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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation assay (two Ames tests): not mutagenic (WoE, rel. 4)
Mammalian cell chromosome aberration study in CHO cells: not clastogenic (K, rel. 2)
Unscheduled DNA synthesis assay in mammalian liver cells: no induction of UDS (K, rel. 2, read-across)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1986
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Published data not GLP with insufficient number of strains but considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
Number of strains used
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene in Salmonella strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9: S9-mix from livers of male Sprague Dawley rat or Syrian hamster treated with Aroclor 1254.
Test concentrations with justification for top dose:
Experiment: (preincubation method): S. typhimurium TA98, TA100, TA1535 and TA1537 strains
Without S9 mix:
Trial 1: 3.3, 10, 33, 100 and 333µg/plate.
Trial 2: 3.3, 10, 33, 100 and 250 µg/plate.

With S9 mix (10 % rat and Hamster S9):
Trial 1: 10, 33, 100, 333 and 1000 µg/plate.
Trial 2: 10, 33, 100, 333 and 800 µg/plate.
Vehicle / solvent:
No data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylenediamine
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: 2 days at 37 °C
NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
Method: Test material toxicity was determined according to one or more of following criteria:
- Appearance of his- pinpoint colonies
- Reduced numbers of revertants colonies per plate
- Thinning or absence of the bacterial lawn by the reduction of the revertant colonies.
Rationale for test conditions:
No data
Evaluation criteria:
The criteria used for data evaluation:
1) Mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold
2) Nonmutagenic response: when no increase in the number of revertants wes elicited by the chemical
3) Questionable response: when there was an absence of a clear -cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.
Statistics:
None
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
≥800 µg/plate was toxic to TA98 with S9-mix and 333 µg/plate was toxic to all the Salmonella tester strains without S9-mix.

None

Conclusions:
Isoeugenol (3.3 to 1,000 µg/plate) was not mutagenic is two independant trials in a bacterial mutagenicity assay conducted with and without exogenous metabolic activation (S9 liver enzymes) in S. typhimurium strains TA98, TA100, TA1535, and TA1537.
Executive summary:

In a reverse gene mutation assay performed similarly to the OECD test guideline No. 471, S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were exposed the test material both in the presence and absence of metabolic activation system (10% rat or hamster liver S9) using the pre-incubation method. The dose range (3.3 to 1000 µg/plate) was based on cytotoxicity. The experiment was repeated.

 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Under the test condition, Isoeugenol was not mutagenic to S. thyphimurium strains TA98, TA100, TA 1535 and TA 1537, in the presence and absence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Published data not GLP with insufficient number of strains but considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Number of strains used
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene in Salmonella strains, tryptophan gene in E.coli
Species / strain / cell type:
other: TA 98, TA 100 and E.coli strain WP2 uvrA/pKM101
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9: S9-mix from livers of male Sprague Dawley rat or Syrian hamster treated with Aroclor 1254.
Test concentrations with justification for top dose:
Experiment (preincubation method):
Without S9 mix:
Trial 1 and 2: 10, 50, 100, 250 and 500 µg/plate in TA98 and TA100 strains; 10, 50, 100, 500, 750 and 1000 µg/plate in E. coli WP2 uvrA/pKM101 strain.
Trial 3: 10, 50, 100, 250 and 500 µg/plate in TA98 strain; 10, 50, 100, 500 and 1000 µg/plate in E. coli WP2 uvrA/pKM101 strain.
With S9 mix (10 % rat S9):
Trial 1: 50, 100, 500, 1000 and 1500 µg/plate in TA98 and TA100 strains; 10, 50, 100, 500, 750 and 1000 µg/plate in E. coli WP2 uvrA/pKM101 strain.
Trial 2: 50, 100, 500, 1000 and 2000 µg/plate in TA98 strain; 50, 100, 500, 1000 and 1500 µg/plate TA100 strain; 10, 50, 100, 500, 750 and 1000 µg/plate in E. coli WP2 uvrA/pKM101 strain.
Trial 3: 50, 100, 500, 1000 and 2000 µg/plate in TA100 strain; 50, 100, 500, 1000 and 1500 µg/plate in TA98 strain; 10, 50, 100, 500 and 1000 µg/plate in E. coli WP2 uvrA/pKM101 strain.
Vehicle / solvent:
No data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylenediamine
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation
DURATION
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: 2 days at 37 °C

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Test material toxicity was determined by the reduction of the revertant colonies.
Rationale for test conditions:
Not reported
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
None
Key result
Species / strain:
other: S. typhimurium TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
other: E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
500 µg/plate was toxic to TA98 without S9-mix; 1000 µg/plate was toxic to E.coli WP2uvrA/pKM101 without S9-mix; 2000 µg/plate was toxic to TA98 and TA100 with S9-mix.Please refer the attached table for further details.

None

Conclusions:
Trans-Isoeugenol (10 to 2,000 µg/plate) was not mutagenic in three independent experiments in a bacterial mutagenicity assay conducted with and without exogenous metabolic activation (S9 liver enzymes) in S. typhimurium strains TA98, TA100 and E. coli strain WP2 uvrA/pKM101.
Executive summary:

In a reverse gene mutation assay performed similarly to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 98, TA 100 and E.coli strain WP2 uvrA/pKM101 were exposed the test material both in the presence and absence of metabolic activation system (10% rat liver S9 in standard co-factors) using the pre-incubation method. The dose range (10 to 2000 µg/plate) was based on cytotoxicity. The experiment was repeated three times.

 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Under the test condition, Trans-isoeugenol was not mutagenic to S. thyphimurium strains TA98, TA100, andE.coliWP2 uvrA/pKM101, in the presence and absence of metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Not a GLP study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No short-term exposure in absence of S9
Principles of method if other than guideline:
None
GLP compliance:
yes
Remarks:
The NTP conducts its studies in compliance with FDA Good Laboratory Practice Regulations
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
0, 50, 100, 200, 300, 400, 500 without S9
0, 150, 160, 170, 180, 190, 200 with S9
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Mitomycin C and Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 10 hours without S9, 2 hours with S9
- Expression time (cells in growth medium): 2 hours without S9, 10 hours with S9
- Fixation time (start of exposure up to fixation or harvest of cells): 12 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Presence of scorable metaphases
Rationale for test conditions:
Not reported
Evaluation criteria:
Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P#0.05) difference for one dose point and a significant trend (P#0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.
Statistics:
No data
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the high dose was limited by toxicity.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Additional information on results:
Isoeugenol (in medium concentrations up to 200 µg/mL) did not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation

See the attached document for information on tables of results

Conclusions:
Isoeugenol (in medium concentrations up to 200 µg/mL) did not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation.
Executive summary:

Testing was performed as reported by Gallowayet al.(1987). Isoeugenol was sent to the testing laboratory as a coded aliquot. It was tested in cultured Chinese hamster ovary (CHO) cells for induction of chromosomal aberrations (Abs), both in the presence and absence of Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of isoeugenol; the high dose was limited by toxicity. A single flask per dose was used. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with isoeugenol for 10 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with isoeugenol and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Isoeugenol (in medium concentrations up to 200 µg/mL) did not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD Guideline No. 482 with deviations: details of test material, individual and summary results not reported.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
yes
Remarks:
details of test material, individual and summary results not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
Not applicable
Species / strain / cell type:
hepatocytes: rat and mouse
Details on mammalian cell type (if applicable):
- Type and identity of media: William’s medium E supplemented with bovine calf serum (10 % for rats, 1 % for mice), and l-glutamine (200 mM) at 5 x 10^5 cells/mL.
- Properly maintained: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0.1, 1, 10, 100 and 1000 µM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive control substance:
other: aminofluorene
Details on test system and experimental conditions:
PROCEDURE: UDS assay was conducted as described by McQueen (1989) and Williams (1977) with some modifications.

HEPATOCYTE ISOLATION: Rat and mouse were anesthetized with sodium pentobarbital prior to the isolation of hepatocytes. Isolations were performed using a two step perfusion technique as described by McQueen (1989). Briefly, the portal vein was cannulated and the liver perfused with HBSS buffered with EGTA and HEPES for 4 min, followed by a collagenase solution for 6.5 min for the rat, and for 5.5 min for the mouse. Flow rates for the rat perfusion were 40 and 20 mL/min for the HBSS solution and the collagenase solution, respectively. Flow rates for the mouse were 8 mL/min for each solution. The livers were removed from the animals and dissociated through sterile gauze. The hepatocytes were washed twice with media and counted. Only those cell preparations with 85 % viability or greater (as determined by trypan blue exclusion) were used for primary culture. Cells were suspended in William’s medium E supplemented with bovine calf serum (10 % for rats, 1 % for mice), and l-glutamine (200 mM) at 5 x 10^5 cells/mL.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 h; incubations were conducted in 95 % air, 5 % CO2 at 37 °C.
- Selection time (if incubation with a selection agent): Hepatocytes were simultaneously exposed to [3H]thymidine (1 mL of 89.4 mCi/mL per 100 mL medium).

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release. LDH activity was expressed as the percent of LDH released into the medium (LDH in the medium divided by total LDH in cells and medium, multiplied by 100). Following incubation, cells were evaluated under an inverted microscope for gross evidence of cytotoxicity to the hepatocytes.

NUMBER OF REPLICATIONS: 3 independent experiments, triplicate wells for rat and duplicate wells for mouse.

PREPARATION AND SCORING OF SLIDES
- The cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting. Results were quantified using an Artek colony counter. Slides were examined under oil immersion. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results are expressed as mean grain counts per nucleus.

NUMBER OF CELLS EVALUATED: Twenty cells per slide, with three slides per dose level were counted when possible.
Rationale for test conditions:
Not reported
Evaluation criteria:
Positive grain counts indicate UDS. A compound is defined as positive if there is an increase in positive grain counts over two or more doses.
Statistics:
None
Key result
Species / strain:
hepatocytes: rat and mouse
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY MEASUREMENT: Test material had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves.

UDS RESULTS: Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Under the test conditions, test material did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.
Executive summary:

In an UDS assay, rat and mouse hepatocytes were incubated with test material at the concentrations ranging from 0.1 to 1000 µM for 18 h. Hepatocytes were simultaneously exposed to [3H]thymidine (1 mL of 89.4 mCi/mL per 100 mL medium). Following incubation, cells were evaluated under an inverted microscope for gross evidence of cytotoxicity to the hepatocytes. The cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting. Results were quantified using an Artek colony counter. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results are expressed as mean grain counts per nucleus. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 500 to 5000 µM.

Test material had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves. Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice.

Under the test conditions, test material did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.

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

Genetic toxicity in vivo

Description of key information

In vivo chromosome aberration (micronucleus) test in mouse peripheral erythrocytes: not clastogenic, not aneugenic (K, rel. 2)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 18 to July 19, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Basic data given. Positive control group not included, number of micronucleated immature erythrocytes for each animal not reported.
Principles of method if other than guideline:
According to methodology of MacGregor et al. (1990).
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc., Germantown, NY
- Age at study initiation: 6-7 weeks
- Assigned to test groups randomly: Yes; animals were distributed randomly into groups of approximately equal initial mean body weights.
- Housing: Male animals were housed individually and females were housed 5/cage in polycarbonate cages.
- Diet: NTP-2000 irradiated wafer or pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water: Tap water (Columbus, OH, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), ad libitum
- Acclimation period: Females: 13 days; males: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F
- Humidity: 50 ± 15 %
- Air changes: ≥ 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES:
From: April 18, 2001 To: July 19, 2001
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amounts of test material and corn oil were placed in a glass mixing container, capped, and thoroughly mixed with a paint shaker for approximately 5 minutes. Dose formulations were prepared approximately monthly during the study.

ANALYSIS OF FORMULATION:
Test material formulations were analysed three times during the study period. Homogeneity studies of 0.2 and 120 mg/mL formulations and stability studies of the 0.2 mg/mL formulation were performed by the analytical chemistry laboratory using GC. Homogeneity was confirmed, and the 120 mg/mL dose formulation was found to be suitable for gavage. Stability was confirmed for up to 35 days for dose formulations stored in amber glass bottles with Teflon® -lined lids at – 20 °C, 5 °C, and room temperature, as well as for 3 h under simulated animal room conditions. All the formulations analysed were within 10 % of the target concentrations.

DOSE VOLUME: 10 mL/kg bw/day
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days/week
Post exposure period:
No data
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
37.5 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (except vehicle control in females where 8 animals used)
Control animals:
yes, concurrent vehicle
Positive control(s):
None
Tissues and cell types examined:
- Frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each animal per treatment group was determined.
- Percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
- At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female animals.

DETAILS OF SLIDE PREPARATION:
- Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS:
- Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each animal per treatment group.
- Percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.
Evaluation criteria:
- In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.
- A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.
- Statistical as well as biological factors are considered.
Statistics:
- The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean.
- The frequency of micronucleated cells among NCEs was analysed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group.
- In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
- Significance was considered at p≤ 0.025.
Key result
Sex:
male/female
Genotoxicity:
positive
Remarks:
female mice
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The frequencies of micronucleated erythrocytes were not increased in peripheral blood of male animals exposed to 37.5 to 600 mg/kg bw/day of test material by gavage for 3 months; in contrast, a 3.2-fold increase of micronucleated erythrocytes in females at 600 mg/kg bw/day were observed.

Table 7.6.2/1. Micronucleus data

Dose

(mg/kg bw/day)

No. of animals with erythrocytes scored

Micronucleated

NCEs/1000 NCEs

(mean ± SE)

P value*

PCEs (%)

0 (corn oil)

5M

0.90 ± 0.37

 

2.3

37.5

5M

1.60 ± 0.46

0.0806

2.8

75

5M

0.70 ± 0.25

0.6915

3.1

150

5M

0.90 ± 0.24

0.5000

2.8

300

5M

0.30 ± 0.12

0.9584

2.5

600

5M

0.90 ± 0.19

0.5000

2.9

 

P = 0.841#

 

0 (corn oil)

8F

0.50 ± 0.16

 

2.8

37.5

5F

1.10 ± 0.19

0.0408

3.5

75

5F

0.20 ± 0.12

0.8850

3.1

150

5F

0.70 ± 0.30

0.2568

2.7

300

5F

1.00 ± 0.35

0.0680

3.4

600

5F

1.60 ± 0.40

0.0022

2.4

 

P = 0.001#

 

Keys:

NCE = Normochromatic erythrocyte

PCE = Polychromatic erythrocyte

* Pairwise comparison with the vehicle control; dosed group values are significant at P ≤ 0.005.

# Significance of micronucleated NCEs/1000 NCEs tested by the one-tailed trend test; significant at P≤ 0.025

Conclusions:
Under the test conditions, the test material showed a statistically significant increase in the frequency of micronucleated erythrocytes in female mice. This was concluded by the authors to be evidence of a positive response. However, the female vehicle control group had a particularly low mean and standard deviation for the frequency of micronucleated erythrocytes and there was no clear dose response relationship, therefore the response was considered to an artefact. The test material was not considered to be clastogenic or aneugenic in male or female mice.
Executive summary:

In an in vivo bone marrow micronucleus test, groups of B6C3F1 mice (5/sex/dose) were exposed to test material in corn oil at doses of 37.5, 75, 150, 300 and 600 mg/kg bw/day, 5 days/week for 14 weeks, by gavage. At the end of the study period, peripheral blood samples were obtained from treated animals and smears were prepared immediately. Slides were fixed, stained and coded for analysis. Frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each animal per treatment group was determined. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.

 

No significant changes in the percentage of PCEs were observed over the dose range tested in either males or females, indicating an absence of treatment-related toxicity to the bone marrow. The frequencies of micronucleated erythrocytes were not increased in peripheral blood of male mice at any dose level. In contrast, female mice had a 3.2-fold increase of micronucleated erythrocytes at 600 mg/kg bw/day.

 

Under the test conditions, the test material showed a statistically significant increase in the frequency of micronucleated erythrocytes in female mice. This was concluded by the authors to be evidence of a positive response. However, the female vehicle control group had a particularly low mean and standard deviation for the frequency of micronucleated erythrocytes and there was no clear dose response relationship, therefore the response was considered to an artefact. The test material was not considered to be clastogenic or aneugenic in male or female mice.

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

Additional information

The genotoxicity of isoeugenol has been extensively studied. Only five studies are reported in this dossier but a complete evaluation was performed by HERA (2005) and EMEA (2011).

Bacterial gene mutation assay (Ames Test)

Two independent assays for bacterial mutagenicity were conducted. The first study was performed as reported by Mortelmans et al.(1986) on an analogue substance (Isoeugenol, mixture of cis- and trans-Isoeugenol, see Iuclid section 13 for read-across justification). The test material was incubated with the Salmonella typhimurium tester strains TA98, TA100, TA1535,and TA1537, either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague Dawley rat or Syrian hamster liver) for 20 minutes at 37° C. The second assay performed with Trans-isoeugenol, used a slightly modified protocol (activation only with rat liver S9) and also employed Escherichia coli strain WP2uvrA/pKM101 as a bacterial tester strain in addition to S. typhimurium strains TA98 and TA100. Trans-isoeugenol was incubated with the bacterial tester strains either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague Dawley rat livers) for 20 minutes at 37° C. Top agar supplemented with L-histidine and d-biotin was added to the cultures, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C. Each trial consisted of triplicate plates of concurrent positive and negative controls and of at least five doses of Trans-isoeugenol. The high dose was limited by toxicity. All trials were repeated.

Isoeugenol (Trans- or mixture of trans- and cis- isomers) (3.3 to 2,000 µg/plate) was not active in either of two independent assays for mutagenicity in S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA conducted with and without exogenous metabolic activation (S9 liver enzymes).

Mammalian Cell Chromosome Aberration Test in CHO Cells

Testing was performed as reported by Galloway et al.(1987). Trans-Isoeugenol was sent to the testing laboratory as a coded aliquot. It was tested in cultured Chinese hamster ovary (CHO) cells for induction of chromosomal aberrations (Abs), both in the presence and absence of Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of Trans-isoeugenol; the high dose was limited by toxicity. A single flask per dose was used. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with Trans-isoeugenol for 10 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with isoeugenol and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9.Cells were selected for scoring on the basis of good morphology and completeness of karyotype(21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Trans-Isoeugenol (in medium concentrations up to 200 µg/mL) did not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation.

Unscheduled DNA Synthesis Assay in Rat and Mouse Hepatocytes in vitro

In an UDS assay (Buckey, 1998), rat and mouse hepatocytes were incubated with the supporting substance (Isoeugenol, mixture of cis- and trans-Isoeugenol, see Iuclid section 13 for read-across justification) at the concentrations ranging from 0.1 to 1000 µM for 18 h. Hepatocytes were simultaneously exposed to [3H]thymidine (1 mL of 89.4 mCi/mL per 100 mL medium). Following incubation, cells were evaluated under an inverted microscope for gross evidence of cytotoxicity to the hepatocytes. The cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting. Results were quantified using an Artek colony counter. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results are expressed as mean grain counts per nucleus. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 500 to 5000 µM. Isoeugenol had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves. Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice. Under the test conditions, test material did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.

Micronucleus Test in the Peripheral Erythrocytes of Mice in vivo

In an in vivo bone marrow micronucleus test, groups of B6C3F1 mice (5/sex/dose) were exposed to Trans-Isoeugenol in corn oil at doses of 37.5, 75, 150, 300 and 600 mg/kg bw/day, 5 days/week for 14 weeks, by gavage. At the end of the study period, peripheral blood samples were obtained from treated animals and smears were prepared immediately. Slides were fixed, stained and coded for analysis. Frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each animal per treatment group was determined. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.   No significant changes in the percentage of PCEs were observed over the dose range tested in either males or females, indicating an absence of treatment-related toxicity to the bone marrow. The frequencies of micronucleated erythrocytes were not increased in peripheral blood of male mice at any dose level. In contrast, female mice had a 3.2-fold increase of micronucleated erythrocytes at 600 mg/kg bw/day.  

Under the test conditions, Trans-Isoeugenol showed a statistically significant increase in the frequency of micronucleated erythrocytes in female mice. This was concluded by the authors to be evidence of a positive response. However, the female vehicle control group had a particularly low mean and standard deviation for the frequency of micronucleated erythrocytes and there was no clear dose response relationship, therefore the response was considered to an artefact. Trans-Isoeugenol was not considered to be clastogenic or aneugenic in male or female mice.

In a newly performed study (ref. in EMEA evaluation), which was an in vivo micronucleus test in male and female mice, there was no indication of genotoxic potential of isoeugenol when dosed up to 2000 mg/kg/day and 1500 mg/kg/day in males and females, respectively. In addition, no evidence of genotoxic potential could be observed in the rat in a newly performed vivo UDS test in male and female rats administered doses of up to 2000 mg/kg (males) and 1250 mg/kg (females).

Other Studies (summary taken from HERA)

Isoeugenol has been chosen for a full risk assessment by HERA (Human and Environmental Risk Assessment on ingredients of household cleaning products) program

Bacterial tests. Ten studies of the mutagenicity of isoeugenol in various Salmonella typhimurium strains (TA 98,TA 97, TA 100, TA 102, TA 1535, TA 1537 and TA 1538) gave no effects either with or without metabolic activation. In most of these studies, doses up to the limits of toxicity (around 0.6 mg/plate) was carried out. Negative results were also obtained in Escherichia coli strain WP2 uvrA trp- with and without metabolic activation and in the SOS Chromotest using Escherichia colistrain PQ37. No effects were also seen in a yeast gene conversion assay with and without metabolic activation.The Rec-assay/DNA-repair test in Bacillus subtilis strains H 17 (Rec+) and M 45 (Rec-) was negative in one study but gave positive effects in another (Sekizawa andShibamoto, 1982). In the latter study the zone of inhibition at a dose of 0.8 mg isoeugenol/disk was 23.4 mm for M 45 (Rec-) and 18.2 for H 17 (Rec+) giving a difference of only 5.2 mm. In this study, isoeugenol was administered neat. The authors explain that the oily nature of the test material did not permit ready diffusion in an aqueous agar layer. Furthermore, the Rec+ cells grew faster (doubling in 48 minutes) than the Rec- cells (doubling at 75 minutes). The Rec+ cells may therefore have grown too fast, giving a visible lawn of bacteria before the sample diffused effectively thereby giving rise to a smaller inhibition zone than the Rec- cells. In the study that was negative, isoeugenol was administered in dimethylsuphoxide and hence would be expected to have diffused more efficiency that the neat test material that was administered in the study reported as positive. The significance of this positive response in the Rec assay is therefore doubtful and may be attributed to an artifact.

In vitro studies in mammalian cells. Key studies, where there were no confounding factors, were negative. No effects were seen in unscheduled DNA synthesis assays with hepatocytes isolated from male Fischer 344 rats and from male Fischer 344 rats and female B6C3F1 mice exposed to isoeugenol by 18-hour incubation at concentrations up to 1.0 mM of this substance. A Sister Chromatid Exchange (SCE) assay was conducted using human lymphocytes gave positive effects at isoeugenol concentrations of 0.5 mM (82 mg/L). The number of SCE per treated cells were 10.3 at 0.25 mM (44 mg/l) and 14.0 at 0.5 mM (88 mg/l). These effects were significantly less than for other substances that were also tested in the same study such as vanillin. In another SCE study, isoeugenol had no effect on the frequency of SCEs induced by mitomycin C in cultured Chinese hamster ovary cells at concentrations of 10, 33.3, 100 uM (1.8 -17.6 mg/l) with cytotoxicity being observed at 333 µM (58.6 mg/l). Positive results in a SCE assay are generally not regarded as evidence of a mutagenic response, especially in cases of high toxicity where lysosome breakdown due to cytotoxicity, and not from the direct action of the test substance on DNA. These effects are observed at concentrations of test substance that produce high levels of cytotoxicity, involving lysosomal breakdown and release of DNAase which induces increased exchanges, chromosome aberrations and DNA double-strandbreaks. Indeed, isoeugenol gave no evidence of an increase in the frequency of chromatid breaks and sister chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells at lower concentrations, but showed an increase in sister chromatid exchanges in human lymphocytes at higher concentrations of 0.25 mM (44 mg/l)and 0.5 mM (88 mg/l) 0.5 mM (82 mg/ml).

In conclusion, the genotoxicity of Isoeugenol has been extensively studied in in vitro and in vivo genotoxicity assays. Overall, the weight of evidence is sufficient to conclude that Isoeugenol is not genotoxic.

References

EMEA (2011). Committee for Medicinal Products for Veterinary Use. European public MRL assessment report (EPMAR). Isoeugenol (fin fish). 14 April 2011. EMA/CVMP/405186/2010

HERA (2005) Human and Environmental Risk Assessment on ingredients of Household Cleaning Products (Isoeugenol)

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.