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Carcinogenicity

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Description of key information

Chronic toxicity/carcinogenicity studies were conducted with Trans-isoeugenol in Fischer 344 rats and B6C3F1 mice as part of the US government-sponsored NTP.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
Good quality studies although some weaknesses in the study design, but not sufficient to invoke an additional assessment factor.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Carcinogenicity study in rats (NTP, 2010):

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

 

Carcinogenicity study in mice (NTP, 2010):

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

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding carcinogenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).