(E)-2-Methoxy-4-prop-1-en-1-ylphenyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation assay (Ames test) on Isoeugenyl acetate (reaction-mass of cis- and trans-isomers): not mutagenic (K, rel. 1);

Mammalian cell chromosome aberration study in CHO cells on Isoeugenyl acetate (reaction-mass of cis- and trans-isomers): not clastogenic (K, rel. 2);

Unscheduled DNA synthesis assay in mammalian liver cells on trans-Isoeugenol: no induction of UDS (K, rel. 2). The UDS assay is considered to be an acceptable surrogate for the in vitro gene mutation assay in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 29 to June 12, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted according to OECD test guideline No. 482 with deviation: purity of test material not reported.

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

purity of test material not reported

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Complete WME (Williams' Media E buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 µg/mL gentamicin and 10 % fetal bovine serum)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Preliminary cytotoxicity assay: 6, 10, 30, 60, 100, 300, 600, 1000, 3000 and 4000 µg/mL
UDS test:
- parallel cytotoxicity assay: 5, 10, 15, 20, 25, 30, 40 and 50 µg/ml based on cytotoxicity.
- Main UDS assay: 5, 10, 15, 20 and 25 µg/mL based on excessive toxicity at 30, 40 and 50 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was determined to be the solvent of choice based on solubility of the test article and compatibility with the target cells. The test material was soluble in DMSO at a maximum concentration of approximately 400 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: dimethylbenzanthracene

Remarks:

3 and 10 µg/mL

Details on test system and experimental conditions:

HEPATOCYTE ISOLATION: Primary rat hepatocytes were derived from the liver of a normal adult male Sprague-Dawley rat. The procedure used for obtaining rat hepatocyte cultures was essentially that of Williams (1977 and 1979). 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 WME (Williams' Media E buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 µg/mL gentamicin and 10 % fetal bovine serum) and approximately 5 x 10^5 cells were seeded into 35 mm tissue culture dishes containing complete WME. The cultures were incubated at 37 ± 1 °C in a humidified 5 ± 1 % CO2 incubator for 90 to 180 minutes, washed with complete medium refed with serum-free medium and used in the test.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18-20 h
- Selection time (if incubation with a selection agent): Hepatocytes were simultaneously exposed to [3H]thymidine (10 µCi/mL).

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release. In the cytotoxicity assays, relative LDH activities were obtained by subtracting the LDH activity of the negative control cultures from the LDH activity of the treated cultures. Relative toxicities were obtained by comparing the relative LDH activities of the treated cultures to the relative LDH activity of the 100 % lysis control cultures.

NUMBER OF REPLICATIONS: Triplicate

PREPARATION AND SCORING OF SLIDES
- After 18-20 h of exposure, the cells in the autoradiography dishes were washed in serum-free WME, swelled in 1 % sodium citrate and fixed in ethanol-glacial acetic acid fixative. The coverslips were air-dried, mounted cell side up on glass slides, and allowed to dry. The slides were coated with Kodak NTB-2 emulsion and stored in a refrigerator for 5-12 days in light tight boxes with desiccant. The slides were then developed in Kodak D-19 developer, fixed in Kodak fixer and stained in hematoxylin-eosin stain.

NUMBER OF CELLS EVALUATED:
- Slides were counted without knowledge of treatment group on an automated colony counter interfaced with the microscope. Where possible, nuclear grains were counted in 50 cells in random areas on each of three coverslips for a total of 150 cells per treatment.

OTHER:
- The net nuclear counts were determined by counting three nucleus-sized areas adjacent to each nucleus and subtracting the average cytoplasmic count from the nuclear count. Replicative synthesis was identified by nuclei completely blackened with grains and such cells were not counted. Nuclei exhibiting toxic effects of treatment such as dark or uneven staining, disrupted membranes or irregular shape were not counted.

Rationale for test conditions:

UDS evaluations were only performed at 15 to 25 mg/ml test item due to excessive cytotoxicity at higher concentrations.

Evaluation criteria:

- If the mean net nuclear count was increased by at least five counts over the negative control, the results for that dose level were considered significant. A test article was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the negative control. In the absence of a dose response, a test article which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive.
- If a test article showed a significant increase in the mean net nuclear grain count at one dose level without a dose response, the activity of the test article was considered to be equivocal.
- The test article was considered negative if no significant increase in the mean net nuclear grain counts was observed.

Statistics:

None

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the initiation and at the termination of treatment precipitate in treatment medium was observed at test material concentrations ranging from 600 to 4000 µg/mL. No precipitate in treatment medium was observed at concentrations ranging from 6 to 300 µg/mL.
- Other confounding effects: None

PRELIMINARY TOXICITY ASSAY: Measurement of released LDH indicated relative toxicity of between 62.1 and 95.9% in cultures exposed to ≥ 30 µg/mL. Relative toxicity was ≤ 15.5% at 10 and 6 µg/mL. A microscopic examination of the hepatocyte cultures at the termination of treatment indicated a moderate level of toxicity in cultures treated with ≥ 600 µg/mL and a low level of toxicity in cultures treated with 30 to 300 µg/mL. Normal morphology was observed at concentrations of 6.0 and 10 µg/mL. Based on the results of the toxicity assay, the highest concentration of test material selected for use in the UDS assay was 50 µg/mL.

MAIN ASSAY: At the initiation and at the termination of treatment, no precipitate was observed at any concentrations ranging from 5 to 50 µg/mL. Due to the total absorbance readings of the 100% lysis of the solvent control cultures falling outside of the standard curve for the LDH assay, the relative toxicity values for the remaining cultures are expressed as s a percentage based on a maximum LDH value of 7100 units/L, the uppermost value of the standard curve. An increase in LDH release in the 100% lysed samples would decrease the relative toxicity of the test material treated samples. Measurement of released LDH indicated relative toxicity of between ≤ 13.4 and 34.8% was indicated in test material treated cultures ranging from 20 to 50 µg/mL. All remaining cultures indicated relative toxicity of < 10%. A microscopic examination of the hepatocyte cultures at the termination of treatment indicated a high level of toxicity in cultures treated with ≥ 30 µg/mL, a moderate level of toxicity in cultures treated with 15 to 25 µg/mL, and a low level of toxicity at 10 µg/mL. Normal morphology was observed at 5 µg/mL. Examination of the fixed and stained cells indicated that cells treated with 30, 40 and 50 µg/mL could not be evaluated for UDS due to excessive toxicity. The dose levels of 5, 10, 15, 20 and 25 µg/mL were evaluated for UDS. None of the test material concentrations produced a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) when compared to the negative control.

None

Conclusions:

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

Executive summary:

In an UDS assay performed according to the OECD test guideline No. 482 and in compliance with GLP, rat hepatocytes were incubated with test material at the concentrations of 5, 10, 15, 20 and 25 µg/mL for 18-20 h. Vehicle and positive control groups treated with DMSO and Dimethylbenz(a)anthracene, respectively. Hepatocytes were simultaneously exposed to [3H]thymidine (10 µCi/mL). Following incubation, cells were evaluated for gross evidence of cytotoxicity to the hepatocytes. After exposure, 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. Slides were counted without knowledge of treatment group on an automated colony counter interfaced with the microscope. Where possible, nuclear grains were counted in 50 cells in random areas on each of three coverslips for a total of 150 cells per treatment. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 6 to 4000 µg/mL and 5 to 50 µg/mL.

None of the test material concentrations produced a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) when compared to the negative control. Therefore, the test material is considered to be negative in this study. Positive control induced the UDS and indicating the validity of the study.

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

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 23 to December 02, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD GLP (inspected on June 11 and 13, 2001/ signed on January 14, 2002)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

15 % v/v S9-mix: S9-mix from the livers of male Wistar Han lbm rats received three consecutive daily doses of phenobarbital and β-naphthoflavone.

Test concentrations with justification for top dose:

- Preliminary toxicity study: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA98 and TA100, with and without S9-mix, using plate incorporation and preincubation method.
- Experiment-1 (plate incorporation method): 3, 10, 33, 100, 333, 1000 and 2000 µg/plate in TA1537 and TA102, with and without S9-mix as well as in TA1535, without S9-mix; 3, 10, 33, 100, 333, 1000 and 2500 µg/plate in TA98 and TA100, with and without S9-mix as well as in TA1535, with S9-mix based on cytotoxicity.
- Experiment-2 (preincubation method): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA1537, with S9-mix; 3, 10, 33, 100, 333, 1000 and 2500 µg/plate in TA98 and TA100, with and without S9-mix as well as in TA1535 with S9-mix; 3, 10, 33, 100, 333, 1000 and 2000 µg/plate in TA1535 and TA1537, without S9-mix as well as in TA102, with and without S9-mix based on cytotoxicity.
- Experiment-3 (plate incorporation method): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA1535, with and without S9-mix based on the absence of toxic effects observed at 2000 µg/plate in Experiment 1 and 2.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Remarks:

Without S9-mix

Untreated negative controls:

yes

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: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes at 37 °C
- Exposure duration: Approximately 48 h at 37 °C

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of a reduction in number of spontaneous revertants or a clearing of the bacterial background lawn.

Rationale for test conditions:

Experiment 1 & 2- Maximum concentration was 2000 μg/plate (limited by cytotoxicity).
Experiment 3 - Since no toxic effects were observed at 2000 µg/plate in strain TA 1535, an additional test was performed with this strain at concentrations up to 5000 µg/plate in a plate incorporation test.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY: Toxicity (decrease in the number of revertants/plate) was observed at higher concentrations with and without metabolic activation in TA 98 and TA 100 strains (≥333 µg/plate). No visible reduction of the background growth was observed up to the highest concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: The comparison was made with the historical control ranges since 1995 of the corresponding testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment -1: Toxicity was observed at ≥1000 µg/plate in TA98, TA100 and TA102, with and without S9-mix; at 2000 µg/plate in TA1537 with and without S9-mix and at 5000 µg/plate in TA1535, with S9-mix.
Experiment-2: Toxicity was observed at ≥333 µg/plate in TA98, with and without S9-mix; at ≥1000 µg/plate in TA102, with and without S9-mix; at 2500 and ≥1000 µg/plate in TA100, without and with S9-mix, respectively; at 2000 and 5000 µg/plate in TA1537, without and with S9-mix, respectively and at ≥1000 µg/plate in TA1535, with S9-mix.
Experiment-3: Toxicity was observed at 5000 µg/plate in TA1535, with S9-mix.
Irregular background growth was observed in strain TA 1535 in experiment 1 and 3 (2500 µg/plate with S9 mix) and in experiment 2 (1000 µg/plate). In experiment 2, irregular background growth was observed in strain TA 1537 at 1000 and 2500 µg/plate with metabolic activation and at 2500 µg/plate without metabolic activation.

See the attached file for table of results

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 strain according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were exposed to test material diluted in ethanol, both in the presence and absence of metabolic activation system (15 % liver S9-mix). The dose range was determined in a preliminary toxicity assay and ranged between 3-2000 µg/plate for TA1537 and TA102 , with and without S9-mix as well as for TA1535, without S9-mix; and 3-2500 µg/plate for TA98 and TA100, with and without S9-mix as well as for TA1535, with S9-mix using plate incorporation method (Experiment-1). In Experiment-2, dose ranged between 3-2000 µg/plate for TA1535 and TA1537, without S9-mix as well as for TA102, with and without S9-mix; 3-2500 µg/plate for TA98 and TA100, with and without S9-mix as well as in TA1535 with S9-mix; and 3-5000 µg/plate in TA1537, with S9-mix, using preincubation method. In Experiment-3, dose ranged between 3-5000 µg/plate for TA1535, with and without S9-mix using plate incorporation method. Vehicle (ethanol), negative and positive control groups were also included in mutagenicity tests.

The number of revertants for the vehicle, negative and positive controls was as specified in the acceptance criteria. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In Experiment 1, toxicity was observed at ≥1000 µg/plate in TA98, TA100 and TA102, with and without S9-mix; at 2000 µg/plate in TA1537 with and without S9-mix and at 5000 µg/plate in TA1535, with S9-mix. In Experiment 2, toxicity was observed at ≥333 µg/plate in TA98, with and without S9-mix; at ≥1000 µg/plate in TA102, with and without S9-mix; at 2500 and ≥1000 µg/plate in TA100, without and with S9-mix, respectively; at 2000 and 5000 µg/plate in TA1537, without and with S9-mix, respectively and at ≥1000 µg/plate in TA1535, with S9-mix. In Experiment 3, toxicity was observed at 5000 µg/plate in TA1535, with S9-mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 strain according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint. The supporting substance is considered adequate for read-across purpose.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
- In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on the source substance.
The Ames test on the source substance provided in this dossier was performed according to OECD TG 471 and in compliance with GLPs. The test material was clearly identified (purity = 99.7 % w/w) although the isomer ratio was not reported. It is however considered to represent the source substance in terms of constituents and impurities. The result of the study is adequate for classification and labelling
- In vitro gene mutation study in mammalian cells
An unscheduled DNA synthesis (UDS) test was performed on the source substance. The source substance did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes, under the conditions of this test.
The UDS test on the source substance provided in this dossier was performed according to OECD TG 482 and in compliance with GLPs. The test material was not clearly identified but it is assumed to represent the source substance in terms of constituents and impurities. The result of the study is adequate for classification and labelling
Therefore, based on the considerations above, it can be concluded that the result of the Ames and the UDS test conducted with the source substance are highly likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.4.1 and 8.4.3.

4. DATA MATRIX
Cf. Iuclid Section 13. 

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY: Toxicity (decrease in the number of revertants/plate) was observed at higher concentrations with and without metabolic activation in TA 98 and TA 100 strains (≥333 µg/plate). No visible reduction of the background growth was observed up to the highest concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: The comparison was made with the historical control ranges since 1995 of the corresponding testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment -1: Toxicity was observed at ≥1000 µg/plate in TA98, TA100 and TA102, with and without S9-mix; at 2000 µg/plate in TA1537 with and without S9-mix and at 5000 µg/plate in TA1535, with S9-mix.
Experiment-2: Toxicity was observed at ≥333 µg/plate in TA98, with and without S9-mix; at ≥1000 µg/plate in TA102, with and without S9-mix; at 2500 and ≥1000 µg/plate in TA100, without and with S9-mix, respectively; at 2000 and 5000 µg/plate in TA1537, without and with S9-mix, respectively and at ≥1000 µg/plate in TA1535, with S9-mix.
Experiment-3: Toxicity was observed at 5000 µg/plate in TA1535, with S9-mix.
Irregular background growth was observed in strain TA 1535 in experiment 1 and 3 (2500 µg/plate with S9 mix) and in experiment 2 (1000 µg/plate). In experiment 2, irregular background growth was observed in strain TA 1537 at 1000 and 2500 µg/plate with metabolic activation and at 2500 µg/plate without metabolic activation.

See the attached file for table of results

Conclusions:

Based on the available data on the source substance, the target substance is considered to be not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 strain.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were exposed to the source substance ( 2-Methoxy-4-prop-1-en-1-ylphenyl acetate) diluted in ethanol, both in the presence and absence of metabolic activation system (15 % liver S9-mix). The dose range was determined in a preliminary toxicity assay and ranged between 3-2000 µg/plate for TA1537 and TA102 , with and without S9-mix as well as for TA1535, without S9-mix; and 3-2500 µg/plate for TA98 and TA100, with and without S9-mix as well as for TA1535, with S9-mix using plate incorporation method (Experiment-1). In Experiment-2, dose ranged between 3-2000 µg/plate for TA1535 and TA1537, without S9-mix as well as for TA102, with and without S9-mix; 3-2500 µg/plate for TA98 and TA100, with and without S9-mix as well as in TA1535 with S9-mix; and 3-5000 µg/plate in TA1537, with S9-mix, using preincubation method. In Experiment-3, dose ranged between 3-5000 µg/plate for TA1535, with and without S9-mix using plate incorporation method. Vehicle (ethanol), negative and positive control groups were also included in mutagenicity tests.

The number of revertants for the vehicle, negative and positive controls was as specified in the acceptance criteria. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In Experiment 1, toxicity was observed at ≥1000 µg/plate in TA98, TA100 and TA102, with and without S9-mix; at 2000 µg/plate in TA1537 with and without S9-mix and at 5000 µg/plate in TA1535, with S9-mix. In Experiment 2, toxicity was observed at ≥333 µg/plate in TA98, with and without S9-mix; at ≥1000 µg/plate in TA102, with and without S9-mix; at 2500 and ≥1000 µg/plate in TA100, without and with S9-mix, respectively; at 2000 and 5000 µg/plate in TA1537, without and with S9-mix, respectively and at ≥1000 µg/plate in TA1535, with S9-mix. In Experiment 3, toxicity was observed at 5000 µg/plate in TA1535, with S9-mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Based on the available data on the source substance, the target substance is considered to be not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 strain.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

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:

Trans-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). 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 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.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because the target substance is hydrolysed to the source substance by tissue esterases.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances are both mono-constituent substance (trans-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
- In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances (for both: reaction-mass of cis- and trans-isomers instead of trans-isomer).
- In vitro gene mutation study in mammalian cells
An unscheduled DNA synthesis (UDS) test was performed on both the source and the target substances (for both: reaction-mass of cis- and trans-isomers instead of trans-isomer). The substances did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes, under the conditions of these tests.
- In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance, no significant increases in the frequency of cells with aberrations was induced.
The CAT test on the source substance provided in this dossier was performed according to OECD TG 473 and in compliance with GLPs. The test material was clearly identified although the impurity profile was not reported. It is however assumed to represent the source substance in terms of constituents and impurities. The result of the study is adequate for classification and labelling

Therefore, based on the considerations above, it can be concluded that the result of the CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2.

4. DATA MATRIX
Cf. Iuclid Section 13. 

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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:

Based on the available data on the source substance, the target substance does not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation.

Executive summary:

Testing was performed as reported by Gallowayet al.(1987). The source substance (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 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. Based on the available data on the source substance, the target substance does not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
- In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on the source substance.
The Ames test on the source substance provided in this dossier was performed according to OECD TG 471 and in compliance with GLPs. The test material was clearly identified (purity = 99.7 % w/w) although the isomer ratio was not reported. It is however considered to represent the source substance in terms of constituents and impurities. The result of the study is adequate for classification and labelling
- In vitro gene mutation study in mammalian cells
An unscheduled DNA synthesis (UDS) test was performed on the source substance. The source substance did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes, under the conditions of this test.
The UDS test on the source substance provided in this dossier was performed according to OECD TG 482 and in compliance with GLPs. The test material was not clearly identified but it is assumed to represent the source substance in terms of constituents and impurities. The result of the study is adequate for classification and labelling
Therefore, based on the considerations above, it can be concluded that the result of the Ames and the UDS test conducted with the source substance are highly likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.4.1 and 8.4.3.

4. DATA MATRIX
Cf. Iuclid Section 13. 

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the initiation and at the termination of treatment precipitate in treatment medium was observed at test material concentrations ranging from 600 to 4000 µg/mL. No precipitate in treatment medium was observed at concentrations ranging from 6 to 300 µg/mL.
- Other confounding effects: None

PRELIMINARY TOXICITY ASSAY: Measurement of released LDH indicated relative toxicity of between 62.1 and 95.9% in cultures exposed to ≥ 30 µg/mL. Relative toxicity was ≤ 15.5% at 10 and 6 µg/mL. A microscopic examination of the hepatocyte cultures at the termination of treatment indicated a moderate level of toxicity in cultures treated with ≥ 600 µg/mL and a low level of toxicity in cultures treated with 30 to 300 µg/mL. Normal morphology was observed at concentrations of 6.0 and 10 µg/mL. Based on the results of the toxicity assay, the highest concentration of test material selected for use in the UDS assay was 50 µg/mL.

MAIN ASSAY: At the initiation and at the termination of treatment, no precipitate was observed at any concentrations ranging from 5 to 50 µg/mL. Due to the total absorbance readings of the 100% lysis of the solvent control cultures falling outside of the standard curve for the LDH assay, the relative toxicity values for the remaining cultures are expressed as s a percentage based on a maximum LDH value of 7100 units/L, the uppermost value of the standard curve. An increase in LDH release in the 100% lysed samples would decrease the relative toxicity of the test material treated samples. Measurement of released LDH indicated relative toxicity of between ≤ 13.4 and 34.8% was indicated in test material treated cultures ranging from 20 to 50 µg/mL. All remaining cultures indicated relative toxicity of < 10%. A microscopic examination of the hepatocyte cultures at the termination of treatment indicated a high level of toxicity in cultures treated with ≥ 30 µg/mL, a moderate level of toxicity in cultures treated with 15 to 25 µg/mL, and a low level of toxicity at 10 µg/mL. Normal morphology was observed at 5 µg/mL. Examination of the fixed and stained cells indicated that cells treated with 30, 40 and 50 µg/mL could not be evaluated for UDS due to excessive toxicity. The dose levels of 5, 10, 15, 20 and 25 µg/mL were evaluated for UDS. None of the test material concentrations produced a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) when compared to the negative control.

None

Conclusions:

Based on the available data on the source substance, the target substance is not expected to induce unscheduled DNA synthesis (UDS) in rat hepatocytes.

Executive summary:

In an UDS assay performed according to the OECD test guideline No. 482 and in compliance with GLP, rat hepatocytes were incubated with the source substance (2-Methoxy-4-prop-1-en-1-ylphenyl acetate) at the concentrations of 5, 10, 15, 20 and 25 µg/mL for 18-20 h. Vehicle and positive control groups treated with DMSO and Dimethylbenz(a)anthracene, respectively. Hepatocytes were simultaneously exposed to [3H]thymidine (10 µCi/mL). Following incubation, cells were evaluated for gross evidence of cytotoxicity to the hepatocytes. After exposure, 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. Slides were counted without knowledge of treatment group on an automated colony counter interfaced with the microscope. Where possible, nuclear grains were counted in 50 cells in random areas on each of three coverslips for a total of 150 cells per treatment. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 6 to 4000 µg/mL and 5 to 50 µg/mL.

None of the test material concentrations produced a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) when compared to the negative control. Therefore, the test material is considered to be negative in this study. Positive control induced the UDS and indicating the validity of the study.

Based on the available data on the source substance, the target substance is not expected to induce unscheduled DNA synthesis (UDS) in rat hepatocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No study was identified on the target substance (Trans-Isoeugenyl acetate). However, three studies were available on souce substances (Isoeugenyl acetate (reaction-mass of cis- and trans-isomers) and Trans-Isoeugenol, see Iuclid section 13 for read-across justification).

 

Bacterial Gene Mutation Assay

In a reverse gene mutation assay performed on the source substance Isoeugenyl acetate (reaction-mass of cis- and trans-isomers) according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were exposed to test material diluted in ethanol, both in the presence and absence of metabolic activation system (15 % liver S9-mix) (RCC, 2002, Rel.1). The dose range was determined in a preliminary toxicity assay and ranged between 3-2000 µg/plate for TA1537 and TA102 , with and without S9-mix as well as for TA1535, without S9-mix; and 3-2500 µg/plate for TA98 and TA100, with and without S9-mix as well as for TA1535, with S9-mix using plate incorporation method (Experiment-1). In Experiment-2, dose ranged between 3-2000 µg/plate for TA1535 and TA1537, without S9-mix as well as for TA102, with and without S9-mix; 3-2500 µg/plate for TA98 and TA100, with and without S9-mix as well as in TA1535 with S9-mix; and 3-5000 µg/plate in TA1537, with S9-mix, using preincubation method. In Experiment-3, dose ranged between 3-5000 µg/plate for TA1535, with and without S9-mix using plate incorporation method. Vehicle (ethanol), negative and positive control groups were also included in mutagenicity tests. The number of revertants for the vehicle, negative and positive controls was as specified in the acceptance criteria. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. 

In Experiment 1, toxicity was observed at ≥1000 µg/plate in TA98, TA100 and TA102, with and without S9-mix; at 2000 µg/plate in TA1537 with and without S9-mix and at 5000 µg/plate in TA1535, with S9-mix. In Experiment 2, toxicity was observed at ≥333 µg/plate in TA98, with and without S9-mix; at ≥1000 µg/plate in TA102, with and without S9-mix; at 2500 and ≥1000 µg/plate in TA100, without and with S9-mix, respectively; at 2000 and 5000 µg/plate in TA1537, without and with S9-mix, respectively and at ≥1000 µg/plate in TA1535, with S9-mix. In Experiment 3, toxicity was observed at 5000 µg/plate in TA1535, with S9-mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.  

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

 

Mammalian Cell Chromosome Aberration Test in CHO Cells

Testing was performed on the source substance Trans-Isoeugenol (NTP, 2010, Rel.2). Trans-Isoeugenol 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. The supporting substance is considered adequate for read-across purpose.

 

Unscheduled DNA Synthesis Assay In Vitro

In an UDS assay performed on the source substance Isoeugenyl acetate (reaction-mass of cis- and trans-isomers (BioReliance, 2002, Rel.2) according to the OECD test guideline No. 482 and in compliance with GLP, rat hepatocytes were incubated with test material at the concentrations of 5, 10, 15, 20 and 25 µg/mL for 18-20 h. Vehicle and positive control groups treated with DMSO and Dimethylbenz(a)anthracene, respectively. Hepatocytes were simultaneously exposed to [3H]thymidine (10 µCi/mL). Following incubation, cells were evaluated for gross evidence of cytotoxicity to the hepatocytes. After exposure, 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. Slides were counted without knowledge of treatment group on an automated colony counter interfaced with the microscope. Where possible, nuclear grains were counted in 50 cells in random areas on each of three coverslips for a total of 150 cells per treatment. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 6 to 4000 µg/mL and 5 to 50 µg/mL.

None of the test material concentrations produced a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) when compared to the negative control. Therefore, the test material is considered to be negative in this study. Positive control induced the UDS and indicating the validity of the study.

 

Under the test conditions, the test material did not induce unscheduled DNA synthesis (UDS) in rat hepatocytes.The supporting substance is considered adequate for read-across purpose.

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 on the supporting substance, 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.