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Description of key information

A sub-chronic (90 days) oral toxicity study in the rat was performed during September 1989 until May 1990 according to OECD Guideline 408, Guideline EPA OTS 798.2650 and GLP. Groups of 10 male and 10 female rats were given 0, 50, 150, or 500 mg/kg of AAEM by oral gavage. The results of this oral gavage study indicate that the test item has no potential to produce toxic effects when administered to rats at dosage of 500 mg/kg/day. Based on the lack of treatment-related effects in clinical signs, ophthalmic examinations, feed consumption, weight gain, clinical pathology, organ weights, gross pathology, microscopic pathology, and FOB results, the no-observed-effect level (NOEL) for sub-chronic exposure to AAEM was considered to be 500 mg/kg/day for both male and female rats when administered 5 days per week for 13 weeks.

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11/September/1989 - 06/May/1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conduct in accordance with international guidelines.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals
Male and female rates (CD (SD)BR)
Source: Charles River Laboratories, Kingston, NY
Age at study initiation: 46 (male) and 51 (female) days old
Weight at study initiation: Male: 235 to 243 g - Female: 179 to 186 grams
Females were nulliparous and nonpregnant.
Housing: individually housed during experiment in stainless steel wire mesh cages.
Ten male and ten female rats were randomly assigned to each of three test groups and one control group.
Identification: by uniquely numbered metal ear tags

Environmental conditions:
Temperature (°C): 21-23 °C.
Humidity (%): 40-58 %
Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

Diet: Agway Prolab Animal diet (RMH 3200) - ad libitum
Water: ad libitum
Acclimation period: 14 days
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Doses were administered daily, five days per week for 13 weeks
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material was analyzed for purity and stability by HPLC prior to and during the study period. HPLC analysis determined that the test
material was of 100% purity prior to and during the test.

A stability and dose verification was performed on the test chemical in corn oil. The test substance was analyzed by HPCL, Perkin Elmer. The lower limit of detection was determined to be 0.42 gm/mL %.
Duration of treatment / exposure:
90 days (13 weeks)
Frequency of treatment:
5 days per week
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
other: corn oil
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
other: corn oil
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
other: corn oil
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
other: corn oil
No. of animals per sex per dose:
15/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Three test groups and a control group were given either corn oil or graded doses of AAEM in corn oil by gavage. The test solutions were prepared
so that animals from all groups received equal volumes of solution on a mg/kg basis. Controls received doses of corn oil at the same dose factor used for the test groups. Doses were administered daily, five days per week for 13 weeks, and were adjusted on Days 4 and 7, and weekly thereafter for
changes in body weight. The gavage route of administration was used since it is an accepted method of administering test materials for toxicity
studies. The dose levels used in the 13-week study were selected based on results from a preliminary study.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
On all work day mornings, each rat was removed from its cage and examined. Immediately after dosing and again in the afternoon,
cageside observations were conducted. Observations included, but were not limited to, examination of behavior patterns, motor activity, respiratory
patterns, hair, skin, eyes, feces, and urine. On weekends, rats were observed for mortality. When the Functional Observational Battery (FOB) was performed, an entry was made in the clinical observation record.

BODY AND FEED WEIGHT DETERMINATION:
Body weights were collected on Days -3 (pretreatment), 0, 1, 4, 7, and at least weekly thereafter. Feed consumption was determined on Days 0, 4, 7,
and at least weekly thereafter.

HAEMATOLOGY AND CLINICAL CHEMISTRY EXAMINATIONS:
Hematology and clinical chemistry tests were performed on blood from all rats. At the time of necropsy, blood was collected from the right ventricle
while the rats were under sodium pentobarbital anesthesia. Blood was collected after thoracotomy and just prior to systemic perfusion.
Hematology tests include: hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, differential white blood cell count,
platelet count, red blood cell indices, and examination of the blood smears for cellular morphology. Clinical chemistry tests include: aspartate
aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, gamma glutamyl transpeptidase, triglycerides, cholesterol, creatinine, urea nitrogen, glucose, total bilirubin, total protein, albumin, A/G ratio, sodium, potassium, chloride, calcium, and phosphorus. Because the animals were heparinized prior to blood collection, clinical chemistry results were determined using plasma.

OPHTHALMOSCOPIC EXAMINATION:
The eyes of all rats were examined by indirect ophthalmoscopy prior to the start of the study. During the last week of exposure, the animals from the
control and high-dose groups were examined. Since no abnormalities were observed in the high-dose group, the low- and middle-dose groups were not examined for ocular effects at the end of the study.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
A Functional Observational Battery (FOB) was performed on all animals pretreatment (Day -3) ,and 1, 7, 14, 28, 56, and 91 days after the
administration of the initial dose. The battery includes procedures to detect unusual body position or activity level, compromised
coordination of movement or gait, and any unusual or bizarre behavior.

Sacrifice and pathology:
PATHOLOGY EXAMINATIONS:
At the end of the exposure period, all rats from each group were perfused systemically with aldehyde fixatives. The animals were anesthetized with
sodium pentobarbital containing heparin (10% by volume) and perfused through the ascending aorta. Each rat received a complete gross examinationat necropsy. Histopathology was performed on the organs and tissues for all the animals in the control and high-dose groups.
Histopathology was performed on gross lesions from all dose groups. Tissues for neuropathology examination included: cross sections through the
forebrain, cerebrum, midbrain, cerebellum, pons, medulla oblongata, cervical spinal cord swelling, lumbar spinal cord swelling, cervical and lumbar dorsal root ganglia, dorsal and ventral spinal roots (cervical and lumbar), sciatic nerve at mid-thigh, and tibial nerve. Spinal cord and peripheral nerve
sections were also cut in the longitudinal plane. High-dose and control tissues were processed by routine neuropathological techniques for paraffin
embedment, hematoxylin-eosin staining, and light microscopic examination.
Statistics:
Mean values with standard deviations were calculated where appropriate on quantitative data. Data were evaluated using the following computer-generated statistical tests: Bartlett's test (p < 0.01), one-way analysis of variance (ANOVA) (p < 0.05), and Duncan's multiple range test (p < 0.05) to indicate statistical significance.

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
No treatment-related clinical abnormalities were observed for any male or female AAEM-dosed group. Other clinical abnormalities are described below.
Five male rats [two control (Rats 555 & 556), two 50 mg/kg (Rats 562 & 568), one 500 mg/kg (Rat 558)] and one control female rat (Rat 608) developed incisor malocclusion or fractures during the study. Additionally, one 50 mglkg male rat (Rat 562:) had an open wound on the palate and one 500 mg/kg female rat (Rat 635) had a raised area on the palate. Clinical signs associated with dental or palate abnormalities consisted of porphyrin tears, porphyrin nasal discharge, swollen muzzle, urine staining of the inguinal haircoat, unkempt haircoat, and poor body condition.
Abnormal clinical signs were sporadically observed, but all were independent of dose and number of exposures at the time of their appearance, and were therefore considered unrelated to the test material.

MORTALITY
No mortality occurred during the study.

BODY WEIGHT AND FEED CONSUMPTION
Mean body weights for male and female rats from the all dose groups were comparable to their respective controls throughout the study.
A slight reduction in mean daily feed consumption was observed in male rats from all dose groups including the control, starting at
Day 63; this reduction most likely represented a decreased nutritional requirement due to a plateauing of the body weight of the male rats.
The feed consumption differences that were observed were not considered to be related to AAEM exposure.
OPHTHALMOSCOPIC EXAMINATION
All rats were examined prior to the start of the study to insure that no animals had ophthalmic abnormalities. When animals from the control and
500 mg/kg groups were reexamined during the last week of treatment, no abnormalities were observed. Based on the lack of ophthalmic effects in the
500 mg/kg group, animals from the 50 and 150 mg/kg groups were not reexamined.

FUNCTIONAL OBSERVATION BATTERY (FOB)
The FOB examinations performed on all animals prior to the first treatment with AAEM and all FOB examinations on all control animals throughout thestudy were unremarkable.

QUANTITATIVE GRIP STRENGTH
During the pre-exposure FOB examination, the mean hindlimb grip strength for the female rats from the 150 mg/kg group was statistically significantly lower than that of the control group. There were no other statistically significant differences in mean forelimb or mean hindlimb grip strength between the treatment and control groups for the remainder of the study.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Differences between groups in haematology and clinical chemistry were limited to a slightly higher mean value for one of the red blood cell indices
in the 50 and 500 mg/kg female groups, and a slightly higher mean plasma chloride level in the 150 and 500 mg/kg female groups. Neither of the differences was dose-dependent.

ORGAN WEIGHTS
All mean absolute, relative organ/body weight, and relative organ/brain weights for treated male and female rats were comparable to the respective
mean control values.

PATHOLOGY EXAMINATION
No treatment-related lesions were identified at necropsy or on histological examination of tissues. No AAEM-related lesions were detected during the
neuropathology examinations.
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Because of the lack of treatment-related effects on clinical signs, ophthalmic examinations, feed consumption, weight gain, clinical pathology, organ weights, gross or microscopic pathology, neuropathology and the FOB.
Critical effects observed:
not specified

Dose levels for this study were based on available data and a preliminary study using the same route of exposure (oral gavage). The acute oral LD50 for AAEMA is >5000 mg/kg/bw in male/female rats.

Conclusions:
The results of this oral gavage study indicate that the test item has no potential to produce toxic effects when administered to rats at dosage of
500 mg/kg/day. Based on the lack of treatment-related effects in clinical signs, ophthalmic examinations, feed consumption, weight gain, clinical pathology, organ weights, gross pathology, microscopic pathology, and FOB results, the no-observed-effect level (NOEL) for subchronic exposure to
AAEM was considered to be 500 mg/kg/day for both male and female rats when administered 5 days per week for 13 weeks.
Executive summary:

The subchronic oral toxicity study in the rat was performed during September 1989 until May 1990 according to OECD Guideline 408, Guideline EPA OTS 798.2650 and GLP. The results of this oral gavage study indicate that the test item has no potential to produce toxic effects when administered to rats at dosage of 500 mg/kg/day. Based on the lack of treatment-related effects in clinical signs, ophthalmic examinations, feed consumption, weight gain, clinical pathology, organ weights, gross pathology, microscopic pathology, and FOB results, the no-observed-effect level (NOEL) for subchronic exposure to AAEM was considered to be 500 mg/kg/day for both male and female rats when administered 5 days per week for 13 weeks.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A sub-chronic (90 days) oral toxicity study in the rat was performed during September 1989 until May 1990 according to OECD Guideline 408, Guideline EPA OTS 798.2650 and GLP. No treatment-related clinical abnormalities were observed for any male or female AAEM-dosed group. Five male rats [two control (Rats 555 & 556), two 50 mg/kg (Rats 562 & 568), one 500 mg/kg (Rat 558)] and one control female rat (Rat 608) developed incisor malocclusion or fractures during the study. Additionally, one 50 mg/kg male rat (Rat 562) had an open wound on the palate and one 500 mg/kg female rat (Rat 635) had a raised area on the palate. Clinical signs associated with dental or palate abnormalities consisted of porphyrin tears, porphyrin nasal discharge, swollen muzzle, urine staining of the inguinal haircoat, unkempt haircoat, and poor body condition. Abnormal clinical signs were sporadically observed, but all were independent of dose and number of exposures at the time of their appearance, and were therefore considered unrelated to the test material. No mortality occurred during the study. Mean body weights for male and female rats from the all dose groups were comparable to their respective controls throughout the study. A slight reduction in mean daily feed consumption was observed in male rats from all dose groups including the control, starting at Day 63; this reduction most likely represented a decreased nutritional requirement due to a plateauing of the body weight of the male rats. The feed consumption differences that were observed were not considered to be related to AAEM exposure. All rats were examined prior to the start of the study to insure that no animals had ophthalmic abnormalities. When animals from the control and 500 mg/kg groups were reexamined during the last week of treatment, no abnormalities were observed. Based on the lack of ophthalmic effects in the 500 mg/kg group, animals from the 50 and 150 mg/kg groups were not reexamined. The FOB examinations performed on all animals prior to the first treatment with AAEM and all FOB examinations on all control animals throughout the study were unremarkable. During the pre-exposure FOB examination, the mean hindlimb grip strength for the female rats from the 150 mg/kg group was statistically significantly lower than that of the control group. There were no other statistically significant differences in mean forelimb or mean hindlimb grip strength between the treatment and control groups for the remainder of the study. Differences between groups in haematology and clinical chemistry were limited to a slightly higher mean value for one of the red blood cell indices in the 50 and 500 mg/kg female groups, and a slightly higher mean plasma chloride level in the 150 and 500 mg/kg female groups. Neither of the differences was dose-dependent. All mean absolute, relative organ/body weight, and relative organ/brain weights for treated male and female rats were comparable to the respective mean control values. No treatment-related lesions were identified at necropsy or on histological examination of tissues. No AAEM-related lesions were detected during the neuropathology examinations.The results of this oral gavage study indicate that the test item has no potential to produce toxic effects when administered to rats at dosage of 500 mg/kg/day. Based on the lack of treatment-related effects in clinical signs, ophthalmic examinations, feed consumption, weight gain, clinical pathology, organ weights, gross pathology, microscopic pathology, and FOB results, the no-observed-effect level (NOEL) for subchronic exposure to AAEM was considered to be 500 mg/kg/day for both male and female rats when administered 5 days per week for 13 weeks.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP compliant study conduct in accordance with international guidelines, reliable study without restictions.

Justification for classification or non-classification

Based on the data available the substance is not classified and labeled according to Regulation 1272/2008/EEC (CLP).