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Repeated dose toxicity: oral

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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-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 08, 2002 to May 06, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
None reported
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at initiation: 5-7 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 per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: May 08, 2002 To: May 06, 2004
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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 and 30 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:
- Dose formulations were analyzed approximately every 3 months; animal room samples were also analyzed. 27 of 28 formulations were within 10 % of the target concentrations. All nine animal room samples analyzed for mice were within 10 % of the target concentrations.
- Homogeneity studies of 0.2 and 120 mg/mL formulations and stability studies of the 0.2 mg/mL formulation were performed; 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 hours under simulated animal room conditions.
Duration of treatment / exposure:
104 (female mice) or 105 (male mice) weeks
Frequency of treatment:
5 days per week
Post exposure period:
None
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)
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Mice exposed to test material in corn oil by gavage for 3 months showed significant decreases in body weights of males and increases in liver weights of the 600 mg/kg bw/day group. Although increased incidences of olfactory lesions in 600 mg/kg bw/day males and females were moderate, they were statistically significant. These nasal lesions were considered to have no effect on survival in longer exposures; however, the importance of olfaction in rodent feeding behavior increased concern that they might affect weight gain, so 300 mg/kg bw/day was selected as the highest dose for the 2-year study. The dosing regimen for the 2-year study of isoeugenol in mice was 0, 75, 150, and 300 mg/kg bw/day.
- Rationale for animal assignment: Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily. Clinical findings were recorded during week 5, every 4 weeks thereafter, and at the end of the exposure phase.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, weekly for the first 13 weeks, every 4 weeks thereafter, and at the end of the exposure phase.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Animals were sacrificed by carbon dioxide asphyxiation.
GROSS PATHOLOGY: Yes; Necropsies were performed on all animals.
At necropsy, all organs and tissues were examined for grossly visible lesions and all major tissues were fixed and preserved in 10% neutral buffered formalin (eyes were fixed in Davidson’s solution for up to 72 hours 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 for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined.
HISTOPATHOLOGY: Yes; Complete histopathology was performed on all mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, gallbladder, Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, 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.
Other examinations:
None
Statistics:
Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. 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.

Analysis of neoplastic and non-neoplastic lesion incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and non-neoplastic lesion prevalence.
Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented as 1– P with the letter N added (e.g., P = 0.99 is presented as P = 0.01N).

Analysis of continuous variables: Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

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.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical findings related to test material exposure were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
- The survival rate of 300 mg/kg bw/day males was significantly decreased compared to that of the vehicle controls. The survival rates of all other exposed groups were similar to those of the vehicle controls.
- Twenty-three of the 50 male mice exposed to 300 mg/kg bw/day test material died before terminal sacrifice; 16 were natural deaths and seven were sacrificed in moribund condition. Liver neoplasia was the likely cause of death for 16 of these mice, as follows: 13 had hepatocellular carcinoma, one had hepatoblastoma, one had hepatocholangiocarcinoma, and one had hepatocellular adenoma. In the 300 mg/kg bw/day group of female mice, half of the 16 early deaths occurred between days 553 and 555. Examination of gross observations made at necropsy and histopathologic diagnoses recorded for these eight animals indicated that the likely cause of death for five of them was moderate or marked liver necrosis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
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:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nose: Incidences of olfactory epithelial respiratory metaplasia in all exposed groups, atrophy and hyaline droplet accumulation in all exposed groups of males and in 150 and 300 mg/kg bw/day females, and degeneration in 150 and 300 mg/kg bw/day males were significantly increased. Small increases of atrophy and hyaline droplet accumulation were also observed in 75 mg/kg bw/day females. The severity of respiratory metaplasia generally increased with increasing dose; severity of the other olfactory lesions were minimal to mild and similar to those of the vehicle controls. The incidences of minimal to moderate hyperplasia of the Bowman’s gland were significantly increased in all exposed groups; severities increased with increasing dose.

Kidney: Low incidences of minimal to moderate necrosis of the renal papilla and mild to moderate renal tubule necrosis in 300 mg/kg bw/day females were significantly greater than those in the vehicle controls.

Stomach: Low incidences of forestomach squamous hyperplasia, inflammation, and ulceration in male mice increased with increasing dose and were significantly increased in the 300 mg/kg bw/day group. Similarly, low incidences of squamous hyperplasia and inflammation in the forestomach of female mice were increased in the 75 and 150 mg/kg bw/day groups and were significantly increased in the 300 mg/kg bw/day group. In males, the three forestomach lesions often were present in the same mouse. Low incidences of ulcers of the glandular stomach were statistically significant in 300 mg/kg bw/day males and females. Ulcers were usually accompanied by minimal to moderate inflammation, and the incidence of inflammation in 300 mg/kg bw/day females was significantly increased.

Spleen: In 300 mg/kg bw/day female mice, the incidence of splenic cellular depletion was significantly increased. The lesion consisted of minimal to marked reductions in lymphocytes accompanied by variable decreases in hematopoietic cells. These findings were considered secondary to stress because they occurred in mice that were found dead or sacrificed moribund and seven of the nine female mice with cellular depletion also had liver and/or kidney necrosis.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: In all exposed male groups, incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) were significantly greater than those in the vehicle control group; the 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, and the incidences of hepatocellular adenoma or carcinoma (combined) exceeded the range for controls by all routes. Metastases of carcinomas to the lungs occurred in all dosed groups of male mice and in 150 and 300 mg/kg bw/day females and were often multiple. 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 males. The incidences of eosinophilic foci were also increased in these two groups, but the differences from vehicle controls were not significant. Incidences of basophilic foci were similar for all male groups. A positive trend in liver necrosis in females was significant, but the incidence in the 300 mg/kg bw/day group was not significant compared to the vehicle control group. Moderate to marked liver necrosis was the likely cause of death for 5/8 female mice in the 300 mg/kg bw/day group that died between days 553 and 555. The proximate cause of this liver necrosis was undetermined.

Histiocytic Sarcoma: The positive trend in the incidences of histiocytic sarcoma in females was statistically significant. Histiocytic sarcoma has not been observed in vehicle controls in corn oil gavage studies, 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. These histiocytic sarcomas were invasive with a variety of distributions in multiple organs, including liver, ovary, uterus, spleen, lung, lymph nodes, kidney, thymus, and bone marrow.
Relevance of carcinogenic effects / potential:
Under the conditions, the authors concluded that there was clear evidence of carcinogenic activity in male 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 mice based on increased incidences of histiocytic sarcoma. However, other interpretations of the data are possible. The EMEA considered that In the mouse carcinogenicity study males and females were exposed to isoeugenol in corn oil by gavage at doses of 0, 75, 150, or 300 mg/kg bw, 5 days per week, except holidays, for 104 (females) or 105 (males) weeks.  Hepatocellular adenoma, hepatocellular carcinoma and combined hepatocellular adenoma or carcinoma were seen in all dose groups, 75 to 300 mg/kg bw/day. In female mice, histiocytic sarcoma (all organs) was noted in the high dose group, 300 mg/kg bw/day. 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.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Key result
Dose descriptor:
LOAEL
Effect level:
75 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.:
75 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:
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.
Executive summary:

In a 2-year carcinogenicity study, performed similarly to OECD guideline 451 and in compliance with GLP, test material 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.

Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 22, 2002 to April 22, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
None reported
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS (F344/N)
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at initiation: 5-7 weeks
- Housing: Rats were housed three (males) or five (females) per 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 per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: April 22, 2002 To: April 22, 2004
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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: 15, 30 and 60 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:
- Dose formulations were analyzed approximately every 3 months; animal room samples were also analyzed. All 27 dose formulations for rats were within 10% of the target concentrations. All nine animal room samples analyzed for rats were within 10% of the target concentrations.
- Homogeneity studies of 0.2 and 120 mg/mL formulations and stability studies of the 0.2 mg/mL formulation were performed; 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 hours under simulated animal room conditions.
Duration of treatment / exposure:
105 weeks
Frequency of treatment:
5 days per week
Post exposure period:
None
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)
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Rats administered test material in corn oil by gavage for 3 months showed minimal toxicity. The olfactory epithelium was the most prominent site of toxicity in both males and females. In females, liver cytoplasmic alterations were noted as well in the 300 and 600 mg/kg bw/day groups. These nasal and liver lesions were not considered to affect survival in the subsequent 2-year study; however, the importance of olfaction in rodent feeding behavior increased concern that they might affect body weight gain. Because effects were generally similar in magnitude in the 300 and 600 mg/kg bw/day groups of both sexes, 300 mg/kg bw/day was selected as the highest dose for the 2-year study. The dosing regimen for the 2-year study in rats was 0, 75, 150, and 300 mg/kg bw/day.
- Rationale for animal assignment: Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily. Clinical findings were recorded during week 5, every 4 weeks thereafter, and at the end of the exposure phase.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, weekly for the first 13 weeks, every 4 weeks thereafter, and at the end of the exposure phase.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Animals were sacrificed by carbon dioxide asphyxiation.
GROSS PATHOLOGY: Yes; Necropsies were performed on all animals.
At necropsy, all organs and tissues were examined for grossly visible lesions and all major tissues were fixed and preserved in 10% neutral buffered formalin (eyes were fixed in Davidson’s solution for up to 72 hours 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 for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined.
HISTOPATHOLOGY: Yes; Complete histopathology was performed on all rats. 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, larynx, 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.
Other examinations:
None
Statistics:
Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. 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.

Analysis of neoplastic and non-neoplastic lesion incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and non-neoplastic lesion prevalence.
Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented as 1– P with the letter N added (e.g., P = 0.99 is presented as P = 0.01N).

Analysis of continuous variables: Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

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.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical findings related to the administration of test material were observed.
Mortality:
no mortality observed
Description (incidence):
Survival rates of dosed male and female rats were similar to those of vehicle controls
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
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:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nose: Incidences of minimal to mild atrophy of the olfactory epithelium and respiratory metaplasia of the olfactory epithelium were increased in 75 mg/kg bw/day males and significantly increased in 150 mg/kg bw/day males and 300 mg/kg bw/day males and females. Olfactory epithelial atrophy was characterized by decreased numbers of epithelial cells that resulted in thinning of the olfactory epithelium. It primarily affected epithelium lining the dorsal meatus at nasal Level II. Atrophy of the olfactory epithelium was frequently associated with atrophy of the olfactory nerves in the adjacent submucosal tissues. This nerve atrophy was considered to be secondary to the loss of sensory neurons in the olfactory epithelium and was not given a separate diagnosis in this study. In respiratory metaplasia of the olfactory epithelium, the normal olfactory epithelium was replaced by ciliated columnar epithelium similar in appearance to normal respiratory epithelium and was observed in the dorsal meatus of Level II and along the ethmoid turbinates of Level III. The incidence of minimal to mild olfactory epithelium degeneration in 300 mg/kg bw/day males was also significantly increased. Degeneration of the olfactory epithelium was characterized by disorganization of the layers of sensory neurons and vacuolization of the epithelium.

Liver: Incidences of basophilic focus were significantly decreased in all exposed groups of male rats, while those of eosinophilic focus decreased significantly in 75 and 150 mg/kg bw/day males. In 300 mg/kg bw/day males, incidences of clear cell focus and bile duct hyperplasia also decreased significantly. These decreases were not present in female rats and have unknown significance. The incidences of mixed cell foci in male groups exposed to test material were not significantly different from the vehicle control group. Eosinophilic, mixed, basophilic, and clear cell foci consisted of localized areas of hepatocytes with characteristic altered tinctorial properties. Otherwise, the hepatocytes were generally similar in appearance. An eosinophilic focus was composed of cells with abundant eosinophilic cytoplasm. A mixed cell focus was composed of a mixture of cells with different staining properties, generally a mixture of eosinophilic cells and clear cells. A basophilic focus consisted of hepatocytes with basophilic cytoplasm, occasionally with basophilic linear (tigroid) intracytoplasmic aggregates. A clear cell focus was composed of cells having clear cytoplasm. Other than tinctorial differences, hepatocytes in foci were generally somewhat larger than hepatocytes in adjacent parenchyma and were arranged in relatively normal lobular patterns. The hepatic cords at the periphery of these foci generally merged imperceptibly with the surrounding normal liver, resulting in an indistinct border and little or no compression of the adjacent liver parenchyma.

Bile duct hyperplasia consisted of multifocal proliferations of small bile ducts within portal areas. These bile ducts were similar in appearance to those seen in portal triads of normal liver lobules. The only distinctive feature was increased numbers of ducts. They consisted of uniform basophilic, flattened to cuboidal epithelium surrounded by scant to moderate amounts of collagenous stroma.

Other Findings: Pancreas acinus atrophy occurred with a negative trend (P=0.010) with a significantly decreased incidence in 300 mg/kg bw/day males (0, 75, 150 and 300 mg/kg bw/day: 22/50, 21/50, 17/50 and 10/49, respectively). Testicular interstitial cell hyperplasia occurred with a negative trend (P=0.045) with a significantly decreased incidence in 300 mg/kg bw/day males (0, 75, 150 and 300 mg/kg bw/day: 7/50, 2/50, 4/50 and 1/50, respectively). The cause of these decreased incidences of pancreas acinus atrophy and testicular interstitial cell hyperplasia was not evident.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Thymus: 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.

Mammary Gland: 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 (4%) was equal to the highest rate for controls by all routes [8/1,199 (1 ± 1%), range 0-4 %]. These mammary gland carcinomas were not accompanied by increased incidences of adenoma or hyperplasia. In one animal, the mammary gland carcinoma consisted of a single highly cellular mass of neoplastic epithelial cells with small numbers of alveolar structures. Irregular cords of neoplastic cells were invading the adjacent tissues. Numerous mitotic figures were evident. The other animal had several nodules of malignant neoplastic glandular epithelium in multiple glands. The cellular patterns varied from nodule to nodule but generally had irregularly shaped tubular structures formed by multiple layers of densely packed, cuboidal cells. Mitotic figures were frequent. These cells were invading the adjacent tissues.

Skin: Keratoacanthoma occurred with a negative trend in male rats, and the incidences were significantly decreased in the 150 and 300 mg/kg bw/day groups (0, 75, 150 and 300 mg/kg bw/day; 7/50, 5/50, 1/50 and 1/50, respectively). The incidences in the 150 and 300 mg/kg bw/day groups were below the historical range for vehicle controls in corn oil gavage studies [9/100 (9% ± 7%), range 4-14 %] and at the lower end of the historical range for controls by all routes [71/1,199 (6% ± 5%), range 0-20 %] (Table A3c). The keratoacanthomas were benign neoplastic proliferations of basal and squamous cells of the epidermis that formed craters or invaginated into the dermis and subcutis. They were often cystic masses filled by layers of keratin and had characteristic thick walls of stratified squamous epithelium.
Relevance of carcinogenic effects / potential:
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.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
LOAEL
Effect level:
75 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.:
75 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:
In conclusion, Trans-isoeugenol is not considered to be carcinogenic in rats.
Executive summary:

In a 2-year carcinogenicity study, performed similarly to OECD guideline 451 and in compliance with GLP, test material 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

Reason / purpose:
reference to same study
Reference
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:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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 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.

Reason / purpose:
reference to same study
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Not a GLP study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No short-term exposure in absence of S9
Principles of method if other than guideline:
None
GLP compliance:
yes
Remarks:
The NTP conducts its studies in compliance with FDA Good Laboratory Practice Regulations
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
0, 50, 100, 200, 300, 400, 500 without S9
0, 150, 160, 170, 180, 190, 200 with S9
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Mitomycin C and Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 10 hours without S9, 2 hours with S9
- Expression time (cells in growth medium): 2 hours without S9, 10 hours with S9
- Fixation time (start of exposure up to fixation or harvest of cells): 12 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Presence of scorable metaphases
Rationale for test conditions:
Not reported
Evaluation criteria:
Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P#0.05) difference for one dose point and a significant trend (P#0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.
Statistics:
No data
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the high dose was limited by toxicity.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Additional information on results:
Isoeugenol (in medium concentrations up to 200 µg/mL) did not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation

See the attached document for information on tables of results

Conclusions:
Trans-Isoeugenol (in medium concentrations up to 200 µg/mL) did not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation.
Executive summary:

Testing was performed as reported by Gallowayet al.(1987). Trans-Isoeugenol was sent to the testing laboratory as a coded aliquot. It was tested in cultured Chinese hamster ovary (CHO) cells for induction of chromosomal aberrations (Abs), both in the presence and absence of Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of isoeugenol; the high dose was limited by toxicity. A single flask per dose was used. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with trans-isoeugenol for 10 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with isoeugenol and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Trans-Isoeugenol (in medium concentrations up to 200 µg/mL) did not induce chromosomal aberrations in cultured CHO cells, with or without S9 activation.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2010
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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

Reference
Name:
Unnamed
Type:
Constituent
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 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 / concentrationsopen allclose all
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

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

open allclose all
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

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.