Registration Dossier

Administrative data

Description of key information

The NOEL for subchronic oral exposure of rats is 5 mg/kg bw based on dose-dependent findings especially in the mesenteric lymph nodes, spleen and gastrointestinal tract.

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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Covance Laboratories Inc.
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., Portage, Michigan.
- Age at study initiation: Approximately 7 weeks old.
- Weight at study initiation:
Main study: 160-227 g (males); 140-190 g (females).
Study with satellite animals: 169-215 g (males); 142-177 g (females).
- Housing: Individually housed in suspended, stainless-steel, hanging, wire-mesh cages measuring 24.2 x 22.0 x 17.3 cm.
- Diet (e.g. ad libitum): PMI Certified Rodent Diet #5002, ad libitum during the acclimation and study periods. Due to feed spillage by the animals, food followers were placed on the jars on Week 3, and used throughout the remainder of the study.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C.
- Humidity (%): 41.2-67.9%.
- Air changes (per hr): 10 or greater air changes per hr.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dietary levels were not adjusted to 100% compound activity. For each dose level, the calculated amount of the test substance was weighed on an appropriate balance (mg) and premixed in a Waring blender with approximately 200 g of feed for approximately 2 minutes or until a homogeneous mix was achieved. The premix was then added to the appropriate amount of feed for each level and mixed at a rate of 1 minute/kg in a Patterson-Kelly twin-shell blender fitted with an intensifier bar to ensure a homogeneous mixture. After the mixing was completed, the diets were placed into containers and sent directly to the technical group.

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared twice weekly (based upon initial 4-day stability information) through Week 5 and at least once weekly thereafter (based upon subsequent 7 and 10 day stability data).
- Mixing appropriate amounts with (Type of food): PMI Certified Rodent Diet #5002
- Storage temperature of food: Prepared diet was stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical method: Diet preparations were analyzed using a method provided by the Sponsor and validated by Covance (testing lab).
- Homogeneity: Duplicate samples (100 g) were taken from the top, middle, and bottom of a full-size batch at the low- and high-dose levels prior to feeding. One sample was analyzed in duplicate, and the other was frozen at approximately -20 °C and retained at Covance-Vienna. Homogeneity analyses were performed on full-size batches at low and high levels prior to initiation of dosing and during Week 1. Smaller batch sizes were used for the partial Week 14 mixes; however, in order to conserve test material, homogeneity sampling was not performed.
- Stability: The stability was determined following 4, 7, and 10 days at room temperature for the low and high levels; samples were taken from mixes prepared prestudy and during Week 3 and from a dietary formulation prepared expressly for determination of stability.
- Routine Analyses: Analyses were performed on one of the duplicate samples per test level collected from the middle of each batch during the first 28 days and at Weeks 7 and 13.
Duration of treatment / exposure:
Exposure for 90 days, followed by a treatment-free recovery period of 28 days (only control and high dose groups)
Frequency of treatment:
Daily (ad libitum diet).
Dose / conc.:
2.5 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- Main study:
Group 1 (control) and 5 (100.0 mg/kg/day) : 30/sex (10 animals/sex were kept for an additional 28 day after substance application)
Group 2 (2.5 mg/kg/day), Group 3 (5.0 mg/kg/day), Group 4 (10.0 mg/kg/day): 20/sex

- satellite animals:
Groups 2 and 5: 12/sex
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected from a preliminary 28-day oral toxicity study, with the recognition that the previous study was conducted with a different strain of rat (Tif: RAIF [SPF], hybrid of RII/1 x RII/2). Doses were spaced appropriately to produce test groups with a range of toxic effects. The highest dose level should ideally result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation. The intermediate dose should ideally produce minimal observable effects. The lowest dose should not produce any evidence of toxicity.
- Post-exposure recovery period:
After terminal necropsy (20 rats/sex/group), the remaining 10 rats/sex/group in Groups 1 and 5 were kept for an additional 28-day posttreatment recovery period prior to necropsy. The recovery animals were fed PMI Certified Rodent Diet #5002 in the same manner as the test animals.
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: All animals were observed for mortality and moribundity twice daily (at least 6 hours between observations), except on 1 day when the evening mortality check was not documented. Once daily for the Main Study animals, a clinical observation for obvious indications of a toxic effect was performed in conjunction with the morning (a.m.) mortality check.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Detailed clinical observations were made at least once prior to treatment and weekly thereafter, in conjunction with the body weight intervals.

BODY WEIGHT: Yes.
- Time schedule for examinations: At least once prior to treatment and weekly thereafter for all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Time schedule for examinations: Once prior to treatment for all animals, during Week 13 for Main Study males, and on the last day of Week 12 for the Main Study females.
- Dose groups that were examined: Control and High-dose animals.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: During Weeks 5, 9, and 14, and from the recovery animals at Weeks 14, 15, 16, and 18.
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen inhalation).
- Animals fasted: Yes.
- How many animals: Samples were collected from 15/sex/group (14/sex/group for Group 2 and 3 males were bled at Week 14 due to deaths) during Weeks 5, 9, and 14 (prior to terminal sacrifice) and from the recovery animals (5/sex) at Weeks 14 (at the same time, samples were obtained from the terminal-sacrifice animals), 15, 16, and 18 (prior to recovery sacrifice).
- Parameters examined: Differential leukocyte count and cell morphology, haemoglobin, leukocyte count, erythrocyte count, platelet count, and hematocrit.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: During Weeks 5, 9, and 14, and from the recovery animals at Weeks 14, 15, 16, and 18.
- Animals fasted: Yes.
- How many animals: Samples were collected from 15/sex/group (14/sex/group for Group 2 and 3 males were bled at Week 14 due to deaths) during Weeks 5, 9, and 14 (prior to terminal sacrifice) and from the recovery animals (5/sex) at Weeks 14 (at the same time, samples were obtained from the terminal-sacrifice animals), 15, 16, and 18 (prior to recovery sacrifice).
- Parameters examined: Alanine aminotransferase, glucose, albumin, inorganic phosphorus, albumin/globulin ratio, potassium, alkaline phosphatase, sodium, aspartate aminotransferase, total bilirubin, calcium, total cholesterol, chloride, total protein, creatinine, triglycerides, gamma glutamyltransferase, urea nitrogen, and globulin.

URINALYSIS: Yes.
- Time schedule for collection of urine: At Week 14, during Weeks 5, 9, and 14, and at Weeks 14, 15, 16, and 18.
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: Yes.
- Parameters examined: Microscopic sediment and specific gravity.

NEUROBEHAVIOURAL EXAMINATION: No.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Necropsies of the unscheduled-death, terminal-sacrifice, and recovery-sacrifice animals of the Main Study included examination of the following:
-all orifices;
-carcass (external surface of the body);
-cranial cavity;
-external surface of the brain (at necropsy); the external surface of the spinal cord and cut surfaces of the brain and spinal cord were examined whenever tissue trimming was performed;
-cervical tissues and organs;
-thoracic, abdominal, and pelvic cavities and their viscera;
-external body surface; and
-nasal cavity and paranasal sinuses.

ORGAN WEIGHTS:
Animals sacrificed at study termination and after recovery, the following organs (when present) were weighed wet (thyroid/parathyroid weighed post fixation) as soon as possible after careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner: adrenals, thymus, liver, kidneys, spleen, testes with epididymides, thyroid/parathyroid brain with brainstem. Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes.
The following tissues were preserved: adrenals, aorta, bone marrow (femur), brain with brainstem (medulla/pons, cerebellar cortex, and cerebral cortex), colon, cecum, rectum, duodenum, jejunum, ileum, esophagus, exorbital lacrimal gland, eyes (with optic nerves), femur including articular surface, harderian gland, heart, kidneys, lesions, liver, lungs, mammary gland, mesenteric lymph node, nasal turbinates, ovaries, pancreas, pituitary, prostate, salivary glands (mandibular), seminal vesicles, sciatic nerve, skin, spinal cord (cervical, midthoracic, and lumbar), spleen, stomach, testes with epididymides, thigh musculature, thymus, thyroid (parathyroids), trachea, urinary bladder, uterus with cervix and vagina.
The preserved tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from all animals in the control and high-dose groups and from all animals that died or were sacrificed in a moribund condition during the study. Gross lesions were examined microscopically from all animals. Adrenal glands, lungs, liver, kidneys, spleen, ovary, lymph nodes, duodenum, jejunum, ileum, colon, cecum, heart, and all gross lesions were examined microscopically from all dosed animals. For the recovery group, histopathology was performed on the target organs: spleen, mesenteric lymph node, jejunum, and ileum.
Other examinations:
Toxicokinetic Analyses: Samples (1 mL) of sodium heparinized blood were collected during Weeks 2 and 13 by jugular venipuncture from three animals/sex/group from Groups 2 and 5 of the Satellite animals at approximately 16:00, 20:00, 0:00, 4:00, and 8:00 o clock over 2 designated days. Plasma was separated by centrifugation, collected into labeled containers, and stored at approximately -20°C. Samples were discarded as per Amendment No. 4.
Statistics:
- Absolute body weights and body weight changes, weekly and total food consumption, clinical pathology data, and organ weights were analyzed by analysis of variance techniques; data from Satellite animals were not analyzed. Tests for homogeneity of variances and ANOVA were evaluated at the 5.0% probability level. Control versus treated group mean comparisons were evaluated at the 5.0% two-tailed probability level.
- If variances of transformed 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.
Clinical signs:
no effects observed
Description (incidence and severity):
In general, the findings noted for those animals which survived to their respective sacrifice occurred sporadically and/or were of the type commonly seen in this species at this laboratory. There were no test material-related differences between the control and test groups.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three male rats died during the study. One control rat from the 28-day post treatment recovery group died after bleeding during Week 16 (Day 110); this was classified as an accidental death. One Group 2 animal from the terminal sacrifice was found dead during Week 4 (Day 26); after microscopic examination, inflammation of various organs was determined as the cause of death. One group 3 animal from the terminal sacrifice was found dead during the Week 5 (Day 31) and the cause of death was not determined microscopically.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For Main Study animals, mean body weight values were comparable between treated and control groups. Mean body weight change values were significantly increased for Group 5 females for Weeks 2-3, Group 2 and 5 females for Weeks 3-4, Group 3, 4 and 5 males for Weeks 6-7, and Group 5 males for Weeks 10-11. Body weight change means were significantly decreased for Group 4 and 5 males for Weeks 12-13. Total body weight change mean values were significantly increased for Group 5 females for Weeks 1-14. With the exception of the higher body weight gains for the Group 5 females during the study, there were no changes in body weight attributed to the test article administration. Body weights and body weight changes during the recovery period were similar between control and treated groups (despite the frequent fasting for clinical pathology blood collections), with the exception of a significant increase noted for Group 5 females during Week 15. For Satellite animals, body weight mean values and body weight change mean values (not statistically analyzed) were comparable between the Group 2 and 5 animals for Weeks 1-13.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption values were significantly increased for Group 5 females for Weeks 5-8, but total food consumed for this group during the study was not significantly different from the control value, although slightly elevated. All other food consumption values were similar between control and treated groups throughout the study.
Mean compound consumption was generally within 10% of the target dose level (mg/kg/day), with the exception of Group 4 males and females during Week 9 (13% and 12% below targeted level, respectively).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Retinal linear atrophy was observed, being the most common incidental finding noted in the eyes of rats. In addition, chromodacryorrhea was recorded, which is often a sign of stress within the animal and is not compound related. Corneal opacity and anterior synechia were observed which appear to be related to iatrogenic trauma, most likely associated with orbital bleeding. The cupping of the optic nerve seen in one Group 1 female is an unusual aberration which is most likely due to physiologic cupping. However, cupping due to increased intraocular pressure could not be ruled out. None of the observations noted were attributed to test material administration.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The effects on the clinical pathology data were noted primarily in the 100 mg/kg bw/day animals but also in the 10 mg/kg bw/day animals. Alterations in hematology data which are attributed to treatment and which were significant at one or more studied intervals, included small decreases in erythrocyte count, hemoglobin and hematocrit values in 100 mg/kg bw/day animals, significantly at one or more intervals, including recovery. In addition, significantly higher mean values for total leukocyte count and segmented neutrophils in Group 5 animals were recorded; these were attributed to the administration of the test substance. A significantly higher mean lymphocyte count was seen in Group 5 females at Week 9 and this change may be related to treatment, but the mechanism of the change is not apparent from the data examined. Furthermore, the mean platelet counts were significantly higher in Group 5 males at Week 15R (R= recovery) and Group 5 females at Weeks 14 and 14A (R). The remaining significant changes in the hematology data are considered incidental to treatment due to the lack of correlating data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower mean values for total protein in Group 3 and 4 females at Week 9 and for albumin in Group 3 and 4 females and Group 5 animals at Week 9 and Group 3 and 5 females at Week 14 were recorded, however they were not dose dependent. They were accompanied by significantly higher globulin values in Group 5 males at Weeks 14A (R), 15R, and 16R and Group 5 females at Week 14A (R). The mean globulin values remained consistently higher in Group 5 animals relative to control values during the recovery intervals; however, they were not significantly different from control values at Week 18R. The elevated globulin concentrations are suggestive of an inflammatory response; the higher individual globulin values generally correlated with the increased total leukocyte and segmented neutrophil counts, the enlarged mesenteric lymph nodes observed at necropsy, and the histologic evidence of mild-to-moderate inflammation in these nodes in these groups at recovery sacrifice. The significant increases observed in the mean values for alanine aminotransferase (ALT) in Group 5 males at Week 5 and for aspartate aminotransferase (AST) in Group 5 males at Weeks 14 and 18R were of low magnitude and not accompanied by findings in necropsy or organ weight data. The significant differences observed in the remaining biochemical analytes are not attributed to the administration of the test substance due to the low magnitude of the changes and the lack of a dose response.
Urinalysis findings:
no effects observed
Description (incidence and severity):
All findings in the urinalysis are considered unremarkable or incidental to treatment.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The mean spleen-to-body-weight ratios and spleen-to-brain weight ratios in males and the mean absolute spleen weights and spleen-to-brain-weight ratios in females were increased in Group 5 and correlated to microscopic findings of foci of reticuloendothelial cell hyperplasia in the germinal centers of the spleen. The additional significant organ weight changes (changes in thyroid, kidney, thymus and adrenal weights) did not correlate to microscopic findings and are considered incidental to test article administration.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related observations noted at the terminal sacrifice included enlarged spleen in one Group 5 male, raised area of the spleen in one Group 3 female, and enlarged mesenteric lymph node in four Group 5 males and eleven Group 5 females. Test article-related observations at the recovery sacrifice included enlarged spleen in one Group 5 male, enlarged mesenteric lymph nodes in five Group 5 males and four Group 5 females, and enlarged lymph node (other) in one Group 5 male.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related histomorphologic findings were seen in the sections of spleen, jejunum, ileum, and mesenteric lymph node.
The treatment-related histomorphologic finding in the spleen was the presence of foci of reticuloendothelial (RE) cell hyperplasia. The incidence of this lesion was increased marginally in animals which received 100 mg/kg bw/day, although the severity was judged minimal. The 28-day post-treatment recovery period failed to resolve this lesion. In general, these foci of reticuloendothelial cells were seen in the germinal centers of the spleen.
In the jejunum, the incidence of foamy macrophages in the lamina propria was increased in animals which received 100 mg/kg bw/day. The ileum showed an increased incidence of foamy macrophages in the lamina propria of animals which received 10 mg/kg bw/day, although the severity was judged minimal. At 100 mg/kg bw/day, both the incidence and severity of this finding were increased. After 28 days of post-treatment recovery, this lesion was still present in the lamina propria of jejunum and ileum of animals which received 100 mg/kg bw/day, though at a decreased severity.
In the mesenteric lymph node, the incidence and severity of foci of reticuloendothelial cell hyperplasia was increased in animals which received 10 mg/kg bw/day although the severity was generally considered minimal. At 100 mg/kg bw/day, both the incidence and severity of this finding were increased. The incidence of chronic active inflammation and foamy macrophages in the mesenteric lymph node was also increased in animals which received 100 mg/kg bw/day. Chronic active inflammation was focal and/or multi focal in nature and was associated with foamy macrophages. All these mesenteric lymph node lesions were still present after 28 days of post-treatment recovery period in animals which received 100 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Based upon the histomorphologic findings of foci of reticuloendothelial cell hyperplasia in the mesenteric lymph node and an increased incidence of foamy macrophages in the lamina propria of the ileum, the no-observable-effect level for the test substance when fed in the diet to rats for 90 days is 5 mg/kg/day. These lesions were still present in animals fed 100 mg/kg/day after a 28-day post treatment recovery period.
Executive summary:

The toxicity of the test article after repeated oral administration was assessed in a 90 day feeding study with rats according to GLP principles and OECD guideline 408. Male and female Sprague-Dawley Crl:CD®BR rats were assigned to 5 groups (30/sex/group in Groups 1 and 5; 20/sex/group in Groups 2, 3, and 4) and designated as the Main Study animals; an additional 12 animals/sex/Groups 2 and 5 were designated as Satellite animals and used to determine absorption of the test article. A sixth group of 10 rats/sex was used for prestudy clinical pathology determinations; after blood and urine collection, these animals were sacrificed and discarded.

Treated animals received dose preparations containing the basal diet and the test material at 2.5, 5, 10, or 100 mg/kg/day (ranges of 25.6 to 54.3, 51.2 to 113.9, 102.4 to 233.2, and 1024.3 to 2289.6 ppm for Groups 2-5, respectively); control animals received standard animal diet. A standard battery of tests was performed on Main Study animals and included daily clinical observations, weekly physical examinations, weekly body weight and food consumption determinations, ophthalmologic examinations (prior to treatment for all animals and during the final week for control and high-dose main study animals), and clinical pathology determinations (prior to initiation of dosing and at Weeks 5, 9, 14 [Days 30, 60, and prior to the terminal sacrifice, respectively] and 14A (R), 15R, 16R, and 18R [7, 14, and 29 days post-treatment, respectively]).

After at least 90 days of treatment (at terminal sacrifices), 20 rats/sex/group were sacrificed and subjected to a gross necropsy; protocol-specified organs/tissues were weighed, collected, and examined microscopically from each terminal-sacrifice animal in the control and high-dose groups and from each animal that died at an unscheduled interval. In addition the adrenal glands, lungs, liver, kidneys, spleen, ovary, lymph nodes, duodenum, jejunum, ileum, colon, cecum and heart were microscopically examined from all dosed animals; gross lesions were examined from all animals. After an additional 28-day post treatment recovery period, the surviving animals were sacrificed and subjected to a gross necropsy; protocol-specified organs/tissues were weighed and collected. Target organs designated at the terminal sacrifice were examined microscopically from the recovery animals.

Three animals died prior to scheduled sacrifice (Group 1 male B89342 - accidental death after bleeding; Group 2 male B89405 - found dead; Group 3 male B89458 - found dead). They were subjected to a gross postmortem examination, and all protocol-specified tissues were examined microscopically. After microscopic examination, inflammation of various organs in Group 2 animal B89405 was determined the cause of death. The cause of death for group 3 animal B89458 was not determined microscopically.

Body weights and food consumption were not affected by treatment, except for sporadic significant increases in body weight gain and food consumption in the 100 mg/kg/day females. There were no test material-related ophthalmological findings. The effects the test substance on the clinical pathology and anatomical pathology data were noted primarily in the 100 mg/kg/day animals but to a lesser degree in the 10 mg/kg/day animals. Alterations in hematology data which are attributed to treatment included small decreases in the erythrocyte count, hemoglobin, and hematocrit values and increases in total leukocyte and segmented neutrophil counts in the 100 mg/kg/day animals, significantly at one or more intervals, including recovery. The significant elevation in globulin concentration noted for the 100 mg/kg/day animals at one or more intervals was suggestive of inflammation; the higher individual globulin values generally correlated with the increased total leukocyte and segmented neutrophil counts, the enlarged mesenteric lymph nodes observed at necropsy, and the histologic evidence of mild-to moderate inflammation in these nodes in these groups at recovery sacrifice.

Gross observations at necropsy included enlarged mesenteric lymph nodes in a high proportion of the 100 mg/kg/day animals at both the terminal and recovery sacrifices. These correlated with the observation of foci of reticuloendothelial cell hyperplasia in the mesenteric lymph nodes of rats treated with 10 mg/kg/day at the terminal sacrifice and rats treated with 100 mg/kg/day at both the terminal and recovery sacrifices; the incidence of chronic active inflammation associated with foamy macrophages was also increased (both sacrifices) in animals that received 100 mg/kg/day.

The mean spleen-to-body weight ratios and spleen-to-brain weight ratios in the males and the mean absolute spleen weights and spleen-to-brain weight ratios in the females were significantly increased at the 100 mg/kg/day dose and correlated to microscopic findings of foci of reticuloendothelial Cell hyperplasia in the germinal centers of the spleen. The additional significant organ weight changes are considered incidental to test article administration.

Treatment-related histomorphologic findings included those previously discussed for the mesenteric lymph node and spleen and an increased incidence of foamy macrophages in the lamina propria of the jejunum of animals which received 100 mg/kg/day of test material. In the ileum, the incidence and severity of foamy macrophages in the lamina propria were increased in animals which received 10 and 100 mg/kg/day. After the 28-day recovery period, this lesion was still present in the jejunum and ileum of animals which received 100 mg/kg/day.

Based upon the histomorphologic findings of foci of reticuloendothelial cell hyperplasia in the mesenteric lymph node and an increased incidence of foamy macrophages in the lamina propria of the ileum, the no-observable-effect level for the test article when fed in the diet to rats for 90 days is 5 mg/kg/day. These lesions were still present in animals fed 100 mg/kg/day after a 28-day post treatment recovery period.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Organ:
mesenteric lymph node

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

Repeated dose oral toxicity of the test article was assessed by two 90 day feeding studies and one 28 day feeding study performed in rats.

 

In the key study the test article was administered in the diet to rats for at least 90 days to determine the no-observable effect level. The study was designed to comply with GLP principles and with the OECD guideline No. 408. Male and female Sprague-Dawley Crl:CD®BR rats were assigned to 5 groups (30/sex/ group for Groups 1 and 5; 20/sex/group for Groups 2 to 4) and designated as the Main Study animals; an additional 12 animals/sex/Groups 2 and 5 were designated as Satellite animals and were used to obtain plasma for possible determination of absorption of the test article. Treated animals received the dose preparations containing the basal diet and the test material at concentrations that would yield a dosage of 2.5, 5, 10, and 100 mg/kg/day; control animals received standard animal diet.

There were three unscheduled deaths during the course of the study. One control rat died during bleeding and was classified as an accidental death. After microscopic examination, inflammation of various organs in a Group 2 animal was determined as the cause of death. The cause of death for the animal of Group 3 was not determined microscopically. Body weights and food consumption were not affected by treatment with the test article except for sporadic significant increases in body weight gain and food consumption in the 100 mg/kg/day females. There were no test material-related ophthalmological findings. The effects of the test substance on the clinical pathology and anatomical pathology data were noted primarily in the 100 mg/kg/day animals but also in the 10 mg/kg/day animals. Alterations in hematology data which are attributed to treatment, included small decreases in erythrocyte count, hemoglobin and hematocrit values and increases in total leukocyte and segmented neutrophil counts in 100 mg/kg/day animals, significantly at one or more intervals, including recovery. The elevations are consistent with active inflammation and may be related to the enlarged mesenteric lymph nodes observed in Group 5 animals at terminal and recovery sacrifices. The remaining significant changes in the hematology data are considered incidental to treatment due to the lack of correlating data. There was a significant elevation in globulin concentration noted for the 100 mg/kg/day animals at one or more intervals. The elevated globulin concentrations are suggestive of an inflammatory response; the higher individual globulin values generally correlated with the increased total leukocyte and segmented neutrophil counts, the enlarged mesenteric lymph nodes observed at necropsy, and the histologic evidence of mild-to-moderate inflammation in these nodes in these groups at recovery sacrifice. All other findings in the serum chemistry and urinalysis are considered unremarkable or incidental to treatment.

Gross observations at necropsy included enlarged mesenteric lymph nodes in a high proportion of the 100 mg/kg/day animals at both the terminal and recovery sacrifices. These correlated with the observation of foci of reticuloendothelial cell hyperplasia in the mesenteric lymph nodes of rats treated with 10 mg/kg/day at the terminal sacrifice and rats treated with 100 mg/kg/day at both the terminal and recovery sacrifices; the incidence of chronic active inflammation associated with foamy macrophages was also increased (both sacrifices) in animals which received 100 mg/kg/day. The remaining gross observations noted at the terminal and recovery sacrifices are considered incidental to treatment, as they were without pathologic correlates. The mean spleen-to-body weight ratios and spleen-to-brain weight ratios in males and the mean absolute spleen weights and spleen-to-brain weight ratios in females were significantly increased at the 100 mg/kg/day dose and correlated to microscopic findings of foci of reticuloendothelial cell hyperplasia in the germinal centers of the spleen. Any other significant organ weight changes are considered incidental to test article administration.

Treatment-related histomorphologic findings included those previously discussed for the mesenteric lymph node and spleen and an increased incidence of foamy macrophages in the lamina propria of the jejunum of animals which received 100 mg/kg/day of test material. In the ileum, the incidence and severity of foamy macrophages in the lamina propria were increased in animals which received 10 and 100 mg/kg/day. After the 28-day recovery period, this lesion was still present though at a lower incidence and lesser degree of severity in the jejunum and ileum of animals which received 100 mg/kg/day.

Based upon the histomorphologic findings of foci of reticuloendothelial cell hyperplasia in the mesenteric lymph node and an increased incidence of foamy macrophages in the lamina propria of the ileum, the no-observable-effect level for the test item when fed in the diet to rats for 90 days is 5 mg/kg/day. These lesions were still present in animals fed 100 mg/kg/day after a 28-day post treatment recovery period.

 

In the second 90-day study the test article was administered in the diet for 3 months at doses of 0, 150, 800, 3000 and 12000 ppm (= mg/kg food) to a total of 100 albino rats, 10 males and 10 females per dose group. Corrected for the analytically found test article concentrations in the diet, the mean daily intake of test material was 9.70, 47.9, 201 and 826 mg/kg bodyweight in males, and 9.92, 50.4, 210 and 817 mg/kg bodyweight in females.

No relevant clinical symptoms and no signs of toxicity were observed during the study. No treatment-related death occurred during the study. Trends to decreased mean bodyweights were recorded for both males and females at the two upper dose levels. This was in line with a decrease in the mean food consumption of males from group 5 (12000 ppm) and group 4 (3000 ppm). In females decreased mean food consumption was recorded for group 4 and 5 (3000 and 12000 ppm) from weeks 1 to 3 and for group 5 (12000 ppm) from week 10 onwards. Decreased mean food consumption ratios were recorded for animals of group 5 (12000 ppm) at treatment week 1, while from week 3 onwards increased mean food consumption ratios were noted for these animals. The mean water consumption of males treated at the top dose level (12000 ppm) was markedly decreased, and to a lesser extent in females of group 5 (12000 ppm). In clinical chemistry analyses, hypochromasia and microcytosis of red blood cells, substantiated by lower values for MCH and MCV, were noted in males and females of the high dose group (12000 ppm). In addition, a decrease of hemoglobin and packed cell volume occurred in the males of this group. A dose-related leucocytosis with neutrophilia, hypersegmentation of neutrophils and relative lymphopenia occurred in group 4 and 5 (3000 and 12000 ppm). Neutrophilia and relative lymphopenia were also observed in group 3 animals (800 ppm). An increased number of thrombocytes was noted in males and females of the high dose group (12000 ppm). Lower plasma glucose and cholesterol levels and a decrease of the plasma albumin concentration, associated with lower plasma protein concentration and decreased albumin to globulin ratio, occurred in group 4 and 5 (3000 and 12000 ppm). Slightly higher plasma levels of sodium, potassium and chloride were recorded in the high dose animals (12000 ppm). Increased activities of aspartate ciminotransferase and alkaline phosphatase were observed in group 5 (12000 ppm). In addition, aspartate aminotransferase activity was slightly higher in the females of group 3 and 4 (800 and 3000 ppm). Bilirubinuria and occurrence of blood in the urine were observed among males and females of group 5 (12000 ppm). Additionally, the males of group 5 (12000 ppm) excreted a more acid urine, and the females of this group excreted more but less concentrated urine.

Decreased mean carcass weights were recorded for animals of group 4 and 5 (3000 and 12000 ppm). In females of group 5 (12000 ppm) the liver/bodyweight ratio was markedly increased, achieving statistical significance at the 1% level. Absolute and relative spleen weights were significantly increased in females of group 4 and 5 (3000 and 12000 ppm). Treatment-related macroscopical findings include enlarged mesenteric lymph nodes in animals of either sex from groups 3, 4 and 5 (800 ppm, 3000 ppm and 12000 ppm), fluid contents and/or dilatation of the small intestine and the caecum in animals of either sex from group 5 (12000 ppm), and mottled liver in one male and one female animal from group 5 (12000 ppm). Primary histopathological changes consisted of the presence of foamy macrophages (foam cells) in the mucosa of the small intestine (animals of either sex from group 3, 800 ppm), and of the small and large intestine (animals of either sex from groups 4 and 5, 3000 ppm and 12000 ppm), in the mesenteric lymph node (animals of either sex from group 2, 150 ppm, and from groups 3, 4 and 5), in the axillary lymph node (one male from group 5), in the liver (one male from group 4, animals of either sex from group 5), in the spleen (one female from group 3, animals of either sex from groups 4 and 5), in the ovary (females from groups 4 and 5), in the adrenal cortex (one male from group 5), in the kidney glomerulus (animals of either sex from group 5), and of acute tubular lesion in the kidney (animals of either sex from group 5). The above lesions were accompanied by the following reactive changes: necrotizing and inflammatory changes and/or cystic dilatation of sinusoids in the mesenteric lymph node, sometimes associated with peritoneal inflammation (animals of either sex from groups 3, 4 and 5), focal necrosis and/or ulceration of the small and/or large intestine (animals of either sex from group 5), granuloma formation and / or inflammatory cell infiltrates in the liver (one female from group 3, animals of either sex from groups 4 and 5), cytoplasmic vacuolization of intrahepatic bile duct epithelium (animals of either sex from group 5), diffuse polymorphonuclear infiltration of the lung (animals of either sex from groups 4 and 5), focal necrosis (one female), haemosiderosis and/or atrophy of the white pulp of' the spleen (animals of either sex from group 5), hypercellularity of the bone marrow (females from groups 4 and 5). It is concluded that the foam cell reaction involving parts of the reticulo-endothelial system and a variety of epithelial tissues was probably due to the accumulation of test material and/or metabolites absorbed from the intestine. All other morphological changes observed in control and treated animals were considered to be incidental and unrelated to the treatment.

Under the conditions of this test, treatment with the test article for 3 months at concentrations as low as 150 ppm resulted in toxic effects as substantiated by the presence of foamy macrophages in a number of organs such as lymph nodes, small and large intestine, liver, spleen, ovary, adrenal gland and kidney. This foam cell reaction was accompanied by a variety of secondary histopathologic findings such as inflammatory and necrotizing changes. Equally laboratory investigations revealed significant changes in animals treated at 800, 3000 and 12000 ppm. Hypochromic microcytic anemia, leucocytosis with neutrophilia and relative lymphopenia were the main hematological findings. Blood chemistry and urine analysis showed changes suggesting a disturbance in liver and kidney function and in electrolyte homeostasis. Reduced mean bodyweight was correlated with reduced mean food consumption at the two upper dose levels (3000 and 12000 ppm). The mean water consumption was decreased at the top dose level (12000 ppm). It can be inferred from the observations made during the above study that a "no observable effect level" for the test substance when offered to rats continuously in their food over a period of 3 months is below 150 ppm, corresponding to a mean daily intake of less than 9.70 mg/kg bodyweight for males and less than 9.92 mg/kg bodyweight in females.

A subacute oral study performed according to OECD 401 (1981) and GLP, resulted in a NOEL of 34 mg/kg bw showing liver and kidney as potential target organs based on hematological, clinical chemistry changes and organ weight changes. However, no histological lesions were observed in either of the two potential target organs. Significant body weight decreases in the high dose group without decrease in the food consumption was considered as an adverse effect, especially in males where the body weight decreased up to 15 %. So based on the effects on body weight, NOAEL was considered to be 134 mg/kg bw in males and 706 mg/kg bw in females.

To summarize, the two subchronic feeding studies with rats showed a dose-dependent effect on the lymphatic system, starting with mild to moderate inflammation of the mesenteric lymph nodes and leading to secondary inflammation and partial necrosis. Specifically, reticuloendothelial cell hyperplasia occurred in the mesenteric lymph node accompanied by an increased incidence of foamy macrophages both in the mesenteric lymph nodes and the lamino propria at 10 and 100 mg/kg bw. At higher doses the lesions were also observed in the large intestine, the liver, the kidney, the adrenal cortex, the spleen and the ovaries. In some cases this effect led to secondary inflammatory lesions and/or partial necrosis (in parallel to slight to moderate dysfunctional changes). In the blood, an activation of the white blood cell system activity was observed, reflected by the increased segmented neutrophilic granulocyte counts. The findings were not completely reversible within the recovery period.

Justification for classification or non-classification

Classification according to GHS (1272/2008/EEC, as amended by Commission Regulation (EC) No 790/2009/EEC)

The substance is assigned to category 2 because the effects observed at higher doses, specifically at doses of equal or less than 100 mg/kg bw, are considered to be serious and irreversible. The primary target organ of systemic toxicity is identified as the gastrointestinal lymphatic system.