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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2010 to 28 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3550
Principles of method if other than guideline:
In addition, the procedures in this study essentially conformed to the following guidelines.

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
EC Number:
619-508-4
Cas Number:
381209-09-2
Molecular formula:
C13 H24 O2
IUPAC Name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
Details on test material:
- Name of test material (as cited in study report): CAT-Acid
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- pH: 5.4-5.2 (1% in water)
- Density: 0.98 g/ml

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: weight range at start of treatment was 291-323 gr (males) and 181-217 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (ad libitum): Tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 4 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.3
- Humidity (%): 37-73
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on oral exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (1.036) and test substance (0.98).

Storage conditions of formulations: at ambient temperature

Justification for use and choice of vehicle: based on trial formulations performed at NOTOX

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase according to a validated method (NOTOX Project 495403). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of
the Group 1 formulation prepared for use during treatment. It was not considered to derive from the formulation since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected. The maximum contribution to the other samples was 0.12% based on peak area.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Group 2 and Group 4 formulations were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 30-44 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Pups were only treated indirectly. Three females of group 1 and one female of group 4 were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 10-Day dose range finding study (NOTOX 495417, See attached results).

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were made in all animals, at least 1-2 hours after dosing (on the peak period of anticipated effects after dosing), and if considered relevant also beyond this period. At weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. To monitor the health status, additional body weight measurements were performed for all females on Day 12 of study (data are retained in the raw data, but are not reported).

FOOD CONSUMPTION: Yes.
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : Yes
Quantitative assessment of water consumption was performed daily from Day 13 of the premating period onwards.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked in table were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation period (all before blood sampling).
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity test (recording period: 1-hour under normal laboratory light conditions, using a computerised monitoring system; Kinder Scientific LLC, Poway, USA).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All males and 5 females/group were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7
Females which failed to deliver (2): Post-coitum days 25 (females with evidence of mating)
Females with total litter loss (3): within 24 hours of liter loss
Euthanized in extremis (1): when pain, distress or discomfort is considered not tranisent in nature or is likely to become more severe.
Males: following completion of the mating period (minimum of 28 days)

Samples of the following tissues and organs were collected:
- Selected 5 animals/sex/group and all animals that were killed in extremis:
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Caecum, Pituitary gland, Cervix, Preputial gland, Clitoral gland, Prostate gland, Colon, Rectum, Coagulation gland, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, (Eyes with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar-, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, (Larynx), Thyroid including parathyroid (if detectable),(Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung (infused with formalin), Urinary bladder, Lymph nodes -mandibular, mesenteric-, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions

Tissues/organs mentioned in parentheses in the above list were not examined by the pathologist, as there were no signs of toxicity or target organ involvement.

- All remaining animals, females which failed to deliver and females with total litter loss:
Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes, Epididymides, Mammary gland area, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions


ORGAN WEIGHTS
- Selected 5 animals/sex/group:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY: Yes
All organs and tissues of Groups 1 and 4, all gross lesions of all animals (all dose groups), liver and kidneys of groups 2 and 3 males, liver, thymus and mammary gland of groups 2 and 3 females, organs and tissues of animal killed in extremis, additional slides of testes of group 1 and 4 males and all males suspected to be infertiel to examine staging of spermatogenesis. Reproductive organs of all surviving animals that failed to mate, conceive, sire or deliver healthy pups were checked.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

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):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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:
MORTALITY
One female at 1000 mg/kg was sacrificed in extremis on Day 14 of the post-coitum phase probably caused by a haemorrhage in the right uterine horn. Three other females at 1000 mg/kg were scheduled to be sacrificed within 24 hours after total litter loss, and hence were not directly related to treatment with the test substance. No cause of a total litter loss was found histopathologically in these females.
One of the females of this dose group with total litter loss was also noted with dark red contents in the vagina (most probably representing vaginal bleeding). In combination with the reproductive effects (described under reproduction), it was considered that this death was related to treatment with the test substance.

CLINICAL SIGNS
At 1000 mg/kg, all females were noted with hunched posture, piloerection and uncoordinated movements primarily during the premating period, and at lower incidence lean appearance, lethargy and flat posture by some females. Piloerection was also shown by several females at 300 mg/kg
towards the end of their pregnancy. Uncoordinated movements were noted by four males at 1000 mg/kg during the premating period.
Incidental findings that were noted included chromodacryorrhoea, scabs, alopecia, salivation and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence
observed, these were considered to be signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test
period.

BODY WEIGHTS
During the last few days of the post-coitum phase, a slightly lower body weight gain was recorded for three females at 1000 mg/kg. This resulted in a statistically significant lower mean body weight gain.
The weight loss (up to 10% from Day 1 values) noted for three females at 1000 mg/kg between Days 1 and 8 of the pre-mating period, was of a temporary nature and had recovered to control values towards the end of the premating period. Hence, no toxicological relevance was ascribed to this finding.

FOOD CONSUMPTION
A slightly lower absolute and relative food consumption (statistically significant) was noted for females at 1000 mg/kg over Days 1-4 of the lactation period.
Food consumption of females at 1000 mg/kg also appeared lower during week 1 of the premating period, which corresponded to the lower body weights in that period. Since this was a temporary and slight change, this was considered to be without toxicological relevance.

WATER CONSUMPTION
Water consumption was significantly higher for females at 300 and 1000 mg/kg during the premating and post coitum period, and to a lesser extent during the lactation phase (not always statistically significant). At 1000 mg/kg, mean over mean values were 105%, 88% and 18% higher than controls
during the premating, post coitum and lactation phase respectively. At 300 mg/kg, mean over mean values were 43%, 47% and 20% higher than controls during the premating, post coitum and lactation phase, respectively. However, no histopathological kidney findings were noted in these females. Therefore, this higher water intake could not be ascribed to renal effects with certainty, and at least at 300 mg/kg this finding was considered not to represent an adverse effect. Water consumption was also higher in males at 1000 mg/kg during the premating and mating period, albeit to a lesser extent than females with mean over means being approximately 40% higher than controls in both periods.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. The significantly higher platelet counts and significantly lower prothrombin time (PT) seen for males at 1000 mg/kg were not considered to be toxicologically relevant because they remained within the range
of data considered normal for rats of this age and strain. Also, platelet counts in the control group were considered to be slightly low.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher creatinine level in males at 300 and 1000 mg/kg, suggests that renal filtration was affected. However, in the absence of supportive morphological correlates indicative of renal dysfunction, and since the mean creatinine level only slightly exceeded the range considered normal in these type of rats, this effect was not considered to be adverse in nature.
- Higher cholesterol level in females at 1000 mg/kg (may reflect a higher functional capacity of the liver)
The higher bile acid level in females at 100, 300 and 1000 mg/kg (not statistically significant at 300 mg/kg) occurred in the absence of a dose-related trend and means remained within the range considered normal for rats of this age and strain. The statistically significant higher calcium level in
females at 1000 mg/kg occurred in conjunction with lower control values, and no dose-related trend was apparent. No toxicological significance was therefore ascribed to these changes.

MACROSCOPIC EXAMINATION
At 1000 mg/kg, a reduced size of the thymus was noted for three females. Enlargement of the liver was observed for two females at this dose level.
The one female at 1000 mg/kg sacrificed in extremis was noted with dark red fluid in the right uterine horn. One of the females with total litter loss was noted with one dead fetus in the left uterine horn and 2 dead fetuses in the right uterine horn, and had dark red contents in the vagina.
The black-brown discolouration of the liver noted in two females at 1000 mg/kg and one female at 300 mg/kg had no (treatment-related) histopathological correlates. Other incidental findings among control and treated animals included a yellowish soft nodule on the tail of the left epididymis, tan, reddish or a red-brown focus on or red-brown discoloration of the clitoral gland, reddish focus on the colon, alopecia on the chest and/or genital region, reddish foci on the stomach glandular mucosa or thymus, a watery-clear cyst on the left kidney, and pelvic dilation of the kidneys. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These findings were therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
The following (statistically significant) changes in organ weights and organ to body weight ratios distinguished treated animals from control animals:
- Higher absolute liver weight and liver to body weight ratio in both sexes at 300 and 1000 mg/kg (not statistically significant for absolute weights of males at 300 mg/kg). Liver to body weight ratio was 45% and 30% higher than controls at 1000 mg/kg for males and females, respectively. At
300 mg/kg, this was 12 and 14%, respectively. At 1000 mg/kg, this correlated to a minimal to slight degree of centrilobular hepatocyte hypertrophy of the liver of some males and most females. There were no microscopic changes in the liver suggestive of hepatotoxicity. Although centrilobular hepatocyte hypertrophy is commonly seen as an adaptive response associated with the metabolism of xenobiotics or their metabolites, the degree
of liver weight increase at 1000 mg/kg (i.e. notably exceeding a 10% increase from control levels) was considered to reflect an adverse effect. At 300 mg/kg, the liver weight increase was much less pronounced (i.e. just exceeding a 10% increase from control levels), and in combination with absence
of histopathological signs of hepatotoxicity considered not to be adverse. (see below)
- Higher absolute kidney weight and kidney to body weight ratio in males at 1000 mg/kg (22% higher than controls for kidney to body weight ratio).
- Lower thymus weight and thymus to body weight ratio in females at 1000 mg/kg (not statistically significant; 28% lower than controls for thymus to body weight ratio). No histopathological correlates were observed, and hence was considered to be of no toxicological relevance.
The statistically significant higher adrenal to body weight ratio of males at 300 and 1000 mg/kg and the higher ovary weight and ovary to body weight ratio of females at 300 and 1000 mg/kg occurred in the absence of a clear dose-related trend and remains within the range considered normal for rats of this age and strain. The statistically significant higher spleen to body weight ratio of males at 1000 mg/kg was also slight in nature and the mean also remained within normal ranges. The higher spleen weight of females at 100 mg/kg occurred in the absence of a dose-related trend. Moreover, no histopathological correlates were noted to support the above changes. No toxicological significance was therefore ascribed to these changes.

MICROSCOPIC EXAMINATION
There were treatment-related microscopic findings at 1000 mg/kg:
- Liver: Hypertrophy of the hepatocytes in the centrilobular area (minimal) was recorded in 2/5 males and 5/7 females which was considered to be an adaptive response.
- Kidneys (males): Tubular granular casts were present in 4/5 males (minimal-slight). An increased incidence and severity of cortical hyaline droplets was seen in 5/5 males (minimal-slight). The hyaline droplets were considered to represent alpha2µglobulin, a normal protein in male rats which
undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. The occurrence of hyaline droplets is a specific male rat response which is not observed in normal female rats, and higher species of either sex, including humans, therefore not toxicologically relevant.
No abnormalities were seen in the reproductive organs of the suspected non-fertile animals of Group 4 which could account for their infertility. The remaining microscopic findings were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Effect levels

Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Higher kidney (m) and liver (m,f) weights, higher cholesterol level (f), higher water intake (m,f) presence of renal granular casts (m), and hypertrophy of hepatocytes (m,f).

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an OECD 422/421 oral repeated dose/reproduction screening study, a NOAEL of 300 mg/kg bw/day was determined based on effects at 1000 mg/kg bw/day: higher kidney (m) and liver (m,f) weights, higher cholesterol level (f), higher water intake (m,f) presence of renal granular casts (m), and hypertrophy of hepatocytes (m,f).
Executive summary:

CAT-ACID was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 30-44 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature. Parental toxicity revealed at 1000 mg/kg bw/day, which consisted of higher kidney (m) and liver (m,f) weight, higher cholesterol level (f), higher water intake (m,f) and renal granular casts (f). No clear and consistent pattern of occurrence of clinical signs was noted during the inlife phase. Also slight changes in body weight and food intake levels were noted, each during different phases of the treatment period. Functional observation tests did not reveal treatment-related changes. Therefore, a parental NOAEL of 300 mg/kg bw/d was derived.