Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
batch no. TD-4024548 (99.8% purity)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ± 3 oc; relative humidity range: 30-70 %). Values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

Rats were housed in groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

Rats were fed pelleted standard Provimi Kliba 3433 (batch no. 39/05, 63/05) rat maintenance diet (Provimi Kliba AG, CH- 4303 Kaiseraugst, Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

Community tap-water from ltingen was available ad libitum in water bottles. Bacteriological, chemical and contaminant analyses were performed on representative samples.

None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Each rat was given a single daily oral dose of test article by gavage in a volume of 5 ml per kg of body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 4 hours and 7 days) of the dose formulations were determined in samples taken before experimental start. Analyses were performed by the laboratory staff of the Principal Investigator of the analytical phase, according to a HPLC analytical method supplied by the Sponsor and previously adapted at RCC Ltd. Details of the analytical method were documented in the raw data generated by the Principal Investigator (and/or his/her staff) and the Principal Investigator provided an analytical phase report. Concentration and homogeneity of the dose formulations were determined in samples taken monthly during the treatment period.
Duration of treatment / exposure:
once
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg b.w.
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg b.w.
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg b.w.
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg b.w.
Basis:
actual ingested
No. of animals per sex per dose:
ten
Control animals:
yes, concurrent vehicle
Details on study design:
A total of 104 rats were ordered for this study, and 50 male and 50 female rats were randomly assigned to one of four groups, using a computer generated random algorithm. Fifteen male and 15 female rats were assigned to groups 1 and 4 (including a recovery group of 5 rats per sex per group), while 10 male and 10 female rats were assigned to groups 2 and 3. Rats were 6 weeks of age at the time of receipt at the facility, and the range of body weights for male and female rats was 135 to 160 grams (mean = 148 grams) and 116 to 133 grams (mean = 125 grams) respectively. Rats were allowed a 7-day acclimation period before initiation of treatments.

Examinations

Observations and examinations performed and frequency:
MORTALITY/VIABILITY:
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (DAILY):
The animals were observed for clinical signs once daily before commencement of administration; and once daily during the treatment and recovery periods.

DETAILED CLINICAL OBSERVATIONS (WEEKLY):
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-12 and 14-16) thereafter. These were entered manually into the computer system and evaluated under a subproject number (A58814) for technical reasons.

FOOD CONSUMPTION:
The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

BODY WEIGHTS:
Body weights were recorded weekly during the acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATIONS:
The ophthalmoscopic examinations of both eyes of all animals were performed after the application of a mydriatic solution using an ophthalmoscope. A description of any abnormality was recorded. For unilateral findings unless otherwise indicated in the tables, the contralateral eye was without abnormalities. The examinations were performed at acclimatization in all animals (Allocation A and B), during week 13 in control and high dose animals (Allocation A and B), and at week 17 in control and high dose animals (Allocation B)

FUNCTIONAL OBSERVATIONAL BATTERY:
Relevant parameters from a modified Irwin screen test wereevaluated during weeks 13 (Allocation A and B) and 17 (Allocation B). These were entered manually into the computer system and evaluated under a subproject number (A58814) for technical reasons.

GRIP STRENGTH:
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

LOCOMOTOR ACTIVITY:
Locomotor (decreased or increased) activity was measured quantitatively with AMS Fohr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

CLINICAL LABORATORY INVESTIGATIONS:
Blood and urine samples were collected from all study rats after 13 weeks and from rats in recovery groups after 17 weeks

Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a microhematocrit glass capillary tube.

Urine was collected during the 18-hour fasting period into a specimen vial. The assays were performed at RCC Ltd (Fullinsdorf) under internal laboratory quality control conditions to assure reliable test results. Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

HEMATOLOGY:
The following hematology parameters were determined:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Platelet (thrombocyte) count
Reticulocyte count
Reticulocyte maturity index
Methemoglobin
Heinz bodies
Total leukocyte count
Differential leukocyte count
Coagulation:
Thromboplastin time
Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY:
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Creatine kinase
Alkaline phosphatase
Gamma-glutamyl-transferase
Sodium
Potassium
Chloride
Calcium
Phosphorus inorganic
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS:
The following urinalysis parameters were determined:
Volume (18 hours)
Specific gravity (relative density)
Osmolality
Color
Appearance
pH
Nitrite
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Blood
Sacrifice and pathology:
NECROPSY:
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless
otherwise indicated):
Adrenal glands
Aorta
Bone (sternum, femur incl. joint)
Bone marrow (femur)
Brain (4 levels)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Nasal cavity (turbinates)
Ovaries
Pancreas
Pituitary gland
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Exorbital lacrimal glands
Eyes with optic nerve (fixed in Davidson's
solution)
Harderian gland (fixed in Davidson's solution)
Heart
Ileum, w/Peyer's patches
Jejunum w/Peyer's patches
Kidneys
Larynx
Lacrimal gland, exorbital
Liver
Lungs (infused with formalin)
Lymph nodes- mesenteric, mandibular
Mammary gland area
Skeletal muscle
Skin
Spinal cord- cervical, midthoracic, lumbar
Spleen
Stomach
Testes (fixed in E3ouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Tongue
Trachea
Urinary bladder (infused w/formalin)
Uterus
Vagina
Gross lesions

ABSOLUTE AND RELATIVE ORGAN WEIGHTS:
The following organ weights were recorded on the scheduled dates of necropsy:
Brain
Heart
Liver
Thyroids w/parathyroids
Thymus
Kidneys
Adrenals
Uterus
Spleen
Testes
Epididymides
Ovaries

The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE:
All organ and tissue samples, as defined under Histopathology, were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY:
Slides of all organs and tissues that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. As possible test item-related morphologic changes were detected in the organs of high-dose animals, the same organs from animals of the mid- and low-dose groups were examined.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, food consumption, body weight, clinical laboratory data, organ weights and ratios, as well as:

• The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables were assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex. Alternatively, the t-test was used for grip strength and locomotor activity.
• The Steel-test (many-one rank test) were applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the ophthalmoscopic findings.

References :
C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
S.C. Gad and C.S. Weil: Statistics and Experimental Design for Toxicologists. The Telford Press, Caldwell, New Jersey, 43-45 (1986).
O.J. Dunn: Multiple comparisons using rank sums. Technometrics 6, 241-252 (1964).
R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
VIABILITY /MORTALITY
No test item-related deaths occurred. Control male no. 12 was removed from the study for ethical reasons during the recovery phase of the study. The poor condition of this rat was found to be due to a kidney nephroblastoma. Control female rat no. 58 died during the treatment period. The macroscopical findings in the lungs correlated microscopically with edema and congestion, and were considered to confirm that the animal died from a dosing error.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
No test item-related clinical signs of toxicological relevance were noted at any dose level. Soft feces were noted in rats at all dose levels. This finding is commonly associated with the use of PEG 300 as vehicle and is considered to be a passive finding rather than one of systemic toxicity. Pale feces were noted in male and female rats treated with 1000 mg/kg/day. In the absence of any concomitant findings which could be related to this change, this was considered to be due to the light-colored test item and is of no toxicological relevance. Soft and pale feces both resolved during the recovery period. All other clinical signs noted during daily observations (such as crusts, alopecia, salivation, kinked tail, half-closed eyes, exophthalmia, rales, etc) were considered to be typical background changes and are of no toxicological relevance.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
No test item-related clinical signs of toxicological relevance were noted during weekly detailed clinical observations (weeks 1-12, 14-16) at any dose level. A small number of typical background findings were noted in males and females. These changes included bedding consumption, ruffled fur, rales, localized erythema or alopecia, exophthalmia (bilateral) and/or miosis (bilateral). None of the observed findings was
considered to be of toxicological relevance.

NEUROBEHAVIOR
FUNCTIONAL OBSERVATIONAL BATTERY:No test item-related clinical signs of toxicological relevance were noted during functional observational battery (week 13 of treatment or week 4 of recovery) at any dose level. A small number of typical background findings were noted in males and females. These changes included bedding consumption, ruffled fur, localized erythema or alopecia, exophthalmia (bilateral), paresis, eye discoloration and/or miosis (bilateral). None of the observed findings was considered to be of toxicological relevance. GRIP STRENGTH: No test item-related differences in the mean fore- and hindlimb grip strength values were noted at any dose level when assessed during week 13 of treatment and week 4 of recovery. In males treated with 300 mg/kg/day, significantly higher forelimb grip strength values were seen (p<0.05), whereas significantly lower hindlimb grip strength values were recorded (p<0.01 ). These contrary changes were considered to be unrelated to the test item. In males treated with 1000 mg/kg/day, significantly lower hindlimb grip strength values were seen when compared with controls. In the absence of similar differences in the forelimb grip strength values, these differences were considered to be incidental. LOCOMOTOR ACTIVITY: No test item-related effects upon locomotor activity were noted after 13 weeks of treatment or 4 weeks of recovery. In males treated with 100 mg/kg/day, significantly elevated total mean locomotor activity (0-60 minutes) was noted when compared with control males. In males treated with 300 mg/kg/day, the mean locomotor activity was significantly reduced (p<0.01) during a single measurement interval (40-50 minutes). An initial increase in locomotor activity (0-1 0 minutes) was noted in males treated with 1000 mg/kg/day (p<0.05). In females treated with 100 mg/kg/day, elevated locomotor activity was noted during 0-10 minutes (p<0.01), 20-30 minutes (p<0.01), 30-40 minutes (p<0.05) , 40-50 minutes (p<0.05) and 50-60 minutes (p<0.05), as well as overall locomotor activity from 0-60 minutes (p<0.05). In females treated with 300 mg/kglday, locomotor activity was elevated during 30-40 minutes (p<0.05).
These incidental differences were either unrelated to dose, transient or within the typical range of variation. After the recovery period, females treated previously with 1000 mglkglday had reduced locomotor activity during 30-40 minutes (p<0.01) and 40-50 minutes (p<0.05). These differences were not considered to be late effects of the test item.

FOOD CONSUMPTION
The mean daily food consumption of the test item-treated rats compared favorably with those of the control rats during the treatment and recovery period. The mean relative daily food consumption was accordingly similar.

BODY WEIGHTS
Reductions in mean body weight noted in rats treated with 1 000 mglkglday were considered to be a slight test item-related effect. The reductions noted in females were more clearly expressed than the reductions noted in males. In males treated with 1000 mglkglday, marginally lower mean body weights were noted (ca. 5-7%) during the treatment period, beginning around day 36 of treatment. This was also reflected in the mean body weight gain values. The body weights and body weight gain increased slightly during the recovery period. In females treated with 1000 mglkglday, reductions in mean body weight attained statistical significance on day 43 (p<0.05), day 50 (p<0.05), day 64 (p<0.05), day 71 (p<0.01), day 78 (p<0.05) and day 91 (p<0.05) of treatment. The reductions in mean body weight gain attained statistical significance throughout the treatment period (p<0.01 or p<0.05), with the exception of day 57. Lower mean body weights persisted during the recovery period in females treated previously with 1 000 mglkglday, attaining statistical significance on days 3 and 15 (p<0.05). Mean body weight gain was also lower than control females but the differences did not attain statistical significance. The transient significant reductions in mean body weight which were noted in females treated with 100 mglkglday (days 36 and 43 (p<0.05)) were considered to be incidental. Similarly, the transient significant reductions in mean body weight gain (days 8, 36 and 43 (p<0.01 or p<0.05)) were considered to be incidental.

OPHTHALMOSCOPIC EXAMINATIONS
13 Weeks: No test item-related ophthalmoscopic findings were noted at any dose level during 13 weeks of treatment. The benchmark observations performed in all· animals during the acclimatization period revealed only typical background findings common to rats of this strain and age.
17 Weeks: No test item-related ophthalmoscopic findings were noted at any dose level during 13 weeks of treatment and 4 weeks of recovery.

CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY (AFTER 13 WEEKS OF TREATMENT):
No test item-related differences in the hematological parameters were noted in males or females at any dose level. In males treated at 300 mg/kg/day, the mean absolute basophil count differed significantly from that of the control males (p<0.05), but this difference was so small that it was lost in the rounding process (note: calculations used the exact raw data value and were thereafter rounded for printing of tables). Such a difference is considered to be mathematical and is of no toxicological relevance. In males treated with 1000 mg/kg/day, the mean relative reticulocyte count was marginally but significantly elevated (p<0.05) when compared with the controls. The difference remained within the limits of the historical control data and was considered to be incidental. A significant reduction in mean absolute lymphocyte count noted in males treated with 1000 mg/kg/day (p<0.05) remained within the ranges of the historical control data and was considered to be unrelated to the test item as this parameter is subject to moderate variation even in control animals. A statistically significant reduction of platelets noted in the males treated with 1000 mg/kg/day (p<0.01) was also considered to be of no toxicological relevance and was most likely due to an incidental increase in the control value. The significant reduction of methemoglobin seen in the males treated with 1000 mg/kg/day (p<0.05) was considered to be incidental; such changes are not considered to be toxicologically relevant. In females treated with 100 or 300 mg/kg/day, a number of incidental test item-unrelated findings were noted which remained within the limits of the historical control values and/or which were dose independent and considered to be incidental. Females treated with 1000 mg/kg/day had significantly elevated mean corpuscular volume (p<0.01) and mean corpuscular hemoglobin (p<0.05), as well as significantly reduced mean
middle fluorescence reticulocyte count (p<0.05) when compared with the controls. All values remained within the ranges of the historical control data and were considered to be incidental.
HEMATOLOGY (AFTER 13 WEEKS OF TREATMENT PLUS 4 WEEKS OF RECOVERY): After the 4-week recovery period, the hematology parameters of the control and previously test item-treated males were similar. Although a small number of statistically significant differences were noted in the females previously treated with 1000 mg/kg/day (increased mean corpuscular volume, red cell distribution width, mean corpuscular hemoglobin and increased mean corpuscular hemoglobin concentration), these differences mostly remained within the ranges of the historical control data, were not supported by concomitant changes in related parameters and were considered to be incidental.
CLINICAL BIOCHEMISTRY (AFTER 13 WEEKS OF TREATMENT): No test item-related changes of toxicological relevance were noted in males or females
treated with the test item. At 1000 mg/kg/day, minor changes were seen in some parameters. Glucose levels were marginally lower in male and females treated with 1000 mg/kg/day (significant in females, P<0.05) when compared with controls. The values remained within the ranges of the respective historical control data and considered to be of no toxicological relevance. In males and females at 1000 mg/kg/day, significantly decreased urea levels (p<0.05 and p<0.01, respectively) and significantly elevated creatinine levels (p<0.01 and p<0.05, respectively) were noted. Alkaline phosphatase was elevated in males treated with 1000 mg/kg/day (p<0.05) and glutamate dehydrogenase levels were reduced in females treated with 1000 mg/kg/day (p<0.05), but both within the range of the respective historical control data. Creatine kinase levels in males and females treated with 1000 mglkg/day significantly exceeded (p<0.05 and p<0.01, respectively) those of the respective controls. This parameter is subject to wide variation and the differences remained within the ranges of the historical control values. At 1000 mg/kg/day, calcium levels were elevated in males (p<0.01) and females (p<0.05) when compared with the control values. Although the females remained within the historical control ranges, the males very slightly exceeded the upper range. This difference was not considered to represent a test item-related change, as all male groups (including the control
males) were slightly higher than usual. Significant changes in some electrolytes were seen. Sodium levels were elevated in test item-treated males and females (p<0.05 at 100 mg/kg/day, p<0.01 at 300 mg/kg/day and 1000 mg/kg/day) and chloride levels were elevated in males (p<0.05 at 300 mg/kg/day and p<0.01 at 1000 mg/kg/day. These changes were considered to be due to slightly low control values. Markedly elevated mean lactate dehydrogenase levels were seen in females treated with 1000 mg/kg/day (p<0.01), but this parameter varies widely and is generally considered to be indicative of hemolysis of individual samples. Some changes seen in the low- and/or mid-dose groups attained statistical significance but were not dose dependent and therefore considered to be incidental. Such changes included reduced aspartate aminotransferase activity in males at 300 mg/kg/day (p<0.05), increased chloride in females at 300 mg/kg/day (p<0.01) and increased phosphorus in females at 100 mg/kg/day (p<0.05) and 300 mg/kg/day (p<0.01 ).
CLINICAL BIOCHEMISTRY (AFTER 13 WEEKS OF TREATMENT PLUS 4 WEEKS OF RECOVERY): No test item-related differences in the clinical biochemistry values were noted after 4 weeks' recovery. After the 4-week recovery period, the clinical biochemistry parameters of the control and previously test item-treated males were similar. Although a small number of statistically significant differences were noted 1n the females previously treated with 1000 mg/kglday (reduced cholesterol, reduced triglycerides, reduced phospholipids, reduced aspartate aminotransferase activity, reduced alanine aminotransferase activity, reduced glutamate dehydrogenase, increased sodium, increased phosphorus), these represented incidental differences and were not considered to be late or persistent effects of the test item.
URINALYSIS (AFTER 13 WEEKS OF TREATMENT): Male and female rats treated with 1000 mglkglday had significantly elevated urine output (p<0.05) when compared with the respective controls. In females at this dose level, significant reductions in relative density (p<0.05), osmolality (p<0.01) and ketones (p<0.05) were noted, whereas males had significantly lower pH (p<0.05) and leukocytes (p<0.05) than the control males. Of these changes, only the elevated urine output was considered to be a marginal test item-related effect.
URINALYSIS (AFTER 13 WEEKS OF TREATMENT PLUS 4 WEEKS OF RECOVERY): After the 4-week recovery period, the urinalysis parameters of the control and previously test item-treated rats were similar.

PATHOLOGY
ORGAN WEIGHTS (AFTER 13 WEEKS OF TREATMENT): In males treated at 1000 mglkglday, statistically significant reductions of the mean spleen-to-brain weight ratio (p<0.05) were noted in males. This finding was accompanied by reduced mean absolute thymus weights (p<0.05), reduced thymus-to-body weight ratio (not significant) and reduced thymus-to-brain weight ratio (p<0.05). The reduction in mean and absolute thymus weights correlated with microscopical changes (lymphoid depletion). These findings were considered to be related to the test item. The mean kidney-to-body weight ratio of males treated with 1000 mg/kg/day was significantly elevated (p<0.01) when compared with the controls, but without microscopical correlation. This difference was considered to be unrelated to the test item. The significant difference noted in mean brain-to-body weight ratio weight of males treated with 1000 mg/kg/day was considered to be incidental, as no correlating microscopic finding was found. The significant reduction in heart-to-brain weight ratio (p<0.01) noted in males treated with 1000 mg/kg/day was also considered to be incidental; no correlating microscopic findings were found. In females treated with 1000 mg/kg/day, slightly reduced mean absolute and relative ovary weights (not significant) were noted, together with reduced mean absolute and relative uterus weights (p<0.05 or P<0.01 ). These differences were considered to be test item-related changes. The significantly elevated liver-to-body weight ratio (p<0.01) noted in these females was considered to be a test item-related adaptive change. In males treated at 300 mg/kg/day, no test item-related changes in mean absolute or relative organ weights were ascertained . In females treated with 300 mg/kg/day, reduced mean absolute and relative uterus weights (p<0.05) were considered to be test item-related changes. The significantly elevated liver-to-body weight ratio (p<0.05) noted in these females was considered to be test item-related adaptive change. In males treated with 100 mg/kg/day, the mean absolute testes weight and mean testes-to-body weight ratio was significantly elevated (both (p<0.05) when compared with the controls. This finding was considered to be incidental, as no correlating microscopic finding was found. In females treated at 100 mg/kg/day, no test item-related changes in mean absolute or relative organ weights were ascertained.
ORGAN WEIGHTS (AFTER 13 WEEKS OF TREATMENT PLUS 4 WEEKS OF RECOVERY): In males treated previously with 1 000 mg/kg/day, test item-related differences in the spleen and thymus were reversible after the recovery period. In females treated previously with 1000 mg/kg/day, reduced mean uterus weights persisted after the recovery period. Test item-related adaptive differences in the liver were reversible after the recovery period. The incidental elevation of mean brain-to-body weight ratio noted in males treated with 1000 mg/kg/day (p<0.05) persisted after the recovery period. Females had a marginal but significant increase in the mean brain-to-body weight ratio (p<0.05) after the recovery period. Neither of these differences was accompanied by any microscopical changes and both were considered to be incidental.
MACROSCOPIC FINDINGS (AFTER 13 WEEKS OF TREATMENT): Liquid contents in the cecum were recorded at necropsy in 10 of 10 males and in 4 of 10 females treated with 1000 mg/kg/day. One control male and female, and one group 3 female also had liquid contents in the cecum. In control female rat no. 58 which died during the treatment period, the lung was not collapsed and was discolored. Foamy fluid was released from the bronchi.The few other findings recorded, such as pelvic dilation of the kidneys or dark red foci in the thymus were regularly distributed among control and treatment groups, and were considered to be within the range of normal background changes which may be seen in rats of this strain and age in 13-week studies. These were considered incidental and to reflect the usual individual variability.
MACROSCOPIC FINDINGS (AFTER 13 WEEKS OF TREATMENT PLUS 4 WEEKS OF RECOVERY): There were no macroscopic findings related to the treatment with the test item. In the male no. 12 of control group that was killed for ethical reasons during the recovery period, the right kidney had a nodule which also adhered to the right adrenal gland.
MICROSCOPIC FINDINGS (AFTER 13 WEEKS OF TREATMENT): Test item-related microscopic lesions were observed in the Liver and Thymus. In the Liver, minimal or slight centrilobular hypertrophy of the hepatocytes was observed in all females treated with 1000 mglkglday. In the Thymus, a minimal or slight lymphoid depletion was observed in 2 of 10 males treated with 300 mg/kg/day and in 6 of 10 males treated with 1000 mg/kg/day. Liquid contents in the cecum recorded at necropsy in all males and some females treated with 1000 mg/kg/day (as well as one control male and female, and one female treated with 300 mg/kg/day) did not correlate microscopically as the cecum morphology did not differ between control and test item-treated animals. Microscopically the gastro-intestinal tract was without test item-related findings. In control female rat no. 58 which died during the treatment period, the macroscopical findings correlated with edema and congestion, and were considered to confirm that the animal died from a dosing error. The remaining microscopic findings were regularly distributed among the groups and were within the range and severity of spontaneous background lesions that may be observed in rats of this strain and age in this laboratory. They were considered to be of no toxicological significance.
MICROSCOPIC FINDINGS (AFTER 13 WEEKS OF TREATMENT PLUS 4 WEEKS OF RECOVERY): At the end of the recovery period, the liver and thymus morphology returned to normal. Control male no. 12 was killed for ethical reasons during the recovery phase of the study due to a kidney nephroblastoma. This rare tumor occurs spontaneously in young rats of this strain.

Effect levels

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Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of 1 ,4-CYCLOHEXANEDICARBOXYLIC ACID (1 ,4-CHDA) to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for at least 90 days resulted in no mortalities of toxicological relevance, only passive and/or incidental clinical signs during daily or weekly clinical observations (weeks 1-12). No changes of relevance were seen during functional observational battery (week 13) and the mean fore- or hindlimb grip strength values and mean locomotor activity did not show test item-related changes. No effects of toxicological relevance were recorded in mean daily food consumption, ophthalmoscopic changes, hematology parameters, clinical biochemistry parameters or macroscopical findings Test item-related findings noted in rats treated with 1000 mg/kg/day included marginally lower mean body weights and mean body weight gain in males and females, and slightly elevated urine output in males and females. In males, changes in organ weights included reduced thymus weights and reduced spleen weights. The differences in the thymus weights of males correlated with lymphoid depletion seen microscopically in these animals. In females, changes included reduced uterus weights and reduced ovary weights. Elevated liver weights were noted in females only, and correlated microscopically with minimal or slight centrilobular hypertrophy of the hepatocytes. This microscopic finding is generally associated with metabolic adaptation and is considered to be non-adverse. Test item-related findings noted in male rats treated with 300 mg/kg/day included lymphoid depletion in the thymus. Test item-related findings noted in female rats treated with 300 mg/kg/day included slightly elevated liver-to-body weight ratios, which are generally associated with metabolic adaptation and considered to be non-adverse. Reduced uterus weights were noted in females at this dose level. No test item-related findings of toxicological relevance were noted at 100 mg/kg/day. After the recovery period, reduced uterus weights persisted in females previously treated with 1000 mg/kg/day, whereas the reduced thymus and spleen weights noted in males compared favorably with the control values. Insofar as the findings noted in the females treated with 300 mg/kg/day were reversible, these changes could not be considered to be adverse. Based on the results of this study, 100 mg/kg body weight/day of 1,4-CYCLOHEXANEDICARBOXYLIC ACID (1,4-CHDA) was established as the no-observedeffect-level (NOEL), and 300 mg/kg body weight/day was considered to be the no-observed adverse-effect-level (NOAEL).
Executive summary:

GENERAL

In this subchronic toxicity study, 1 ,4-CYCLOHEXANEDICARBOXYLIC ACID (1 ,4-CHDA) was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of at least 90 days. A control group was treated similarly with the vehicle, PEG 300, only. The groups comprised 1 0 animals per sex, which were sacrificed after at least 90 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These

animals were treated for 90 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Ophthalmoscopic examinations were performed at acclimatization, the end of the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13 and during week 17 (treatment and recovery, respectively). At the end of the dosing and the treatment-free recovery period, blood samples were

withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals, as well as selected organs from low- and mid-dose animals.

MORTALITY I VIABILITY

There were no test item-related deaths.One control male was killed for ethical reasons during the recovery period and one control

female died during the treatment period as the result of a dosing error.

CLINICAL SIGNS

No test item-related clinical signs of toxicological relevance were noted at any dose level during daily clinical observations or during weekly detailed clinical observations (weeks 1-12). Daily clinical observations showed soft feces in rats of all groups, including controls. This finding is commonly associated with the use of PEG 300 as vehicle. Pale feces were noted in male and female rats treated with 1000 mg/kg/day, but were considered to be due to the light-colored test item. Both resolved during the recovery period and were considered to be passive findings of no toxicological relevance. During the weekly detailed clinical observations (weeks 1-12), observations included ruffled fur, rales, localized erythema or alopecia, exophthalmia (bilateral) and/or miosis (bilateral).

None of the observed findings was considered to be of toxicological relevance. Bedding consumption was noted after administration and although this finding was considered to be related to the test item, it was not considered to be of toxicological relevance. During the post-treatment weekly detailed clinical observations (weeks 14 -16), observations included soft and/or pale feces, rales, paresis, salivation, exophthalmia (bilateral) and/or ptosis (bilateral).

FUNCTIONAL OBSERVATIONAL BATTERY

During the functional observational battery (week 13), observations included ruffled fur, rales, localized erythema or alopecia, exophthalmia (bilateral) and/or miosis (bilateral). None of the observed findings was considered to be of toxicological relevance. Bedding consumption was noted after administration and although this finding was considered to be related to the test item, it was not considered to be of toxicological relevance. During the post-treatment functional observational battery (week 17), no signs were evident. No test item-related differences in the mean forelimb and hindlimb grip strength values or locomotor activity were noted at any dose level after 13 weeks of treatment or 4 weeks of recovery.

FOOD CONSUMPTION

The mean daily food consumption and mean relative daily food consumption of the test item-treated rats were unaffected during the treatment and recovery period.

BODY WEIGHT

The mean body weights and mean body weight gain of the rats treated with 100 mg/kg/day or 300 mg/kg/day were considered to be unaffected by treatment. Reduced mean body weights noted in rats treated with 1000 mg/kg/day were considered to be a slight test item-related effect. These changes were more distinct in the females. The lower mean body weights and mean body weight gain were noted during treatment in males treated with 1000 mg/kg/day improved slightly during the recovery period, whereas lower mean body weights and lower mean body weight gain persisted during the recovery period in females.

OPHTHALMOSCOPIC EXAMINATIONS

No test item-related ophthalmoscopic findings were noted at any dose level after 13 weeks of treatment or 4 weeks of recovery.

CLINICAL LABORATORY INVESTIGATIONS

No test item-related differences in the hematological or biochemical parameters were noted during treatment and recovery in males or females at any dose level. Urinalysis indicated that male and female rats treated with 1000 mg/kg/day had elevated urine output during

treatment when compared with the respective controls. This was considered to be a marginal test item-related effect. Urine output reverted to control levels during recovery.

PATHOLOGY - ORGAN WEIGHTS

After 13 Weeks' Treatment at 1000 mg/kg/day, males showed reduced mean spleen-to-brain weight ratios accompanied by reduced mean absolute thymus weights, reduced thymus-to-body weight ratio and reduced thymus-to-brain weight ratio. The differences noted in the latter organ correlated with lymphoid depletion seen microscopically. These findings were considered to be test item-related. Females showed slightly reduced mean absolute ovary weights as well as reduced ovary-to-body weight ratios and ovary-to-brain weight ratios, together with reduced mean absolute uterus weights, reduced uterus-to-body weight and reduced uterus-to-brain weights. These differences were considered to be test item-related changes. The elevated liver-to-body weight ratio noted in these females was considered to be a test item-related adaptive change. After 13 weeks of treatment at 300 mg/kg/day, no test item-related changes in mean absolute or relative organ weights were ascertained in males. Females had reduced mean absolute uterus weights, reduced uterus-to-body weight ratios and reduced uterus-to-brain weight ratios which were considered to be test item-related. The elevated liver-to-body weight ratio noted in these females was considered to be test item-related adaptive change.

At 100 mg/kg/day, no test item-related changes of toxicological relevance were ascertained. All other differences in the mean absolute and relative organ weights were considered to be incidental. In rats treated previously with 1000 mg/kg/day and allowed to recover for 4 weeks, test item-related differences noted in the spleen and thymus of males were reversible after the recovery period. In females, the test item-related reduction of mean uterus weights persisted after the recovery period in females. Test item-related adaptive differences in the liver of females were reversible after the recovery period.

PATHOLOGY - MACROSCOPIC FINDINGS

During necropsy after 13 weeks of treatment, liquid cecal contents were noted in all males and four females treated with 1000 mg/kg/day. This macroscopic change did not correlate with any microscopic change in the cecum morphology. After 4 weeks of recovery, there were no macroscopical findings related to the treatment with the test item.

PATHOLOGY - MICROSCOPIC FINDINGS

After 13 weeks of treatment period, minimal or slight centrilobular hypertrophy of the hepatocytes was observed in all females at 1000 mg/kg/day, while lymphoid depletion was seen in the thymus in males treated with 300 or 1000 mg/kg/day with dose-related incidence and severity. After 4 weeks of recovery, the liver and the thymus morphology returned to normal.