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

Endpoint summary

Description of key information

Repeated dose toxicity oral: systemic NOAEL = 188 mg/kg bw/day in female mice (similar to OECD 408, read-across to trans-isoeugenol K, rel. 2) 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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 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

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

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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

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)

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

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

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.

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

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

See the attached document for information on tables of results

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

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
The source substance is the hydrolysis product of the target substance, formed via hydrolysis by tissue esterases. The source substance is expected to be a worst-case compared to the target substance considering the respective physico-chemical and toxicological properties.
The study design (Similar to OECD 408, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the sub-chronic toxicity studies conducted in both the rat and the mouse with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.6.2.

4. DATA MATRIX
Cf. Iuclid Section 13. 

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Principles of method if other than guideline:

Not applicable

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

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Remarks:

corrected for MW

Sex:

male

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Remarks:

corrected for MW

Sex:

female

Basis for effect level:

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

Key result

Critical effects observed:

no

See the attached document for information on tables of results

Conclusions:

Under the test conditions, the systemic NOAEL for the source substance 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.
A correction factor based on the ratio between the molecular mass of the target substance (206.24 g/mol) and the source substance ((E)-2-Methoxy-4-prop-1-en-1-ylphenol - 164.21 g/mol) was applied to extrapolate the corrected NOAEL for the target substance. A systemic NOAEL was considered to be 377 mg/kg bw/day for male mice, and 754 mg/kg bw/day for females. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

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 the source substance 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.

A correction factor based on the ratio between the molecular mass of the target substance (206.24 g/mol) and the source substance ((E)-2-Methoxy-4-prop-1-en-1-ylphenol - 164.21 g/mol) was applied to extrapolate the corrected NOAEL for the target substance. A systemic NOAEL was considered to be 377 mg/kg bw/day for male mice, and 754 mg/kg bw/day for females. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 16 to July 17, 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

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

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

Deviations:

yes

Remarks:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Not reported

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS (F344/N)
- Source: Taconic Farms, Inc., Germantown, NY
- Age at study initiation: 6-7 weeks
- Housing: Animals 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: Males: 11 days; females: 12 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 16, 2001 To: July 17, 2001

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: 7.5, 15, 30, 60 and 120 mg/mL
- Amount of vehicle (if gavage): 5 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 per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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

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.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: 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:
Haematology: 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: Urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsies were performed on all animals by Carbon dioxide asphyxiation.

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, 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 and clinical chemistry 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)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical findings related to test material were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Dosing accidents resulted in the early death of one male treated with 600 mg/kg bw/day and one female treated with 37.5 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Decreases in mean body weights and body weight gains of all dosed groups of males were statistically significant compared to those of the vehicle controls; however, only the decrease in the 600 mg/kg bw/day treated group was clearly related to test material exposure.
- Mean body weights of dosed females were similar to that of the vehicle control group.

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):

- Minor changes in haematology variables were observed. All changes were within physiological normal levels, and there was no evidence of a dose relationship; they were not considered biologically important or toxicologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Minor changes in clinical chemistry variables were observed. All changes were within physiological normal levels, and there was no evidence of a dose relationship; they were not considered biologically important or toxicologically relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Absolute and relative liver weights were significantly increased in 300 and 600 mg/kg bw/day treated female rats. The EMEA considered that, in this study, the changes in liver weight were attributed to centrilobular hyperplasia and, therefore, are not considered as adaptative effects.
- Absolute and relative kidney weights were significantly increased in 600 mg/kg bw/day treated female rats.
- No other changes in organ weights of male or female animals related to test material exposure were observed.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Incidences of minimal atrophy of the olfactory epithelium were increased in all exposed groups and were significantly increased in males administered 150 mg/kg bw/day or greater and females administered 300 or 600 mg/kg bw/day.
- Minimal to mild atrophy of olfactory nerve bundles was observed in all exposed groups of males and in females exposed to 150 mg/kg bw/day or greater; the incidence was significantly increased in 600 mg/kg bw/day females.
- Olfactory epithelial atrophy was characterized by loss of cilia, altered orientation of affected cells, and decreased numbers of epithelial cells that resulted in thinning of the olfactory epithelium. The regions affected included the ventral nasal septum at Level III and less frequently along the junction of the nasal septum with the dorsal wall of the nasal meatus in the middle nasal section, Level II. Atrophy of the olfactory epithelium was accompanied by chronic active inflammation in a few males. Glands under the affected olfactory epithelium were unremarkable. Atrophy of olfactory nerve bundles was observed in areas beneath the atrophic epithelium. Nerve atrophy with concomitant reductions in the number and size of nerve bundles was considered secondary to the atrophy of the overlying olfactory epithelium.
- Incidences of minimal to mild periportal hepatocellular cytoplasmic alteration were significantly increased in females exposed to 300 or 600 mg/kg bw/day. The liver alteration consisted of decreased eosinophilic cytoplasmic staining with increased microvacuolation and accentuated basophilic granulation of periportal hepatocytes, similar to what is commonly associated with glycogen depletion.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

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

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 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

See the attached document for information on tables of results

Special study results:

-Stomach pH was significantly decreased in female rats treated with 150 mg/kg bw/day or greater.

-In microscopic examination, no treatment related changes were observed in stomach.

- Dose-related decreases to 70 % of EROD (CYP1A1) activity and to 72 % of PROD (CYP2B) activity were observed in males treated with 75 mg/kg bw/day or greater.

- Although a small decrease in acetanilide-4-hydroxylase (A4H) (CYP1A2) activity in the males treated with 600 mg/kg bw/day was statistically significant, the response across groups did not correlate with dose.

Conclusions:

Under the test conditions, systemic NOAEL was considered to be 150 mg/kg bw/day in female rats based on the effects in the liver weight increase due to centrilobular hyperplasia at 300 and 600 mg/kg bw/day. The systemic NOAEL was considered to be 300 mg/kg bw/day in males based on Mean body weight change decrease at 600 mg/kg bw/day. The local NOAEL was considered to be 75 and 150 mg/kg bw/day in male and female rats, respectively based on the histological changes in olfactory epithelium.

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 F344/N rats (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 rats were given the vehicle alone. Clinical signs and bodyweight development were monitored during the study. Haematology and clinical chemistry were performed. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed in control and high dose group.

Dosing accidents resulted in the early death of one male treated with 600 mg/kg bw/day and one female treated with 37.5 mg/kg bw/day. No clinical findings related to isoeugenol exposure were observed. Decreases in mean body weights and body weight gains of all dosed groups of males were statistically significant compared to those of the vehicle controls; however, only the decrease in the 600 mg/kg bw/day treated group was clearly related to test material exposure (body weight reduced by 13%, and body weight gain by 16% whereas only reduced by < 10% in other groups). Mean body weights of dosed females were similar to that of the vehicle control group.

Minor changes occurred throughout the hematology and clinical chemistry variables in the rats. All changes were within physiological normal levels, and in general, there was no evidence of a dose relationship; they were not considered biologically important or toxicologically relevant.

Absolute and relative liver weights were significantly increased in 300 (Abs. 9.5%/ Rel. 6.1%) and 600 mg/kg bw/day treated female rats (Abs. 19.3% / Rel. 13.9%) as were kidney weights in 600 mg/kg bw/day treated female rats (Abs. 11% / Rel. 10.6%). Incidences of minimal to mild periportal hepatocellular cytoplasmic alteration were significantly increased in females exposed to 300 and 600 mg/kg bw/day. The liver alteration consisted of decreased eosinophilic cytoplasmic staining with increased microvacuolation and accentuated basophilic granulation of periportal hepatocytes, similar to what is commonly associated with glycogen depletion.

Incidences of minimal atrophy of the olfactory epithelium were increased in all exposed groups and were significantly increased in males treated with 150 mg/kg bw/day or greater and females treated with 300 and 600 mg/kg bw/day. Minimal to mild atrophy of olfactory nerve bundles was observed in all exposed groups of males and in females exposed to 150 mg/kg bw/day or greater; the incidence was significantly increased in 600 mg/kg bw/day females.

 

The EMEA considered that, in this study, the changes in liver weight observed in females rats were attributed to centrilobular hyperplasia. Therefore, the systemic NOAEL for this study was 150 mg/kg bw/day based on increased hyperplasia in liver in female rats. In male rat, a systemic NOAEL of 300 mg/kg bw/day can be determined based on body weight effects at 600 mg/kg.

 

The EMEA also suggested that the effects on the nose were a result of direct contact rather than systemic exposure. Based on these data, the local NOAEL was considered to be 75 and 150 mg/kg bw/day in male and female rats, respectively, based on the histological changes in olfactory epithelium.

Reference 4

Endpoint:

sub-chronic toxicity: oral

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
The source substance is the hydrolysis product of the target substance, formed via hydrolysis by tissue esterases. The source substance is expected to be a worst-case compared to the target substance considering the respective physico-chemical and toxicological properties.
The study design (Similar to OECD 408, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the sub-chronic toxicity studies conducted in both the rat and the mouse with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.6.2.

4. DATA MATRIX
Cf. Iuclid Section 13. 

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Limit test:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical findings related to test material were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Dosing accidents resulted in the early death of one male treated with 600 mg/kg bw/day and one female treated with 37.5 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Decreases in mean body weights and body weight gains of all dosed groups of males were statistically significant compared to those of the vehicle controls; however, only the decrease in the 600 mg/kg bw/day treated group was clearly related to test material exposure.
- Mean body weights of dosed females were similar to that of the vehicle control group.

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):

- Minor changes in haematology variables were observed. All changes were within physiological normal levels, and there was no evidence of a dose relationship; they were not considered biologically important or toxicologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Minor changes in clinical chemistry variables were observed. All changes were within physiological normal levels, and there was no evidence of a dose relationship; they were not considered biologically important or toxicologically relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Absolute and relative liver weights were significantly increased in 300 and 600 mg/kg bw/day treated female rats. The EMEA considered that, in this study, the changes in liver weight were attributed to centrilobular hyperplasia and, therefore, are not considered as adaptative effects.
- Absolute and relative kidney weights were significantly increased in 600 mg/kg bw/day treated female rats.
- No other changes in organ weights of male or female animals related to test material exposure were observed.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Incidences of minimal atrophy of the olfactory epithelium were increased in all exposed groups and were significantly increased in males administered 150 mg/kg bw/day or greater and females administered 300 or 600 mg/kg bw/day.
- Minimal to mild atrophy of olfactory nerve bundles was observed in all exposed groups of males and in females exposed to 150 mg/kg bw/day or greater; the incidence was significantly increased in 600 mg/kg bw/day females.
- Olfactory epithelial atrophy was characterized by loss of cilia, altered orientation of affected cells, and decreased numbers of epithelial cells that resulted in thinning of the olfactory epithelium. The regions affected included the ventral nasal septum at Level III and less frequently along the junction of the nasal septum with the dorsal wall of the nasal meatus in the middle nasal section, Level II. Atrophy of the olfactory epithelium was accompanied by chronic active inflammation in a few males. Glands under the affected olfactory epithelium were unremarkable. Atrophy of olfactory nerve bundles was observed in areas beneath the atrophic epithelium. Nerve atrophy with concomitant reductions in the number and size of nerve bundles was considered secondary to the atrophy of the overlying olfactory epithelium.
- Incidences of minimal to mild periportal hepatocellular cytoplasmic alteration were significantly increased in females exposed to 300 or 600 mg/kg bw/day. The liver alteration consisted of decreased eosinophilic cytoplasmic staining with increased microvacuolation and accentuated basophilic granulation of periportal hepatocytes, similar to what is commonly associated with glycogen depletion.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Remarks:

corrected for MW

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Remarks:

corrected for MW

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

See the attached document for information on tables of results

Special study results:

-Stomach pH was significantly decreased in female rats treated with 150 mg/kg bw/day or greater.

-In microscopic examination, no treatment related changes were observed in stomach.

- Dose-related decreases to 70 % of EROD (CYP1A1) activity and to 72 % of PROD (CYP2B) activity were observed in males treated with 75 mg/kg bw/day or greater.

- Although a small decrease in acetanilide-4-hydroxylase (A4H) (CYP1A2) activity in the males treated with 600 mg/kg bw/day was statistically significant, the response across groups did not correlate with dose.

Conclusions:

Under the test conditions, systemic NOAEL for the source substance was considered to be 150 mg/kg bw/day in female rats based on the effects in the liver weight increase due to centrilobular hyperplasia at 300 and 600 mg/kg bw/day. The systemic NOAEL was considered to be 300 mg/kg bw/day in males based on Mean body weight change decrease at 600 mg/kg bw/day. The local NOAEL was considered to be 75 and 150 mg/kg bw/day in male and female rats, respectively based on the histological changes in olfactory epithelium.
A correction factor based on the ratio between the molecular mass of the target substance (206.24 g/mol) and the source substance ((E)-2-Methoxy-4-prop-1-en-1-ylphenol - 164.21 g/mol) was applied to extrapolate the corrected NOAEL for the target substance. A systemic NOAEL was considered to be 188 mg/kg bw/day for female rats, and 377 mg/kg bw/day for males. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

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 F344/N rats (10/sex/dose) were exposed to the source substance 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 rats were given the vehicle alone. Clinical signs and bodyweight development were monitored during the study. Haematology and clinical chemistry were performed. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed in control and high dose group.

Dosing accidents resulted in the early death of one male treated with 600 mg/kg bw/day and one female treated with 37.5 mg/kg bw/day. No clinical findings related to isoeugenol exposure were observed. Decreases in mean body weights and body weight gains of all dosed groups of males were statistically significant compared to those of the vehicle controls; however, only the decrease in the 600 mg/kg bw/day treated group was clearly related to test material exposure (body weight reduced by 13%, and body weight gain by 16% whereas only reduced by < 10% in other groups). Mean body weights of dosed females were similar to that of the vehicle control group.

Minor changes occurred throughout the hematology and clinical chemistry variables in the rats. All changes were within physiological normal levels, and in general, there was no evidence of a dose relationship; they were not considered biologically important or toxicologically relevant.

Absolute and relative liver weights were significantly increased in 300 (Abs. 9.5%/ Rel. 6.1%) and 600 mg/kg bw/day treated female rats (Abs. 19.3% / Rel. 13.9%) as were kidney weights in 600 mg/kg bw/day treated female rats (Abs. 11% / Rel. 10.6%). Incidences of minimal to mild periportal hepatocellular cytoplasmic alteration were significantly increased in females exposed to 300 and 600 mg/kg bw/day. The liver alteration consisted of decreased eosinophilic cytoplasmic staining with increased microvacuolation and accentuated basophilic granulation of periportal hepatocytes, similar to what is commonly associated with glycogen depletion.

Incidences of minimal atrophy of the olfactory epithelium were increased in all exposed groups and were significantly increased in males treated with 150 mg/kg bw/day or greater and females treated with 300 and 600 mg/kg bw/day. Minimal to mild atrophy of olfactory nerve bundles was observed in all exposed groups of males and in females exposed to 150 mg/kg bw/day or greater; the incidence was significantly increased in 600 mg/kg bw/day females.

 

The EMEA considered that, in this study, the changes in liver weight observed in females rats were attributed to centrilobular hyperplasia. Therefore, the systemic NOAEL for this study was 150 mg/kg bw/day based on increased hyperplasia in liver in female rats. In male rat, a systemic NOAEL of 300 mg/kg bw/day can be determined based on body weight effects at 600 mg/kg.

 

The EMEA also suggested that the effects on the nose were a result of direct contact rather than systemic exposure. Based on these data, the local NOAEL was considered to be 75 and 150 mg/kg bw/day in male and female rats, respectively, based on the histological changes in olfactory epithelium.

A correction factor based on the ratio between the molecular mass of the target substance (206.24 g/mol) and the source substance ((E)-2-Methoxy-4-prop-1-en-1-ylphenol - 164.21 g/mol) was applied to extrapolate the corrected NOAEL for the target substance. A systemic NOAEL was considered to be 188 mg/kg bw/day for female rats, and 377 mg/kg bw/day for males. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

188 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

No study was available on Trans-Isoeugenyl acetate. The mouse study on trans-Isoeugenol was selected as the key study (lowest NOAEL compared to rat study). The key study is GLP-compliant and of high quality (Klimisch score = 2).

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral route:

No study was available on trans-Isoeugenyl acetate. Therefore, the repeated dose toxicity was investigated across several key studies performed on the source substance, trans-Isoeugenol (see Iuclid section 13 for read-across justification) to assess carcinogenicity and for toxicity to reproduction, in rats and/or mice exposed by oral (gavage) route.

A correction factor based on the ratio between the molecular mass of trans-Isoeugenyl acetate (206.24 g/mol) and the supporting substance (trans-Isoeugenol - 164.21 g/mol) was applied to extrapolate the corrected NOAEL for the registered substance.

 

Sub-chronic toxicity in mice (NTP, 2010):

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 trans-Isoeugenol 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 female mice based on the histological changes in olfactory epithelium.

After MW correction, the systemic NOAEL for Trans-Isoeugenyl acetate was considered to be 377 mg/kg bw/day for male mice, and 754 mg/kg bw/day for females. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

 

Sub-chronic toxicity in rats (NTP, 2010):

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 F344/N rats (10/sex/dose) were exposed to trans-Isoeugenol 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 rats were given the vehicle alone. Clinical signs and bodyweight development were monitored during the study. Haematology and clinical chemistry were performed. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed in control and high dose group.

Dosing accidents resulted in the early death of one male treated with 600 mg/kg bw/day and one female treated with 37.5 mg/kg bw/day. No clinical findings related to isoeugenol exposure were observed. Decreases in mean body weights and body weight gains of all dosed groups of males were statistically significant compared to those of the vehicle controls; however, only the decrease in the 600 mg/kg bw/day treated group was clearly related to test material exposure (body weight reduced by 13%, and body weight gain by 16% whereas only reduced by < 10% in other groups). Mean body weights of dosed females were similar to that of the vehicle control group.

Minor changes occurred throughout the hematology and clinical chemistry variables in the rats. All changes were within physiological normal levels, and in general, there was no evidence of a dose relationship; they were not considered biologically important or toxicologically relevant.

Absolute and relative liver weights were significantly increased in 300 (Abs. 9.5%/ Rel. 6.1%) and 600 mg/kg bw/day treated female rats (Abs. 19.3% / Rel. 13.9%) as were kidney weights in 600 mg/kg bw/day treated female rats (Abs. 11% / Rel. 10.6%). Incidences of minimal to mild periportal hepatocellular cytoplasmic alteration were significantly increased in females exposed to 300 and 600 mg/kg bw/day. The liver alteration consisted of decreased eosinophilic cytoplasmic staining with increased microvacuolation and accentuated basophilic granulation of periportal hepatocytes, similar to what is commonly associated with glycogen depletion.

Incidences of minimal atrophy of the olfactory epithelium were increased in all exposed groups and were significantly increased in males treated with 150 mg/kg bw/day or greater and females treated with 300 and 600 mg/kg bw/day. Minimal to mild atrophy of olfactory nerve bundles was observed in all exposed groups of males and in females exposed to 150 mg/kg bw/day or greater; the incidence was significantly increased in 600 mg/kg bw/day females.

 

The EMEA considered that, in this study, the changes in liver weight observed in females rats were attributed to centrilobular hyperplasia. Therefore, the systemic NOAEL for this study was 150 mg/kg bw/day based on increased hyperplasia in liver in female rats. In male rat, a systemic NOAEL of 300 mg/kg bw/day can be determined based on body weight effects at 600 mg/kg.

 

The EMEA also suggested that the effects on the nose were a result of direct contact rather than systemic exposure. Based on these data, the local NOAEL was considered to be 75 and 150 mg/kg bw/day in male and female rats, respectively, based on the histological changes in olfactory epithelium.

 

After MW correction, the systemic NOAEL for Trans-Isoeugenyl acetate was considered to be 188 mg/kg bw/day for female rats, and 377 mg/kg bw/day for males. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

 

Carcinogenicity study in rats (NTP, 2010):

In a 2-year carcinogenicity study, performed similarly to OECD guideline 451 and in compliance with GLP, trans-Isoeugenol was administered through gavage to groups of F344/N rats (50/sex/dose) at dose levels of 0, 75, 150 and 300 mg/kg bw/day, 5 days per week for 105 weeks. Parameters evaluated included survival, clinical observations, body weight, necropsy and histopathological examination.

Survival rates of dosed male and female rats were similar to those of vehicle controls. No clinical findings related to the administration of test material were observed. The mean body weight of 300 mg/kg bw/day males was greater than that of the vehicle controls after week 64 of exposure, and their final mean body weight was 9% greater than that of the vehicle controls. The mean body weights of all other exposed groups were similar to those of the vehicle control groups throughout the study.

 

Non-neoplastic effects:

Nose:

- Males: olfactory epithelium, atrophy (1/50, 5/48, 9/49, 13/49); olfactory epithelium, metaplasia, respiratory (4/50, 6/48, 10/49, 15/49); olfactory epithelium, degeneration (1/50, 0/48, 2/49, 6/49) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

- Females: olfactory epithelium, atrophy (0/50, 0/49, 0/49, 4/49); olfactory epithelium, metaplasia, respiratory (5/50, 5/49, 9/49, 12/49) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

Low incidences of minimal atrophy and minimal to mild respiratory metaplasia of the olfactory epithelium were increased in 150 mg/kg bw males and 300 mg/kg bw males and females. Similar incidences of minimal to mild olfactory epithelial degeneration in 300 mg/kg bw males were also increased.

 

Equivocal findings:

Thymus:

- Males: thymoma, benign or malignant (0/47, 0/43, 0/49, 2/48 [4%]).

The incidence of thymoma in exposed male rats was not statistically significant compared to vehicle controls, but the trend across all groups was significant. The incidence of benign or malignant thymoma (combined) in the 300 mg/kg bw/day males exceeded the historical range for vehicle controls in corn oil gavage studies and for controls by all routes [0-2%]. The two thymomas were proliferative lesions consisting of neoplastic epithelial and lymphoid components. One was benign and the other was malignant.

 

Mammary gland:

- Males: carcinoma (0/50, 0/50, 0/50, 2/50 [4%]) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

Rare, malignant carcinomas occurred in two 300 mg/kg bw/day male rats. The trend was statistically significant, no carcinomas occurred in corn oil vehicle controls in the current historical database, and the rate was equal to the highest rate for controls by all routes [0%-4%]. These mammary gland carcinomas were not accompanied by biological evidence, specifically by increased incidences of adenoma or hyperplasia.

 

Conclusions:

Under the test conditions, NTP concluded that there was equivocal evidence of carcinogenic activity in male rats based on increased incidences of rarely occurring thymoma and mammary gland carcinoma. There was no evidence of carcinogenic activity in female rats.

The EMEA considered that the conduct of the study had significant weaknesses related to the route of administration (oral gavage) and the dosing regime (week days only). A slight increase in the incidences of thymoma in thymus and carcinoma in mammary gland were seen in male rats at the highest tested dose, 300 mg/kg/day. These findings were, however, within the historical control range. In conclusion, Trans-Isoeugenol is not considered to be carcinogenic in rats.

Therefore, a systemic NOAEL of 300 mg/kg bw can be determined. A local NOAEL of 75 mg/kg bw/day can be determined based on effects on the nasal epithelium

After MW correction, a systemic NOAEL of 377 mg/kg bw can be determined for Trans-Isoeugenyl acetate. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

 

Carcinogenicity study in mice (NTP, 2010):

In a 2-year carcinogenicity study, performed similarly to OECD guideline 451 and in compliance with GLP, trans-Isoeugenol was administered through gavage to groups of B6C3F1 mice (50/sex/dose) at dose levels of 0, 75, 150 and 300 mg/kg bw/day, 5 days per week for 104 (female mice) or 105 (male mice) weeks. Parameters evaluated included survival, clinical observations, body weight, necropsy and histopathological examination.

The survival rate of 300 mg/kg bw/day males was significantly decreased compared to vehicle controls. The decreasing trend in survival across all groups were statistically significant. Liver neoplasms were the likely cause of death for many of the early-death animals. Exposure to isoeugenol had no effect on survival of female mice.

Mean body weights of 300 mg/kg bw/day male and female mice were less than 95 % of the vehicle controls after 60 and 28 weeks, respectively, of exposure. At this dose, mean body weights were reduced by 10% in males, and by 14% in females at the end of the study. Values for lower exposed groups were similar to those of vehicle controls.

Non-neoplastic effects:

Nose:

- Males: olfactory epithelium, atrophy (5/50, 13/50, 36/50, 41/50); olfactory epithelium, metaplasia, respiratory (4/50, 31/50, 47/50, 49/50); olfactory epithelium, degeneration (1/50, 1/50, 7/50, 6/50); olfactory epithelium, accumulation, hyaline droplet (0/50, 6/50, 26/50, 19/50); glands, hyperplasia (3/50, 34/50, 49/50, 48/50) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

- Females: olfactory epithelium, atrophy (3/48, 8/50, 36/50, 43/50); olfactory epithelium, metaplasia, respiratory (6/48, 37/50, 49/50, 50/50); olfactory epithelium, accumulation, hyaline droplet (0/48, 4/50, 18/50, 12/50); glands, hyperplasia (6/48, 38/50, 49/50, 49/50) at 0, 75, 150 and 300 mg/kg bw/day, respectively

Incidences of respiratory metaplasia in olfactory epithelium in all exposed groups and of atrophy and hyaline droplet accumulation in all exposed groups except 75 mg/kg bw/day females were significantly greater than those in corresponding vehicle control groups. Incidences of minimal to marked hyperplasia of Bowman’s gland were increased significantly in all exposed groups.

 

Forestomach:

- Males: hyperplasia, squamous (7/50, 8/49, 8/50, 14/49); inflammation (5/50, 8/49, 9/50, 14/49); ulcer (1/50, 4/49, 4/50, 9/49) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

- Females: hyperplasia, squamous (2/48, 8/50, 5/49, 8/50); inflammation (2/48, 8/50, 5/49, 8/50) 0, 75, 150 and 300 mg/kg bw/day, respectively

Incidences of forestomach squamous hyperplasia, inflammation, and ulceration (males only) increased with exposure and were significant in the 300 mg/kg bw/day groups

 

Glandular stomach:

- Males: ulcer (0/50, 1/49, 4/49, 5/44) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

- Females: ulcer (0/46, 1/48, 1/47, 7/48) at 0, 75, 150 and 300 mg/kg bw/day, respectively

The incidence of glandular stomach ulcers was low but significantly increased in the 300 mg/kg bw/day groups.

 

Kidney:

- Females: papilla, necrosis (including bilateral) (0/47, 1/50, 1/49, 18/49); renal tubule, necrosis (0/47, 1/50, 0/49, 6/49) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

Incidences of minimal to mild necrosis of renal papilla and mild to moderate necrosis of renal tubules were increased significantly in 300 mg/kg bw/day females

 

Neoplastic effects in males:

Liver: hepatocellular adenoma (24/50 [48%], 35/50 [70%], 37/50 [74%], 33/50 [66%]); hepatocellular carcinoma (8/50 [16%], 18/50 [36%], 19/50 [38%], 18/50 [36%]); hepatocellular adenoma or carcinoma (28/50 [56%], 43/50 [86%], 43/50 [86%], 43/50 [86%]) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

In all groups of exposed males, the incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) were significantly greater than those in the vehicle control group; incidences of multiple hepatocellular adenoma were also significantly increased. The incidences of these neoplasms exceeded the historical control range for corn oil vehicle control groups [Adenoma: 48-52% / Carcinoma: 16%-28% / Combined: 56-66%], and the incidences of hepatocellular adenoma or carcinoma (combined) exceeded the range for controls by all routes [Adenoma: 14-72% / Carcinoma: 8-48% / Combined: 20-84%].

In contrast to the effect in males, incidences of hepatocellular adenoma in females occurred with a negative trend, increases in the incidences of hepatocellular carcinoma were not significant, and incidences of these neoplasms in exposed females individually or combined, were within their respective historical control (all routes) ranges.

Incidences of clear cell focus were significantly increased in 75 and 150 mg/kg bw/day male mice.

 

Equivocal findings in females:

All organs: histiocytic sarcoma (0/49 [0%], 1/50 [2%], 1/50 [2%], 4/50 [8%]) at 0, 75, 150 and 300 mg/kg bw/day, respectively.

There was a significant positive trend in the incidences of histiocytic sarcoma in females, and this neoplasm occurred in multiple tissues. Histiocytic sarcoma has not been observed in vehicle controls in corn oil gavage studies (small historical sample size), and the incidence in the 300 mg/kg bw/day group was at the upper end of the historical range for controls by all routes [0-8%].

 

Conclusions:

Under the conditions, the authors concluded that there was clear evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined). There was equivocal evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of histiocytic sarcoma.

However, other interpretations of the data are possible. The EMEA considered that these findings were close to the historical control range and could represent a random effect. Furthermore, no dose response was identified in hepatic tumour incidence in male mice. It was noted that the conduct of the study had significant weaknesses related to the route of administration (oral gavage), the dosing regime (week days only) and a number of dead females (9) observed in the high dose group over a period of 8 days. These deficiencies could have affected the final results. It was also noted that the mouse strain used (B6C3F1) is known to have a high incidence of spontaneous liver tumours and lymphomas. The CVMP concluded that although equivocal positive findings were identified in the carcinogenicity study in mice the deficiencies identified in the study and the comparison with historical data did not allow a firm conclusion to be drawn. Overall, the findings of the mouse carcinogenicity study were considered equivocal and their relevance for the human consumer remains unclear.

The NTP concluded that no NOAEL could be determined from this study as effects were seen at all doses. However, since the neoplastic observations are not conclusive then a systemic NOAEL of 150 mg/kg bw can be determined based on the adverse effects on the kidney in the females at 300 mg/kg bw/day. Effects in the stomach are considered as adaptive responses (adaptation to a bolus of a substance that is shown to have clearly irritant properties). A local NOAEL of 75 mg/kg bw/day can be determined based on effects on the nasal epithelium.

After MW correction, a systemic NOAEL of 188 mg/kg bw can be determined for Trans-Isoeugenyl acetate. The local NOAEL was not considered adequate for read-across purpose since the target substance is neither an eye nor a skin irritant, therefore upper respiratory tract irritation is not expected to occur.

 

 

Two-generation reproduction toxicity study in rats (NIEHS, 2003):

In a two-generation reproduction toxicity study, Isoeugenol, was administered to rats daily via oral gavage at dose levels of 0, 70, 230 or 700 mg/kg bw/day. Body weight, food consumption, clinical signs, organ weight, gross and microscopic pathology information allowed to assess the repeated dose toxicity potential of Isoeugenol across the generations. Based on an expert judgement a LOAEL for parental toxicity was determined at 70 mg/kg bw/day, based on decreased body weight gains and hyperkeratosis and hyperplasia in the non-glandular stomach at all dose levels and in both sexes of the F0 and F1 animals. It should be pointed out that the appearance of hyperkeratosis and hyperplasia in non-glandular stomachs and decreased body weight may be due to the route of administration whereby the test material is administered by intubation thereby delivering a bolus of a substance that is shown to have clearly irritant properties at high concentrations.

After MW correction, a systemic LOAEL of 88 mg/kg bw can be determined for Trans-Isoeugenyl acetate.

 

Pre-natal developmental toxicity study in rats (NTP, 1999)

In an pre-natal developmental toxicity study, Isoeugenol was administered to female rats via oral gavage at dose levels of 0, 250, 500 and 1000 mg/kg bw/day. The LOAEL was reported to be the low dose level of 250 mg/kg bw/day based on a statistically and dose-dependent reduced gestation body weight gain. Other effects in maternal animals included several types of clinical effects at higher doses and lower maternal relative food intake at the highest dose (1000 mg/kg bw/day).

After MW correction, a systemic LOAEL of 314 mg/kg bw can be determined for Trans-Isoeugenyl acetate.

 

 

Table 7.5: Overall NOAEL and LOAEL (corrected for MW) for repeated dose toxicity identified across key studies

Study Type	Species	Route of exposure	NOAEL (mg/kg bw/day) Corrected for MW	LOAEL (mg/kg bw/day Corrected for MW	Basis
90-day	Rat	Gavage	F: 188 M: 377	F: 377 M: 754	F: Increased hyperplasia in liver at 300 and 754. M: Reduced body weight and body weight gain of 11.6% at 754.
90-day	Mouse	Gavage	M: 377 F: 754 (highest-dose)	M: 754 (highest-dose)	M: Reduced body weight and body weight gain at 754
Carcinogenicity	Rat	Gavage	M: 377 F: 377 (highest-dose		None
Carcinogenicity	Mouse	Gavage	F/M: 188*	F/M: 377 (highest-dose	F: adverse effects in kidney + reduced body weight gain of 10% at 377 M: reduced body weight gain of 15% at 377
Two-generation reproduction toxicity study	Rat	Gavage	Not identified	M/F: 88 (lowest dose)	Decreased body weight gains + hyperkeratosis and hyperplasia in non-glandular stomachs at all dose levels
Pre-natal developmental toxicity study	Rat	Gavage	Not identified	Female: 314 (lowest dose)	Dose-dependent reduced gestation body weight gain

 

*Relevant Systemic NOAEL determined from expert judgement according to the following criteria.

In a weight of evidence approach, i.e. taking into account the whole database (see Table 7.5), the following criteria were considered to determine the most relevant systemic NOAEL for repeated dose toxicity of trans-Isoeugenyl acetate:

- the duration of the study: long-term studies preferred to short-term studies to detect delayed systemic effect,

- the effects observed: obvious relevant systemic effects observed with dose-related changes preferred to transient effects inducing equivocal results.

 

 

In conclusion, results from the mouse carcinogenicity study conducted on trans-Isoeugenol allowed to identify the most relevant systemic NOAEL at 188 mg/kg bw/d for repeated dose toxicity of trans-Isoeugenyl acetate. This value was used to extrapolate the DNELs for worker and general population.

 

Reference

EMEA (2011). Committee for Medicinal Products for Veterinary Use. European public MRL assessment report (EPMAR). Isoeugenol (fin fish). 14 April 2011.EMA/CVMP/405186/2010 

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure.