Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG471): Negative

Chromosome aberration study (OECDTG473): Positive

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June, 1999 - 16 February, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9.

Test concentrations with justification for top dose:

Direct plate:
- Dose range finding test (all 5 strains, without and with S9): 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg/plate
Based on the results of the preliminary test, the following dose levels were used:
- Experiment 1:
TA 98, TA 1537 and WP2uvrA (without and with S9), TA 100 and TA 1535 (without S9): 25, 75, 200, 600, 1800 and 5000 μg/plate
TA 100 and TA 1535 (with S9): 7.5, 25, 75, 200, 600, 1800 and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: Acetone was selected as the solvent of choice based on solubility of the test substance and compatibility with the target cells. The test substance was soluble in acetone at approximately 500 mg/L, the maximum concentration tested.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; all Salmonella strains with S9: 1 μg/plate; WP2uvrA with S9: 10 μg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance and controls were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

OTHER EXAMINATIONS:
- Precipitate was evaluated by visual examination without magnification.

Evaluation criteria:

Evaluation of results:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.
For the test article to evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article.
Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value.
Data sets for strains TA98, TA100 and WP2uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Criteria for a valid test:
The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehivle control. A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met:
(1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count.
(2) A reduction in the background lawn.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 600, 1800 and 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 600, 1800 and 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 600, 1800 and 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1800 and 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1800 and 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was generally observed at 5000 μg/plate.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity assay, the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 100 mg/mL and 50 μL platinf aliquot. Precipitate was observed at ≥ 3333 μg per plate and toxicity was observed at ≥ 667 to 5000 μg per plate. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 μg per plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached illustration
- Negative historical control data: see attached illustration

ADDITIONAL INFORMATION:
The study was concluded to be nagative without conducting an independent repeat assay because no unique metabolism requirements were known about the article and because no equivocal responses were observed in the assay that would suggest further testing is warranted.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed equivalent to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated equivalent to OECD 471 guideline and according to GLP principles. The test was performed in one direct plate experiment up to and including 5000 μg/plate, in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding test (≥ 667 to 5000μg). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. The study was concluded to be negative without conducting an independent repeat assay because no unique metabolism requirements were known about the article and because no equivocal responses were observed in the assay that would suggest further testing is warranted. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July, 1999 - 06 September, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 1997.

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Manassas, VA.
- Suitability of cells: The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981)
- Number of passages if applicable: Stocks were not used beyond passage 20.
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: A modal chromosome number of 20
- Normal (negative control) cell cycle time: Average cell cycle time of 10-14 hours.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5A medium supplemented with 10% Foetal Bovine serum, 100 units penicillin and 100 μg streptomycin/mL, and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (using the Hoechst staining procedure)
- Periodically checked for karyotype stability: no data.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9.

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 4hr exposure; 16 hr recovery: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL
Without S9-mix, 20 hr exposure; 0 hr recovery: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL
First cytogenetic test:
With and without S9-mix, 4hr exposure; 16 hr recovery: 12.5, 25, 50, 100, 120, 150 and 180 μg/mL
Without S9-mix, 20 hr exposure; 0 hr recovery: 5, 10, 20, 40, 60, 80, 100 and 120 μg/mL
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 4 hr exposure, 16 hr recovery: 100, 120 and 150 μg/ mL
With S9-mix, 4 hr exposure, 16 hr recovery: 50, 100 and 120 μg/ mL

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: Acetone was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in acetone at approximately 500 mg/L, the maximum concentration tested.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5x10^5 cells/25 cm^2 flask

DURATION
- Preincubation period: 16-24 hours
- Exposure duration: 4 hr (with and without S9-mix), 20 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hr

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 (100 per duplicate flask)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 500 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Evaluation of test results:
The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell was calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but were not included in the total percentage of cells with one or more aberrations or in the frequency of sturctural aberrations per cell.
All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test articel was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-reponsive manner with one or more concentrations being statistically significant (p≤0.05). Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with metabolic activation may need to be confirmed on a case-by-case basis. In those cases where confirmation of nagative results is not necessary, justification was provided.

Criteria of a valid test:
The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fisher's exact test) relative to the solvent control.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher;s test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test substance in treatment medium was approximately 7.5.
- Effects of osmolality: The osmolality in treatment medium of the highest concentration tested, 180 μg/mL, was 287 mmol/kg (solvent (acetone) in tretament medium was 312 mmol/kg)
- Precipitation: No precipitaion was observed in treatment medium.

RANGE-FINDING/SCREENING STUDIES:
Visible precipitate was observed in treatment mediumat concentrations of 500, 1500 and 5000 μg/mL. Concentrations of ≤ 150 μg/mL were soluble in treatment medium. The osmolality in treatment medium of the highest concentration tested, 5000 μg/mL, was 264 mmol/kg. The osmolality of the solvent (acetone) in treatment medium was 283 mmol/kg. The pH of the highest concentration of test substance in tretament medium was approximately 7.5. Cell growth inhibition relative to the solvent control was 100% at 5000 μg/mL, the highest concentration tested in both the non-activated and S9-activated 4 hour exposure groups, respectively. Cell growth inhibition relative to the control was 100% at 5000 μg/mL, the highest concentration tested in the non-activated 20 hour continuous exposure group.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see illustration
- Negative (solvent/vehicle) historical control data: see illustration

ADDITIONAL INFORMATION ON CYTOTOXICITY AND GENOTOXICITY:
The mitotic index at the highest dose level without S9 evaluated for chromosome aberrations, 150 μg/mL, was 22% reduced relative to the solvent control.
The percentage of cells with structural aberrations in the test substance groups was significantly increased above the solvent control at all test substance dose levels ((p≤0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in test substance treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally dameged cells in the MMC group was found to be statistically significant (15%).
The mitotic index at the highest dose level with S9 evaluated for chromosome aberrations, 120 μg/mL, was 90% reduced relative to the solvent control.
The percentage of cells with structural aberrations in the test substance groups was significantly increased above the solvent control at 100 and 120 μg/mL ((p≤0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in test substance treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally dameged cells in the CP group was found to be statistically significant (25%).
In the presence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were not evaluated for chromosome aberrations.
The study was concluded to be positive. An independent repeat assay was not required because no unique metabolic requirements were known about the test substance and because no equivocal reponses were observed.

Conclusions:

A chromosome aberration study with the substance was performed equivalent to OECD 473 guideline and GLP principles, in cultured Chinese hamster ovary (CHO) cells in one experiment. It is concluded that the substance is clastogenic in Chinese hamster ovary (CHO) cells.

Executive summary:

In a chromosome aberration study, cultured Chinese hamster ovary (CHO) cells were exposed to different concentrations of the substance (dissolved in acetone), in the presence and absence of S9-mix equivalent to OECD 473 guideline and GLP principles. In the cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 150 and 120 μg/mL for a 4 h exposure time with a 16 h recovery time in the absence and presence of S9 -mix, respectively. Reliable positive and negative controls were included.

The mitotic index at the highest dose level without S9 evaluated for chromosome aberrations, 150 μg/mL, was 22% reduced relative to the solvent control. The percentage of cells with structural aberrations in the test substance groups was significantly increased above the solvent control at all test substance dose levels ((p≤0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in test substance treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally dameged cells in the MMC group was found to be statistically significant (15%). The mitotic index at the highest dose level with S9 evaluated for chromosome aberrations, 120 μg/mL, was 90% reduced relative to the solvent control. The percentage of cells with structural aberrations in the test substance groups was significantly increased above the solvent control at 100 and 120 μg/mL ((p≤0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in test substance treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally dameged cells in the CP group was found to be statistically significant (25%). In the presence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were not evaluated for chromosome aberrations.

The study was concluded to be positive. An independent repeat assay was not required because no unique metabolic requirements were known about the test substance and because no equivocal reponses were observed. Finally, it is concluded that the substance is clastogenic to Chinese hamster ovary (CHO) cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus study (OECDTG474): Negative

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:

19 October, 1999 - 19 November, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD.
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Males: 26.7 - 31.2 g; females: 24.8 - 30.0 g
- Assigned to test groups randomly: yes
- Housing: Five per cage in polycarbonate cages
- Diet: Free access to certified laboratory rodent chow
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS set to maintain
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Solvent used: corn oil
- Justification for choice of solvent: Corn oil was determined to be the solvent of choice based on a request by the Sponsor and compatibility of the vehicle with the test system animals.
- Concentration of test material in vehicle: The test article was workable in corn oil at 50 mg/mL, the maximum concentration tested in the study.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was workable in corn oil at 50 mg/mL, the maximum concentration tested in the study. Dosing concentrations at 35 and 50 mg/mL were delivered to the test system as light yellow suspensions and concentrations below 35 mg/mL as light yellow solutions.

VOLUME: 20 mL/kg body weight

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Single injection

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

24 and 48 hours exposure time

Dose / conc.:

95 mg/kg bw (total dose)

Remarks:

24 hours exposure time

Dose / conc.:

190 mg/kg bw (total dose)

Remarks:

24 hours exposure time

Dose / conc.:

380 mg/kg bw (total dose)

Remarks:

24 and 48 hours exposure time

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: intraperitoneal at 20 mL/kg body weight
- Doses / concentrations: Dissolved in sterile distilled water at a concentration of 2.5 mg/mL

Tissues and cell types examined:

Polychromatic erythrocytes (2000) were scored for the presence of micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the observed mortality in the toxicity assay, dose levels were selected for the micronucleus assay. Intraperitoneal injection was selected to maximize delivery of the test substance to the test system.

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice times, mice were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cellw were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staing nuclear fragments, having a sharp contour with diameters usually 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:

Evaluation of the results:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analysis were performed separately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test substance was considered to induce a positive response if a dose-reponsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤0.05, Kastenbaum-Bowman tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test substance was considered negative if no statistically increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

Criteria for a valid test:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocyte in the positive control group must be significantly increased relative to the vehicle control group (p≤0.05, Kastenbaum-Bowman tables).

Statistics:

See evaluation criteria.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: The substance was administered by intraperitoneal injection to male and female mice at 300, 500, 700 or 1000 mg test substance/kg body weight which was administered in a total volume of 20 mL test substance-vehicle mixture/kg body weight. Mortality occurred after dose administration as follows: 2/5 males and 4/5 females at 500 mg/kg bw and in all males and females at 700 and 1000 mg/kg bw. The LD50/3 was calculated by probit analysis to be approximately 487 mg/kg bw for male and female mice. The high dose for the micronucleus test was set at 380 mg/kg bw for male and female mice which was estimated to be approximately 80% of the LD50/3.
- Solubility: The test article was workable in corn oil at 50 mg/mL, the maximum concentration tested in the study.
- Clinical signs of toxicity in test animals: Clinical signs, which were noted after dose administration, included: lethargy and piloerection in males and females at 300 and 500 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Mortality/clinical signs: No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs, which were noted after dose administration, included: lethargy and piloerection in males and females at 380 mg/kg bw. All other mice treated with test and control substances appeared normal during the study.
- Induction of micronuclei (for Micronucleus assay): No siginificant increase in micronucleated polychromatic erythrocytes in test substance-treated groups relative to the repsective vehicle control groups was observed in male or female mice at 24 hours and in males at 48 hours after dose administration (p≤0.05, Kastenbaum-Bowman). A statistically significant increase was observed in female mice at 48 hours after treatment with 380 mg/kg bw (p≤0.05, Kastenbaum-Bowman tables). However, this increase was not biologically significant based on the lack of dose response at 24 hour harvest time and the maximum number of micronuclei observed per animal. The number of micronuclei in two females (3/2000 polychromatic erythrocytes) was the same as in the control female animal at 24 hour harvest time and was within the historical solvent control range. Based on these facts, the increase was judged not to be biologically significant. CP induced a siginificant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman tables).
- Ratio of PCE/NCE (for Micronucleus assay): Slight reductions of 1% to 17% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance treated groups relative to their respective vehicle controls. Reductions were observed in male dose group 24 hours after treatment with 95 mg/kg bw, in female dose group 24 hours after treatment with 190 mg/kg bw and in male and female dose groups 24 and 48 hours after treatment with 380 mg/kg bw. The reductions in the frequency of polychromatic erythrocytes in the bone marrow suggest that the sunstance did not inhibit erythropoiesis.
- Appropriateness of dose levels and route: Intraperitoneal injection was selected to maximize delivery of the test substance to the test system.

Conclusions:

A micronucleus study with the substance was performed equivalent to OECD 474 guideline and GLP principles, in male and female mice. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

Executive summary:

In an in vivo micronucleus study, male and female mice were exposed to 95, 190 and 380 mg/kg bw of the substance, performed equivalent to OECD 474 guideline and GLP principles.

No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs, which were noted after dose administration, included: lethargy and piloerection in males and females at 380 mg/kg bw. All other mice treated with test and control substances appeared normal during the study. Reliable positive and negative controls were included.

No siginificant increase in micronucleated polychromatic erythrocytes in test substance-treated groups relative to the repsective vehicle control groups was observed in male or female mice at 24 hours and in males at 48 hours after dose administration (p≤0.05, Kastenbaum-Bowman). A statistically significant increase was observed in female mice at 48 hours after treatment with 380 mg/kg bw (p≤0.05, Kastenbaum-Bowman tables). However, this increase was not biologically significant based on the lack of dose response at 24 hour harvest time and the maximum number of micronuclei observed per animal. The number of micronuclei in two females (3/2000 polychromatic erythrocytes) was the same as in the control female animal at 24 hour harvest time and was within the historical solvent control range. Based on these facts, the increase was judged not to be biologically significant. CP induced a siginificant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman tables).

Slight reductions of 1% to 17% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance treated groups relative to their respective vehicle controls. Reductions were observed in male dose group 24 hours after treatment with 95 mg/kg bw, in female dose group 24 hours after treatment with 190 mg/kg bw and in male and female dose groups 24 and 48 hours after treatment with 380 mg/kg bw. The reductions in the frequency of polychromatic erythrocytes in the bone marrow suggest that the substance did not inhibit erythropoiesis.

It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Veramoss is an ester and due to the aromatic ring with OH groups attached the ester is electrophilic and may hydrolyse quickly, resulting in a relative strong acid (pKa of 3.21 estimated with SPARC). The steep dose-response for cytotoxicity and absence of dose response in the positive result may further support this.

Additional information

Ames test

The mutagenic activity of the substance was evaluated equivalent to OECD 471 guideline and according to GLP principles. The test was performed in one direct plate experiment up to and including 5000 μg/plate, in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding test (≥ 667 to 5000μg). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. The study was concluded to be negative without conducting an independent repeat assay because no unique metabolism requirements were known about the article and because no equivocal responses were observed in the assay that would suggest further testing is warranted. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.

In vitro Chromosome aberration:

Introduction and Method: In a chromosome aberration study, cultured Chinese hamster ovary (CHO) cells were exposed to different concentrations of the substance (dissolved in acetone), in the presence and absence of S9-mix equivalent to OECD 473 guideline and GLP principles. In the cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 150 and 120 μg/mL for a 4 h exposure time with a 16 h recovery time in the absence and presence of S9 -mix, respectively. Reliable positive and negative controls were included.

Results without S9: The mitotic index at the scored dose levels (100, 120 and 150 ug/ml) without S9 was reduced with 34 -57% compared to the solvent control and showed a steep dose effect curve. The percentage of cells with structural aberrations in the test substance groups was significantly increased above the solvent control at all test substance dose levels ((p≤0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in test substance treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test) found to be statistically significant and fulfilled the criteria. The percentage of structurally damaged cells in the MMC group was found to be statistically significant (15%).

Results with S9: For the test with S9, the mitotic index at the three highest dose levels that were scored (50, 100 and 120 ug/ml) was reduced with 16, 42 and 72% relative to the solvent control. At 100 ug/ml the percentage of cells with structural aberrations in the test substance groups was significantly increased above the solvent control. A similar increase was also seen at 120 μg/mL but the toxicity exceeded the criterion of 50% ((p≤0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). In view of the high cytotoxicity this result is of doubtful toxicological relevance. The percentage of cells with numerical aberrations in test substance treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the CP group was found to be statistically significant (25%). In the presence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were not evaluated for chromosome aberrations.

Repeat test: An independent repeat assay was not required because no unique metabolic requirements were known about the test substance and because no equivocal responses were observed.

Conclusion: The results of this study show that the substance is positive in this cytogenicity assay. Finally, it is concluded that the substance is in vitro clastogenic to Chinese hamster ovary (CHO) cells.

In vivo micronucleus assay

Introduction and Method: In an in vivo micronucleus study, male and female mice were exposed to 95, 190 and 380 mg/kg bw of the substance, performed equivalent to OECD 474 guideline and GLP principles.

Result-Clinical signs: No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs, which were noted after dose administration, included: lethargy and piloerection in males and females at 380 mg/kg bw and was therefore set as the maximum tolerable dose. All other mice treated with test and control substances appeared normal during the study. Reliable positive and negative controls were included.

Results: No significant increase in micronucleated polychromatic erythrocytes in test substance-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 hours and in males at 48 hours after dose administration (p≤0.05, Kastenbaum-Bowman). A statistically significant increase was observed in female mice at 48 hours after treatment with 380 mg/kg bw (p≤0.05, Kastenbaum-Bowman tables). However, this increase was not biologically significant based on the lack of dose response at 24 hour harvest time and the maximum number of micronuclei observed per animal. The number of micronuclei in two females (3/2000 polychromatic erythrocytes) was the same as in the control female animal at 24 hour harvest time and was within the historical solvent control range. Based on these facts, the increase was judged not to be biologically significant. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman tables).

Result-toxicity: Slight reductions of 1% to 17% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance treated groups relative to their respective vehicle controls. Reductions were observed in male dose group 24 hours after treatment with 95 mg/kg bw, in female dose group 24 hours after treatment with 190 mg/kg bw and in male and female dose groups 24 and 48 hours after treatment with 380 mg/kg bw. These results indicate that the bone marrow was reached.

Conclusion: The substance is negative in this in vivo mouse micronucleus assay with an indication that the bone marrow was reached.

Justification for classification or non-classification

Based on the results of the negative Ames test and negative in vivo micronucleus study, the substance does not have to be classified for genotoxicity in accordance with EU CLP (EC No 1272/2008 and its amendments).