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Administrative data

Description of key information

Oral NOAEL (28d) rat, males and females, systemic toxicity: 75 mg/kg bw/day

Oral NOAEL (28d) rat, males and females, neurological effects: 225 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: the male rats were received from Charles River Laboratories, Inc., Raleigh, North Carolina, the female rats were received from Charles River Laboratories, Inc., St. Constant, Quebec.
- Animal Husbandry: animal housing and care were based on the standards established by the Association for Assessment and Accreditation of Laboratory Animal Care, international (AAALAC) and the guidelines set forth in the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 96-03, 1996.
- Weight at study initiation: 246-285 grams for males, 164-191 grams for females.
- Age at study initiation: 8 weeks
- Housing: animals were housed individually during acclimation and while on study in suspended stainless steel cages.
- Diet: PMI Cerified Rodent Meal #5002 (Purina Mills), ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: municipal tap water, ad libitum. The tap water was purifief by reversed osmosis and UV irradiation and supplied to the animals by an automatic watering system or water bottles. Water supplying the facility is analyzed for contaminants according to SLI Standard Operating Procedures.
- Acclimation period: the animals were acclimated to the laboratory conditions for a period of 36 days prior to initiation of dosing.
- Health check: the animals were examined upon receipt and daily thereafter during acclimation for signs of physical or behavioral abnormalities. General health/mortality checks were performed twice daily, in the morning and afternoon. Individual body weights were recorded on the day following receipt and prior to randomization on day -1.

ENVIRONMENTAL CONDITIONS
- Temperature: 18-26 °C
- Humidity: 30-70 %
- Air changes: 10-15 air changes per hr.
- Photoperiod: 12 hrs light/12 hours dark cycle.
Route of administration:
oral: gavage
Details on route of administration:
All doses were administered using a plastic syringe with an attached gavage needle. Individual doses were based on the most recent body weight data.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
For each dose group, a specified amount of test item was weighed into a pre-calibrated beaker. A sufficient quantity of corn oil was added to the beaker to achieve the desired concentration and the mixture was stirred for 30 minutes. Each test article solution was prepared fresh weekly, dispensed into daily aliquots and stored in amber glass containers at ambient conditions. During each preparation, corn oil was dispensed into daily aliquots for administration to control animals. The physical state of the control and each dosing solution was recorded during each preparation. The vehicle was a clear yellow liquid and each test article preparation was a clear yellow solution. All dosing solutions were prepared and dispensed under a fume hood. The dosing solutions were stirred continuously prior to and during dosing.Dosage Volume: 2 ml/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation of the study,homogeneity and stability analyses were performed on concentrations of the test article in the vehicle which encompassed the low- and high-dose concentrations. Homogeneity analyses were performed on duplicate samples taken from the top, middle and bottom of the two mixtures. Stability of the test article in the vehicle was evaluated on duplicate samples on day 0 and at 3 days and 10 days of room temperature storage. Concentration verification analysis was performed on the vehicle and each test article dosing solution prepared for study weeks 0, 2, 4 and 6.
Duration of treatment / exposure:
The animals were dosed for a minimum of four weeks, up to and including the day prior to scheduled euthanasia.
Frequency of treatment:
Single dose, daily.
Remarks:
Dose level: 0, 75, 225, 750 mg/kg bw/day (nominal)
Remarks:
Dose concentration: 0, 37.5, 112.5, 375 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 5 female rates per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosage levels were selected by the Sponsor and Study Director in an attempt to produce graded responses to the test article. The high-dose level was expected to produce some toxic effects, but not excessive lethality. The mid-dose level was expected to produce no to minimal observable effects and the low-dose level was expected to produce no observable effects.
- Preliminary study: prior to initiation of the toxicity and reproduction/developmental screening studies, a 14-day range-finding study was conducted in rats to aid in selection of dosage levels.
- Rationale for animal assignment: animals determined to be suitable test subjects were randomly assigned to groups using a computer randomization program. The program ranked the animals according to day -1 body weights and randomly assigned the animals to study groups in a stratified block design.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
Cage-side observations for overt signs of toxicity were performed once daily within approximately one-half hour to two hours following dosing.

DETAILED CLINICAL OBSERVATIONS
Mortality/general health checks were performed twice daily, in the morning and afternoon.Detailed clinical observations were performed a minimum of weekly and on the day of scheduled euthanasia.

BODY WEIGHT
Individual body weights were recorded on days 0, 3, 7, 12, 16, 20, 23, 27 and 30. A final body weight was recorded prior to scheduled euthanasia (day 33/34).

FOOD CONSUMPTION
Individual food consumption was recorded on the same days as body weights.

FUNCTIONAL OBSERVATION BATTERY
A functional observation battery (FOB), including home cage, removal from home cage and open field evaluation, was performed for all animals beginning on day -1 and weekly thereafter (days 7, 14 and 21). A full FOB assessment, including home cage, removal from home cage, open field evaluation, manipulative tests and motor activity measurements, was performed following 28 days of treatment. On day 28, observations for home cage, removal from home cage and open field evaluation were recorded for all animals. On day 29, manipulative tests were performed for ten males each in groups 1 and 2, five males each in groups 3 and 4 and all females. On day 30, motor activity measurements were performed for five males and five females from each group.
The following parameters were evaluated:
- home cage observations: body posture, clonic involuntary motor movements, tonic involuntary motor movements and vocalization;
- removal from home cage observations: ease of removal, reactivity to being handled, ocular discharge, eyelid closure, salivation and piloerection;
- open field observations: clonic involuntary motor movements, tonic involuntary motor movements, gait score, gait abnormalities, mobility score, arousal, stereotypy, bizarre behavior, urination, defecation and rearing;
- manipulative tests: approach response, touch response, startle response, tail pinch, pupil response, righting ability, forelimb grip strength, hlndlimb grip strength, handing foot splay, body temperature.

MOTOR ACTIVITY
Each animal was evaluated for behavioral changes in an open field chamber (San Diego lnstruments, San Diego, California). For each animal, the test consisted of an one-hour observation period in the open field chamber. Data included the total number of squares entered during the testing interval. A white noise generator was activated prior to open field testing to reduce auditory disturbance to the animals. The white noise sound leve! in each enclosure ranged from 74 to 75 dB prior to motor activity measurements. In addition, the motor activity test was performed under red lighting to reduce observational disturbances to the animals. Light readings for each enclosure prior to testing ranged from 0.30 to 0.33 foot candles.

CLINICAL PATHOLOGY
Blood was collected from five males and five females from each group on the day of scheduled euthanasia (day 33 for males and day 34 for females) for evaluation of selected hematology, coagulation and clinical chemistry parameters. Blood samples for hematology and clinical chemistry parameters were obtained via the orbital plexus while the animals were under light isoflurane anesthesia and blood samples for coagulation parameters were obtained from the vena cava following carbon dioxide inhalation at scheduled euthanasia. Feed was withheld overnight prior to blood collection; however, water was available.

HAEMATOLOGY
Parameters examined: erythrocyte count (RBC), Hematocrit (Hct), Hemoglobin concentration (Hgb), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Platelet count, Total and differentialleukocyte counts.Coagulation parameters: Prothrombin time (PT), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY
Parameters examined: Alanine aminotransferase (ALT), Albumin, Albumin/globulin ratio (calculated), Alkaline phosphatase, Aspartate aminotransferase (AST), Calcium Cholesterol Creatinine, Globulin (calculated) Glucose, Electrolytes (sodium, potassium and chloride), Phosphorus, Total bilirubin, Total serum protein, Triglycerides, Urea nitrogen.
Sacrifice and pathology:
GROSS PATHOLOGY
All animals were subjected to a complete gross necropsy at scheduled euthanasia. The necropsy examination included examination of the external surfaces ofthe body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. The males were euthanized on day 33 and the females were euthanized on day 34 by carbon dioxide inhalation followed by exsanguination. All abnormalities were recorded. The animals were fasted overnight prior to euthanasia.Fresh organ weights were obtained at scheduled euthanasia for the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus. Paired organs were weighed together.
The following organs and tissues were preserved in 10 % neutral buffered formalin for possible future histopathological examination: Accessory genital organs (epididymides, seminai vesicles and prestate or uterus and vagina), Adrenals, All gross lesions, Aorte, Brain (including sections of medulla/pons, cerebellar cortex and cerebral cortex), Cecum, Colon, Duodenum, Esophagus, Exorbitallachrymal glands, Eyes with optic nerve, Femur (including articular surface) and bene marrow, Heart, lleum, Jejunum, Kidneys, Liver (three sections collected), Lungs (infused with formalin) with bronchi, Mammary gland, Mandibular lymph node, Mediastinal lymph node, Mesenteric lymph node, Ovaries, Pancreas, Peripheral nerve (sciatic), Pituitary, Rectum, Skeletal muscle (thigh), Skin, Spinal cord (cervical, midthoracic and lumbar), Spleen, Sternum with bene marrow ,Stomach (glandular/nonglandular), Submaxillary salivary gland, Testes (preserved in Bouin's fixative for 48 to 96 hours, rinsed and then retained in 70% ethanol), Thymus Thyroid/parathyroid Tongue, Trachea, Urinary bladder.

HISTOPATHOLOGY
All tissues and organs collected at necropsy from five males and five females from each group and gross lesions from ali animals were processed for histopathological examination. The tissues were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin.
Other examinations:
- Prior to initiation of the toxicity studiy, a 14-day range-finding study was conducted in rats to aid in selection of dosage levels.
- After a minimum of 14 days of treatment, males were cohabited with females in the reproduction/developmental screening study.
Statistics:
Statistical analyses for the toxicity study were performed using the SLI Alpha GenTox computer system. Data, including body weights, body weight changes and food consumption were analyzed by One-Way Analysis of Variance (ANOVA). lf significance was detected with ANOVA (p<0.05), pair wise group comparisons proceeded using the Tukey-Kramer test. Descriptive (categorical) and quanta data were analyzed by Fisher's Exact Test. When significance was observed, group by group comparisons were performed using Fisher's Exact Test. Absolute and relative organ weights, and clinica! pathology data were analyzed for homogeneity of variance using Levene's test. lf significance was detected with Levene's test (p<0.01), multiple group comparisons proceeded using the Kruskai-Wallis non-parametric ANOVA, followed by Dunn's test, when p<0.05. lf significance was not detected with Levene's test, parametric procedures were used to analyzethe data, i.e., ANOVA followed by Tukey-Kramer test when p<0.05. All analyses were two-tailed with a minimum significance level of 5 % (p<0.05).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs were observed for males and females in the 225 and 750 mg/kg/day groups.
Mortality:
no mortality observed
Description (incidence):
No mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant body weight loss in males at 750 mg/kg/day during days 0-3.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption statistically lower than control during days 0-3.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically meaningful differences between the control and dosed groups in the hematology and coagulation parameters.
Description (incidence and severity):
Several statistically significant differences in biochemistry data were noted. However, with the exception of higher mean total protein, calcium and cholesterol far males in the 750 mg/kg/day group and higher mean cholesterol far females in the 750 mg/kg/day group, the mean values remained within the range of SLI historical contrai data.
Description (incidence and severity):
Mean forelimb drip strength and motor activity were decreased in the high dose males.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically meaningful differences in absolute or relative organ weights between the control and dosed groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No remarkable findings were noted at scheduled necropsy.
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
Mean forelimb grip strength and mean motor activity of males in the 750 mg/kg/day group were statistically lower than controls on day 29.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No toxicologically significant lesions were noted in any of the tissues examined.
Details on results:
CLINICAL SIGNS AND MORTALITY
All males and females survived to scheduled euthanasia at the study termination.
Clinical signs were observed far males and females in the 225 and 750 mg/kg/day groups. In males, the clinical signs included a low incidence of predose salivation and postdose salivation in the 225 mg/kg/day group, and predose and postdose salivation, postdose wobbly gait and a low incidence of postdose urine stain in the 750 mg/kg/day group. No remarkable clinical signs were observed for males in the 75 mg/kg/day group.In females, clinical signs included a low incidence of urine stain and postdose salivation in the 225 mg/kg/day group, and hair loss, urine stain, predose and postdose salivation and postdose wobbly gait in the 750 mg/kg/day group. Additional clinical signs for one female in the 750 mg/kg/day group included a low incidence of dark material around the nose, urine stain, ocular discharge, impaired mobility, cool to the touch, increased activity and nasal discharge following dosing. Remarkable clinical signs for females in the 75 mg/kg/day group were limited to a single incidence of postdose salivation in one female.

FUNCTION OBSERVATION BATTERY
Mean forelimb grip strength and mean motor activity of males in the 750 mg/kg/day group were statistically lower than controls on day 29. No other toxicologically meaningful differences were notedtin the functional observation battery. Body posture of males in the 75 mg/kg/day group and females in the 750 mg/kg/day group was statistically different than controls on day 21. These latter differences were not considered toxicologically meaningful since they did not follow a consistent pattern or dose response.

BODY WEIGHT AND BODY WEIGHT CHANGES
Statistically significant body weight loss occurred for males in the 750 mg/kg/day group during days 0-3. No other statistically significant differences in mean body weight change were noted for males or females in the 75, 225 or 750 mg/kg/day groups during the study. However, there was an apparent mean body weight loss noted in all dose groups, including the control from days 12-16. The mean body weight loss in males ranged from 19-26 grams and in the females it ranged from 5-17 grams. A compensatory rebound weight gain was noted during the following body weight change interval (days 16-20), where animals in all dose groups gained a relatively large amount of body weight, which ranged in the males from 22-36 grams and in the females from 10-25 grams.There were no statistically significant differences in mean body weights between the control, 75, 225 and 750 mg/kg/day groups. However, mean body weights of males in the 750 mg/kg/day group were approximately 5-10 % lower than controls beginning on day 3 and continuing through day 30. Mean body weights of males in the 75 and 225 mg/kg/day groups and females in the 75, 225 and 750 mg/kg/day groups were comparable to controls (within 3 % of controls) throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Mean food consumption (grams/animal/day) of males and females in the 750 mg/kg/day group was statistically lower than controls during study days 0-3. In addition, mean food consumption appeared to be slightly, but not statistically lower than controls for females in the 225 mg/kg/day group during days 0-3 and males in the 750 mg/kg/day group during days 16-30. No other toxicologically meaningful differences in mean food consumption were noted between the contrai, 75, 225 and 750 mg/kg/day groups during the study.

HAEMATOLOGY AND COAGULATION
There were no toxicologically meaningful differences between the control, 75, 225 and 750 mg/kg/day groups in the hematology and coagulation parameters evaluated at study termination (days 33/34). A few statistically significant differences were noted; however, none of the differences were considered toxicologically meaningful since they did not follow a consistent pattern and the mean values remained well within the range of SLI historical contrai data. In males, statistically significant differences in hematology and coagulation data included a lower mean prothrombin time in the 75 mg/kg/day group and a higher mean MCV value in the 750 mg/kg/day group compared to controls. In females, statistically significant differences in hematology and coagulation data were limited to lower mean erythrocyte and hematocrit values in the 750 mg/kg/day group compared to controls.

CLINICAL CHEMISTRY
Several statistically significant differences in biochemistry data were noted between the control, 75, 225 and 750 mg/kg/day groups at study termination (days 33/34). However, with the exception of higher mean total protein, calcium and cholesterol far males in the 750 mg/kg/day group and higher mean cholesterol far females in the 750 mg/kg/day group, the mean values remained within the range of SLI historical contrai data.
The toxicological significance of the higher total protein and calcium in the 750 mg/kg/day males and higher cholesterol in 750 mg/kg/day males and females is notclear; however, it should be noted that the mean values far these parameters were outside the range of SLI historical control data.Additional statistically significant differences in male biochemistry data included higher mean total protein, albumin, calcium and phosphorus in the 225 mg/kg/day group and higher albumin, globulin, glucose, sodium, potassium and phosphorus in the 750 mg/kg/day group compared to controls. In females, additional statistically significant differences in biochemistry data included higher mean total bilirubin the 75 mg/kg/day group, higher mean total protein, albumin and globulin in the 225 mg/kg/day group, and hfgher mean ALT, total protein, albumin and potassium in the 750 mg/kg/day group compared to controls. The mean values of these additional differences were within the range of SLI historical control data. Even though these values were within the historical control range, a common finding to both male and female animals included a dose-related increase in total protein, albumin and globulin that appeared to correlate with a dose-related increase in liver weight. These changes may be related to a compensatory reaction to the test article, possibly due to its metabolism.

GROSS PATHOLOGY
No remarkable findings were noted for males or females in the control, 75, 225 or 750 mg/kg/day groups at scheduled necropsy.

ORGAN WEIGHTS
There were no toxicologically meaningful differences in absolute or relative organ weights between the control, 75, 225 and 750 mg/kg/day groups. A few statistically significant differences in organ weights were noted; however, the findings did not correlate with any abnormal biochemistry or histopathology.Statistically significant differences in organ weight data included lower absolute heart and epididymides weight and higher liver weight relative to final body weight for males in the 750 kg/kg/day group, and higher absolute liver weight and liver weight relative to final body weight and higher kidney weight relative to final body weight for females in the 225 and 750 mg/kg/day groups compared to controls.

HISTOPATHOLOGY
No toxicologically significant lesions were noted in any of the tissues examined. A minimal to mild hyaline droplet nephropathy was observed for males in the 75, 225 and 750 mg/kg/day groups. The hyaline droplets were sometimes accompanied by minimal tubular epithelial degeneration and regeneration. Hyaline droplets can be induced by a variety of chemical compounds in this strain of male rats and is frequently associated with the accumulation of a2 -globulin within the hyaline droplets. However, this finding is not considered toxicologically significant for humans. Minimal to mild vacuolar change was observed in individuai hepatocytes in males from the 750 mg/kg/day group. This change was interpreted as a background lesion based on the nature and distribution of changes. In addition, similar changes have been seen in control animals in other toxicity studies. Other microscopic changes were considered to be normal findings of rats of this age and strain.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology
Critical effects observed:
no
Lowest effective dose / conc.:
225 mg/kg bw/day (nominal)
System:
other: no target organ identified

Results of the homogeneity analyses revealed that the average recoveries of the 37.5 and 375 mg/ml dosing formulations were within 4.9 % of the nominal concentration, indicating that the formulations were homogeneous. Results of the stability analyses revealed that the 37.5 and 375 mg/ml dosing formulations were stable for 10 days following room temperature storage. The average results of the concentration verification analyses were within 5.3 % of the nominal concentrations. No substance was detected in the vehicle control samples analyzed during the course of the study.

 

Conclusions:
NOAEL (28d) rat, males and females, systemic toxicity: 75 mg/kg bw/day
NOAEL (28d) rat, males and females, neurological effects: 225 mg/kg bw/day
Executive summary:

The study was conducted to provide initial information on the repeated-dose systemic toxicity of test item, with emphasis on potential neurological effects, and serve as a screening study for potential reproductive and developmental effects in male and female rats.

The test article was administered once daily by oral gavage for a minimum of four weeks, up to and including the day prior to scheduled euthanasia. Detailed clinical observations were performed a minimum of weekly and on the day of scheduled euthanasia. Blood samples were collected from five males and five females from each group on the day of scheduled euthanasia for evaluation of selected hematology, coagulation and clinical chemistry parameters. All animals were subjected to a complete gross necropsy examination at scheduled euthanasia. Fresh organ weights were recorded and selected tissues and organs were preserved from all animals. All tissues and organs collected at necropsy from five males and five females from each group and gross lesions from all animals were examined microscopically.

Clinical signs were observed for males and females in the 225 and 750 mg/kg/day groups. The clinical signs included urine stain, predose salivation and postdose salivation in the 225 and 750 mg/kg/day groups, and postdose wobbly gait and postdose urine stain in the 750 mg/kg/day group. Remarkable clinical signs in the 75 mg/kg/day group were limited to a single incidence of postdose salivation in one female. Mean forelimb grip strength and mean motor activity of males in the 750 mg/kg/day group were statistically lower than controls on day 29. No other toxicologically meaningful differences were noted in the functional obsen/ation battery. Statistically significant body weight loss occurred for males in the 750 mg/kg/day group during days 0-3. An apparent body weight loss was noted in all dose groups for both male and female animals during days 12-16 and this was followed by a compensatory rebound in weight gain the following body weight change interval (days 16-20). The definitive reason for this occurrence could not be determined, but it may be related to the initiation of mating. There were no statistically significant differences in mean body weights; however‘ mean body weights of males in the 750 mg/kg/day group were approximately 5-10 % lower than controls on days 3-30. No other toxicologically meaningful differences in mean body weights or body weight change were noted during the study.

Mean food consumption of males and females in the 750 mg/kg/day group was statistically lower than controls during study days 0-3. In addition, mean food consumption appeared to be slightly but not statistically lower than controls for females in the 225 rng/kg/day group during days 0-3 and males in the 750 mg/kg/day group during days 16-30. No other toxicologically meaningful differences in mean food consumption were noted during the study. There were no toxicologically meaningful differences between the control, 75, 225 and 750 mglkg/day groups in the hematology and coagulation parameters evaluated at study termination. With the possible exception of higher mean total protein, calcium and cholesterol for males in the 750 mg/kg/day group and higher mean cholesterol for females in the 750 mg/kg/day group, there were no toxicologically meaningful differences in the biochemistry parameters evaluated. The toxicological significance of the higher total protein, calcium and cholesterol values is not clear; however, the mean values for these parameters were outside the range of SLI historical control data. There was a common finding to both male and female animals that included a dose-related increase in total protein, albumin and globulin that appeared to correlate with a dose-related increase in liver weight. These changes may be related to a compensatory reaction to the test article, possibly due to its metabolism. No remarkable gross necropsy findings were noted in the control, 75, 225 or 750 mglkg/day groups and there were no toxicologically meaningful differences in absolute or relative orgar weights between the groups. No toxicologically significant test article-related microscopii lesions werenoted in any of the tissues examined. A nondose-related, minimal to mili hyaline droplet nephropathy was observed for males in the 75, 225 and 750 mglkg/day groups; however, this finding is not considered toxicologically significant for humans.

Based on the results, a dosage level of 75 mg/kg/day was considered a no-observed-adverse-effect level (NOAEL) for systemic toxicity. Other than possible indications of central nervous system (CNS) depression, such as wobbly gait, reduced forelimb strength and decreased motor activity in the high-dose group, there was no evidence of more severe CNS effects in this study. Thus, a dosage level of 225 mg/kg/day was considered the NOAEL for neurological effects.

Conclusion

NOAEL (28d) rat, males and females, systemic toxicity: 75 mg/kg bw/day

NOAEL (28d) rat, males and females, neurological effects: 225 mg/kg bw/day

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
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

The preparatory work for toxicological evaluations of food additives and contaminants by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) on propriophenone has been conducted. The data on uses and intake requested by the Committee at its fiftythird meeting were provided and raised no safety concern for propiophenone (WHO, 2002).

No specific experiments conducted on propiophenone are available, however there are assays conducted using the similar substance acetophenone, which have been taken into account in order to perform an assessment as more complete as possible. The Read Across approach can be considered appropriate for the assessement of repeated dose toxicity. Details can be found in the Read Across justification document attached in section 13 of IUCLID.

A study, commissioned by Acetophenone Task Force (2003), was conducted to provide initial information on the repeated-dose systemic toxicity of test item, with emphasis on potential neurological effects, and serve as a screening study for potential reproductive and developmental effects in male and female rats.

The test article was administered once daily by oral gavage for a minimum of four weeks, up to and including the day prior to scheduled euthanasia. Detailed clinical observations were performed a minimum of weekly and on the day of scheduled euthanasia. Blood samples were collected from five males and five females from each group on the day of scheduled euthanasia for evaluation of selected hematology, coagulation and clinical chemistry parameters. All animals were subjected to a complete gross necropsy examination at scheduled euthanasia. Fresh organ weights were recorded and selected tissues and organs were preserved from all animals. All tissues and organs collected at necropsy from five males and five females from each group and gross lesions from all animals were examined microscopically.

Clinical signs were observed for males and females in the 225 and 750 mg/kg/day groups. The clinical signs included urine stain, predose salivation and postdose salivation in the 225 and 750 mg/kg/day groups, and postdose wobbly gait and postdose urine stain in the 750 mg/kg/day group. Remarkable clinical signs in the 75 mg/kg/day group were limited to a single incidence of postdose salivation in one female. Mean forelimb grip strength and mean motor activity of males in the 750 mg/kg/day group were statistically lower than controls on day 29. No other toxicologically meaningful differences were noted in the functional obsen/ation battery. Statistically significant body weight loss occurred for males in the 750 mg/kg/day group during days 0-3. An apparent body weight loss was noted in all dose groups for both male and female animals during days 12-16 and this was followed by a compensatory rebound in weight gain the following body weight change interval (days 16-20). The definitive reason for this occurrence could not be determined, but it may be related to the initiation of mating. There were no statistically significant differences in mean body weights; however‘ mean body weights of males in the 750 mg/kg/day group were approximately 5-10 % lower than controls on days 3-30. No other toxicologically meaningful differences in mean body weights or body weight change were noted during the study.

Mean food consumption of males and females in the 750 mg/kg/day group was statistically lower than controls during study days 0-3. In addition, mean food consumption appeared to be slightly but not statistically lower than controls for females in the 225 rng/kg/day group during days 0-3 and males in the 750 mg/kg/day group during days 16-30. No other toxicologically meaningful differences in mean food consumption were noted during the study. There were no toxicologically meaningful differences between the control, 75, 225 and 750 mglkg/day groups in the hematology and coagulation parameters evaluated at study termination. With the possible exception of higher mean total protein, calcium and cholesterol for males in the 750 mg/kg/day group and higher mean cholesterol for females in the 750 mg/kg/day group, there were no toxicologically meaningful differences in the biochemistry parameters evaluated. The toxicological significance of the higher total protein, calcium and cholesterol values is not clear; however, the mean values for these parameters were outside the range of SLI historical control data. There was a common finding to both male and female animals that included a dose-related increase in total protein, albumin and globulin that appeared to correlate with a dose-related increase in liver weight. These changes may be related to a compensatory reaction to the test article, possibly due to its metabolism. No remarkable gross necropsy findings were noted in the control, 75, 225 or 750 mglkg/day groups and there were no toxicologically meaningful differences in absolute or relative orgar weights between the groups. No toxicologically significant test article-related microscopii lesions werenoted in any of the tissues examined. A nondose-related, minimal to mili hyaline droplet nephropathy was observed for males in the 75, 225 and 750 mglkg/day groups; however, this finding is not considered toxicologically significant for humans.

Furthermore, in an FDA feeding study, 10000 ppm fed to rats in the diet for 17 weeks produced no effects (Haganet al, 1967). No effects on growth, hematological values or macroscopic tissue changes were observed in groups of 10 male and 10 female Osborne-Mendel rats exposed to 0, 1000, 2500 and 10000 ppm acetophenone. Microscopic examination of the 10000 ppm group revealed no effects. Thus, the 10000 ppm level was the highest concentration at which no effects were observed. Some loss of the compound from the feed due to volatilization was reported; therefore, the dietary concentration of 10000 ppm was multiplied by a factor of 0.845 (based on data provided by the investigators) yielding a NOAEL of 8450 ppm or 423 mg/kg/day assuming that a rat consumes a daily amount of food equivalent to 5 % of its body weight/day as food. Dividing the dose by an uncertainty factor of 3000 yields the RfD of 0.4 mg/kg/day or 7 mg/day for a 70 kg person (IRIS).

REFERENCE

Hagan, E. C , Hansen, W. H., Fitzhugh, O. G., Jenner, P. M., Jones, W. I., Taylor, Jean M., Long, Eleanor L., Nelson, A. A. & Brouwer, J. B. (1967). Food flavourings and compounds of related structure. II. Subacute and chronic toxicity. Fd Cosmet. Toxicol. 5, 141.

Integrated Risk Information System (IRIS), U.S. Environmental Protection Agency. Last Revised — 08/22/1988. https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_nmbr=321

World Health Organization (WHO) (2002). Joint FAO/WHO Expert Committee on Food Additives (2001, Rome, Italy) Evaluation of certain food additives and contaminants: fifty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives. (WHO technical report series ; 909) ttp://apps.who.int/iris/bitstream/10665/42578/1/WHO_TRS_909.pdf

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values which take into account the duration of exposure and the dose/concentration which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

 

In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified (Category 1 or Category 2), dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. Nevertheless, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by- case basis). For example, for 28-day study the guidance values are increased by a factor of three. The guidance values and ranges mentioned in the specific CLP paragraphs are intended only for guidance purposes: they are not intended as strict demarcation values.

Statistically significant effects were recorded at 225 mg/kg bw/day in females and males, which were treated for 28 days. Thus the structural analogous specific profile of toxicity occurs in repeat-dose animal study at a dose/concentration below the guidance value (i.e. < 300 mg/kg bw/day by the oral route, 28 days study); however, taken into account all the available information and the evaluation of propiophenone by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) a classification has been considered not appropriate.