Carvacrol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from publication.

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity evaluation of the given test chemical in rats using micronucleus assay

Author:

Llana-Ruiz-Cabello et al.

Year:

2016

Bibliographic source:

Food and Chemical Toxicology

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The mammalian erythrocyte MN test was performed using the given test chemical.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Carvacrol

EC Number:

207-889-6

EC Name:

Carvacrol

Cas Number:

499-75-2

Molecular formula:

C10H14O

IUPAC Name:

2-methyl-5-(propan-2-yl)phenol

Details on test material:

- Name of test material : 2-methyl-5-(propan-2-yl)phenol
- Common name : Carvacrol
- Molecular formula : C10H14O
- Molecular weight : 150.2196 g/mol
- Smiles notation : CC(C)c1ccc(C)c(O)c1
- InChl : 1S/C10H14O/c1-7(2)9-5-4-8(3)10(11)6-9/h4-7,11H,1-3H3
- Substance type: Organic
- Physical state: Liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

strain RjHan:WI

Details on species / strain selection:

The number of animals included in each group was selected according to the OECD 474 guideline a minimum of 5 analyzable animals of each sex for group (OECD 474) was selected.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: University of Sevilla Center for Animal Production and Experimentation (Espartinas, Spain).
- Age at study initiation: between 8 and 10 week-old when purchased and the animals were acclimatized to the environmental conditions for one week before the experiments (i.e. 9-11 week-old)
- Weight at study initiation: Animals were weighed after arrival
- Assigned to test groups randomly: yes
- Fasting period before study: No data
- Housing: housed in polycarbonate cages with stainless steel covers
- Diet (e.g. ad libitum): standard diet (Harlan, 2014; Harlan Laboratories, Barcelona, Spain), ad libitum.
- Water (e.g. ad libitum): water ad libitum.
- Acclimation period: the animals were acclimatized to the environmental conditions for one week before the experiments,

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1°C
- Humidity (%): 55 ± 10%,
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h dark/light cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Test chemical was soluble in corn oil
- Concentration of test material in vehicle: 1 mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: All doses were prepared in corn oil at a final volume of 1 mL

Duration of treatment / exposure:

0, 24 and 45 h

Frequency of treatment:

Single

Post exposure period:

3 h

Doses / concentrations

Remarks:

0 (vehicle), 81, 256 or 810 mg/kg bw

No. of animals per sex per dose:

23 male and 23 female rats

Control animals:

yes, concurrent vehicle

Positive control(s):

- ethylmethanesulphonate
- Route of administration: gavage
- Doses / concentrations: 200 mg/kg bw

Examinations

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:the bone marrow cells were smeared on a glass slide, fixed in absolute methanol air dried and stained with Giemsa.

METHOD OF ANALYSIS: The polychromatic erythrocytes (PCE) among total erythrocytes (normochromatic erythrocytes (NCE) þ PCE) ratio and the PCE among NCE ratio were calculated by counting 500 erythrocytes per animal. The incidence of micronucleated immature erythrocytes (MNPCE) expressed as % MN was analyzed by counting a total of 5000 cells per animal. Five male and 5 female animals were analyzed for the negative control and the dosed groups. Moreover, 3 males and 3 females were analyzed in the case of the positive group.

Statistics:

For the MN assay, data are presented as the mean ± SD for each group of animals and statistical analysis was performed using the analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: Test chemical did not increase the % MN in immature erythrocytes at any dose tested on both sexes.

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: The MN assay OECD protocol (OECD 474) recommend a preliminary range-finding study to identify the maximum tolerated dose (MTD), which was defined by Derelanko (2000) as the highest dose to produce toxic effects without causing death and to decrease body weight (bw) by no more than 10% relative to controls. For this purpose, the Acute oral toxicity OECD 425 guideline for testing of chemicals (OECD, 2008) was followed, taking into account the historical report of the LD50 in rats described by Jenner et al. (1964). Moreover, considering the OECD 474 (2014a) guideline, the doses used in the combined MN assay were the MTD and two lower doses (Bowen et al., 2011) appropriately spaced by less than √10.

Applicant's summary and conclusion

Conclusions:

In vivo MN assay protocol demonstrates that the given test chemical did not induce genotoxic effects in bone marrow cells of orally exposed (81-810 mg/kg bw) Wistar rats.

Executive summary:

The mammalian erythrocyte micronucleus test was conducted for the given test chemical on 23 male and 23 female rats. Animals were randomly divided into five groups, two control and three treatment groups: the negative control group (C-) (5 males and 5 females rats) was treated with corn oil (vehicle); the positive control group (C+) (3 males and 3 females rats) was exposed to 200 mg/kg bw ethylmethanesulfonate (EMS) and three exposed groups (5 males and 5 females rats per group) received 81, 256 or 810mg/kg bw. The number of animals included in each group was selected according to the OECD 474 and OECD 489 guidelines: a minimum of 5 analyzable animals of each sex for group (OECD 474) and a minimum of 3 animals of each sex treated with a positive control (OECD 489). All doses were prepared in corn oil at a final volume of 1 mL. Animals were dosed at 0, 24 and 45 h by gavage using anenteral feeding tube. Then, animals were sacrificed 3 h after the final dose administration. During the treatment period, clinical signs, body weight, and food and water consumption were recorded daily. Briefly, the bone marrow cells were smeared on a glass slide, fixed in absolute methanol air dried and stained with Giemsa. The polychromatic erythrocytes (PCE) among total erythrocytes (normochromatic erythrocytes (NCE) + PCE) ratio and the PCE among NCE ratio were calculated by counting 500 erythrocytes per animal. The incidence of micronucleated immature erythrocytes (MNPCE) expressed as % MN was analyzed by counting a total of 5000 cells per animal. Five male and 5 female animals were analyzed for the negative control and the dosed groups. Moreover, 3 males and 3 females were analyzed in the case of the positive group. 3 h after the final administration (45 h) of test chemical, significant differences in the PCE/total erythrocytes ratio were observed from 256 mg/kg bw with respect to the negative control group in males (p < 0.01) and females (p < 0.05). Regarding PCE/NCE ratio in male rats, a significant (p < 0.01) decrease was detected from 256 mg/kg bw. Female rats exposed to test chemical also showed a significant (p < 0.05) decrease of PCE/NCE, but only at the highest dose assayed (810 mg/kg bw). Moreover, it did not increase the % MN in immature erythrocytes at any dose tested on both sexes. Treatment with EMS as positive control induced significant decreases (p < 0.01) in the PCE/total erythrocytes and PCE/NCE ratios, and significant increases (p < 0.01) in the % MN with respect to the negative control. Hence, results from MN assay revealed that the given test chemical did not induce in vivo genotoxicity in bone marrow cells investigated.