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Administrative data

Description of key information

Repeated administration of the test item up to 90 consecutive days to rats did not reveal any adverse effects to the test species. Therefore, a NOAEL of 1000 mg/kg/day has been established.

Key value for chemical safety assessment

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:
18-Sep-1996 to 04-Feb-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OPP 82-1, OECD 408)
Qualifier:
according to
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on 12-May-1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc, Raleigh, North Carolina, USA
- Age at study initiation: approximately 4 weeks old
- Weight at initiation of dosing: 139-168 g (males), 104 to 138 g (females)
- Housing: individually, in stainless steel cages
- Diet: PMI Certified Rodent Diet #5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 64.9-77.6 °F (approximately 18.3-25.3 °C)
- Humidity: 31.3-63.3 %
- Air changes: at least 10 changes/hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: 18-Sep-1996 to 04-Feb-1997
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily. The appropriate amount of test item was weighed on an analyical balance and transferred to a beaker. The vehicle, corn oil, was added to give the appropriate concentrations. Formulations were mixed using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: standard vehicle for studies of this type
- Amount of vehicle: dose volume of 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity analyses were conducted prior to dose initiation. Concentration analyses were conducted at weeks 1, 4, 8 and 13.
Duration of treatment / exposure:
at least 90 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
2, 10, 100 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
main study: 10 animals (all groups)
recovery: 5 animals (0 and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses were selected from a preliminary 28-day oral toxicity study and were spaced appropriately to produce test groups with a range of toxic effects.
- Post-exposure recovery period in satellite groups: 28 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: mortality was assessed twice daily, observations for clinical signs were perfomed once daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once prior to treatment and weekly thereafter

BODY WEIGHT:
- Time schedule: once prior to treatment and weekly thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: yes

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: once prior to treatment and once during week 13
- Dose groups that were examined: all groups prior to treatment, control and high dose group animals during week 13

HAEMATOLOGY:
- Time schedule for collection of blood: from 10 animals/sex/dose group at week 5, from all animals at week 14 and from 5 recovery animals/sex at week 18
- Anaesthetic used for blood collection: yes (carbon dioxide)
- Animals fasted: yes
- Parameters examined: differential leukocyte count and cell morphology, haemoglobin concentration, haematocrit; leukocyte, erythrocyte and platelet count; activated partial thromboplastin time and prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: from 10 animals/sex/dose group at week 5, from all animals at week 14 and from 5 recovery animals/sex at week 18
- Animals fasted: yes
- Parameters examined: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and gamma glutamyltransferase activity; albumin, calcium, chloride, creatinine, globulin, glucose, inorganic phosphorus, potassium, sodium, total bilirubin, total cholesterol, total protein, triglycerides and urea nitrogen concentration; albumin/globulin ratio
Sacrifice and pathology:
GROSS PATHOLOGY:
Macroscopical examination of all orifices, carcass, cervical tissues and organs, cranial cavity, cut surfaces of the brain, external surface of the body, external surface of the brain, external and cut surfaces of the spinal cord, nasal cavity and paranasal sinuses; thoracic, abdominal and pelvic cavities/viscera

ORGAN WEIGHTS:
The following organs were weighed wet after careful dissection and trimming of fat and other contiguous tissue: adrenals, liver, spleen, thyroid/parathyroids, thymus, kidneys, testes, epididymides, brain with brainstem

HISTOPATHOLOGY:
Examinations were performed on the following tissues from all terminal-sacrifice animals in Groups 1 and 5 and from the animal that was found dead: adrenals, aorta, bone marrow, brain with brain stem, colon, caecum, rectum, duodenum, jejunum, ileum, epididymides, esophagus, exorbital lacrimal gland, eyes with optic nerves, femur, Harderian gland, heart, kidneys, lesions, liver, lung, mammary gland, mesenteric lymph node, nasal turbinates, ovaries, pancreas, pituitary, prostate, salivary glands, seminal vesicles, sciatic nerve, skin, spinal cord, spleen, stomach, testes, thigh musculature, thymus, thyroid (parathyroids) trachea, urinary bladder, uterus with cervix and vagina. The lungs, liver, kidneys, heart, mesenteric lymph nodes, ileum,
spleen, thymus, and bone marrow (femur) were examined microscopically from all terminal-sacrifice animals in Groups 2, 3, and 4. Gross lesions were examined from all terminal-sacrifice animals.
Other examinations:
staging of spermatogenesis
Statistics:
Weekly body weights, weekly and total body weight change, weekly and total food consumption, clinical pathology data (except haemolysis and cellular morphology gradings), fasted terminal body weights, and organ weight data of the treated groups were compared statistically to the data from the same sex of the control group. If variances of untransformed data were heterogeneous, a rank transformation of the data was performed to achieve variance homogeneity. If the transformation did not achieve variance homogeneity, the analyses were still performed on the rank-transformed data.
Details on results:
CLINICAL SIGNS AND MORTALITY
No test item-related mortality occurred during the study. One control male was found dead during the treatment period. No cause of death was established for this animal. Abnormal clinical observations noted during the course of the study included skin/pelage sores, alopecia, crust on the skin/pelage, chromodacryorrhea, malocclusion, and missing body part (a digit on the front left foot of one Group 5 male). These findings occurred sporadically and/or were of the type commonly observed in this species at this laboratory. There were no test article-related differences between the control and test groups.

BODY WEIGHT AND WEIGHT GAIN
There was no effect of the treatment with the test item on body weight or body weight gain throughout the treatment period; however, during the recovery period, a significant increase in the mean body weight gain was noted for the males previously treated with 1000 mg/kg bw/day. For females of the same group, the mean body weight change value was lower (although not significantly) than that of the control animals. These changes in body weight during the recovery period were not biologically relevant and not related to test article toxicity.

FOOD CONSUMPTION
Mean food consumption was comparable between the control and treated groups throughout the treatment phase. During the recovery phase, a significant increase in the mean food consumption was noted for the Group 5 males at Weeks 14-15 in comparison to the control value. The total mean food consumption was significantly decreased for the Group 5 females at Weeks 14-18 (recovery) in comparison to the control value. These changes in food consumption during the recovery period were not biologically relevant and not related to test article toxicity.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmological findings related to the treatment with the test item.

HAEMATOLOGY
The mean prothrombin time value was significantly higher in Group 5 males at Weeks 5 and 14; the mean value was higher at Week 18, but not significantly different from the concurrent control value. It is possible that the aforementioned change is the result of treatment, but it is of low magnitude and of no biologic importance. The mechanism of the difference is not apparent from the remainder of the data examined (clinical observations, body weights, food consumption, necropsy findings, organ weights, and clinical laboratory data). The slight, but significant, decrease in mean lymphocyte count
noted for Group 5 males at Week 5 is not attributed to the administration of test material due to the low magnitude of the change. The significantly
lower mean monocyte count seen in Group 5 males at Week 18 is considered incidental to the administration of the test material due to the low magnitude of the change and the occurrence of the difference only at recovery Week 18. The remaining hematology and coagulation data, differential leukocyte counts, and cellular morphology were generally unremarkable and comparable between the groups at Weeks 5 and 14 and between Groups 1 and 5 at Week 18.

CLINICAL CHEMISTRY
The significantly higher mean globulin value observed in Group 5 males at Week 5 was not of a magnitude great enough to cause a concurrent significant elevation in the mean total protein concentration for this group. The cause of the increase is not apparent from the data examined; for one Group 5 male, the elevation is likely related to the thoracic adhesions (i.e., inflammation) observed at terminal sacrifice. The significant differences noted in the remainder of the clinical biochemistry data are not attributed to treatment due to the low magnitude of the change, the lack of dose dependence, and/or the occurrence of the difference only at recovery Week 18.

ORGAN WEIGHTS
Evaluation of the terminal body weight and organ weight data from the terminal sacrifice revealed significant increases in the mean organ to-body- and organ-to-brain-weight ratios for the thyroid/parathyroid in the Group 5 males. Evaluation of the terminal body weight and organ weight data from
the recovery sacrifice revealed a significant increase in the mean kidney-to-body weight ratio for Group 5 females. These organ weight changes are considered unrelated to test article administration.

GROSS PATHOLOGY
Gross lesions noted for the Group 1 male that was found dead during the study included dark-red lobes of the lungs and moderately enlarged, dark-brown lobes of the liver. Gross lesions noted for the terminal-sacrifice animals included interlobar adhesion of the lung in one Group 5 male; thickened pericardial sac in the heart of one Group 5 male; granular/pitted/rough spleen in one Group 4 male; enlarged liver in two Group 2 males and one male each in Groups 3-5; pale liver in three males each in Groups 1-5 and one-Group 1 female; depressed area of the kidney in one Group 5 male; dark area of the glandular stomach in one Group 1 male, two Group 2 males, and one female each in Groups 2-4; distended uterus in two Group 1 females and one Group 3 female; fluid in the lumen of the uterus in two Group 1 females and one Group 3 female; and dark thymus in one Group 2 male. There were no gross lesions noted at the recovery sacrifice. The reported findings were without histological correlates and are therefore considered unrelated to test article administration.

HISTOPATHOLOGY
No microscopical findings related to the treatment with the test item were recorded. Nearly all microscopic changes were inflammatory in nature (graded minimal [+1] to slight [+2]), occurred in both control and treated rats with a similar incidence and severity, and are considered to be common microscopic findings for this age and strain of rat. Microscopic examination of the right testis and epididymis revealed essentially normal spermatogenic activity in the rats that received the test material, with no histologic evidence of altered spermatogenesis.
There was no microscopic correlate to the significantly increased thyroid-to-body- and thyroid-to-brain-weight ratios present in the Group 5 male terminal-sacrifice animals. Although not significantly different, thyroid weights were variable among the Group 2-4 male rats (Group 2 male weight was higher than that of the Group 4 males), with no evidence of a dose response. In addition, individual thyroid weights were lower in the Group 5 recovery animals compared to those of the control group. Therefore, because of the absence of specific microscopic correlation and the overall variability of thyroid weights among the groups, the significantly increased thyroid weights present in the Group 5 males was likely a spurious finding unrelated to test item administration.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test item-related effects noted up to and including the high dose level of 1000 mg/kg bw/day
Critical effects observed:
not specified

CHEMICAL ANALYSIS OF DOSE FORMULATIONS

The analysed test item concentrations were acceptable. The test item was homogeneously distributed in the dose formulations.

Conclusions:
No test item-related effects were noted during the study and consequently no target organ was identified. Based on this study outcome, the oral no-observed-effect-level for the test item was established at 1000 mg/kg bw/day.
Executive summary:

A GLP compliant 90-day gavage study following OECD guideline 408 was conducted in order to assess the test item's toxicity after repeated oral administration. Groups of 10 Sprague-Dawley rats per sex and dose (control and high dose groups contained 5 additional animals per sex for recovery investigations) received a freshly prepared suspension of the test article in corn oil at doses of 0, 2, 10, 100, and 1000 mg/kg/day (groups 1 -5, respectively). A standard battery of tests was performed and included daily clinical observations, weekly physical examinations, weekly body weight and food consumption determinations, ophthalmologic examinations and clinical pathology determinations. After at least 90 days of treatment, all animals scheduled for sacrificed were subject to a gross necropsy and protocol-specified tissues were weighed and collected for histological examination. Recovery animals in the control and high-dose groups were sacrificed after a 28-day recovery period. All tissues were examined from control and high-dose animals and selected tissues were examined from low- and mid-dose animals. There was no test article-related mortality; one male in group 1 was found dead during the study, however the cause of death could not be established. The abnormal clinical observations noted are considered unrelated to test article treatment, as they occurred infrequently and without relationship to treatment. Body weight and food consumption means were similar between the test article-treated and control animals during the treatment period. During the recovery phase, significant increases in the mean body weight gain and food consumption values were noted for the Group 5 males at Weeks 14-15 in comparison to the respective control values. Mean total mean food consumption was significantly decreased for the Group 5 females at Weeks 14-18 (recovery) in comparison to the control value. These changes in body weight and food consumption during the recovery period were not biologically relevant and not related to test article toxicity. There was no evidence in the clinical pathology data of an effect from the administration of the test material. Although a variety of lesions was noted grossly at the terminal necropsy, they were without histological correlates and are therefore considered unrelated to test article administration. Terminal body weight and organ weight data from the terminal sacrifice revealed a significant increase in the mean organ-to-body- and organ-to-brain-weight ratios for the thyroid/parathyroid in the Group 5 males, but this was without histological correlates. Evaluation of the terminal body weight and organ weight data from the recovery sacrifice revealed a significant increase in the mean kidney-to-body-weight ratio for Group 5 females, which, similarly, did not have histological correlates. These organ weight changes are considered unrelated to test article administration. There was no microscopic evidence of specific test material related histopathologic changes associated with test article administration. In conclusion, administration of test material to Sprague-Dawley rats by oral gavage in corn oil at doses of 2, 10, 100, and 1000 mg/kg/day was without effect on mortality, clinical observations (including ophthalmologic examinations), bodyweight and food consumption, and clinical and microscopic pathology. Under the conditions of this study, the no-observable-effect level is therefore 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 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 GLP compliant 90-day gavage study following OECD guideline 408 was conducted in order to assess the test item's toxicity after repeated oral administration. Groups of 10 Sprague-Dawley rats per sex and dose (control and high dose groups contained 5 additional animals per sex assigned to a 28-day recovery period) received a freshly prepared suspension of the test article in corn oil at doses of 0, 2, 10, 100, and 1000 mg/kg/day (groups 1 -5, respectively). A standard battery of tests was performed and included daily clinical observations, weekly physical examinations, weekly body weight and food consumption determinations, ophthalmologic examinations, and clinical pathology determinations, gross necropsy and histopathology. There was no test article-related mortality; one male in group 1 was found dead during the study, however the cause of death could not be established. Some abnormal clinical observations were noted, however they were considered unrelated to test article treatment, as they occurred infrequently and without relationship to treatment. Body weight and food consumption means were similar between the test article-treated and control animals during the treatment period. During the recovery phase in group 5, significant increases in the mean body weight gain (males) and decreases in food consumption values (males and females). These changes were considered not biologically relevant and not related to test article toxicity. There was no evidence in the clinical pathology data of an effect from the administration of the test material. Although a variety of lesions was noted grossly at the terminal necropsy, they were without histological correlates and are therefore considered unrelated to test article administration. Examinations during terminal sacrifice revealed a significant increase in the mean organ-to-body- and organ-to-brain-weight ratios for the thyroid/parathyroid in group 5 males and during recovery sacrifice examinations a significant increase in the mean kidney-to-body-weight ratio for Group 5 females was reported. These organ weight changes were without histopathological correlate and were considered unrelated to test article administration. There was no microscopic evidence of specific test material related histopathological changes associated with test article administration. In conclusion, under the conditions of this study, the no-observable-effect level is therefore 1000 mg/kg/day.

Above result is supported by a previously conducted 28-day gavage study performed in rats. In this GLP-compliant study following OECD guideline 408 groups of Sprague-Dawley rats were dosed with test material in corn oil at 0, 10, 100, or 1000 mg/kg body weight (Groups 1-4, respectively). A standard battery of in-life observations and data were collected and included daily clinical observations, weekly body weight and food consumption data, weekly physical examinations, and ophthalmoscopic examinations prior to initiation of dosing and prior to sacrifice (terminal sacrifice, group 1 and 4 animals only). Prior to sacrifice, blood was collected for determination of clinical pathology values. After 29 days of dosing, 10 rats/sex/group were sacrificed and necropsied, and protocol-specified tissues were weighed and collected for histological examination; five rats/sex/group in Groups 1 and 4 were sacrificed after an additional 14 days of recovery (without dosing). All tissues were examined from control and high-dose animals (terminal and recovery) and selected tissues were examined from low- and mid-dose animals. All animals survived to their scheduled sacrifice and gained weight during the course of the study; mean body weight gain and food consumption values were comparable between test article-treated and control groups. There were no treatment-related clinical observations or ophthalmoscopic findings. Gross observations at necropsy were comparable among the groups, and the changes in organ weights are considered incidental to treatment due to either the lack of a dose response or the low magnitude of the change. There was no evidence in the clinical pathology or histopathology data of treatment-related effects. The no-observable-effect level is therefore 1000 mg/kg/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study (OPP 82-1, OECD 408)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at doses of less than 100 mg/kg bw upon subchronic oral exposure in rats. Specifically, no adverse effects were noted at the limit dose of 1000 mg/kg bw. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.