 200, 600 and 2000 µg/mL. The highest concentration of 2000 µg/mL was the limit cocnentration for this assay. Concentrations tested in the micronucleus assay were 2, 6, 20, 60, 200 µg/mL and were based upon post-treatment toxicity (cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) or visible precipitate in the treatment medium at the conclusion of the treatment period.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells.

In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Vinblastine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION: Exposure duration: HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
STAIN (for cytogenetic assays): Acridine orange
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange.
NUMBER OF CELLS EVALUATED: A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:¿ the micronucleus should have the same staining characteristics as the main nucleus¿ the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)¿ the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus
DETERMINATION OF CYTOTOXICITY- Method: other: cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control
Rationale for test conditions:
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986).
Evaluation criteria:
The test substance was considered to have induced a positive response if at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ¿ 0.05), and the increase was concentration-related (p ¿ 0.05), and¿ results were outside the 95% control limit of the historical negative control data.The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ¿ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Effects of pH: pH values were considered acceptable
Effects of osmolality: Osmolality was considered acceptable
Precipitation: In the preliminary toxicity assay, at the conclusion of the treatment period, visible precipitate was observed at doses ¿ 200 µg/mL in all three exposure groups. In the micronucleus assay, at the conclusion of the treatment period, visible precipitate was observed at 200 µg/mL in all three exposure groups.- Definition of acceptable cells for analysis: Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:¿ the micronucleus should have the same staining characteristics as the main nucleus¿ the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)¿ the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus
Remarks on result:
other: Negative

Summary of results

 

4/24h (-S9)

4/24h (+S9)

24/24h (-S9)

CBPI

Cytotoxicity

MNBN

CBPI

Cytotoxicity

MNBN

CBPI

Cytotoxicity

MNBN

DMSO

1.579

-

0.60%

1.602

-

0.40%

1.659

-

0.45%

6 µg/mL

1.543

6%

0.55%

1.509

15%

0.50%

1.567

14%

0.60%

60 µg/mL

1.473

18%

0.65%

1.474

21%

0.50%

1.467

29%

0.30%

200 µg/mLp

1.463

20%

0.60%

1.387

36%

0.45%

1.295

55%

0.35%

CP 5 µg/mL

1.420

27%

 -

1.285

53%

1.65%**

 -

VB 7.5 ng/mL

1.320

45%

 -

 -

1.574

13%

1.95%**

MNBN: micronucleated binuclear cell

CBPI: cytokinesis block proliferation index

pvisible precipitate

**significantly different to controls (p<0.01)

Conclusions:
The results of the study indicate that [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was negative for the induction of micronuclei in the presence and absence of metabolic activation.
Executive summary:

The test substance [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes in the absence and presence of an exogenous metabolic activation system (induced rat liver S9 fraction).  Dimethyl sulphoxide (DMSO) was used as the vehicle. In a preliminary toxicity assay, the concentrations tested ranged from 0.2-2000 µg/mL, the limit concentration for this assay.  Cytotoxicity (¿50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed at concentrations of ¿600 µg/mL in the non-activated and S9-activated 4-hour exposure groups and at concentrations of¿200µg/mL in the non-activated 24-hour exposure group.  At the conclusion of the treatment period, visible precipitate was observed at concentrations of ¿200 µg/mL in all three exposure groups.  Based upon these results, the concentrations chosen for the micronucleus assay ranged from 2 to 200 µg/mL for all three exposure groups. In the micronucleus assay, cytotoxicity (¿50% CBPI relative to the vehicle control) was not observed at any concentration in the non-activated and S9-activated 4-hour exposure groups.  Cytotoxicity was observed at 200 µg/mL in the non-activated 24-hour exposure group.  At the conclusion of the treatment period, visible precipitate was observed at 200 µg/mL in all three exposure groups.  The concentrations selected for evaluation of micronuclei were 6, 60, and 200 µg/mL for all three exposure groups. No significant or concentration-dependent increases in micronuclei induction were observed in treatment groups with or without S9. The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.  The results of the study indicate that [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was negative for the induction of micronuclei in the presence and absence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames test

Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was tested in an Ames test to evaluate its mutagenic potential by measuring its ability to induce reverse mutations Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia coli strain WP2uvrA, in the presence and absence of an exogenous metabolic activation system (induced rat liver S9 fraction).  Dimethyl sulphoxide (DMSO) was used as the vehicle. In an initial assay, concentrations of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 ¿g/plate were tested. No toxicity was observed. Precipitation of the test material was observed at concentrations of 1500 and 5000 ¿g/plate. No positive responses were observed with any of the tester strains in either the presence or absence of S9 activation. In a confirmatory mutagenicity assay, the concentrations tested were 15.0, 50.0, 150, 500, 1500 and 5000 ¿g/plate. No toxicity was observed. Precipitate was observed beginning at 1500 and 5000 ¿g/plate. No positive responses were observed with any of the tester strains in either the presence or absence of S9 activation. The results of this study indicate that the test material [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] is not mutagenic under the conditions of the assay.

Micronucleus assay

Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes in the absence and presence of an exogenous metabolic activation system (induced rat liver S9 fraction).  Dimethyl sulphoxide (DMSO) was used as the vehicle. In a preliminary toxicity assay, the concentrations tested ranged from 0.2-2000 µg/mL, the limit concentration for this assay.  Cytotoxicity (¿50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed at concentrations of ¿600 µg/mL in the non-activated and S9-activated 4-hour exposure groups and at concentrations of¿200µg/mL in the non-activated 24-hour exposure group.  At the conclusion of the treatment period, visible precipitate was observed at concentrations of ¿200 µg/mL in all three exposure groups.  Based upon these results, the concentrations chosen for the micronucleus assay ranged from 2 to 200 µg/mL for all three exposure groups. In the micronucleus assay, cytotoxicity (¿50% CBPI relative to the vehicle control) was not observed at any concentration in the non-activated and S9-activated 4-hour exposure groups.  Cytotoxicity was observed at 200 µg/mL in the non-activated 24-hour exposure group.  At the conclusion of the treatment period, visible precipitate was observed at 200 µg/mL in all three exposure groups.  The concentrations selected for evaluation of micronuclei were 6, 60, and 200 µg/mL for all three exposure groups. No significant or concentration-dependent increases in micronuclei induction were observed in treatment groups with or without S9. The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.  The results of the study indicate that [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was negative for the induction of micronuclei in the presence and absence of metabolic activation.

HPRT assay

Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine. Dimethyl sulphoxide (DMSO) was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.  The highest concentration evaluated approximated the limit concentration for this assay.  Visible precipitate was observed at concentrations ¿125 µg/mL at the beginning of treatment and at concentrations ¿31.3 µg/mL by the end of treatment.  Adjusted relative survival was 11.79% at a concentration of 1000 µg/mL with S9 and 18.94% at a concentration of 250 µg/mL without S9.  Adjusted relative survival approximated to 0% at all higher concentrations with and without S9.  Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 2.00, 4.00, 8.00, 16.0 and 32.0 µg/mL with and without S9. In the definitive mutagenicity assay, visible precipitate was observed at a concentration of 32.0 µg/mL at the beginning and end of treatment.  The average adjusted relative survival was 101.86 and 82.82% at a concentration of 32.0 µg/mL with and without S9, respectively.  Cultures treated at all concentrations with and without S9 were chosen for mutant selection.  No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p >0.01).  The positive controls induced significant increases in mutant frequency (p <0.01). These results indicate that the test material [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] was negative for the ability to induce forward mutations at the HPRT locus of CHO cells in the presence and absence of an exogenous metabolic activation system.

Justification for classification or non-classification

Based on the negative results in three studies in vitro, the submission substance [Epoxypropyl neodecanoate, oligmeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] does not require classification for germ cell mutagenicity according to the CLP criteria.