(E)-2-methoxy-4-(prop-1-enyl)phenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 18 to July 19, 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Food and water consumption not monitored; clinical biochemistry, ophthalmological and neurobehavioral examination not performed.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2010

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(food and water consumption not monitored; clinical biochemistry, ophthalmological and neurobehavioral examination not performed)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(E)-2-methoxy-4-(prop-1-enyl)phenol

EC Number:

227-678-2

EC Name:

(E)-2-methoxy-4-(prop-1-enyl)phenol

Cas Number:

5932-68-3

Molecular formula:

C10H12O2

IUPAC Name:

2-methoxy-4-prop-1-en-1-ylphenol

Test material form:

liquid

Details on test material:

- Physical state: Yellow liquid
- Stability under test conditions: Stable
- Storage condition of test material: Stored at or below - 20° C, protected from light, in 1-L Teflon® bottles.

Test animals

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Not reported

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc., Germantown, NY
- Age at study initiation: 6-7 weeks
- Housing: Male animals were housed individually and females were housed 5/cage in Polycarbonate cages.
- Diet: NTP-2000 irradiated wafer or pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Females: 13 days; males: 14 days

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

IN-LIFE DATES: From: April 18, 2001 To: July 19, 2001

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was chosen because it is the major route of human exposure, and gavage was chosen after preliminary studies showed that isoeugenol in feed was unpalatable to both rats and mice and the concentration in feed decreased when stored at room temperature.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The dose formulations were prepared by mixing test material with corn oil to give the required concentrations. The appropriate amounts of test material and corn oil were placed in a glass mixing container, capped, and thoroughly mixed with a paint shaker for approximately 5 minutes. Dose formulations were prepared approximately monthly and stored at room temperature in amber glass bottles with Teflon® -lined lids for up to 35 days.

VEHICLE
- Concentration in vehicle: 3.75, 7.5, 15, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Test material formulations were analysed three times during the study period by GC.
- Homogeneity and stability of test material formulations were determined. Homogeneity studies of 0.2 and 120 mg/mL formulations and stability studies of the 0.2 mg/mL formulation were performed using GC on a different lot of test material (Penta International Corporation, Lot No. 46928).
- Homogeneity was confirmed, and the 120 mg/mL dose formulation was found to be suitable for gavage. Stability was confirmed for up to 35 days for dose formulations stored in amber glass bottles with Teflon® -lined lids at - 20 °C, 5 °C, and room temperature, as well as for 3 h under simulated animal room conditions.
- All the formulations analysed were within 10% of the target concentrations.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

37.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings for core study animals were recorded initially, then weekly and at the end of the exposure phase.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, then weekly and at the end of the exposure phase (Day 85).

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Day 93 of the study from the retroorbital sinus.
- Anaesthetic used for blood collection: Yes; animals were anesthetized with carbon dioxide.
- Animals fasted: No
- How many animals: All survival animals
- Parameters checked: Haematocrit; haemoglobin; erythrocyte, reticulocyte, nucleated erythrocyte, and platelet counts; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte counts and differentials.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals were sacrificed by carbon dioxide asphyxiation and necropsies were performed.

ORGAN WEIGHTS:
Heart, right kidney, liver, lungs, right testis, and thymus were weighed.

HISTOPATHOLOGY: Yes
- Histopathology was performed on animals from vehicle control and 600 mg/kg bw/day dosed group.
- In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain,
clitoral gland, esophagus, eyes, gall bladder, Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin,
spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. Tissues were examined in the remaining dosed groups to a no-effect level.
- Tissues for microscopic examination were fixed and preserved in 10 % neutral buffered formalin (eyes were fixed in Davidson’s solution for up to 72 h and then transferred to 10 % neutral buffered formalin), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.

Other examinations:

None

Statistics:

- The probability of survival was estimated by the product- limit procedure of Kaplan and Meier (1958).
- Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
- Organ and body weight data, which historically have approximately normal distributions, were analysed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Haematology data, which have typically skewed distributions, were analysed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
- Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
- Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.
- Probability values (p) are presented as follows:
p ≤ 0.01 **
p ≤ 0.05 *
p > 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean body weight and body weight gain were significantly decreased in males treated with 600 mg/kg bw/day compared to the vehicle control group (- 12%).
- After 85 days of exposure, mean body weights of females treated with 75 and 150 mg/kg bw/day exceeded that of the vehicle control group by 10 and 8 %, respectively, while that of 600 mg/kg bw/day treated females was 7 % less than vehicle controls; however, the differences were not statistically significant.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No haematological effects were observed.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- The only exposure-related organ weight change was an increase in liver weights. As compared to vehicle control group, significant increases were observed in relative liver weights of all male groups (+10.6% at 37.5 mg/kg bw/day ; +14.0% at 75 mg/kg bw/day ; + 17.0% at 150 mg/kg bw/day ; +26.4% at 300 mg/kg bw/day ; +33.1% at 600 mg/kg bw/day) and absolute liver weights of males treated with 300 (+21.6%) and 600 mg/kg bw/day (+14.3%). The EMEA considered that, in this study, some changes in liver to bodyweight ratios were observed, but these changes were not considered treatment related because the magnitude of the changes was small and there was no clear dose response relationship.
- As compared to vehicle control group, significant decreases were observed in absolute kidney weights of males treated with 150 (-7.8%), 300 (-7.4%) and 600 mg/kg bw/day (-9.5%) and absolute lung weights of females treated with 600 mg/kg bw/day were significantly decreased (-18.2%) as compared to vehicle control group and considered related to exposure. The relative weights of these organs were not significantly different from controls and were therefore considered non-adverse.
- Moreover, the EMEA considered the body weight change as the only treatment related adverse systemic effects in this study.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- No microscopic findings in liver, kidney, and lung related to treatment.
- Incidences of mild to moderate olfactory epithelial atrophy and minimal to mild atrophy of olfactory nerve bundles increased significantly in males and females treated with 600 mg/kg bw/day. Olfactory epithelial atrophy was characterized by a decrease in the number of cells, resulting in thinning of the olfactory epithelium. The atrophy occurred in the most distal portion of the nasal cavity along the junction of the nasal septum with the dorsal wall of the nasal meatus in Level III. The atrophic epithelium was simple or pseudostratified, ciliated, and columnar and resembled normal respiratory epithelium. Glands within the lamina propria under the affected olfactory epithelium were often slightly dilated. Some of these glands contained secretory material with occasional inflammatory cells and cell debris. Some glands were lined by minimally hyperplastic epithelial cells. Atrophy of olfactory nerves consisted of reductions in the number and size of nerve bundles within the lamina propria beneath areas of atrophic epithelium. The nerve atrophy was considered to be secondary to the loss of sensory neurons in the overlying atrophic olfactory epithelium.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No significant treatment-related systemic adverse effects in females.

3

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (actual dose received)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the systemic NOAEL was considered to be 300 mg/kg bw/day in male mice based on the reduction in bodyweight gain at 600 mg/kg bw/day. The systemic NOAEL was considered to be 600 mg/kg bw/day in the female mice based on the absence of systemic effects. The local NOAEL was considered to be 300 mg/kg bw/day based on the histological changes in olfactory epithelium in males and females at 600 mg/kg bw/day.

Executive summary:

In a repeated dose toxicity study used as range-finding for carcinogenicity study and performed similar to OECD test Guideline No. 408 and in compliance with GLP, groups of B6C3F1 mice (10/sex/dose) were exposed to test material in corn oil by gavage at 37.5, 75, 150, 300 and 600 mg/kg bw/day, 5 days per week for 14 weeks. Control mice were given the vehicle alone. Clinical signs and bodyweight development were monitored during the study. Haematology was performed. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed in control and high dose group.

No mortality nor clinical signs were observed. Mean body weight and body weight gain were significantly decreased in males treated with 600 mg/kg bw/day compared to the vehicle control group (by 12% and 31%, respectively). After 85 days of exposure, mean body weights of 75 and 150 mg/kg bw/day females exceeded that of the vehicle control group by 10% and 8%, respectively, while that of 600 mg/kg bw/day females was 7% less than vehicle controls; however, the differences were not statistically significant. There were no hematological effects in mice exposed to isoeugenol.

Absolute liver weights were increased at 300 and 600 mg/kg bw/day in males (22% and 14%, respectively). Increases in relative liver weights of all male groups correlated with dose and were statistically significant (11%, 14%, 17%, 26% and 33% at 37.5, 75, 150, 300 and 600 mg/kg bw/day, respectively). Absolute kidney weights of males treated with 150, 300 and 600 mg/kg bw/day and absolute lung weights of females treated with 600 mg/kg bw/day were significantly decreased as compared to vehicle control group. However, the differences in liver, kidney, and lung weights were not associated with any microscopic findings were therefore considered non-adverse.

Incidences of mild to moderate olfactory epithelial atrophy and minimal to mild atrophy of olfactory nerve bundles increased significantly in males and females treated with 600 mg/kg bw/day.

 

The EMEA considered that, in this study, a treatment related effect on bodyweight was noted in males in the high dose group. Some changes in liver to bodyweight ratios were observed, but these changes were not considered treatment related because the magnitude of the changes was small and there was no clear dose response relationship. Therefore, the systemic NOAEL for this study was 300 mg/kg bw/day based on body weight effects seen at 600 mg/kg bw/day.

 

The EMEA also suggested that the lesions in the olfactory epithelium were a consequence of local irritation due to contact with the test material. The observed atrophy of olfactory nerves is considered to have been secondary to the effects on the overlying epithelium. Based on these data, the local NOAEL was considered to be 300 mg/kg bw/day in male and female mice based on the histological changes in olfactory epithelium.