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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

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

Test material

Test material information

1

Reference substance name:

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

EC Number:

225-193-0

EC Name:

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

Cas Number:

4707-47-5

Molecular formula:

C10H12O4

IUPAC Name:

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

Test material form:

other: solid

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

24 and 48 hours exposure time

Doses / concentrations 2

Dose / conc.:

95 mg/kg bw (total dose)

Remarks:

24 hours exposure time

Doses / concentrations 3

Dose / conc.:

190 mg/kg bw (total dose)

Remarks:

24 hours exposure time

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.