Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity oral: systemic NOAEL = 150 mg/kg bw/day in female mice (similar to OECD 408, K, rel. 2) 
Repeated dose toxicity oral: local NOAEL = 75 mg/kg bw/day in mice (similar to OECD 408, K, rel. 2)
Repeated dose toxicity inhalation: local LOAEC = 1 mg/m3 in rats (OECD 412, GLP, K, rel.1)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 18 to July 19, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Food and water consumption not monitored; clinical biochemistry, ophthalmological and neurobehavioral examination not performed.
Reason / purpose:
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; clinical biochemistry, ophthalmological and neurobehavioral examination not performed)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
Not reported
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc., Germantown, NY
- Age at study initiation: 6-7 weeks
- Housing: Male animals were housed individually and females were housed 5/cage in Polycarbonate cages.
- Diet: NTP-2000 irradiated wafer or pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Females: 13 days; males: 14 days

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

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


Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was chosen because it is the major route of human exposure, and gavage was chosen after preliminary studies showed that isoeugenol in feed was unpalatable to both rats and mice and the concentration in feed decreased when stored at room temperature.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The dose formulations were prepared by mixing test material with corn oil to give the required concentrations. The appropriate amounts of test material and corn oil were placed in a glass mixing container, capped, and thoroughly mixed with a paint shaker for approximately 5 minutes. Dose formulations were prepared approximately monthly and stored at room temperature in amber glass bottles with Teflon® -lined lids for up to 35 days.

VEHICLE
- Concentration in vehicle: 3.75, 7.5, 15, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Test material formulations were analysed three times during the study period by GC.
- Homogeneity and stability of test material formulations were determined. Homogeneity studies of 0.2 and 120 mg/mL formulations and stability studies of the 0.2 mg/mL formulation were performed using GC on a different lot of test material (Penta International Corporation, Lot No. 46928).
- Homogeneity was confirmed, and the 120 mg/mL dose formulation was found to be suitable for gavage. Stability was confirmed for up to 35 days for dose formulations stored in amber glass bottles with Teflon® -lined lids at - 20 °C, 5 °C, and room temperature, as well as for 3 h under simulated animal room conditions.
- All the formulations analysed were within 10% of the target concentrations.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days/week
Dose / conc.:
37.5 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

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

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

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

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

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Animals were sacrificed by carbon dioxide asphyxiation and necropsies were performed.

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

HISTOPATHOLOGY: Yes
- Histopathology was performed on animals from vehicle control and 600 mg/kg bw/day dosed group.
- In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain,
clitoral gland, esophagus, eyes, gall bladder, Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin,
spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. Tissues were examined in the remaining dosed groups to a no-effect level.
- Tissues for microscopic examination were fixed and preserved in 10 % neutral buffered formalin (eyes were fixed in Davidson’s solution for up to 72 h and then transferred to 10 % neutral buffered formalin), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.
Other examinations:
None
Statistics:
- The probability of survival was estimated by the product- limit procedure of Kaplan and Meier (1958).
- Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
- Organ and body weight data, which historically have approximately normal distributions, were analysed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Haematology data, which have typically skewed distributions, were analysed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
- Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
- Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.
- Probability values (p) are presented as follows:
p ≤ 0.01 **
p ≤ 0.05 *
p > 0.05 (not significant)
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean body weight and body weight gain were significantly decreased in males treated with 600 mg/kg bw/day compared to the vehicle control group (- 12%).
- After 85 days of exposure, mean body weights of females treated with 75 and 150 mg/kg bw/day exceeded that of the vehicle control group by 10 and 8 %, respectively, while that of 600 mg/kg bw/day treated females was 7 % less than vehicle controls; however, the differences were not statistically significant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No haematological effects were observed.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- The only exposure-related organ weight change was an increase in liver weights. As compared to vehicle control group, significant increases were observed in relative liver weights of all male groups (+10.6% at 37.5 mg/kg bw/day ; +14.0% at 75 mg/kg bw/day ; + 17.0% at 150 mg/kg bw/day ; +26.4% at 300 mg/kg bw/day ; +33.1% at 600 mg/kg bw/day) and absolute liver weights of males treated with 300 (+21.6%) and 600 mg/kg bw/day (+14.3%). The EMEA considered that, in this study, some changes in liver to bodyweight ratios were observed, but these changes were not considered treatment related because the magnitude of the changes was small and there was no clear dose response relationship.
- As compared to vehicle control group, significant decreases were observed in absolute kidney weights of males treated with 150 (-7.8%), 300 (-7.4%) and 600 mg/kg bw/day (-9.5%) and absolute lung weights of females treated with 600 mg/kg bw/day were significantly decreased (-18.2%) as compared to vehicle control group and considered related to exposure. The relative weights of these organs were not significantly different from controls and were therefore considered non-adverse.
- Moreover, the EMEA considered the body weight change as the only treatment related adverse systemic effects in this study.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- No microscopic findings in liver, kidney, and lung related to treatment.
- Incidences of mild to moderate olfactory epithelial atrophy and minimal to mild atrophy of olfactory nerve bundles increased significantly in males and females treated with 600 mg/kg bw/day. Olfactory epithelial atrophy was characterized by a decrease in the number of cells, resulting in thinning of the olfactory epithelium. The atrophy occurred in the most distal portion of the nasal cavity along the junction of the nasal septum with the dorsal wall of the nasal meatus in Level III. The atrophic epithelium was simple or pseudostratified, ciliated, and columnar and resembled normal respiratory epithelium. Glands within the lamina propria under the affected olfactory epithelium were often slightly dilated. Some of these glands contained secretory material with occasional inflammatory cells and cell debris. Some glands were lined by minimally hyperplastic epithelial cells. Atrophy of olfactory nerves consisted of reductions in the number and size of nerve bundles within the lamina propria beneath areas of atrophic epithelium. The nerve atrophy was considered to be secondary to the loss of sensory neurons in the overlying atrophic olfactory epithelium.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No significant treatment-related systemic adverse effects in females.
Dose descriptor:
NOAEL
Remarks:
(local)
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

See the attached document for information on tables of results

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

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

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

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

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

 

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

 

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

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
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP-level quality study and of high quality (Klimisch score = 2).
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 20, 2011 to February 07, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: Stored at room temperature and was considered stable under these conditions
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 187-240 g for males; 149-184 g for females at randomization
- Housing: Animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet: Basal diet; PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, ad libitum
- Food and water were withheld during each daily exposure period.
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70.4-71.5 °F (21.3-21.9 °C)
- Humidity: 36.6-52.6 %
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: September 20, 2011 To: February 07, 2012
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1.3 - <= 2.2 µm
Remarks on MMAD:
Mean MMAD: 1.4, 1.3 and 2.2 µm for 1, 10 and 100 mg/m3, respectively
Mean GSD: 3.59, 4.00 and 2.30 for 1, 10 and 100 mg/m3, respectively
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 7.9-L conventional nose-only exposure systems with synthetic rubber grommets in exposure ports to engage animal holding tubes.
- Method of holding animals in test chamber: To perform nose-only exposure, it is necessary to restrain the rats in specially designed nose-only holding tubes. The 6-hour period of restraint was necessary to achieve the exposure duration.
- Source and rate of air: HEPA- and charcoal-filtered air source (WIL Research Inhalation Department supply air source) or breathing-quality in-house compressed air source.
- Method of conditioning air: Charcoal- and HEPA-filters
- System of generating aerosols: Aerosolized test substance was generated using a Collison nebulizer.
- Temperature, humidity in air chamber: Mean temperature ranged from 20-21 °C and the mean relative humidity ranged from 43-56 %.
- Air flow rate: Daily mean ventilation rate ranged from 44.3-47.9 liters per minute (LPM).
- Method of particle size determination: Aerosol particle size determinations were conducted for each test substance exposure system using a 7-stage stainless steel cascade impactor.
- Treatment of exhaust air: All test substance atmosphere exposure system exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consisted of redundant exhaust blowers proceeded by charcoal- and HEPA-filters.

TEST ATMOSPHERE
- Brief description of analytical method used:
Nominal exposure concentrations: Due to the small amount of test substance that was required for the study, a nominal concentration was not calculated.
Actual exposure concentrations: Actual aerosol mass concentrations of the test atmospheres for exposure systems 1, 3, and 4 (control, 10 and 100 mg/m3) were determined using standard gravimetric methods. Due to the volatility of the test material, standard gravimetric methods cannot be used for the analysis of the atmosphere in the Group 2 exposure system.
Analysed exposure concentrations: Analysed exposure concentrations of the total test material (combined vapour and aerosol) within exposure system 2 were determined at approximately 60-minute intervals using an appropriate gas chromatography (GC) method and were expressed as mg/m3.
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample of the test substance was collected from prior to the initiation of exposure for purity and concentration analyses. Analyses of purity and stability were performed using a similar method from a sample collected at the end of the exposure period. All analyses were conducted using a gas chromatography method with flame ionization detection.
Results: Analyses conducted for a sample of the test substance collected prior to exposure initiation resulted in a purity of 90.8%. Following the completion of the exposure phase, another sample was collected that resulted in a purity of 89.3%. Comparison of these purity data indicated that the test substance was stable (98.3% of the initial test substance concentration after up to 34 days), based on the acceptance criteria for stability (i.e., the mean post-storage concentration was not <90% of the pre-storage value).
Mean exposure concentrations were 1.28, 9.8 and 98 mg/m3 for the target exposure concentrations 1, 10 and 100 mg/m3, respectively.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 days per week for 2 weeks (10 total exposures)
Dose / conc.:
1 mg/m³ air (nominal)
Remarks:
0.15 ppm
Dose / conc.:
10 mg/m³ air (nominal)
Remarks:
1.5 ppm
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
14.9 ppm
No. of animals per sex per dose:
10
Control animals:
other: filtered air (control group)
Details on study design:
- Dose selection rationale: Prior to exposure of animals for the main study, preliminary animal exposures were conducted at 750 and then at 300 mg/m3 (89.4 and 44.7 ppm, respectively) for three days to assess tolerability of the test substance. At 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period (-14 and -10% for males and females, respectively, compared to day 0) and significant clinical observations included rales and laboured respiration in 1/3 males and 2/3 females. At 300 mg/m3, the mean body weight was decreased by 8% and 3 % compared to the study day 0 values for males and females, respectively. Therefore, one third of the lowest concentration tested in the preliminary exposures, 100 mg/m3 (i.e.14.9 ppm), was chosen as the high exposure level for the main two-week study.
- Rationale for animal assignment: Animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design.
Positive control:
None
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed daily, prior to exposure, during exposure (at the approximate midpoint for visible signs of animals in nose-only exposure restraint tubes), and 0 to 1 h following the end of exposure. The absence or presence of findings was recorded for individual animals at observations conducted prior to exposure and following the end of exposure. Only significant findings were recorded at the approximate midpoint of exposure observations. On non-exposure days, the animals were observed once daily.
- Survival: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity, except on the days of the scheduled necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were conducted on all animals during pretest prior to randomization, at the time of randomization prior to group assignment, and approximately weekly during the exposure period, including prior to the scheduled necropsy. Observations conducted prior to exposure were not performed on days when detailed physical examinations were conducted, provided they were conducted prior to
exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded during the pretest period, at randomization, prior to the first exposure, and twice weekly during the exposure period (prior to the first and last exposure each 5-day exposure week) including the day before the first scheduled day of necropsy (nonfasted).
- Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION:
- Individual food consumption was recorded during the pretest period (prior to randomization) and twice weekly during the exposure period. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected at the scheduled necropsy (study day 12).
- Anaesthetic used for blood collection: Yes; Blood was collected for haematology and standard serum chemistry evaluations via the retro-orbital sinus and for coagulation parameters via the vena cava from animals anesthetized by inhalation of isoflurane at the scheduled necropsy. (inhalation of isoflurane)
- Animals fasted: Yes; animals were fasted overnight prior to blood collection.
- How many animals: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from all animals, and blood samples for coagulation evaluations were collected from 5 animals/sex/group (Subgroup 1).
- Parameters checked:
HAEMATOLOGY AND COAGULATION: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Haemoglobin Distribution Width (HDW), Differential leukocyte count - Percent and absolute (Neutrophil (NEU), Lymphocyte (LYMPH),Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC)), Platelet estimate, Red cell morphology (RBC Morphology)
CLINICAL CHEMISTRY: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio), [by calculation], Total bilirubin (Total Bili) Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Sorbitol dehydrogenase (SDH) and Lactate dehydrogenase (LDH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Animals were anesthetized by isoflurane inhalation and euthanized by exsanguination. For 5 animals/sex/group (BALF animals), a BAL was performed on the lungs as soon as possible after exsanguination and after completion of the BAL, all BALF animals were subjected to necropsy. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Tissues or gross lesions were not collected from the BALF animals (Subgroup 2).

HISTOPATHOLOGY: Yes; the following tissues and organs were collected from the histopathology animals (remaining 5 animals/sex/group; Subgroup 1) and placed in 10 % neutral-buffered formalin for examination:
Adrenals (2), Aorta, Bone with marrow, Femur (with joint)[a], Sternebrae, Bone marrow smear (from femur), Brain with olfactory blub - Cerebrum level 1, Cerebrum level 2 , Cerebellum with medulla/pons, Cervix, Epididymides (2)[b], Eyes with optic nerve (2) [c], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Harderian glands (2), Heart, Kidneys (2), Lacrimal gland (exorbital [2]), Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by constant pressure inflation with fixative), Lymph nodes - Auricular (2 [if visible]), Axillary (2), Bronchial (if visible), Mandibular (2), Mediastinal and bronchial (if visible), Mesenteric, Mammary glands (females only) [d], Nasal cavity (with turbinates) [e], Olfactory bulbs [e], Ovaries with oviducts (2) [f], Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands (mandibular [2]), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin, Spinal cord (cervical, lumbar, thoracic), Spleen, Testes (2), Thymus, Thyroid (with parathyroids [2]) [f], Tongue, Trachea, Urinary bladder, Uterus, Vagina and Gross lesions

a = Bone marrow smears were obtained at scheduled necropsy from the femur, but not placed in formalin; slides were not examined.
b = Fixed in Bouin’s solution
c = Fixed in Davidson’s solution
d = A corresponding section of skin was taken from the same anatomical area for males.
e = Following the collection of the appropriate protocol-specified tissues, the entire head was removed and preserved (olfactory bulbs were severed from the brain and remained in the skull). Following decalcification, 6 cross-sections of the nasal cavities were prepared for microscopic examination in accordance with the method described by Morgan (1991) and Mery et al. (1994).
f = Oviducts and parathyroids were examined microscopically when in the plane of section and in all cases where a gross lesion was present.

After fixation, lungs with bronchi, bronchus associated lymphoid tissues [BALT], nasal tissues, nasal-associated lymphoid tissue [NALT], larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions) were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with haematoxylin and eosin. Microscopic examination of the lungs with bronchi, BALT, nasal tissues, NALT, larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions were performed for 5 animals/sex/group (Subgroup 1) not assigned for the BAL at the scheduled necropsy.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsy:
Adrenals, Brain, Heart, Kidneys, Liver, Lungs (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids*

Paired organs were weighed together. Designated (*) organs were weighed after fixation. Organ to final body weight and organ to brain weight ratios were calculated.
Other examinations:
Bronchoalveolar lavage fluid (BALF) clinical pathology and serum and BALF cytokine evaluation:
Prior to the scheduled necropsy, the animals were fasted overnight. At the time of necropsy, the 5 animals/sex/group selected for serum cytokine evaluations (Subgroup 2) were anesthetized by inhalation of isoflurane. Blood samples were collected from the vena cava into tubes containing no anticoagulant. A bronchoalveolar lavage (BAL) was performed on the lungs of 5 animals/sex/group (Subgroup 2) at the scheduled necropsy (study day 12).

BALF chemistry and cytology
The following parameters were evaluated:
Total and differential cell counts for: Alveolar macrophages, Neutrophils, Lymphocytes, Eosinophils, Basophils
Lactate dehydrogenase (LDH), Total protein, Alkaline Phosphatase

Serum and BALF cytokine evaluations
The following cytokines were evaluated:
TNF-α, IL-5, IL-10, ICAM-1, IFN-γ, IL-4, TGF-β, MCP-1, IL-1β, IL-13, MIP-2, RANTES

TNF = Tumour necrosis factor, IFN = Interferon, IL = Interleukin, TGF = Transforming growth factor, MIP = Macrophage inflammatory protein, ICAM = Intracellular adhesion molecule, MCP = Macrophage chemotactic protein, RANTES = Regulated upon activation, normal T-cell expressed, and presumably secreted.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Body weight, body weight change, food consumption, clinical pathology data including BALF total protein, lactate dehydrogenase, alkaline phosphatase, and cytology parameters (with the exception of gamma glutamyltransferase), and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations. All clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled necropsy
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- There were no toxicologically significant effects on body weights.
- Transient lower body weight gains were present in the 100 mg/m3 group males and females during the exposure period, reaching statistical significance during study days 0 to 4 and/or 7 to 11. Statistically significantly lower cumulative body weight gains were recorded for the 100 mg/m3 group males from study day 0 to 11. Although lower cumulative body weight gains were noted in the 100 mg/m3 group females by the end of the study, the values were not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- There were no toxicologically significant effects on food consumption.
- Transient slightly lower food consumption was noted for the 100 mg/m3 group males and females during the exposure period. Although the differences were statistically significant compared to the control group from study days 0 to 4 and/or 7 to 11, these effects on food consumption were sporadic and not considered toxicologically relevant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no clear test substance-related differences in haematology parameters when compared to the control group. Reticulocyte counts were slightly lower in the 1, 10, and 100 mg/m3 group females and 100 mg/m3 group males when compared to the control group; however, a relationship with test substance administration was not established due to the lack of dose response in females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related differences in serum clinical chemistry parameters when compared to the control group. Alanine aminotransferase (ALT) was slightly lower in the 100 mg/m3 group males when compared to the control group; however, decreases in ALT have no known toxicological meaning, and this difference was considered to be incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- There were no test substance-related alterations in organ weights.
- Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a test substance-related effect on final body weight. For the 100 mg/m3 group males, mean adrenal gland weights (absolute and relative to brain weight) were lower, but the mean adrenal gland to body weight ratio was not statistically significantly lower. The mean final body weight was lower for this group, and this contributed to the lower adrenal gland weight values. Mean liver weights (absolute and relative to final body and brain weights) were also lower for the 100 mg/m3 group males, but these were affected by the lower final body weights in this male group. The mean liver weight relative to final body weight, which is a more appropriate permutation of the liver weight parameters, was very similar to the value in the 1 mg/m3 group indicating that there really were no differences in these relative weights. Adrenal gland, liver, and final body weights were not significantly altered in the 100 mg/m3 group females. Additionally, the adrenal gland weight values fell within the historical control reference ranges and there were no test substance-related microscopic changes in the livers of the 100 mg/m3 group males.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related macroscopic findings at the scheduled necropsy. All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test substance exposure.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were present in the nasal cavity of males and females exposed to the test substance, but not in the larynx, trachea, lung, liver, or kidney. Nasal cavity changes tended to be more frequent in anterior portions of the nasal cavity (nasal levels II and III) and were also more frequent in males than in females.
Epithelial inflammation and degeneration were most prominent at nasal levels II (1, 10, and 100 mg/m3 group males and females) and III (1, 10, and 100 mg/m3 group males and 100 mg/m3 group females) and showed reduced incidence and severity in the posterior nasal levels (IV, V, and/or VI) in the 1, 10, and/or 100 mg/m3 group males and 10 and/or 100 mg/m3 group females.
- There were no other test substance-related histologic changes. The nasal-associated lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT) were unaffected by test substance exposure. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
LOAEL
Effect level:
1 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels. Therefore, a no-observed-effect level (NOEL) could not be determined for this study.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 mg/m³ air (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

None

Conclusions:
Results showed histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels up to 100 mg/m3. There were no test substance-related microscopic effects in the lower respiratory tract tissues (lungs, trachea, or larynx) or in the liver and kidneys, and there were no toxicologically significant effects on BALF cytokine levels. Therefore a NOEL could not be determined for this study and the LOAEL is 1 mg/m3, based on the microscopic findings in the nasal tissues at all exposure levels.
Executive summary:

In a repeated dose toxicity study conducted according to the OECD Guideline 412 and in compliance with GLP, test material was administered by inhalation-aerosol to groups of Sprague-Dawley rats (10 animals/sex/dose) at the concentrations of 1, 10 and 100 mg/m3(0.15, 1.5, and 14.9 ppm, respectively) for 6 hours per day, 5 days per week for 2 weeks (10 total exposures). A concurrent control group was exposed to humidified, filtered air on a comparable regimen. Examinations during the study included: mortality, clinical observation, body weight change, food consumption, laboratory investigations: haematology, blood clinical chemistry, Bronchoalveolar lavage fluid (BALF) clinical pathology and serum and BALF cytokine evaluation, gross pathology, organ weights and histopathology.

 

Prior to the main study, a preliminary 3-day exposure (6 hours/day) was run at 750 and 300 mg/m3 (89.4 and 44.7 ppm, respectively) to allow selection of appropriate exposure concentrations. At 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period (-14 and -10% for males and females, respectively, compared to day 0) and significant clinical observations included rales and laboured respiration in 1/3 males and 2/3 females. At 300 mg/m3, the mean body weight was decreased by 8% and 3 % compared to the study day 0 values for males and females, respectively. At both concentrations, no animals died. Therefore, one third of the lowest concentration tested in the preliminary exposures, 100 mg/m3 (i.e.14.9 ppm), was chosen as the high exposure level for the main two-week study.

 

There were no test substance related effects on survival, clinical observations, haematology, coagulation, bronchoalveolar lavage clinical chemistry or cytology, serum cytokines, macroscopic findings, or organ weights. There were no toxicologically significant effects on body weight gain, food consumption, or BALF cytokine levels. The only test substance-related changes in BALF cytokine levels were the lower concentrations of sICAM in the 10 and 100 mg/m3group animals and lower concentrations of RANTES in the 100 mg/m3 group animals. No test substance-related microscopic findings were observed in the lungs, trachea, larynx, liver, or kidneys. Portal of entry effects suggestive of irritation were present in the nasal cavity of males and females exposed to test material. Epithelial inflammation and degeneration of the nasal cavity were noted in the 1, 10, and 100 mg/m3 group males and females. A dose-relationship was evident as higher incidences and severity of degeneration and subacute inflammation in males and females affecting transitional epithelium (nasal level II) at the 10 and 100 mg/m3 exposure levels, compared with effects at the 1 mg/m3 exposure. Based on severity and combined incidences for males and females, a similar test substance-relationship was evident for subacute inflammation of the respiratory epithelium at nasal level III. The findings were most prominent at nasal levels II and III and showed reduced incidence and severity in the posterior nasal levels (IV-VI), and were generally more frequent in males than in females at all exposure and nasal cavity levels. All nasal cavity findings were minimal to mild in severity and would be considered reversible with removal of the irritant. 

 

Results showed histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels up to 100 mg/m3. There were no test substance-related microscopic effects in the lower respiratory tract tissues (lungs, trachea, or larynx) or in the liver and kidneys, and there were no toxicologically significant effects on BALF cytokine levels.

 

Therefore a NOEL could not be determined for this study and the LOAEL is 1 mg/m3, based on the microscopic findings in the nasal tissues at all exposure level.

 

Based on the nasal irritation, test material is classified as H335: May cause respiratory irritation according to the Regulation (EC) No. 1272/2008 (CLP).

 

This study is considered as acceptable and satisfies the requirement for repeated dose inhalation toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEC
1 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study of very high quality (Klimisch score = 1).
System:
respiratory system: upper respiratory tract

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 20, 2011 to February 07, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: Stored at room temperature and was considered stable under these conditions
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 187-240 g for males; 149-184 g for females at randomization
- Housing: Animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet: Basal diet; PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, ad libitum
- Food and water were withheld during each daily exposure period.
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70.4-71.5 °F (21.3-21.9 °C)
- Humidity: 36.6-52.6 %
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: September 20, 2011 To: February 07, 2012
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1.3 - <= 2.2 µm
Remarks on MMAD:
Mean MMAD: 1.4, 1.3 and 2.2 µm for 1, 10 and 100 mg/m3, respectively
Mean GSD: 3.59, 4.00 and 2.30 for 1, 10 and 100 mg/m3, respectively
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 7.9-L conventional nose-only exposure systems with synthetic rubber grommets in exposure ports to engage animal holding tubes.
- Method of holding animals in test chamber: To perform nose-only exposure, it is necessary to restrain the rats in specially designed nose-only holding tubes. The 6-hour period of restraint was necessary to achieve the exposure duration.
- Source and rate of air: HEPA- and charcoal-filtered air source (WIL Research Inhalation Department supply air source) or breathing-quality in-house compressed air source.
- Method of conditioning air: Charcoal- and HEPA-filters
- System of generating aerosols: Aerosolized test substance was generated using a Collison nebulizer.
- Temperature, humidity in air chamber: Mean temperature ranged from 20-21 °C and the mean relative humidity ranged from 43-56 %.
- Air flow rate: Daily mean ventilation rate ranged from 44.3-47.9 liters per minute (LPM).
- Method of particle size determination: Aerosol particle size determinations were conducted for each test substance exposure system using a 7-stage stainless steel cascade impactor.
- Treatment of exhaust air: All test substance atmosphere exposure system exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consisted of redundant exhaust blowers proceeded by charcoal- and HEPA-filters.

TEST ATMOSPHERE
- Brief description of analytical method used:
Nominal exposure concentrations: Due to the small amount of test substance that was required for the study, a nominal concentration was not calculated.
Actual exposure concentrations: Actual aerosol mass concentrations of the test atmospheres for exposure systems 1, 3, and 4 (control, 10 and 100 mg/m3) were determined using standard gravimetric methods. Due to the volatility of the test material, standard gravimetric methods cannot be used for the analysis of the atmosphere in the Group 2 exposure system.
Analysed exposure concentrations: Analysed exposure concentrations of the total test material (combined vapour and aerosol) within exposure system 2 were determined at approximately 60-minute intervals using an appropriate gas chromatography (GC) method and were expressed as mg/m3.
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample of the test substance was collected from prior to the initiation of exposure for purity and concentration analyses. Analyses of purity and stability were performed using a similar method from a sample collected at the end of the exposure period. All analyses were conducted using a gas chromatography method with flame ionization detection.
Results: Analyses conducted for a sample of the test substance collected prior to exposure initiation resulted in a purity of 90.8%. Following the completion of the exposure phase, another sample was collected that resulted in a purity of 89.3%. Comparison of these purity data indicated that the test substance was stable (98.3% of the initial test substance concentration after up to 34 days), based on the acceptance criteria for stability (i.e., the mean post-storage concentration was not <90% of the pre-storage value).
Mean exposure concentrations were 1.28, 9.8 and 98 mg/m3 for the target exposure concentrations 1, 10 and 100 mg/m3, respectively.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 days per week for 2 weeks (10 total exposures)
Dose / conc.:
1 mg/m³ air (nominal)
Remarks:
0.15 ppm
Dose / conc.:
10 mg/m³ air (nominal)
Remarks:
1.5 ppm
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
14.9 ppm
No. of animals per sex per dose:
10
Control animals:
other: filtered air (control group)
Details on study design:
- Dose selection rationale: Prior to exposure of animals for the main study, preliminary animal exposures were conducted at 750 and then at 300 mg/m3 (89.4 and 44.7 ppm, respectively) for three days to assess tolerability of the test substance. At 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period (-14 and -10% for males and females, respectively, compared to day 0) and significant clinical observations included rales and laboured respiration in 1/3 males and 2/3 females. At 300 mg/m3, the mean body weight was decreased by 8% and 3 % compared to the study day 0 values for males and females, respectively. Therefore, one third of the lowest concentration tested in the preliminary exposures, 100 mg/m3 (i.e.14.9 ppm), was chosen as the high exposure level for the main two-week study.
- Rationale for animal assignment: Animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design.
Positive control:
None
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed daily, prior to exposure, during exposure (at the approximate midpoint for visible signs of animals in nose-only exposure restraint tubes), and 0 to 1 h following the end of exposure. The absence or presence of findings was recorded for individual animals at observations conducted prior to exposure and following the end of exposure. Only significant findings were recorded at the approximate midpoint of exposure observations. On non-exposure days, the animals were observed once daily.
- Survival: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity, except on the days of the scheduled necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were conducted on all animals during pretest prior to randomization, at the time of randomization prior to group assignment, and approximately weekly during the exposure period, including prior to the scheduled necropsy. Observations conducted prior to exposure were not performed on days when detailed physical examinations were conducted, provided they were conducted prior to
exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded during the pretest period, at randomization, prior to the first exposure, and twice weekly during the exposure period (prior to the first and last exposure each 5-day exposure week) including the day before the first scheduled day of necropsy (nonfasted).
- Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION:
- Individual food consumption was recorded during the pretest period (prior to randomization) and twice weekly during the exposure period. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected at the scheduled necropsy (study day 12).
- Anaesthetic used for blood collection: Yes; Blood was collected for haematology and standard serum chemistry evaluations via the retro-orbital sinus and for coagulation parameters via the vena cava from animals anesthetized by inhalation of isoflurane at the scheduled necropsy. (inhalation of isoflurane)
- Animals fasted: Yes; animals were fasted overnight prior to blood collection.
- How many animals: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from all animals, and blood samples for coagulation evaluations were collected from 5 animals/sex/group (Subgroup 1).
- Parameters checked:
HAEMATOLOGY AND COAGULATION: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Haemoglobin Distribution Width (HDW), Differential leukocyte count - Percent and absolute (Neutrophil (NEU), Lymphocyte (LYMPH),Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC)), Platelet estimate, Red cell morphology (RBC Morphology)
CLINICAL CHEMISTRY: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio), [by calculation], Total bilirubin (Total Bili) Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Sorbitol dehydrogenase (SDH) and Lactate dehydrogenase (LDH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Animals were anesthetized by isoflurane inhalation and euthanized by exsanguination. For 5 animals/sex/group (BALF animals), a BAL was performed on the lungs as soon as possible after exsanguination and after completion of the BAL, all BALF animals were subjected to necropsy. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Tissues or gross lesions were not collected from the BALF animals (Subgroup 2).

HISTOPATHOLOGY: Yes; the following tissues and organs were collected from the histopathology animals (remaining 5 animals/sex/group; Subgroup 1) and placed in 10 % neutral-buffered formalin for examination:
Adrenals (2), Aorta, Bone with marrow, Femur (with joint)[a], Sternebrae, Bone marrow smear (from femur), Brain with olfactory blub - Cerebrum level 1, Cerebrum level 2 , Cerebellum with medulla/pons, Cervix, Epididymides (2)[b], Eyes with optic nerve (2) [c], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Harderian glands (2), Heart, Kidneys (2), Lacrimal gland (exorbital [2]), Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by constant pressure inflation with fixative), Lymph nodes - Auricular (2 [if visible]), Axillary (2), Bronchial (if visible), Mandibular (2), Mediastinal and bronchial (if visible), Mesenteric, Mammary glands (females only) [d], Nasal cavity (with turbinates) [e], Olfactory bulbs [e], Ovaries with oviducts (2) [f], Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands (mandibular [2]), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin, Spinal cord (cervical, lumbar, thoracic), Spleen, Testes (2), Thymus, Thyroid (with parathyroids [2]) [f], Tongue, Trachea, Urinary bladder, Uterus, Vagina and Gross lesions

a = Bone marrow smears were obtained at scheduled necropsy from the femur, but not placed in formalin; slides were not examined.
b = Fixed in Bouin’s solution
c = Fixed in Davidson’s solution
d = A corresponding section of skin was taken from the same anatomical area for males.
e = Following the collection of the appropriate protocol-specified tissues, the entire head was removed and preserved (olfactory bulbs were severed from the brain and remained in the skull). Following decalcification, 6 cross-sections of the nasal cavities were prepared for microscopic examination in accordance with the method described by Morgan (1991) and Mery et al. (1994).
f = Oviducts and parathyroids were examined microscopically when in the plane of section and in all cases where a gross lesion was present.

After fixation, lungs with bronchi, bronchus associated lymphoid tissues [BALT], nasal tissues, nasal-associated lymphoid tissue [NALT], larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions) were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with haematoxylin and eosin. Microscopic examination of the lungs with bronchi, BALT, nasal tissues, NALT, larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions were performed for 5 animals/sex/group (Subgroup 1) not assigned for the BAL at the scheduled necropsy.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsy:
Adrenals, Brain, Heart, Kidneys, Liver, Lungs (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids*

Paired organs were weighed together. Designated (*) organs were weighed after fixation. Organ to final body weight and organ to brain weight ratios were calculated.
Other examinations:
Bronchoalveolar lavage fluid (BALF) clinical pathology and serum and BALF cytokine evaluation:
Prior to the scheduled necropsy, the animals were fasted overnight. At the time of necropsy, the 5 animals/sex/group selected for serum cytokine evaluations (Subgroup 2) were anesthetized by inhalation of isoflurane. Blood samples were collected from the vena cava into tubes containing no anticoagulant. A bronchoalveolar lavage (BAL) was performed on the lungs of 5 animals/sex/group (Subgroup 2) at the scheduled necropsy (study day 12).

BALF chemistry and cytology
The following parameters were evaluated:
Total and differential cell counts for: Alveolar macrophages, Neutrophils, Lymphocytes, Eosinophils, Basophils
Lactate dehydrogenase (LDH), Total protein, Alkaline Phosphatase

Serum and BALF cytokine evaluations
The following cytokines were evaluated:
TNF-α, IL-5, IL-10, ICAM-1, IFN-γ, IL-4, TGF-β, MCP-1, IL-1β, IL-13, MIP-2, RANTES

TNF = Tumour necrosis factor, IFN = Interferon, IL = Interleukin, TGF = Transforming growth factor, MIP = Macrophage inflammatory protein, ICAM = Intracellular adhesion molecule, MCP = Macrophage chemotactic protein, RANTES = Regulated upon activation, normal T-cell expressed, and presumably secreted.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Body weight, body weight change, food consumption, clinical pathology data including BALF total protein, lactate dehydrogenase, alkaline phosphatase, and cytology parameters (with the exception of gamma glutamyltransferase), and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations. All clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled necropsy
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- There were no toxicologically significant effects on body weights.
- Transient lower body weight gains were present in the 100 mg/m3 group males and females during the exposure period, reaching statistical significance during study days 0 to 4 and/or 7 to 11. Statistically significantly lower cumulative body weight gains were recorded for the 100 mg/m3 group males from study day 0 to 11. Although lower cumulative body weight gains were noted in the 100 mg/m3 group females by the end of the study, the values were not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- There were no toxicologically significant effects on food consumption.
- Transient slightly lower food consumption was noted for the 100 mg/m3 group males and females during the exposure period. Although the differences were statistically significant compared to the control group from study days 0 to 4 and/or 7 to 11, these effects on food consumption were sporadic and not considered toxicologically relevant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no clear test substance-related differences in haematology parameters when compared to the control group. Reticulocyte counts were slightly lower in the 1, 10, and 100 mg/m3 group females and 100 mg/m3 group males when compared to the control group; however, a relationship with test substance administration was not established due to the lack of dose response in females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related differences in serum clinical chemistry parameters when compared to the control group. Alanine aminotransferase (ALT) was slightly lower in the 100 mg/m3 group males when compared to the control group; however, decreases in ALT have no known toxicological meaning, and this difference was considered to be incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- There were no test substance-related alterations in organ weights.
- Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a test substance-related effect on final body weight. For the 100 mg/m3 group males, mean adrenal gland weights (absolute and relative to brain weight) were lower, but the mean adrenal gland to body weight ratio was not statistically significantly lower. The mean final body weight was lower for this group, and this contributed to the lower adrenal gland weight values. Mean liver weights (absolute and relative to final body and brain weights) were also lower for the 100 mg/m3 group males, but these were affected by the lower final body weights in this male group. The mean liver weight relative to final body weight, which is a more appropriate permutation of the liver weight parameters, was very similar to the value in the 1 mg/m3 group indicating that there really were no differences in these relative weights. Adrenal gland, liver, and final body weights were not significantly altered in the 100 mg/m3 group females. Additionally, the adrenal gland weight values fell within the historical control reference ranges and there were no test substance-related microscopic changes in the livers of the 100 mg/m3 group males.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related macroscopic findings at the scheduled necropsy. All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test substance exposure.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were present in the nasal cavity of males and females exposed to the test substance, but not in the larynx, trachea, lung, liver, or kidney. Nasal cavity changes tended to be more frequent in anterior portions of the nasal cavity (nasal levels II and III) and were also more frequent in males than in females.
Epithelial inflammation and degeneration were most prominent at nasal levels II (1, 10, and 100 mg/m3 group males and females) and III (1, 10, and 100 mg/m3 group males and 100 mg/m3 group females) and showed reduced incidence and severity in the posterior nasal levels (IV, V, and/or VI) in the 1, 10, and/or 100 mg/m3 group males and 10 and/or 100 mg/m3 group females.
- There were no other test substance-related histologic changes. The nasal-associated lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT) were unaffected by test substance exposure. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
LOAEL
Effect level:
1 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels. Therefore, a no-observed-effect level (NOEL) could not be determined for this study.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 mg/m³ air (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

None

Conclusions:
Results showed histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels up to 100 mg/m3. There were no test substance-related microscopic effects in the lower respiratory tract tissues (lungs, trachea, or larynx) or in the liver and kidneys, and there were no toxicologically significant effects on BALF cytokine levels. Therefore a NOEL could not be determined for this study and the LOAEL is 1 mg/m3, based on the microscopic findings in the nasal tissues at all exposure levels.
Executive summary:

In a repeated dose toxicity study conducted according to the OECD Guideline 412 and in compliance with GLP, test material was administered by inhalation-aerosol to groups of Sprague-Dawley rats (10 animals/sex/dose) at the concentrations of 1, 10 and 100 mg/m3(0.15, 1.5, and 14.9 ppm, respectively) for 6 hours per day, 5 days per week for 2 weeks (10 total exposures). A concurrent control group was exposed to humidified, filtered air on a comparable regimen. Examinations during the study included: mortality, clinical observation, body weight change, food consumption, laboratory investigations: haematology, blood clinical chemistry, Bronchoalveolar lavage fluid (BALF) clinical pathology and serum and BALF cytokine evaluation, gross pathology, organ weights and histopathology.

 

Prior to the main study, a preliminary 3-day exposure (6 hours/day) was run at 750 and 300 mg/m3 (89.4 and 44.7 ppm, respectively) to allow selection of appropriate exposure concentrations. At 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period (-14 and -10% for males and females, respectively, compared to day 0) and significant clinical observations included rales and laboured respiration in 1/3 males and 2/3 females. At 300 mg/m3, the mean body weight was decreased by 8% and 3 % compared to the study day 0 values for males and females, respectively. At both concentrations, no animals died. Therefore, one third of the lowest concentration tested in the preliminary exposures, 100 mg/m3 (i.e.14.9 ppm), was chosen as the high exposure level for the main two-week study.

 

There were no test substance related effects on survival, clinical observations, haematology, coagulation, bronchoalveolar lavage clinical chemistry or cytology, serum cytokines, macroscopic findings, or organ weights. There were no toxicologically significant effects on body weight gain, food consumption, or BALF cytokine levels. The only test substance-related changes in BALF cytokine levels were the lower concentrations of sICAM in the 10 and 100 mg/m3group animals and lower concentrations of RANTES in the 100 mg/m3 group animals. No test substance-related microscopic findings were observed in the lungs, trachea, larynx, liver, or kidneys. Portal of entry effects suggestive of irritation were present in the nasal cavity of males and females exposed to test material. Epithelial inflammation and degeneration of the nasal cavity were noted in the 1, 10, and 100 mg/m3 group males and females. A dose-relationship was evident as higher incidences and severity of degeneration and subacute inflammation in males and females affecting transitional epithelium (nasal level II) at the 10 and 100 mg/m3 exposure levels, compared with effects at the 1 mg/m3 exposure. Based on severity and combined incidences for males and females, a similar test substance-relationship was evident for subacute inflammation of the respiratory epithelium at nasal level III. The findings were most prominent at nasal levels II and III and showed reduced incidence and severity in the posterior nasal levels (IV-VI), and were generally more frequent in males than in females at all exposure and nasal cavity levels. All nasal cavity findings were minimal to mild in severity and would be considered reversible with removal of the irritant. 

 

Results showed histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels up to 100 mg/m3. There were no test substance-related microscopic effects in the lower respiratory tract tissues (lungs, trachea, or larynx) or in the liver and kidneys, and there were no toxicologically significant effects on BALF cytokine levels.

 

Therefore a NOEL could not be determined for this study and the LOAEL is 1 mg/m3, based on the microscopic findings in the nasal tissues at all exposure level.

 

Based on the nasal irritation, test material is classified as H335: May cause respiratory irritation according to the Regulation (EC) No. 1272/2008 (CLP).

 

This study is considered as acceptable and satisfies the requirement for repeated dose inhalation toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
1 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study of very high quality (Klimisch score = 1).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral:

The repeated dose toxicity of Isoeugenol was investigated across several key studies performed to assess carcinogenicity and for toxicity to reproduction, in rats and/or mice exposed by oral (gavage) route.

 

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

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

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

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

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

 

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

 

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

 

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

In a repeated dose toxicity study used as range-finding for carcinogenicity study and performed similar to OECD test Guideline No. 408 and in compliance with GLP, groups of F344/N rats (10/sex/dose) were exposed to trans-Isoeugenol in corn oil by gavage at 37.5, 75, 150, 300 and 600 mg/kg bw/day, 5 days per week for 14 weeks. Control rats were given the vehicle alone. Clinical signs and bodyweight development were monitored during the study. Haematology and clinical chemistry were performed. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed in control and high dose group.

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

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

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

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

 

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

 

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

 

Carcinogenicity study in rats (NTP, 2010):

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

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

 

Non-neoplastic effects:

Nose:

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

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

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

 

Equivocal findings:

Thymus:

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

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

 

Mammary gland:

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

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

 

Conclusions:

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

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

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

 

Carcinogenicity study in mice (NTP, 2010):

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

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

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

Non-neoplastic effects:

Nose:

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

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

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

 

Forestomach:

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

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

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

 

Glandular stomach:

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

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

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

 

Kidney:

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

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

 

Neoplastic effects in males:

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

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

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

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

 

Equivocal findings in females:

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

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

 

Conclusions:

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

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

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

 

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

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

 

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

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

 

Table 7.5: Overall NOAEL and LOAEL for repeated dose toxicity identified across key studies

Study Type

Species

Route of exposure

Type of effect

NOAEL (mg/kg bw/day)

LOAEL

(mg/kg bw/day

Basis

90-day

Rat

Gavage

Systemic

F: 150

M: 300

F: 300

M: 600

F: Increased hyperplasia in liver at 300 and 600.

 

M: Reduced body weight and body weight gain of 11.6% at 600.

Local

M: 75

F: 150

M: 150

F: 300

Histological changes in olfactory epithelium

90-day

Mouse

Gavage

Systemic

M: 300

F: 600 (highest-dose)

M: 600 (highest-dose)

M: Reduced body weight and body weight gain at 600

Local

M/F: 300

M/F: 600 (highest-dose)

Histological changes in olfactory epithelium

Carcinogenicity

Rat

Gavage

Systemic

M: 300

F: 300 (highest-dose

 

None

Local

Not identified

M/F: 75 (lowest-dose)

Dose-related effects on the nasal epithelium

Carcinogenicity

Mouse

Gavage

Systemic

F/M: 150*

F/M: 300 (highest-dose

F: adverse effects in kidney + reduced body weight gain of 10% at 300

 

M: reduced body weight gain of 15% at 300.

 

Local

Not identified

M/F: 75 **(lowest-dose)

Dose-related effects on the nasal epithelium

Two-generation reproduction toxicity study

Rat

Gavage

Systemic

Not identified

M/F: 70 (lowest dose)

Decreased body weight gains + hyperkeratosis and hyperplasia in non-glandular stomachs at all dose levels

Pre-natal developmental toxicity study

 

Rat

Gavage

Systemic

Not identified

Female: 250 (lowest dose)

Dose-dependent reduced gestation body weight gain

 

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

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

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

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

 

**Relevant Local LOAEL determined as the lowest concentration at which effects were observed in the whole database.

 

 

In conclusion, results from the carcinogenicity study conducted in mouse allowed to identify the most relevant systemic NOAEL at 150 mg/kg bw/d for repeated dose toxicity of Isoeugenol. This value was used to extrapolate the different DNELs for worker and general population.

 

Inhalation:

A key study was identified (WIL, 2012, rel.1). In this repeated dose toxicity study conducted according to the OECD Guideline 412 and in compliance with GLP, test material was administered by inhalation-aerosol to groups of rats at the concentrations of 1, 10 and 100 mg/m3 for 6 hours per day, 5 days per week for 2 weeks. A concurrent control group was exposed to humidified, filtered air on a comparable regimen. A preliminary 3-day exposure study (6 hours/day) was run at 750 and 300 mg/m3 to allow selection of appropriate exposure concentrations.

 

There were no test substance related effects on survival, clinical observations, haematology, coagulation, bronchoalveolar lavage clinical chemistry or cytology, serum cytokines, macroscopic findings, or organ weights at 1, 10 and 100 mg/m3.

There were no toxicologically significant effects on body weight gain, food consumption, or BALF cytokine levels. The only test substance-related changes in BALF cytokine levels were the lower concentrations of sICAM in the 10 and 100 mg/m3group animals and lower concentrations of RANTES in the 100 mg/m3 group animals. No test substance-related microscopic findings were observed in the lungs, trachea, larynx, liver, or kidneys. Portal of entry effects suggestive of irritation were present in the nasal cavity of males and females exposed to test material. Epithelial inflammation and degeneration of the nasal cavity were noted in the 1, 10, and 100 mg/m3 group males and females. A dose-relationship was evident as higher incidences and severity of degeneration and subacute inflammation in males and females affecting transitional epithelium (nasal level II) at the 10 and 100 mg/m3 exposure levels, compared with effects at the 1 mg/m3 exposure. Based on severity and combined incidences for males and females, a similar test substance-relationship was evident for subacute inflammation of the respiratory epithelium at nasal level III. The findings were most prominent at nasal levels II and III and showed reduced incidence and severity in the posterior nasal levels (IV-VI), and were generally more frequent in males than in females at all exposure and nasal cavity levels. All nasal cavity findings were minimal to mild in severity and would be considered reversible with removal of the irritant.

 

Therefore, a no observed adverse effect concentration (NOAEC) could not be determined for this study but a LOAEC of 1 mg/m3 for local effects was determined based on the microscopic findings in the nasal tissues at all exposure level.

 

 

Reference

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

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

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