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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several mutagenicity studies have been conducted in in-vitro test systems where the Ames test (OECD 471) and the in vitro gene mutation in Mammalian cells (Mouse Lymphoma test) were both determined to be positive.

One in-vivo mutagenicity assays was performed: Micronucleus test in bone marrow cells of the mouse, the results of the study were negative and the test item ( 1,3-Isobenzofurandione, hexahydro-, reaction products with epichlorohydrin (source) and therefore bis(2-3-epoxypropyl)cyclohex-4-ene-1-2-dicarboxylate (target) is also concluded to have no significant mutagenic potential in-vivo.

It was concludedd that the test substance is mutagenic in vitro and not mutagenic in in-vivo experiment and therefore classifcation in accordance with UN-GHS is not required.

Link to relevant study records

Referenceopen allclose all

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to a published procedure.
Qualifier:
no guideline followed
Principles of method if other than guideline:
method according to Clive, D. et al Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutation Res. 59, 61-108 (1979)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase genes
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse Lymphoma cells L5178Y, heterocygous subline, TK +/- (Obtained from Dr. D. Clive with burroughs wellcome Company Research TRiangle Park, N. C., USA).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
without metabolic activation: 1.2, 2.5, 5, 7.5, 10, 11.5 and 12.5 nl/ml (first experiment)
without metabolic activation: 2, 4, 8, 12, 16, 18 and 20 nl/ml (second experiment)

with metabolic activation: 10, 20, 40, 60, 80, 90 and 100 nl/ml (first experiment)
with metabolic activation: 8, 16, 32, 48, 64, 72 and 80 nl/ml (second and third experiment)
Vehicle / solvent:
DMSO (1%)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate (without S9 activation) and dimethylnitrosamine (with S9 activation)
Details on test system and experimental conditions:
The test substance was dissolved in DMSO, and diluted into the culture medium.
In a preliminary toxicity test the concentration of TGIC causing a 85% reduction of the viability of cells was determined in suspension growth after 4-hour treatment and 72 hour suspension growth of the cells.
For this test cells were taken from exponential growth phase in Fischer’s medium containing 10% horse serum and anti-biotics; the final concentration of DMSO in the cell culture medium was 1%.
Seven concentrations were tested (51.563 – 3300 nl/ml) in the first and second (0.7183 - 50 nl/ml) test.
Suspension growth was compared to control cultures of the same initial cell density. The concentration calculated to cause 85% reduction of relative growth was used as highest dose in the mutagenicity experiment.
The mutagenicity test was carried out using exponentially growing cells (free of TK -/- cells) at 3 x 10E5 cells/ml, in round bottom flasks. After cleaning of spontaneous mutants and an additional three-days incubation period in THG, the cells were treated for four hours with the selected concentrations.
S9-activating mix was prepared from livers of male rats treated with Aroclor 1254.
After the 4-hour treatment, cells were washed and resuspended in growth medium and grown for 72 hours. Cell counts were regularly adjusted to 3 x 10E5 cells/ml. At the end of this expression period, cells were transferred to culture tubes containing 5 ml semi-solid agar cloning medium: for each concentration 8 tubes were seeded with 4 x 10E5 cells, in cloning medium containing BudR at 50 ug/ml, for viability counts 4 tubes of each concentration were seeded with 200 cells each in cloning medium without BudR.
Incubation time was 14 to 16 days at 37°C, and 12 days for the viability tubes.
Colonies were counted with a Colony counter (Fisher Count-All, Model 600), and with the aid of the viability counts the relative number of mutant colonies (TK-/-) was determined.
The results are expressed as induced TK -/- mutants /10E6 surviving cells. Mutagenicity criteria is an increase of 2.5 above control in any of the concentrations tested.
Positive control were ethylmethanesulfonate for the experiment without, and dimethylnitrosamine for the experiment with S9-activation
Evaluation criteria:
The results are expressed as induced TK -/- mutants /10E6 surviving cells. Mutagenicity criteria is an increase of 2.5 above control in any of the concentrations tested.
Statistics:
no data
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation

TK 12 608 caused mutagenic effects in the presence and absence of metabolic activation, and is therefore a direct-acting mutagen under the conditions of the assay.
Executive summary:

TK 12 608 was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 )ug/0.1 ml. In order to confirm the results, the experiments were repeated with the same concentrations.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone backmutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture(rat liver microsomes and co-factors).

In the experiments performed without microsomal activation treatment with TK 12 608 led to a marked increase in the number of back-mutant colonies of strains TA 100 and TA 1535. In the experiments carried out with microsomal activation this effect was more pronounced.

TK 12 608 and its metabolites formed as a result of microsomal activation thus displayed a clear-cut mutagenic effect in this test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to a published procedure.
Justification for type of information:
Please refer to the Read-across justification document enclosed in chapter 13 for more details.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
method according to Clive, D. et al Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutation Res. 59, 61-108 (1979)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase genes
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse Lymphoma cells L5178Y, heterocygous subline, TK +/- (Obtained from Dr. D. Clive with burroughs wellcome Company Research TRiangle Park, N. C., USA).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
without metabolic activation: 1.2, 2.5, 5, 7.5, 10, 11.5 and 12.5 nl/ml (first experiment)
without metabolic activation: 2, 4, 8, 12, 16, 18 and 20 nl/ml (second experiment)

with metabolic activation: 10, 20, 40, 60, 80, 90 and 100 nl/ml (first experiment)
with metabolic activation: 8, 16, 32, 48, 64, 72 and 80 nl/ml (second and third experiment)
Vehicle / solvent:
DMSO (1%)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate (without S9 activation) and dimethylnitrosamine (with S9 activation)
Details on test system and experimental conditions:
The test substance was dissolved in DMSO, and diluted into the culture medium.
In a preliminary toxicity test the concentration of TGIC causing a 85% reduction of the viability of cells was determined in suspension growth after 4-hour treatment and 72 hour suspension growth of the cells.
For this test cells were taken from exponential growth phase in Fischer’s medium containing 10% horse serum and anti-biotics; the final concentration of DMSO in the cell culture medium was 1%.
Seven concentrations were tested (51.563 – 3300 nl/ml) in the first and second (0.7183 - 50 nl/ml) test.
Suspension growth was compared to control cultures of the same initial cell density. The concentration calculated to cause 85% reduction of relative growth was used as highest dose in the mutagenicity experiment.
The mutagenicity test was carried out using exponentially growing cells (free of TK -/- cells) at 3 x 10E5 cells/ml, in round bottom flasks. After cleaning of spontaneous mutants and an additional three-days incubation period in THG, the cells were treated for four hours with the selected concentrations.
S9-activating mix was prepared from livers of male rats treated with Aroclor 1254.
After the 4-hour treatment, cells were washed and resuspended in growth medium and grown for 72 hours. Cell counts were regularly adjusted to 3 x 10E5 cells/ml. At the end of this expression period, cells were transferred to culture tubes containing 5 ml semi-solid agar cloning medium: for each concentration 8 tubes were seeded with 4 x 10E5 cells, in cloning medium containing BudR at 50 ug/ml, for viability counts 4 tubes of each concentration were seeded with 200 cells each in cloning medium without BudR.
Incubation time was 14 to 16 days at 37°C, and 12 days for the viability tubes.
Colonies were counted with a Colony counter (Fisher Count-All, Model 600), and with the aid of the viability counts the relative number of mutant colonies (TK-/-) was determined.
The results are expressed as induced TK -/- mutants /10E6 surviving cells. Mutagenicity criteria is an increase of 2.5 above control in any of the concentrations tested.
Positive control were ethylmethanesulfonate for the experiment without, and dimethylnitrosamine for the experiment with S9-activation
Evaluation criteria:
The results are expressed as induced TK -/- mutants /10E6 surviving cells. Mutagenicity criteria is an increase of 2.5 above control in any of the concentrations tested.
Statistics:
no data
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation

TK 12 608 caused mutagenic effects in the presence and absence of metabolic activation, and is therefore a direct-acting mutagen under the conditions of the assay.
Executive summary:

TK 12 608 was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 )ug/0.1 ml. In order to confirm the results, the experiments were repeated with the same concentrations.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone backmutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture(rat liver microsomes and co-factors).

In the experiments performed without microsomal activation treatment with TK 12 608 led to a marked increase in the number of back-mutant colonies of strains TA 100 and TA 1535. In the experiments carried out with microsomal activation this effect was more pronounced.

TK 12 608 and its metabolites formed as a result of microsomal activation thus displayed a clear-cut mutagenic effect in this test system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date: 12 June 2014 and Experimental completion date: 15 July 2014
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:
Adopted July 21, 1997.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Species / strain / cell type:
other: TA98, TA100, TA1535, TA 1537 and Escherichia coli WP2 uvrA
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
- In the preliminary toxicity assay: 6.7 / 10 / 33 / 67 / 100 / 333 / 667 / 1000 / 3333 and 5000 μg per plate.
In the preliminary assay, neither precipitate nor toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.
- In the mutagenicity assay: 30 / 100 / 300 / 1000 / 2500 and 5000 μg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxyde (DMSO)
- Justification for choice of solvent/vehicle: DMSO wa selected as the solvent of choice based on information provided by the sponsor, solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
Strain TA98 and TA1535.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
Strain TA100 and TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
Strain WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
2-nitrofluorene
Remarks:
Strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in sterile water
Positive control substance:
sodium azide
Remarks:
Strain TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
9-aminoacridine
Remarks:
Strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
methylmethanesulfonate
Remarks:
Strain WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

Number Cells Analyzed/Culture: 1.4 to 5.4 x10E8 cells per plate.

NUMBER OF REPLICATIONS:
- In the preliminary toxicity assay: Single plate per condition.
- In the mutagenicity assay: Triplicate

Preparation of Tester Strain
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.
Rationale for test conditions:
The following criteria must be met for the mutagenicity assay to be considered valid:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.

The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.

A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5)
Evaluation criteria:
Evaluation of Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

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 of test substance.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

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:
Not applicable
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
other: TA100, TA1535 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
positive
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:
Please se "Any other information on results incl. tables"
Remarks on result:
other: positive mutagenic responses were observed with tester strain TA98 in the presence of S9 activation

Solubility Test

DMSO was selected as the solvent of choice based on information provided by the Sponsor, solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes

Tester Strain titer Results

               Tester strain
   TA98 TA100  TA1535  TA1537  WP2 uvrA 
 Experiment              Titer Value (x10E9 cells per mL)
 B1  2.9 1.4  4.1  4.3  5.4 

Preliminary Toxicity Assay

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Although mutagenicity is not formally assessed in the preliminary toxicity assay, increases in revertant counts (ranging from 1.9- to 78.6-fold maximum increases) were observed with tester strains TA98, TA100, TA1535 and WP2 uvrA. Neither precipitate nor toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

Mutagenicity Assay

In Experiment B1 (Mutagenicity Assay), positive mutagenic responses were observed with tester strain TA98 in the presence of S9 activation and with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of S9 activation.

Maximum fold increases are indicated in the following table:

      Activation conditions
   -S9  +S9
 Tester strain     Maximum Fold Increase
 TA98  / 2.3 
 TA100  8.2 8.8 
 TA1535  17.0 49.9 
 WP2 uvrA  5.0 5.4 

The dose levels tested were 30, 100, 300, 1000, 2500 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

HISTORICAL CONTROL DATA:

                                  

Historical Negative and Positive Control Values

2011 -2013

Revertants per plate

                               Activation
                None Rat Liver            
 Strain Control   Mean SD  Min  Max  Mean  SD  Min  Max 

 TA98

 Neg  18 64  24 60 

TA98

 Pos  271 214  34  2274    423 190  47   1711  

TA100

 Neg  98 18  50  251  110  23 

55 

247 

TA100

 Pos  608 143 211  1393    730  260 247  2421   

TA1535

 Neg 11  4 1  43  12  4  1  35

TA1535

 Pos  501 164 20  1593    103  92  20  1472  

TA1537

 Neg  7 28  0 28 

TA1537

Pos  422 386  17  3711    59  56  10  850  

 WP2 uvrA

 Neg 27  10  5  84  30 10  7  80

WP2 uvrA

 Pos  380  160  42  1796    245  98  21  969  

SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

 Metabolic activation  Test substance

 Dose Level

(μg/plate)

Revertant Colony Counts (Mean +/- SD)               
 Without metabolic Activation      TA98 TA100  TA1535  TA1537  WP2 uvrA   
   DMSO  50 microL/plate  10 +/- 1 108 +/-15 10 +/- 5  9 +/-4 24 +/-4  
    Test item 30  14 +/-4 127 +/-2 13 +/-2 6 +/-4  22 +/-9  
    Test item  100  20 +/-3 196 +/-33 16 +/-5  6 +/-3   24 +/-8  
    Test item  300  16 +/-3 369 +/-45 47 +/-5  6 +/-3  41 +/-6  
    Test item  1000  19 +/-13 592 +/-88  89 +/-22 6 +/-0  75 +/-5  
    Test item  2500 24 +/-4  845 +/-97 170 +/-15 8 +/-4 93 +/-24  
   Test item  5000 39 +/-8 890 +/-44  163 +/-23  6 +/-3  119 +/-11   
   2NF  1.0  79 +/-28      
   SA 1.0    391 -/-25  381 +/-41      
   9AAD  75        111 +/-97    


   MMS  1000          264 +/-9  

Key to positive controls:

SA: sodium azide

2AA: 2-aminoanthracene

9AAD: 9-Aminoacridine

2NF: 2-nitrofluorene

MMS: methyl methanesulfonate

 Metabolic activation  Test substance

 Dose Level

(μg/plate)

Revertant Colony Counts (Mean +/- SD)             
 Without metabolic Activation      TA98 TA100  TA1535  TA1537  WP2 uvrA 
   DMSO  50 microL/plate 27 +/-2 105 +/-8 14 +/-3 90 +/-3 23 +/-10
    Test item 30 24 +/-6 129 +/-13 26 +/-9 17 +/-13 25 +/-6
    Test item 100 27 +/-5 139 +/-10 67 +/-12 7 +/-1 28 +/-7
    Test item 300 21 +/-10 238 +/-29 153 +/-17 6 +/-1 25 +/-3
    Test item 1000 26 +/-7 364 +/-132 380 +/-61 10 +/-1 70 +/-12
    Test item 2500 38 +/-11 920 +/-268 565 +/-53 11 +/-4 117 +/-38
   Test item

5000

61 +/-8

834 +/-159 

698 +/-23 

12 +/-3 

124 +/-3 

 

 2AA

1.0

276 +/-16

57 +/-8

 

 

 2AA

2.0

502 +/-131

 41 +/-8

 

 

 2AA

15

 

 

 

 

 122 +/-11

Key to positive controls:

2AA: 2-aminoanthracene

Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test item did cause positive mutagenic responses with tester strain TA98 in the presence of Aroclor-induced rat liver S9 and with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted.
Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test substance.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on information provided by the Sponsor, solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Although mutagenicity is not formally assessed in the preliminary toxicity assay, increases in revertant counts (ranging from 1.9- to 78.6-fold maximum increases) were observed with tester strains TA98, TA100, TA1535 and WP2 uvrA. Neither precipitate nor toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, positive mutagenic responses (ranging from 2.3- to 49.9-fold maximum increases) were observed with tester strain TA98 in the presence of S9 activation and with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of S9 activation. The dose levels tested were 30, 100, 300, 1000, 2500 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Under the conditions of this study, test substance was concluded to be positive in the Bacterial Reverse Mutation Assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

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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:
04-Nov-2011 to 25-Jan-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: first main study 32 - 39 g and for the second main study 31 - 39 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 - 21.8°C
- Humidity (%): 44 - 72%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Test compound was stable in corn oil and an emulsion could be obtained in corn oil. Corn oil has been accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 43.8, 87.5, 150 and 175 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight
Duration of treatment / exposure:
First experiment:
Treatment:
Solvent, positive control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours

Second experiment
Treatment:
Solvent, positive control and dose level: 48 hours
Frequency of treatment:
Once
Remarks:
Doses / Concentrations:
First experiment: 438, 875 and 1750 mg/kg BW
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Second experiment: 1500 mg/kg BW
Basis:
nominal conc.
No. of animals per sex per dose:
At leasr five animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight
Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.
Statistics:
Wilcoxon Rank Sum Test, one-sided, p < 0.05
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500, 1750, 1875 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
In the dose range finding test, one animal per sex was dosed with 2000 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight. The animals died within 21 hours after dosing. One animal per sex dosed with 1500 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight showed the following toxic sign: hunched posture.
In total 3 animals per sex were dosed with 1750 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight. Initially, one male and one female dosed with 1750 mg
bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight showed the following toxic signs after dosing: lethargy, rough coat and hunched posture.

One male and one female animal dosed with 1875 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight died within 20 and 5 hours, respectively.
Two additional males and females dosed with 1750 mg/kg body weight showed the following toxic signs after dosing: lethargy, hunched posture and rough coat (2 males, 1 female).


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
First experiment: The animals dosed with 1750 mg/kg body weight showed the following toxic signs after dosing: hunched posture, rough coat (7 animals), lehargy (7 animals), closed eyes (2 animals), ventral recumbency (2 animals) and a low body temperature (2 animals). Seven animals died within 24 hours after dosing. No treatment related clinical signs or mortality were noted in the animals treated with 875 and 438 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kg body weight
Second experiment:
The animals showed the following toxic signs after dosing: hunched posture and rough coat (3 animals). One animal died within 18 hours after dosing.
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. However, the clinical signs observed were taken to indicate that systemic absorption had occurred
- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via the oral route administration.
Conclusions:
Interpretation of results (migrated information): negative
bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 1750 mg/kg and 1500 mg/kg, respectively (the maximum tolerated dose in accordance with current regulatory guidelines)
Executive summary:

No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate.

 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

 

The groups that were treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
04-Nov-2011 to 25-Jan-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Justification for type of information:
Please refer to the Read-Across justification document enclosed in chapter 13 for more details.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: first main study 32 - 39 g and for the second main study 31 - 39 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 - 21.8°C
- Humidity (%): 44 - 72%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Test compound was stable in corn oil and an emulsion could be obtained in corn oil. Corn oil has been accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 43.8, 87.5, 150 and 175 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight
Duration of treatment / exposure:
First experiment:
Treatment:
Solvent, positive control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours

Second experiment
Treatment:
Solvent, positive control and dose level: 48 hours
Frequency of treatment:
Once
Remarks:
Doses / Concentrations:
First experiment: 438, 875 and 1750 mg/kg BW
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Second experiment: 1500 mg/kg BW
Basis:
nominal conc.
No. of animals per sex per dose:
At leasr five animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight
Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.
Statistics:
Wilcoxon Rank Sum Test, one-sided, p < 0.05
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500, 1750, 1875 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
In the dose range finding test, one animal per sex was dosed with 2000 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight. The animals died within 21 hours after dosing. One animal per sex dosed with 1500 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight showed the following toxic sign: hunched posture.
In total 3 animals per sex were dosed with 1750 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight. Initially, one male and one female dosed with 1750 mg
bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight showed the following toxic signs after dosing: lethargy, rough coat and hunched posture.

One male and one female animal dosed with 1875 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight died within 20 and 5 hours, respectively.
Two additional males and females dosed with 1750 mg/kg body weight showed the following toxic signs after dosing: lethargy, hunched posture and rough coat (2 males, 1 female).


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
First experiment: The animals dosed with 1750 mg/kg body weight showed the following toxic signs after dosing: hunched posture, rough coat (7 animals), lehargy (7 animals), closed eyes (2 animals), ventral recumbency (2 animals) and a low body temperature (2 animals). Seven animals died within 24 hours after dosing. No treatment related clinical signs or mortality were noted in the animals treated with 875 and 438 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kg body weight
Second experiment:
The animals showed the following toxic signs after dosing: hunched posture and rough coat (3 animals). One animal died within 18 hours after dosing.
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. However, the clinical signs observed were taken to indicate that systemic absorption had occurred
- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via the oral route administration.
Conclusions:
Interpretation of results (migrated information): negative
bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 1750 mg/kg and 1500 mg/kg, respectively (the maximum tolerated dose in accordance with current regulatory guidelines)
Executive summary:

No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate.

 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

 

The groups that were treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

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

Additional information

A reliable bacterial reverse mutation test, an in vitro gene mutation in Mammalina cells (Mouse Lymphoma test) and in vivo Micronucleus test are available, the two in-vitro tests were positive for mutagenicity.

The in vivo Micronucleus test was negative. All these three studies are taken into consideration to conclude that the test substance is not classified.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).