7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A number of in vitro genotoxicity studies are available for the substance.

An Ames test (Daicel, 1987) was performed in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli WP2. This study reports positive responses in strains TA100 and TA1535 when tested in the presence of metabolic activation; no response was seen in the absence of metabolic activation. A further Ames test (Machigaki, 1995) was performed in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli WP2. This study also reports positive responses in strains TA100 and TA1535 in the presence of metabolic activation. The positive responses seen in the Ames tests performed with the substance are indicative of a base pair mutagenic response, which is dependent on metabolic activation.

A mouse lymphoma assay (Beilstein, 1984) reports a positive response, both in the absence and presence of metabolic activation.  A clastogenicity assay performed in vitro in CHO cells (Slesinski et al., 1980) reports consistently negative results both in the absence and presence of metabolic activation. Slesinski et al. (1980) also report a positive and ‘highly significant’ result in a sister chromatid exchange (SCE) assay in CHO cells; this assay was performed in the absence of metabolic activation only. The same authors report an equivocal result in an unscheduled DNA synthesis (UDS) assay using cultured primary rat hepatocytes. A very weak increase in the level of DNA damage indicated at lower concentrations in this study was not replicated at higher concentrations.

The results of the in vitro studies therefore indicate a genotoxic potential for the substance.

Link to relevant study records

Reference

Reference 1

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:

3 August 1995 to 2 October 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: Ministry of Labor of Japan 1988

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan genes

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Test concentrations with justification for top dose:

156, 313, 625, 1250, 2500 and 5000ug per plate were used.

Vehicle / solvent:

Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

0.01ug / plate

Positive control substance:

2-acetylaminofluorene

Remarks:

Positive control used without metabolic activation for TA 100 Migrated to IUCLID6: AF-2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

0.5ug / plate

Positive control substance:

sodium azide

Remarks:

Positive control used without metabolic activation for TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

80ug / plate

Positive control substance:

9-aminoacridine

Remarks:

Positive control used without metabolic activation for TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

2ug / plate

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Positive control used without metabolic activation for E coli. WP2 uvrA-

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

0.5ug / plate (TA98), 1ug / plate (TA100), 2ug / plate (TA1535), 2ug / plate (TA1537), 10ug / plate (E.coli WP2 uvrA-)

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Positive control used with metabolic activation for all strains of bacteria.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 2 days
- Expression time (cells in growth medium): Not documented
- Selection time (if incubation with a selection agent): Not documented
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): No information provided.
SPINDLE INHIBITOR (cytogenetic assays): Not relevant
STAIN (for cytogenetic assays): Not relevant

NUMBER OF REPLICATIONS: Three plates per dose level were used for each assay and the test was run twice.

NUMBER OF CELLS EVALUATED: Not documented

DETERMINATION OF CYTOTOXICITY
- Method: other: Number of revertants per plate.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

OTHER: Not documented

Evaluation criteria:

The test substance was positive in this assay when the mean number of revertants was more than double the negative control value and when the increase was significantly dose-dependent.

Statistics:

No information provided

Species / strain:

other: S. typhimurium TA100 and TA1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 98 and TA1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not documented
- Effects of osmolality: Not documented
- Evaporation from medium: Not documented
- Water solubility: Not documented
- Precipitation: Not documented
- Other confounding effects: Not documented

RANGE-FINDING/SCREENING STUDIES:
In the dose finding test, the numbers of revertants induced by the test substance were more than double of the solvent control value in S. typhimurium TA100 and TA1535 with S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
No information provided

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The positive controls induced significant revertants. For the solvent control, the numbers of revertants of all the tester strains were within the normal range of our laboratory's background data respectively. These results assured that the tests were performed correctly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Strain Gene affected Additional mutations Mutation type

Repair LPS R-factor

S. typhimuriurn

TA98 his D uvrB rfa pKM 101 Frameshift

TAlOO his G uvrB rfa pKM101 Base-pair change

TA1535 his G uvrB rfa - Base-pair change

TA1537 his C uvrB rf'a - Frameshift

E. coli

WP2 uvrA- trp E uvrA + - Base-pair change

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation For TA 100 and TA 1535, all other strains with or without metabolic activation gave negative results

Under the conditions employed in this test, the test substance UVR-6110, was considered to be a mutagen in the presence of metabolic activation when tested in the Salmonella strains TA100 and TA1535 which indicated a base pair mutagenic response. Without S-9 there were no positive responses in the strains tested.

Executive summary:

In a study conducted by Machigaki (1991), the test substance, UVR-6110 was examined for its potential to be mutagenic when tested in a bacterial assay using 5 strains of bacteria, including Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and the Escherichia coli WP2 uvrA- strain. The study was performed using the following concentrations, 156, 313, 625, 1250, 2500 and 5000ug per plate. Each concentration was tested in triplicate and each assay was conducted twice. Each strain was tested using the appropriate a suitable positive control, relevant for each bacterial strain. Each assay was done both in the presence and absence of metabolic activation in the form of S9 mix.

Based on the results of this study, UVR-6110 was considered to be mutagenic under the test conditions employed in this study in the presence of metabolic activation when tested in the Salmonella strains TA100 and TA1535 which indicated a base pair mutagenic response. Without S-9 there were no positive responses in the strains tested..

Reference 2

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:

26 April 1979 to 16 May 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No indication of Guidelines complied with or if the study was conducted according to GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was examined in Chinese Hamster Ovary cells in the presence and absence of metabolic activation and appropriate positive, negative and solvent controls.

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Epoxy Resin ERL-4221
- Molecular formula (if other than submission substance): C14H20O4
- Molecular weight (if other than submission substance): 252.30g
- Physical state: Light yellow, viscous liquid.
- Lot/batch No.: 42-136
- Stability under test conditions: Stable, avoid heating over 37.7°C

Target gene:

HGPRT gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium
supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine.
For treatment of cells without metabolic activation, F12 medium with 50 units/ml of penicillin, 50 ug/ml streptomycin and 5% (v/v) of dialyzed bovine serum (F12-D5) was used. For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, was employed.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - determined by a microscopic fluorescence assay employing Hoechst 33258 dye.
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Arochlor 1254 induced, male Sprague Dawley rats.

Test concentrations with justification for top dose:

Five concentrations ranging from 100 x 10E-4% to 6.25 x 10E-4% without activation or 200 x 10E-4% to 12.5 x 10E-4% with activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

glass-distilled dimethyl-sulfoxide (DMSO)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

Positive control used with S9 metabolic activation.

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

glass-distilled dimethyl-sulfoxide (DMSO)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control used without S9 metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: Not documented
- Exposure duration: 16 hours for cells exposed in the absence of metabolic activation and 5 hours for cells exposed in the presence of metabolic activation.
- Expression time (cells in growth medium): 7 - 9 days.
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

NUMBER OF REPLICATIONS:
The experiment was conducted twice.

NUMBER OF CELLS EVALUATED:
Only the top five concentrations which allowed sufficient cell survival were assessed for survival and induction of mutants. 100 cells per dish were evaluated, meaning 200 cells per dose were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: other: the percentage of cells surviving the treatment, the frequencies of mutant colonies and the number of mutants/10E6 viable cells.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

Evaluation criteria:

The criteria for interpretation of the test result as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response following treatment.

Statistics:

The use of Parametric analyses is not justified unless the data can be transformed before application of standard parametric tests. The Box-Cox Transformation transforms data prior to parametric analyses. The mutation frequency for each plate was increased by 1.0 (to eliminate zeros) and raised to the 0.15 power. Parametric analysis of mutation data by the Student's t - test was performed with the transformed data.

Species / strain:

Chinese hamster Ovary (CHO)

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:

RANGE-FINDING/SCREENING STUDIES:
A concentration of 100 x 10E-4% was selected as the maximum concentration for testing with and without S9 activation. In a second repeat experiment, the maximum concentration tested with S9 activation was increased to 200 x 10E-4% to attain a higher level of cytotoxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
No information provided

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cells treated with the test agent together with an S9 activation system were not assessed for mutant induction because the C02 concentration in the incubator used for these plates was abnormally high (due to a malfunction) and this malfunction inhibited or killed the cells. The results of the second experiment indicated a similar cytotoxic response at the highest dose level tested without S9 activation in comparison to the results obtained in the first experiment (a steep dose-response effect with the test agent was suggested from the high degree of cytotoxicity observed for the top concentrations (100 x 10E-4) in comparison to the markedly lower cytotoxicity obtained at only one-half the top dose-level)

In the mutation study, Epoxy Resin ERL-4221 did not produce a dose related increase in the frequency of mutants/10E6 viable cells over the 16-fold range of concentrations tested for potential mutagenic action without the presence of an S9 metabolic activation system. Although no concentration of Epoxy Resin ERL-4221 produced a statistically significant increase in the mutation frequency in the test without S9 activation, the test was repeated because the EMS positive control was also not significantly above the solvent control values. The results obtained in a repeat experiment were consistent with the results obtained in the 1st test. No dose-level of Epoxy Resin ERL-4221 produced an increase which was statistically significant from the solvent control, and there was no indication of a dose-related production of mutants. This second experiment was consistent with the classification of Epoxy Resin ERL-4221 as not active in producing a mutagenic effect detectable in the CHO test system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mutation frequencies for the solvent controls for test in both experiment #l and #2 without S9 activation were in an acceptable and low range based upon experience with historical control values.. The mutation frequency for the solvent control in experiment #2 with S9 activation was numerically higher than our historical control values but similar small increases in the frequency of mutants have been seen in previous experiments in which the S9 liver homogenate itself displays a weak mutagenic activity. Small numerical increases at some treatment levels of Epoxy Resin ERL-4221 were within the range of historical variability encountered for negative and solvent controls using this test.

Highly statistically significant mutation frequencies were obtained for the DMN and EMS positive controls in experiment #2 and these values were within the normally expected range of variation observed in historical control data.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Epoxy Resin ERL-4221 was consistently inactive as a mutagenic agent for CHO cells when tested with or wlthout an S9 metabolic activation system over a 16-fold range of concentrations. Although small increases in the numerical frequency of mutants were obtained at some test concentrations of Epoxy Resin ERL-4221, these results appeared to be a random effect without statistical or probable biological significance.

Executive summary:

In a study conducted by Slesinski et al (1980), the test substance, Epoxy Resin ERL-4221 was evaluated for its potential to induce mutagenic activity in an in vitro gene mutation study using Chinese Hamster Ovary (CHO) cells. The test was conducted in the presence and absence of metabolic activation in the form of S9 mix. The experiment was conducted twice, with concentrations in the 1st experiment ranging from 100 x 10E-4% to 6.25 x 10E-4% and in the 2nd experiment from 200 x 10E-4% to 12.5 x 10E-4%. The tests were conducted alongside appropriate positive, negative and solvent controls. Based on the results of this study, Epoxy Resin ERL-4221 was consistently inactive as a mutagenic agent for CHO cells when tested with or wlthout an S9 metabolic activation system over a 16-fold range of concentrations. Although small increases in the numerical frequency of mutants were obtained at some test concentrations of Epoxy Resin ERL-4221, these results appeared to be a random effect without statistical or probable biological significance. As a result of this, the test substance does not require classification according to Regulation EC No. 1272/2008.

Reference 3

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:

March 19th 1984 to December 17th 1984.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No indications of guidelines adhered to. There is no indication of the number of replicates conducted.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was examined in Mouse Lymphoma L5178Y cells in the presence and absence of metabolic activation and appropriate positive, negative and solvent controls.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): TK 10 310 (ARALDIT CY 179)
- Molecular formula (if other than submission substance): Not documented
- Molecular weight (if other than submission substance): Not documented
- Smiles notation (if other than submission substance): Not documented
- InChl (if other than submission substance): Not documented
- Structural formula attached as image file (if other than submission substance): see Fig. Not documented
- Substance type: Not documented
- Physical state: Not documented
- Analytical purity: Commercial grade
- Impurities (identity and concentrations): Not documented
- Composition of test material, percentage of components: Not documented
- Isomers composition: Not documented
- Purity test date: Not documented
- Lot/batch No.: 608632
- Expiration date of the lot/batch: Not documented
- Radiochemical purity (if radiolabelling): Not documented
- Specific activity (if radiolabelling): Not documented
- Locations of the label (if radiolabelling): Not documented
- Expiration date of radiochemical substance (if radiolabelling): Not documented
- Stability under test conditions: Stable
- Storage condition of test material: Not documented

Target gene:

TK +/-. Mouse lymphoma (L5178Y), heterozygous subline TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK +/- cells from growing stock cultures were cleansed of spontaneous TK-/-mutants by exposure for 24 hours to THMG (a combination of thymidine, hypoxanthine, methotrexate and glycine). This procedure ensured a low background of TK-/- mutant clones . After an additional three-days incubation period in THG (thymidine, hypoxanthine and glycine) medium the cells were ready to be used.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix derived from rat liver.

Test concentrations with justification for top dose:

12.5, 25, 50, 100, 150, 200 and 250 ug/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1%)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

0.75ul/ml

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control used in the absence of metabolic activation

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

0.75ul/ml

Positive control substance:

N-dimethylnitrosamine

Remarks:

Positive control used in the presence of metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: Not documented
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 3 days.
- Selection time (if incubation with a selection agent): 14 days for mutant selection and 11 - 12 days for viability in the experiments with and without metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): 5-Bromodeoxyuridine (BUdR)
SPINDLE INHIBITOR (cytogenetic assays): Not documented
STAIN (for cytogenetic assays): Not documented

NUMBER OF REPLICATIONS: Not documented

NUMBER OF CELLS EVALUATED: 4x10E5 cells per tube were evaluated for mutant selection and 200 cells per tube were evaluated for viability control.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth and mutant frequency

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

OTHER:
Following treatement with the test material, the cells were washed once with 25 ml F10P to remove the test substance, resuspended in F10P
medium and allowed to grow for three days to express the induced forward TK -/-- mutants. Cell counts were performed and registered daily and the cell number in each case was adjusted to the initial count (3 x 10E5 cell/ml ). At the end of the expression period, for mutant selection as well as for viability control cultures were set up in culture tubes containing 5 ml of a semi-solid agar cloning medium. For mutant selection, eight tubes were prepared at each concentration containing 4 x 10E5 cells per tube in cloning medium with BUdR at a final concentration of 50 pg/ml. For viability control four tubes for each concentration were set up containing 200 cells per tube in cloning medium without BUdR.

Evaluation criteria:

The test substance was considered to be mutagenic in this test system if the colony count exceeded that of the solvent control by a factor of more than 2.5 at any concentration.

Statistics:

No information provided

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: Owing to the solubility limit of the test substance, the concentration of 250 ug/ml was used as the highest in the preliminary toxicity test.
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES:
In both toxicity tests an 80-90% reduction in the relative suspension growth was not obtained with any of the concentrations applied. Therefore, for mutagenicity testing with and without microsomal activation the highest concentration (250 ug/ml) applied in the toxicity test together with six further concentrations of 200, 150, 100, 50, 25 and 12.5ug/ml were used.

In the mutagenicity test without metabolic activation, the mutant frequency of the solvent control was 2.9 x 10E-6. At the highest concentration, the mutant frequency values of the treated cultures were 557.7x10-6 and the positive control yielded a mutant frequency value of 274.6x10E-6, meaning a mutant factor of 80.5 was determined.

In the mutagenicity test with metabolic activation, the mutant frequency of the solvent control was 4.6 x 10E-6. At the highest concentration, the mutant frequency values of the treated cultures were 16.9x10E-6. When the mutant factors of the positive control and the test substance were compared, a mutant factor of 11.9 was recorded.

COMPARISON WITH HISTORICAL CONTROL DATA:
No information provided.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No additional information provided.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No additional information

Conclusions:

Interpretation of results (migrated information):
positive with and without metabolic activation.

Under the experimental conditions of this study, the test substance was considered to be mutagenic, both in the presence and absence of metabolic activation.

Executive summary:

In a study conducted by Beilstein (1984), the test substance TK 10 310 (ARALDIT DY 179) was tested for its potential to cause mutagenic effects on L5178Y/TK+/- mouse lymphoma cells in vitro, both in the presence and absence of microsomal metabolic activation and the appropriate positive controls were also used. A mutagenic effect of a substance was demonstrable on comparison of the number of colonies in the treated and control cultures. The test substance was examined at 7 concentrations, 12.5, 25, 50, 100, 150, 200 and 250ug/ml.

The results of this study indicate that the test substance was considered to be mutagenic as the colony count of the test substance was greater than 2.5 at the top three concentrations in the test in the presence of metabolic activation and the 4 highest concentrations in the test conducted in the absence of metabolic activation indicated mutagenic activity.

Based on these results, the test substance was considered to be mutagenic, both in the presence and absence of metabolic activation.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

December 10th 1979 to March 24th 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No indication of whether any specific international test guidelines were followed. Study conducted prior to adoption of GLP. The test was not conducted in the presence of metabolic activation, however a justification was provided.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mammalian cells were exposed to the test substance in the absence of metabolic activation and cultured in BrdU-containing medium. Cells were treated with a spindle inhibitor, harvested and chromosomal preparations were made.
Because there was a highly significant dose-related indication of direct mutagenic effect in this study without the addition of metabolic activation, the assay was not replicated in the presence of S-9.

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

- Name of test material (as cited in study report): Epoxy Resin ERL-4221
- Molecular formula (if other than submission substance): C14H20O4
- Molecular weight (if other than submission substance): 252.30g
- Physical state: Light yellow, viscous liquid.
- Lot/batch No.: 42-136
- Stability under test conditions: Stable, avoid heating over 37.7°C

Target gene:

sister chromatid exchange frequency

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cells were maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine serum, and lacking in hypoxanthine and thymidine.
For treatment of cells without metabolic activation, modified F12 medium with 50 units/ml of penicillin, 50 ug/ml streptomycin and 5% (v/v) of heat inactivated dialyzed bovine serum (F12-D5) was used. For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, was employed.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - determined by a microscopic fluorescence assay employing Hoechst 33258 dye.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentrations ranged from 100 x 10E-4% to 3.125 x 10E-4% (by volume)

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

glass-distilled dimethyl-sulfoxide (DMSO)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control used without metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20hours prior to treatment
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): Selection agent is added 1 - 2 hours prior to harvesting cells.
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.2ug/ml) or Colcemide (0.1ug/ml)
STAIN (for cytogenetic assays): Hoechst 33258 dye

NUMBER OF REPLICATIONS: The test was conducted in triplicate, however, the first two replications were unsuccessful as the cytotoxicity of the test agent reduced the number of mitotic cells and chromosome preparations were not suitable for scoring.

NUMBER OF CELLS EVALUATED: A minimum of 15 cells are recorded for each dose level.

DETERMINATION OF CYTOTOXICITY
- Method: other: frequency of Sister Chromatid Exchanges.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

OTHER: Not documented

Evaluation criteria:

The key determinant in evaluating a positive or negative result was whether a dose dependent increase in SCE's was induced by the test agent.
Clearly positive responses will include any of the following: ( i ) Doubling in the SCE frequency at a minimum of two of the five concentrations tested; (ii) Statistically significant responses of p < 0.05 at three concentrations or at 2 concentrations if p < 0.01;
(iii) Induction of a statistically significant, dose-related increase in the number of SCE.

Statistics:

Data was analyzed using appropriate parametric tests as was determined by the testing laboratory.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: The study was repeated and only the third assay is reported, because of excessive cytotoxcity in the first two assays

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A total of 15 cells/dose level and 5 dose levels, with or without metabolic activation were examined. The experiment was conducted in triplicate but only the final successful results were reported.

Epoxy Resin ERL-4221 produced statistically significant increases in the SCE frequency at three of the six dose-levels tested for direct action in the absence of a metabolic activation system. Also, the increase in the numbers of SCE was dose dependent. The test without S9 activation was consideredan indication of a significant direct mutagenic action of Epoxy Resin ERL-4221.

Induction of SCE by the concurrent EMS positive control was highly statistically significant from the concurrent solvent control and these data indicated an appropriate sensitivity of the test system comparable to our historical positive control data . The numbers of SCE obtained with the H20 solvent and DMSO controls were also in an acceptable range of values included in the variability encountered in our historical experience with this test.

Testing of Epoxy Resin ERL-4221 with S9 metabolic activation was not performed because the highly positive results obtained without S9 indicated that metabolic activation was not required to express mutagenic activity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No additional information

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

Epoxy Resin ERL-4221 produced highly significant increases in the frequency of SCE when tested over a 32-fold range of concentrations in tests without addition of an S9 metabolic activation system. Evidence of a dose-related effect on the SCE frequency following exposure to Epoxy Resin ERL-4221 indicated that the test agent should be considered significantly active in the present in vitro assay.

Executive summary:

In a study conducted by Slesinski et al in 1980, the test substance, Epoxy Resin ERL-4221, was investigated for its potential mutagenic activity in the in vitro test, the Sister Chromatid Exchange test. The test was conducted in the absence of metabolic activation in the form of S-9 mix and the appropriate positive, negative and solvent controls. The test material was tested over a range of 6 concentrations, from 100 x 10E-4% to 3.125 x 10E-4%.

Epoxy Resin ERL-4221 produced highly significant increases in the frequency of SCE when tested aver a 32-fold range of concentrations in

tests without addition of an S9 metabolic activation system. Evidence of a dose-related effect on the SCE frequency following exposure to Epoxy Resin ERL-4221 indicated that the test agent should be considered significantly active in the present in-vitro assay.

Reference 5

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:

1987

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Japanese report; results tables availalble

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The study report is in Japanese; however the basic study design appears to be broadly comparable to OECD 471

Deviations:

not specified

Principles of method if other than guideline:

The study report is in Japanese; however the results table is interpretable and the study appears to be broadly comparable to OECD 471.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): CELLOXIDE-2021P
- Lot/batch No.: CELPS-HF-2
- Storage condition of test material: in the room temperature and shield from the light
- Substance type: liquid

Target gene:

No information provided in report, Histidine and Tryptophan genes are the normal targets for Ames test investigations

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S-9 mixture

Test concentrations with justification for top dose:

Medium control, 100, 200, 500, 1000, 2000 and 5000 (µg/plate)

Vehicle / solvent:

Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-AA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk.
DURATION
- Preincubation period: 11h
- Exposure duration: 48h

Evaluation criteria:

No details provided

Statistics:

No information available

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

This study reports positive responses in strains TA100 and TA1535 when tested in the presence of metabolic activation; no response was seen in the absence of metabolic activation.

Executive summary:

A proprietary Ames test (Daicel, 1987) was performed in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli WP2. This study reports positive responses in strains TA100 and TA1535 when tested in the presence of metabolic activation; no response was seen in the absence of metabolic activation.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 31st 1979 to March 11th 1980.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No indication of whether any specific test guidelines were complied with. Study was not conducted according to GLP. The test was conducted using an inappropriate positive control.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Unscheduled DNA Synthesis (UDS) in primary hepatocytes was conducted to measure the DNA repair synthesis after excision and removal of a stretch of DNA containing the region of damage induced by chemical or physical agents. The test was conducted in the absence of metabolic activation.

GLP compliance:

no

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Specific details on test material used for the study:

- Name of test material (as cited in study report): Epoxy Resin ERL-4221
- Molecular formula (if other than submission substance): C14H20O4
- Molecular weight (if other than submission substance): 252.30g
- Physical state: Light yellow, viscous liquid.
- Lot/batch No.: 42-136
- Stability under test conditions: Stable, avoid heating over 37.7°C

Target gene:

Not documented

Species / strain / cell type:

hepatocytes: rat liver cells

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

As study was conducted using primary hepatocytes, no metabolic activation was required.

Test concentrations with justification for top dose:

Six dose levels, ranging from 1000 x 10E-4% to 1.0 x 10E-4% by volume.

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

Positive control for indirect acting mutagens

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Positive control for direct acting mutagens.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium containing 3H-thymidine, hydroxyurea and appropriate dilutions of Epoxy Resin ERL-4221 prepared in DMSO.

DURATION
- Preincubation period: 1 hour
- Exposure duration: 2 hours
- Expression time (cells in growth medium): Not documented
- Selection time (if incubation with a selection agent): Not documented
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Not documented
STAIN (for cytogenetic assays): Not documented

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

Evaluation criteria:

The classification of a chemical as a positive, active agent depends upon the production of a statistically significant, dose-related increase in the amount of UDS activity. A key determinant of the reliability of the UDS data is the detection of a similar response with both DNA and isolated nuclei determined at two or three consecutive concentrations.

Statistics:

The average DPM is calculated For each dose level and the controls and final results are expreseed as DPM/10E6 viable hepatocytes. Data are also expressed as a percent of the solvent control for purposes of comparison. The original data are statistically analyzed by the appropriate parametric test as determined by the testing laboratory.

Species / strain:

hepatocytes:

Metabolic activation:

without

Genotoxicity:

other: questionable to weak activity

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

other: 4-NQO was an appropriate control to use, however, DMN did not seem to be appropriate.

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not documented
- Effects of osmolality: Not documented
- Evaporation from medium: Not documented
- Water solubility: Not documented
- Precipitation: Not documented
- Other confounding effects: Not documented

RANGE-FINDING/SCREENING STUDIES:
Not documented

COMPARISON WITH HISTORICAL CONTROL DATA:
Not documented

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Nuclear -Bound Radioactive Label:
In hepatocytes treated with Epoxy Resin ERL-4221, only one concentration tested for potential activity induced a statistically significant increase in the amounts of 3H-thymidine incorporation. A gradual decrease in the amounts of radioactive incorporation, over the entire range of concentrations
tested, was considered an indication of the cytotoxicity of the test agent. The production of statistically significant levels of UDS at only the lowest
concentration may suggest that even lower concentrations should be tested. These data were considered equivocal but suggestive of a questionable to-weak activity for Epoxy Resin ERL-4221.

DNA-Bound Radioactive Label:
For hepatocytes treated with Epoxy Resin ERL-4221, two of the six test concentrations induced levels of UDS which were statistically significant from the solvent control. We also observed a similar pattern of responses as obtained in the assessment of nuclei from cells treated with the same range of concentrations; three of the lowest six concentrations produced numerical elevations in UDS similar in magnitude to values obtained with the
DMN positive control. Although there was no indication of a dose-effect relationship due to treatments with the test agent, the UDS values were
sufficiently elevated to suggest a very weak level of mutagenic activity. These several considerations were consistent in the classification of Epoxy
Resin ERL-4221 as a questionable-to -weakly active agent in the induction of DNA damage in the present test with the hepatocyte test system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No additional information

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation questionable to weakly active

Based on the results, Epoxy Resin ERL-4221 appeared to produce a very weak response in the present test with cells treated over a 1000-fold range of test concentrations. Epoxy Resin ERL-4221 was considered questionable-to-weakly active in producing DNA damage in the tests with hepatocytes.

Executive summary:

In a study conducted by Slesinski et al (1980), the test substance, Epoxy Resin ERL-4221, was investigated for its potential to induce Unscheduled DNA Synthesis (UDS) in vitro using primary hepatocytes from rat liver cells. The hepatocytes were exposed to the test substance at 6 concentrations, ranging from 1000 x 10E-4% to 1.0 x 10E-4% by volume.

The results of this study indicate that the lowest 3 concentrations produced highly numerically elevated levels of UDS activity. Because these values were not consistently significant in statistical comparisons to the concurrent solvent control, the results could not be definitively labelled as either positive or negative. The data were considered to be suggestive of a low level of activity and Epoxy Resin ERL-4221 appeared to be questionably-to-weakly active i n the present test with the hepatocyte test system

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A mouse bone marrow micronucleus assay (Wing & Machado, 1991) was performed in groups of Swiss mice (5/sex/time point) using intraperitoneal dosing at dose levels of 500, 1000 and 2250 mg/kg bw. Bone marrow was sampled at 24, 48 and 72 hours following the administration of a single dose. Clear signs of systemic toxicity (including decreased motor activity, collapse, weakness, ataxia and laboured breathing) were observed in mice of both sexes at the highest dose level. Evidence of bone marrow cytotoxicity was observed, confirming exposure of the target tissue. A statistically significant increase in the proportion of micronucleated polychromatic erythrocytes (PCEs) was reported for male mice treated with 1000 mg/kg bw at the 48 hour time point only. This finding is not considered to be biologically significant in the absence of a dose-response relationship. It is concluded, therefore, that the study does not show any evidence of genotoxicity.

A rat liver UDS assay (San & Sly, 1999) used a single gavage administration of the substance at dose levels of 500, 1000 and 2000 mg/kg bw. UDS was assessed in hepatocytes isolated at 2-4 or 12‑16 hours following administration, using autoradiography. No signs of toxicity were observed in this study. Administration of the substance did not result in any significant increases in the number of net nuclear grains (NNG) in isolated hepatocytes. It is concluded, therefore, that the study does not show any evidence of genotoxicity.

Transgenic rodent assay

A more recent guideline (OECD 488) and GLP-compliant rodent transgenic mutagenicity assay (Masumori, 2016) has been performed using the MutaMouse model. This study used gavage dose levels of 0, 250, 500 and 1000 mg/kg bw/dl mice were treated for 28 days. The study investigated the forestomach, liver, male germ cells and nasal tissue. The nasal tissue was specifically investigated in this study following a request from ECHA and based on histopathology seen in a previous 90‑day study.

Nasal tissue: There was no effect of treatment on mutation frequency in the nasal tissue. The study report concludes that the results show an absence of mutagenicity in the nasal tissue.

Forestomach: An increase in mutation frequency was seen in the forestomach of mice administered 1000 mg/kg bw/d. This was statistically significant and met the laboratory’s criterion for a positive response, in that it exceeded the ‘acceptable range’, defined by the laboratory as the historical control mean value ± three standard deviations. However the mean mutation frequency for the forestomach at 1000 mg/kg bw/d (78.5 x10^-6) only marginally exceeds the ‘acceptable range’ defined by the laboratory (mutation frequency of 15.6-78.0 x10^-6) and clearly lies within the background range (mutation frequency 31.1-84.7 x10^-6)

Liver: An increase in mutation frequency was seen in the liver of mice administered 1000 mg/kg bw/d (mutation frequency 78.2 x10^-6); this increase was statistically significant but did not meet the laboratory’s criterion for a positive response as it lies within the ‘acceptable range’ (mutation frequency of 0.6-99.6 x10^-6). The value is also clearly within the background range (mutation frequency of 31.1-84.7 x10^-6). 

Germ cells: There was no effect of treatment on mutation frequency in the male germ cells. The study report concludes that the results show an absence of mutagenicity in germ cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 16th 1998 - June 21st 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary guideline study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Remarks:

Includes Compliance statement

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Name of test material (as cited in study report): Union Carbide Cycloaliphatic Epoxy Resin ERL-4221
- Physical state: clear, colorless, slightly viscous liquid
- Analytical purity: 89% - dose levels were corrected for purity.
- Lot/batch No.: TF3-24462
- Expiration date of the lot/batch: Not documented
- Radiochemical purity (if radiolabelling): Not documented
- Specific activity (if radiolabelling): Not documented
- Locations of the label (if radiolabelling): Not documented
- Expiration date of radiochemical substance (if radiolabelling): Not documented
- Stability under test conditions: Not documented
- Storage condition of test material: Room temperature, protected from exposure to light

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Dublin, VA
- Age at study initiation: Approximately 8 weeks old.
- Weight at study initiation: Dose range-finding assay - 246.5 - 260.0 g
UDS assay - 257.4 - 280.6g
- Assigned to test groups randomly: yes
- Fasting period before study: Not documented
- Housing: Male rats were housed up to five per cage in plastic autoclavable cages. Heat-treated hardwood chips were used for bedding.
- Diet (e.g. ad libitum): Harlan Teklad Rodent Chow 7012C ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: No less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle.

IN-LIFE DATES: From: 16 July 1998 To: Not stated

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was determined to be the solvent of choice based on solubility of the test article and compatibility with the test system. The test article was workable in water at 500 mg/mL, the maximum concentration tested.
- Concentration of test material in vehicle: Not documented
- Amount of vehicle (if gavage or dermal): Not documented
- Type and concentration of dispersant aid (if powder): Not documented
- Lot/batch no. (if required): Not documented
- Purity: Not documented

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article-vehicle mixture and negative control were administered via oral gavage at a constant volume of 10 mL/kg bw and the positive control DMN was administered at a constant volume of 5 mL/kg bw. All rats in the experimental and control groups were weighed immediately prior to dose administration and the dose volume was based on individual body weights.

Duration of treatment / exposure:

Single exposure.

Frequency of treatment:

Single treatment

Post exposure period:

3 days

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

10 males per dose

Control animals:

yes

Positive control(s):

N-dimethylnitrosamine (DMN)
- Doses / concentrations: 35mg/kg bw

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

For preparation of hepatocyte cultures, each rat was anesthetized by inhalation of Metofane and a midventral incision was made to expose the liver. The liver was perfused with 0.5 mM ethylene glycol-bis(β-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) solution followed by collagenase solution (80-100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation, resuspended in complete Williams' medium E (WME; buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 ug/mL gentamicin and 10% fetal bovine serum). Approximately 5 x 10E5 cells were seeded into each of six 35mm tissue culture dishes containing 25mm coverslips and preconditioned complete WME (i.e., complete WME medium in 35mm tissue culture dishes incubated overnight in a Kidified atmosphere of 5±1% CO2, and 37±1°C). A minimum of 6 cultures were set up for each rat.
Ninety to 180 minutes after plating, the cells were washed once with complete WME and refed with serum-free WME containing 10uCi 3H-thymidine/ml. After 4 hours, the radioactive medium was removed, the cultures were washed 3 times in serum-free WME containing 0.25mM thymidine and then refed with serum-free WME containing 0.25mM thymidine and incubated for 17-20 hours.
Following this incubation period and exposure to thymidine, the coverslips bearing cultures were washed once in serum-free WME. The nuclei were swelled in 1% sodium citrate solution and the cultures fixed in three changes of ethanol-glacial acetic acid fixative (3:1, v/v).
At least three of the six slides for each rat were dipped in NTB-2 emulsion (diluted 1 : 1 in deionized H20) at 43-45°C, allowed to drain and dry for at least 1.5 hours at room temperature and were stored for 5-12 days at 2-8°C in light tight boxes with a desiccant. Slides were developed in Kodak D-19 developer (diluted 1:1 in deionized H2O), fixed in Kodak fixer, and stained with hematoxylineosin stain.

Evaluation criteria:

Any mean net nuclear count which was increased by at least five counts over the negative control was considered significant. The test article was judged positive if it induced a dose-related increase with no less than one dose significantly elevated above the negative control. A significant increase in the mean net nuclear grain count in at least two successive doses in the absence of a dose response was considered positive. A significant increase in the mean net nuclear grain count at the high dose group only with no evidence of a dose response was considered suspect. A significant increase in the mean net nuclear grain count at one dose with no evidence of a dose response was judged to be equivocal. The test article was considered negative if no significant increase in the mean net nuclear grains count was evident.

Statistics:

No information provided

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
For the dose range finding assay, the test article was administered via oral gavage to male rats at four treatment levels: 1000, 2000, 4000 and 5000 mg test article/kg bw, administered in a total volume of 10mL/kg bw.
At these concentrations, the clinical signs included lethargy, piloerection, diarrhoea, tremors and crusty eyes. In addition to this, 2 animals in the top dose level were found dead on days 1, 2 and 3 and 1 was found dead on day 4. In the 4000mg/kg bw dose group, 4 animals were found dead on day 2 and 1 was found dead on day 3. For the lower dose groups, all animals had returned to normal by Day 3. Based on these results, the high dose for the UDS assay was set at the maximum tolerated dose which was estimated to be 2000 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
For the UDS assay, the test article was administered via single oral gavage to male rats at three treatment levels: 500, 1000 and 2000 mg test article/kg bw. All animals were observed to be normal for the 2 to 4 hour and 12 to 16 hour exposures immediately following dose administration.
Upon the time of hepatocyte harvest, one animal treated with 2000 mg/kg bw (12 to 16 hour exposure) and one DMN-treated (2 to 4 hour exposure) animal exhibited diarrhoea. All other test article treated and control treated animals appeared normal at the time of hepatocyte harvest.
In hepatocytes isolated 2 to 4 hours post exposure, the means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were 0.1, -0.2 and -0.3, respectively. For the positive and negative controls, the mean net nuclear grain counts were 17.6 and 0.2, respectively.
The mean net nuclear grain counts from the positive control or test article treatment groups were compared to the mean net nuclear grain counts from the negative control group. None of the test article doses caused a significant increase in the mean net nuclear counts.
In hepatocytes isolated 12 to 16 hours post exposure, the means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were -0.4, -0.2 and 0.4, respectively. For the positive and negative controls, the mean net nuclear grain counts were 10.5 and -0.2, respectively.The mean net nuclear grain counts from the positive control or test article treatment groups were compared to the mean net nuclear grain counts from the negative control group. None of the test article doses caused a significant increase in the mean net nuclear counts.

No additional information

Conclusions:

Interpretation of results (migrated information): negative no increase in net nuclear grain count
The results of the unscheduled DNA synthesis test with mammalian liver cells in vivo indicate that, under the test conditions, the test article did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated from treated animals, and was concluded to be negative in the unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo.

Executive summary:

In a study conducted by San and Sly (1999), the test article, Union Carbide Cycloaliphatic Epoxy Resin ERL-4221, was investigated for its ability to induce mutations in an unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo in male Sprague-Dawley rats. The assay was performed in two phases. The first phase, the dose range-finding assay, was used to determine the maximum tolerated dose. The second phase, the UDS assay, was used to evaluate the potential of the test article to induce unscheduled DNA synthesis in hepatocytes of exposed male rats. In the dose range finding assay, male rats were exposed to 1000,2000, 4000 and 5000 mg test article/kg bw and in the definitive UDS test, the highest dose administered was the maximum tolerated dose of 2000 mg/kg bw, in addition to 500 and 1000 mg test article/kg bw.

No mortality was observed in any test article treated or control treated rats. All animals appeared normal following dose administration.

However, diarrhea was observed in one animal treated with 2000 mg/kg bw (12-16 hour exposure) and one DMN treated (2-4 hour exposure) animal prior to sacrifice. The test article did not induce a significant increase in the mean number of net nuclear grain counts in

hepatocytes isolated either 2 to 4 hours or 12 to 16 hours after dose administration.

Based on these results, the test article did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated from treated animals, and was concluded to be negative in the unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo and as a result, does not require classification according to Regulation EC No. 1272/2008.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 22nd 1990 - March 1st 1991.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study not conducted in accordance with any specific internationally adopted test guidelines. The PCE:NCE ratio was low due to evaluating only 1000 PCE per animal (the requirement to assess 2000 PCE per animal was included in test guidelines that were adopted later but the study parameters were correct for the time the study was conducted.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The test substance was administered by intraperitoneal injection to 18 male and female Swiss Albino mice at each of 3 concentration levels. Bone marrow smears were taken from 5 animals per sex per dose group at 24, 48 and 72 hours after treatment. Any clinical signs of toxicity were recorded and the number of micronucleated polychromatic erythrocytes was noted.

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): ERL-4221
- Physical state: Light yellow liquid
- Lot/batch No.: TF3-01564
- Storage condition of test material: Stored at ambient temperature, protected from light.

Species:

mouse

Strain:

other: Swiss Albino Crl:CD-1 (ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: 63 days old
- Weight at study initiation: 19.6 - 35.2g (females) and 27.0 - 37.6g (males)
- Assigned to test groups randomly: yes, under following basis: animals were randomly assigned to treatment groups using the Taussky-Todd overflow procedure to minimize the difference between groups of same sex.
- Fasting period before study: Not documented
- Housing: Housed individually in hanging stainless steel cages with wire mesh floors,
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22°C
- Humidity (%): 35 - 45%
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

IN-LIFE DATES: From: To:

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: peanut oil
- Justification for choice of solvent/vehicle: Not documented
- Concentration of test material in vehicle: Not documented
- Amount of vehicle (if gavage or dermal): Not documented
- Type and concentration of dispersant aid (if powder): Not documented
- Lot/batch no. (if required): 807493B
- Purity: Not documented

Details on exposure:

In order to ensure an adequate survival rate for bone marrow sampling (5 animals/sex/sampling point), an excess number of animals were treated for all concentrations of test material and the vehicle control.
The test material was administered intraperitoneally as a suspension in peanut oil.

The dosing mixtures were prepared by diluting appropriate amounts of test material with peanut oil to produce 100, 200 and 450 mg/ml emulsions approxiamtely 8 hours before dosing. Dosing mixtures were placed on a magnetic stir plate with a stir bar during withdrawal of the dosing aliquots and analytical samples.

Duration of treatment / exposure:

Single injection

Frequency of treatment:

Single exposure

Post exposure period:

72 hours

Remarks:

Doses / Concentrations:
0.5, 1 and 2.25g/kg
Basis:
nominal conc.

No. of animals per sex per dose:

18 animals of each sex. However, in the definitive test, 10 animals were used (5 males and 5 females per dose).

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (positive control) and peanut oil (vehicle control)
- Justification for choice of positive control(s): Cyclophosphamide is known to produce micronuclei in vivo.
- Route of administration: intraperitoneal injection.
- Doses / concentrations: 5ml/kg peanut oil and 25 or 40 mg/kg positive control delivered at a rate of 5ml/kg.

Tissues and cell types examined:

Bone marrow
Polychromatic erythrocytes
Micronuclei

Details of tissue and slide preparation:

Five male and five female mice from each treatment group and the vehicle control were sacrificed for bone marrow sampling at approximately 24, 48 and 72 hours after the start of treatment. Mice were sacrificed by cervical dislocation and a femur was removed from each animal. The marrow was aspirated from the bone and placed into a 0.5ml conical bottom beaker containing approximately 0.4ml foetal bovine serum and centrifuged (~2000 rpm, 5 minutes at room temperature). The supernatant was decanted and the pellet resuspended in the residual serum. Two bone marrow smears weremade from each animal. The slides were fixed in methanol and stained with 5% Giemsa for approximately 20 minutes.

Evaluation criteria:

The test material was considered negative if the highest dose given was the maximum tolerated dose and all dose time-points fell in the negative classification, as outlined by the level of significance achieved following statistical analysis. If no difference was detected at the 5% level of significance, the results were negative. If a difference was detected at the 5% level, the results were considered to be positive. If a trend was not observed, the Study Director evaluated the variability observed in the vehicle control and the nature of the statistically significant responses. The results were declared negative, inconclusive, or positive after consideration of all contributing factors.

Statistics:

One-tailed Fischer exact test and binomial approximation tests to determine if there was a statistically significant increase in the frequency of micronucleated cells in the treated group compared to the vehicle control groups. As multiple comparisons were made, Bonferroni corrections were made to adjust the probablilty value required for significance. If a difference was detected the dose response was analyzed using the one-tailed Cochran-Armitage test for trend in binomial proportions.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.5 - 4.0 g/kg.
- Solubility: Not documented
- Clinical signs of toxicity in test animals: Decreased motor activity, ataxia, collapse and laboured breathing. On day 1, 2 males and 3 females at 2.5 g/kg and 3 amles and 3 females at 4 g/kg died.
- Evidence of cytotoxicity in tissue analyzed: Not documented
- Rationale for exposure: The maximum clastogenic effect was likely to be found at doses near the maximum tolerated dose, the lower doses were spaced at approximately half and a quarter of the upper dose.
- Harvest times: Not documented
- High dose with and without activation: Not documented
- Other: Not documented.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not relevant
- Induction of micronuclei (for Micronucleus assay): The ranges for treated groups were 0.6 to 2.0 and 0.2 to 1.8 MN - PCE/1000 for males and females respectively.
- Ratio of PCE/NCE (for Micronucleus assay): Statistically significant decreases in the treatment group PCE/(NCE + PCE) ratios were detected in females at 0.50 and 2.25 g/kg at 48 hours. No statistically significant decreases in PCE/(NCE + PCE) ratios were detected in other treatment groups.
- Appropriateness of dose levels and route: No information provided
- Statistical evaluation: When the data for five animals within a group were pooled and compared to the concurrent vehicle control, small increases in the frequency of MN-PCE were observed. Only the increase observed in males treated with 1.00 g/kg and sampled at 48 hours was statistically significant when analyzed with the Fisher exact or binomial approximation test with the Bonferroni correction for multiple comparisons. This increase was not dose-responsive when analyzed with the Cochran-Armitage test for trend in binomial proportions. It is likely that the significance of that response is due to the unusually low spontaneous rate (0.0) in the control group for that sampling time; therefore, the response is not considered to be biologically significant.

No additional information

Conclusions:

Interpretation of results (migrated information): negative
Based on the results of this study, the test substance, ERL 4221 did not induce micronuclei in bone marrow erythrocytes of mice under the conditions of this test. As a result of this, the test substance did not require classification according to Regulation EC No. 1272/2008.

Executive summary:

In a study conducted by Wing and Machado (1991), the test substance ERL 4221 was examined for its ability to induce micronuclei in mouse bone marrow erythrocytes. The test material was diluted in peanut oil and administered intraperitoneally to 18 Swiss Albino mice of each sex. The dose levels used were 0 .50, 1.00 and 2.25 g/kg. Bone marrow smears of 5 animals per sex pre treatment group were made at approximately 24, 48 and 72 hours after treatment.

Clinical signs of toxicity, including decreased motor activity, collapse, weakness, ataxia and laboured breathing were observed in both sexes at the 2.25g/kg dose level. Cytotoxicity was noted in females treated with 0.50 and 2.25g/kg and sampled at 48 hours.

A statistically significant increase in micronucleated polychromatic erythrocytes was noted in males treated with 1.00 g/kg and sampled at 48 hours; however, this increase was not dose-responsive and was not considered to be biologically significant.

Based on the results of this study, the test susbtance, ERL 4221 did not induce micronuclei in bone marrow erythrocytes of mice under the conditions of this test. As a result of this, the test substance did not require classification according to Regulation EC No. 1272/2008.

Reference 3

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13th November 2015 - 27th May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Deviations:

no

GLP compliance:

yes

Type of assay:

transgenic rodent mutagenicity assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Daicel Corporation, Lot No.CELP-FB-008
- Purity: 96%

Species:

mouse

Strain:

other: MutaMouse

Remarks:

CD2-LacZ80/HazfBR (MutaTM Mouse) [SPF]

Details on species / strain selection:

CD2-LacZ80/HazfBR mice are commonly used transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Laboratory Animals, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 24.4-28.1 g
- Housing: group (2-3/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0-24.6
- Humidity (%): 40-61
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 November 2015 To: 22 December 2015

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: based on solubility of the test material
- Concentration of test material in vehicle: 25-100 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Corn oil (lot No. WEF2972), the vehicle to prepare the test substance formulations, was used.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Post exposure period:

3 days

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

6 males (5 males per group evaluated)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylnitrosurea
- Route of administration: intraperitoneal
- Doses / concentrations: 100 mg/kg bw/d

Tissues and cell types examined:

Liver, forestomach, nasal epithelium, germ cells

Details of tissue and slide preparation:

The liver, stomach, nasal cavity, testes and vas deferens/cauda epididymis were removed from the animals at termination.

Liver: Two samples were prepared from the left lateral lobe using a biopsy trephine. The samples were separately placed in microtubes and frozen in liquid nitrogen. The other lobes were stored frozen in liquid nitrogen.

Stomach: The greater curvature of the stomach was incised and the stomach contents removed by washing with physiological saline. The forestomach was stored frozen in liquid nitrogen; the glandular stomach was discarded.

Nasal tissue: The nasal cavity was incised and the mucous membrane from the nasal cavity removed and stored frozen in liquid nitrogen.

Germ cells: The seminiferous tubules and vas deferens/cauda epididymis were cut and placed in a Petri dish containing 2 mL cold Dulbecco’s phosphate-buffered saline (PBS). The germ cells that migrated into the PBS were filtered using a cell strainer (pore size 40 µm). About 0.5 mL of the resulting cell suspension was put into each of three microtubes and stored frozen in liquid nitrogen.

Evaluation criteria:

The results were evaluated as positive when the mutation frequency in the test substance-treated group was significantly different from that in the negative control group, and the increase was dose-dependent.

Statistics:

Liver, forestomach and germ cells

Data on the mutant frequency from the negative control group and each treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant using Bartlett’s test), Dunnett’s multiple comparison test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and each test substance treated group. If there was no homogeneity (significant using Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyse the differences.
The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant using F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t-test (two-sided, significance level of 0.05) was performed to analyse the differences.

Nasal tissue
As genomic DNA was extracted from the pooled nasal tissue of each test group, χ2 test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group or the positive control group. If significant difference was observed, dose dependency was analyzed by the Cochran-Armitage trend test (one-sided significance level: 2.5%).

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Germ cells

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: There was no effect of treatment on mutation frequency in the male germ cells. 

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Liver

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: An increase in mutation frequency seen at 1000 mg/kg bw/d does not meet the laboratory’s criterion for a positive response

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

Stomach

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The result for the stomach at 1000 mg/kg bw/d is positive according to the laboratory's evaluation criterion, but is not considered to be of clear relevance to the risk assessment

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Nasal tissue

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: There was no effect of treatment on mutation frequency in the nasal tissue

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 125-1000 mg/kg bw/d (7 days)
- Clinical signs of toxicity in test animals: none

Nasal tissue

There was no effect of treatment on mutation frequency in the nasal tissue. The study report concludes that the results show an absence of mutagenicity in the nasal tissue.

Forestomach

An increase in mutation frequency was seen in the forestomach of mice administered 1000 mg/kg bw/d. This was statistically significant and met the laboratory’s criterion for a positive response, in that it exceeded the ‘acceptable range’, defined by the laboratory as the historical control mean value ± three standard deviations. However the mean mutation frequency for the forestomach at 1000 mg/kg bw/d (78.5 x10^-6) only marginally exceeds the ‘acceptable range’ defined by the laboratory (mutation frequency of 15.6-78.0 x10^-6) and clearly lies within the background range (mutation frequency 31.1-84.7 x10^-6).

Liver

An increase in mutation frequency was seen in the liver of mice administered 1000 mg/kg bw/d (mutation frequency 78.2 x10^-6); this increase was statistically significant but did not meet the laboratory’s criterion for a positive response as it lies within the ‘acceptable range’ (mutation frequency of 0.6-99.6 x10^-6). The value is also clearly within the background range (mutation frequency of 31.1-84.7 x10^-6). 

Germ cells

There was no effect of treatment on mutation frequency in the male germ cells. The study report concludes that the results show an absence of mutagenicity in germ cells.

Mutation frequencies

Tissue	Mutation frequency (/10e6)
	Dose level (mg/kg bw/d)				+ control
	0	250	500	1000	ENU	Background range	Acceptable range
Nasal tissue	53.7	40.6	50.2	54.3	215.7*	-	-
Liver	48.2	62.0	61.2	78.2*	143.8*	16.6 - 95.0	0.6 - 99.6
Forestomach	49.1	52.2	54.9	78.5	624.7*	31.1 - 84.7	15.6 -78.0
Germ cells	32.6	33.7	40.3	42.4	82.1*	12.2 - 83.5	-

*significantly different to the concurrent control group (p<0.05)

Acceptable range defined as historical mean +/- 3sd

Conclusions:

The study report concludes a positive response for CEL2021P based on a marginal (but statistically significant) response seen in the stomach at the highest dose level of 1000 mg/kg bw/d. The conclusion is based on the mean mutation value for the stomach marginally exceeding the laboratory's 'acceptable range', although the mean mutation frequency is clearly within the background control range for this tissue. The result of this study is therefore not considered to be sufficiently convincing to trigger classification of the substance.

Executive summary:

A somatic and germ cell mutation assay was performed with the substance 7-oxabicyclo [4.1.0] hept-3-ylmethyl-7-oxabicyclo [4.1.0] heptane-3-carboxylate; (CEL2021P) using male transgenic mice (MutaMouse). The study was conducted to assess the potential of the test material to induce gene mutations in the liver, stomach, nasal cavity and germ cells, using the lacZ gene as a mutation reporter gene. The dose levels of CEL2021P used in the main study were based on the results of a dose range-finding study in which CD2F1/Slc mice were gavaged for 7 days with 0, 125, 250, 500, or 1000 mg/kg bw/d CEL2021P in corn oil. No signs of toxicity were observed in any of the dose groups. A dose level of 1000 mg/kg bw/d was therefore selected as the high dose level for the main study.

 

The test material was administered by gavage (in corn oil) to groups of six male transgenic mice orally for 28 consecutive days. A control group was treated with the vehicle alone and a positive control group was administered ENU by intraperitoneal injection at 100 mg/kg bw/d for two days. Three days following administration of the final dose, the liver, stomach, nasal cavity, testes and vas deferens/cauda epididymis were removed. Mutation frequencies in the liver, stomach, nasal tissue and germ cells were determined for five mice per group.

 

The mutation frequencies in the nasal tissue and germ cells of all groups treated with CEL2021P did not show any statistically significant increase compared to the vehicle control group. A slight (but statistically significant) increase in the mean mutation frequency was seen in the liver of mice administered 1000 mg/kg bw/d; however the value was within the background control range and was also within the laboratory’s acceptable range (defined as the mean of the background control range ±3sd). A slight (but statistically significant) increase in the mean mutation frequency was seen in the stomach of mice administered 1000 mg/kg bw/d. This value was within the background control range, but marginally exceeded the laboratory’s acceptable range and was therefore considered by the laboratory to represent a positive response. The mutation frequencies in the liver, stomach, nasal tissue and germ cells of the positive control group were statistically significantly increased compared with the negative control group, demonstrating the sensitivity of the assay.

 

The study report concludes that CEL2021P induces gene mutations in the stomach of transgenic mice under the conditions of this study. This interpretation of a marginal response which is clearly within the background control range, is considered to be questionable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Classification under the CLP Regulation is required for substances causing germ cell mutagenicity. This hazard class is primarily concerned with substances having potential to cause mutations in the germ cells of humans that may be transmitted to progeny. The results from tests in vitro and in mammalian somatic and germ cells in vivo are considered when classifying substances in this hazard class. Substances may be classified in Category 1A (appropriate substances known to induce heritable mutations in the germ cells of humans); in Category 1B (substances regarded as if they induce heritable mutations in the germ cells of humans); or in Category 2 (substances which cause concerns owing to the possibility that they may induce heritable mutations in the germ cells of humans).

The criteria used for classification are described below:

Classification in Category 1A is on the basis of human data and is therefore not relevant for this substance.

Classification in Category 1B may be based on a positive result from an in vivo mammalian germ cell mutagenicity test, or on a positive result from an in vivo mammalian somatic cell mutagenicity test together with further toxicokinetic data showing systemic availability and germ cell exposure. Classification in Category 1B is not appropriate for the substance given the clearly negative result in germ cells in the recently conducted transgenic rodent assay.

Classification in Category 2 may be based on positive results in an in vivo mammalian somatic cell mutagenicity test, indicating mutagenic effects in somatic cells; or may be based on positive results in a mammalian somatic cell genotoxicity testin vivosupported by positive in vitro mutagenicity data. The CLP Guidance notes that ‘…where there is evidence of only somatic cell genotoxicity, substances are classified as suspected germ cell mutagens’; i.e. they should be classified in Category 2. It is further stated that ‘…if positive results in vitro are supported by at least one positive local in vivo somatic cell test, such an effect should be considered as enough evidence to lead to classification in Category 2’.

In this case, therefore, consideration focuses on the need for classification of the substance for germ cell mutagenicity in Category 2. In the case of this substance, clearly negative responses are reported in somatic cells in vivo in a mouse bone marrow micronucleus assay, a rat liver UDS assay and in the transgenic mouse assay in the liver and nasal tissue. The transgenic mouse assay, however, reports a positive result for the forestomach, based on the laboratory’s statistical evaluation criterion. Any conclusion on whether the substance is classified in Category 2 therefore hinges on interpretation of the response seen in the forestomach in this study, which may be taken to indicate that the substance has site of contact mutagenic activity.

The result in the forestomach is considered by the laboratory to be positive according to their statistical interpretation criterion, but even this is marginal (the mutation frequency of 78.5e-6 is compared to an ‘acceptable range’ maximum of 78.0e-6. Furthermore, the mutation frequency in the forestomach is well within the historical control range for this tissue (reported by the laboratory to be 31.1-84.7e-6). The interpretation of this result as positive based purely on a statistical analysis is questionable. It is notable that the test guideline (OECD 488) states that there are ‘several criteria for determining a positive result’, further stating that ‘…biological relevance of the results should be the primary consideration’ and that ‘appropriate statistical methods may be used as an aid in evaluating the test results’. The marginal response in the forestomach, seen only at the highest dose level of 1000 mg/kg bw/d and in a tissue with a highly acidic pH, is not considered to be clear biological significance and is also of questionable relevance to other potential (and more relevant) site of contact tissues such as the skin or respiratory tract. In this respect it is notable that a mouse skin-painting carcinogenicity study for the substance demonstrates an absence of site-of-contact carcinogenicity following long-term exposure to high dose levels of the substance.

Guidance on application of the CLP criteria states that the classification of substances ‘shall be based on the total weight of evidence available’ and additionally states that the decision should use expert judgement. For substances classified on the basis of a single study, it is stated that this study ‘shall provide clear and unambiguously positive results’. Furthermore it is stated that ‘the relevance of the route of exposure used in the study….compared to the most likely route of human exposure shall also be taken into account’. In the case of this substance, three in vivo studies of somatic cell mutagenicity are available. Two of these studies provide clearly negative results and a third study (the transgenic mouse study) gives negative results in all of the tissues examined, with the single exception of a marginal response in the forestomach where a positive response is concluded based purely on a statistical criterion. For the reasons discussed, the response in the forestomach is not considered to provide ‘clear and unambiguously positive results’. The relevance of the finding in the forestomach is also questionable compared to the likely routes of occupational exposure (dermal and inhalation), particularly when considered in light of the negative mouse carcinogenicity (skin painting study).

In conclusion, based on a detailed assessment of the genotoxicity data and comparison with the classification criteria, it is concluded that the available data are not sufficient to classify the substance 7‑oxabicyclo [4.1.0] hept-3-ylmethyl-7-oxabicyclo [4.1.0] heptane-3-carboxylate (CAS 2386-87-0 / EC 219-027-4) for germ cell mutagenicity according to CLP.