Registration Dossier

Administrative data

Description of key information

A NOAEL of 450 mg/kg b.w./day was derived from a sub-chronic oral toxicity study with 4-MHHPA.

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:
2018-01-18 to 2018-05-22
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)
Version / remarks:
September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch No. of test material: T404217186
- Manufacturing date: 05 Jul 2017
- Expiration date of the lot/batch: 04 Jul 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; in closed vessels; avoid contact with humidity.
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
Details on species / strain selection:
The test for 90-Day repeated dose toxicity was performed in the rat. Although several mammalian species may be used, the rat is the preferred rodent species.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt, Budapest, Hungary
- Age at study initiation: 51 - 55 days
- Weight at study initiation: Males: 226 - 266 g; Females: 144 - 178 g
- Fasting period before study: No, not applicable
- Housing: Group housed (5 of same sex/cage) in polypropylene/polycarbonate cages
- Diet: Rodent diet (ssniff SM R/M-Z+H) ad libitum
- Water: Municipal drinking water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
Sunflower oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (sunflower oil) in concentrations of 10 mg/mL, 30 mg/mL and 90 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility and stored at 5 - 3 °C until use, but not longer than for three days (based on the stability features of the test item in the vehicle).

VEHICLE
- Justification for use and choice of vehicle:
The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 10, 30 and 90 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations for concentration and homogeneity was performed three times during the study period.
Five samples (5 mL, each) were taken from different places from each concentration for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from different places from the control substance.
Measured concentrations varied between 93 and 108 % of the nominal concentrations and all formulations were found to be acceptably homogeneous.
Duration of treatment / exposure:
90 or 91 days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 male/ 10 female per group
Additional satellite groups of 5 male / 5 female for control and high dose to examine effects of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 50, 150 and 450 mg/kg bw/day is based on findings obtained in a previous 28-days repeated dose toxicity study. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for selecting satellite groups: Groups pre-assigned, animal assignment with groups random as for main study groups
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily, at same time interval
- Cage side observations included: Mortality/morbidity, gross clinical signs/response to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
- Observations included: Detailed clinical examination in an open arena. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma (treatment and recovery periods).

BODY WEIGHT: Yes
- Time schedule for examinations: On the day treatment commenced, twice weekly for the first 4 weeks and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Yes - Weight of food consumed by each cage of rats recorded at weekly intervals. Group mean daily intake per rat calculated.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-dose and during Week 13 of treatment.
- Dose groups that were examined: All animals pre-dose. Control and high-dose animals during Week 13 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On termination of treatment and at end of Week 4 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table No.1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On termination of treatment and at end of Week 4 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table No.3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during last week of exposure
- Dose groups that were examined: All groups at end of treatment phase. As no changes were observed, control and high-dose animals at the end of the recovery phase were not examined.
- Battery of functions tested: Sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity.

EXAMINATION OF OESTRUS CYCLE: Yes
- Time schedule: During last 2 weeks of treatment and during last 2 weeks of recovery
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Detailed post mortem examination including examination of the external surface and orifices.
Organ weights - Adrenal glands, Brain, Coagulating glands, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate gland, Seminal vesicles, Spleen, Testes, Thymus, Uterus.
Tissues fixed and preserved are listed in table No. 4.

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues (table 4) of the animals of the control (Group 1) and high dose (Group 4) groups including recovery groups. Additionally, liver and seminal vesicles in single male animals at 150 mg/kg bw/day were processed and evaluated histologically on the basis of necropsy observations.
Detailed histological examination were performed on the male mammary gland, on the testes, epididymides, prostate (dorsolateral and ventral) and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
After dehydration and embedding in paraffin wax, sections were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
Other examinations:
Sperm Examinations:
Sperm analysis was performed for 10 control and 10 high dose males at the necropsy. One-side testes and epididymides were used for examinations. For the same subset of males, sperm from the ductus deferens was collected for evaluation of sperm motility and sperm morphology.
- Quantitative examination
The total number of homogenization testes sperm was enumerated. Enumeration was performed from frozen tissues after thawing.
- Qualitative examinations
Sperm motility was determined from ductus deferens of the same animals as enumeration at the necropsy. For the determination of the sperm motility the mean percentage of motile sperms was determined. Both numbers of motile and immotile sperms was recorded. Two samples were prepared from each animal.
A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails).

Determination of Serum Levels of Thyroid Hormones:
Serum samples were collected from all animals for determination of T4 and TSH. Samples were stored under appropriate conditions (at approximately -20 °C) until measurement.
The quantitative determination of thyroid hormones (T4 and TSH) in serum samples was performed by electrochemiluminescence immunoassay.
Statistics:
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
Results were evaluated in comparison with values of control group (i.e. control value).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily observation
No adverse signs of systemic toxicity related to the test item were detected at any dose level (50, 150 or 450 mg/kg bw/day) at the daily clinical observations.
Treatment period
The behavior and physical condition of control male animals were considered to be normal during the entire treatment period.
Test item related nuzzling up the bedding material and salivation were observed in male animals at 50, 150 and 450 mg/kg bw/day with different incidence and duration during the treatment period as follows: nuzzling up the bedding material: 6/10 at 50 mg/kg bw/day, 5/10 at 150 mg/kg bw/day and 15/15 at 450 mg/kg bw/day; salivation: 2/10 at 50 mg/kg bw/day, 5/10 at 150 mg/kg bw/day and 12/15 at 450 mg/kg bw/day; 5/15 animals salved before the treatment at 450 mg/kg bw/day.
Noisy breathing (1/15) and porphyrin around the right eye (1/5) were noted for single male animals at 450 mg/kg bw/day as individual signs for few days (3 and 4, respectively) during the treatment period.
The behavior and physical condition female animals were normal in the control and 50 mg/kg bw/day groups (15/15 and 10/10, respectively) during the entire observation period.
In the female animals, nuzzling up the bedding material and salivation were detected at 150 and 450 mg/kg bw/day: nuzzling up the bedding material: 3/10 at 150 mg/kg bw/day and 11/15 at 450 mg/kg bw/day; salivation: 2/10 at 150 mg/kg bw/day, 11/15 at 450 mg/kg bw/day. Some female animals (1/10 at 150 mg/kg bw/day and 8/15 at 450 mg/kg bw/day) also salved before the daily treatment.
The noisy breathing, detected on Day 75 in a single male animal in the high dose group after the treatment, was not anymore detectable at the detailed weekly observation on Day 77. Due to the occurrence in a single animal for a very limited time, this observation was not considered test item related.
Salivation and nuzzling up the bedding material, with a slight dose dependency, were seen shortly after the daily administration of the test item and it always ceased shortly thereafter. Based on the short time frame of observation, these findings were judged to be test item related but not adverse. Control male and female animals did not salivate therefore salivation was considered to be related to the treatment with the test item.
Recovery period
Clinical signs were not detected in animals of the control or 450 mg/kg bw/day (male or female) dose group during the recovery period. The behavior and physical condition of animals were considered to be normal.

Weekly detailed observation
There were no test item related clinical signs during the weekly detailed observations in male or female animals at any dose level during the entire observation period.
Treatment and recovery periods
Porphyrin around the right eye was detected in one male animal at 450 mg/kg bw/day at the weekly clinical observation on Day 21 as described above. Porphyrin is normally secreted by the Harderian gland. Occasional low level of porphyrin staining around the rat's eyes are normal, therefore this observation had no toxicological significance in the present study.
The general physical condition and behavior of animals were considered to be normal at the detailed weekly observations throughout the entire observation period (treatment and recovery periods).

Mortality:
no mortality observed
Description (incidence):
No mortality was observed in any group (control, 50, 150 or 450 mg/kg bw/day) during the course of study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development was unaffected by the test item at 50, 150 and 450 mg/kg bw/day (male and female animals) during the 3-month treatment period.
Treatment period
The mean body weight was similar to the control in male animals treated with 50, 150 and 450 mg/kg bw/day during the entire treatment period.
Some statistically significant difference with respect to the control was detected for the higher mean body weight gain in male animals at 50 and 150 mg/kg bw/day between Days 84 and 89. Furthermore the lower mean body weight gain in male animals at 150 and 450 mg/kg bw/day between Days 63 and 70 was statistically significant. These slight changes in the body weight gain had no influence on the mean body weight or on the summarized body weight gain and therefore had no toxicological relevance.
The mean body weight was comparable in the control and test item treated female animals at 50, 150 and 450 mg/kg bw/day during the entire treatment period. The mean body weight gain was slightly higher than in the control group in female animals at 50 mg/kg bw/day between Days 84 and 89 and at 450 mg/kg bw/day between Days 49 and 56. These minor and transient changes did not influence significantly the mean body weight values and therefore were considered to be not toxicologically relevant.
Recovery period
The mean body weight was similar to the control in male and female animals at 450 mg/kg bw/day during the recovery period.
The mean body weight gain was slightly higher than in the control group both in male and female animals at 450 mg/kg bw/day during the post-treatment observation period. In male animals statistical significances for the mean body weight gain was detected in recovery week 2 (between Day 97 and 104) and for the summarized mean body weight gain of the recovery period (Days 90 – 117). However, the mean body weight of these male animals was similar to the mean body weight of the control animals, therefore the slight, albeit significant, changes in the mean body weight gain were toxicologically not relevant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item influence on the mean daily food consumption of male or female animals at 50, 150 or 450 mg/kg bw/day during the treatment period.
Treatment period
The mean daily food consumption was comparable with the respective controls in male and female animals at 50, 150 and 450 mg/kg bw/day in most cases during the entire treatment period.
Statistically significant differences with respect to the control were observed for the slightly lower mean daily food consumption in male animals at 50 mg/kg bw/day during week 11 (between Days 70 and 77) and for the slightly higher mean daily food consumption of female animals at 450 mg/kg bw/day in week 7 (between Days 42 and 49). These minor and transient differences were indicative of biological variation and not related to the test item.
Recovery period
During the course of the recovery period, the mean daily food consumption was similar in the control and high dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The eyes were without any abnormalities in all animals before the treatment and in animals in the control and high dose groups at termination of the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (50, 150 or 450 mg/kg bw/day).

Treatment period
Compared to the respective control group slight, albeit significant, reductions in the mean percentage of monocytes (MONO) and eosinophil granulocytes (EOS) were detected in male animals at 50 mg/kg bw/day. For MONO the reduction at 50 mg/kg bw/day was -28 % (mean: 1.77 %) compared to the respective control (mean: 2.45 %) and for EOS it was -22 % (mean: 0.86 %) compared to the respective control (mean: 1.1 %). The changes in percentage observed at 50 mg/kg bw/day have no toxicological relevance as similar findings were not detected in animals receiving higher doses, demonstrating an absence of dose response. In addition, all mean values were within the historical control ranges of the most recent historical control data, conducted in 2018, the year the study was performed (HCD-2018).
All examined hematological and blood coagulation parameters were comparable to the respective value in the control group for male animals at 150 and 450 mg/kg bw/day.
The mean values of white blood cell (WBC) counts for male animals showed a slight, statistically not significant reduction for low (-11 %, mean: 6.93x109/L) and mid (-6 %, mean: 7.29x109/L) dose groups compared to the concurrent control (mean: 7.76x109/L). In contrast, the value for the high dose group was increased (+4 %, mean: 8.05x109/L) compared to the control. This clearly demonstrates a lack of dose response on male WBC counts. Furthermore, all mean values were within the mean value ± SD of the HCD-2018 (7.2x109/L ± 1.3) for male animals.
Statistically significant changes were revealed with respect to the control group for the slightly higher mean hemoglobin concentration (HGB) in female animals at 50 mg/kg bw/day (+3 %, mean: 153.7 g/L; control mean: 149.0 g/L) and for the slightly reduced mean activated prothrombin time (APTT) in female animals dosed with 450 mg/kg bw/day (-7 %, mean: 12.17 sec; control mean: 13.09 sec) at the end of the treatment period. The slight change detected for HGB at 50 mg/kg bw/day has no toxicological relevance as similar findings were not detected in female animals receiving 150 and 450 mg/kg bw/d. The individual APPT values were comparable in the control and high dose group in spite of the statistical significance, therefore its biological or toxicological importance is equivocal.
For WBC counts in female animals a non-significant reduction for low (-8 %, mean: 4.97x109/L), mid (-18 %, mean: 4.40x109/L) and high dose (-18 %, mean: 4.42x109/L) groups compared to the concurrent control (mean: 5.38x109/L) group was observed. There was no dose response. Taking into account the mean value of the HCD-2018, a treatment related effect can be excluded as all mean values of the treatment groups were comparable to the mean value of the HCD-2018 (4.60x109/L ± 1.4), whereas the concurrent control value was clearly above the HCD-2018 mean value. Thus, the observed reduction in females was only due to the exceptionally high control value.

Recovery period
All examined hematological parameters were comparable in male animals in the control and high dose group at the end of the recovery period.
In female animals of the recovery group previously treated with 450 mg/kg bw/day, a statistically significant difference with respect to the relevant control was observed for the lower mean percentage of basophil granulocytes (BASO) (-67 %, mean: 0.04 %). However, this significance was due to higher mean percentage of basophil granulocytes in the recovery control (mean: 0.12 %) compared to the treatment control (mean: 0.07 %), while the value for the 450 mg/kg bw/day treated (mean: 0.04 %) and recovery (mean: 0.04 %) females remained identical.

The statistically significant differences noted during the treatment and recovery periods, with respect to the appropriate control, were of small degree. All mean values remained within the most recent historical control values and these changes were therefore considered not to be toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No adverse test item effect was detected at the evaluation of clinical chemistry parameters for all dose groups (50, 150 and 450 mg/kg bw/day).

Treatment period
In the male animals treated with 50 mg/kg bw/day, the mean activity of alkaline phosphatase (ALP), the mean albumin concentration (ALB) and albumin: globulin ratio (A/G) exceeded the control value statistically significantly. For ALP the increase at 50 mg/kg bw/day was +43 % (mean: 122.1 U/L) compared to the respective control (mean: 85.2 U/L), for ALB it was +6 % (mean: 44.56 g/L) compared to the respective control (mean: 41.98 g/L) and the detected increase for A/G was +19 % (mean: 2.28; control mean: 1.92). The changes in percentage observed at 50 mg/kg bw/day have no toxicological relevance as similar findings were not detected in animals receiving higher doses, demonstrating an absence of dose response. In addition, all values remained within the mean value ± SD of the most recent historical control data, conducted in 2018, the year the study was performed (HCD-2018).
The mean glucose concentration (GLUC) was significantly higher (9 %, mean: 5.94 mmol/L) than in the control group (mean: 5.45 mmol/L) in male animals at 150 mg/kg bw/day. However, it remained within the mean value ± SD of the HCD-2018 (6.2 ± 1.0 mmol/L) and no statistical effect was observed in the higher dose group.
Statistical significance was seen for the slightly higher mean creatinine concentration (CREA) in male animals at 450 mg/kg bw/day when compared to the control group (18 %, mean: 31.0 µmol/L, control mean: 26.3 µmol/L). Still, the mean value was within the historical control ranges of the HCD-2018.
In the female animals, some statistically significant differences with respect to the control were detected for the lower mean concentration of inorganic phosphorous (Pi) at 150 and 450 mg/kg bw/day (150 mg/kg bw/d: -16 %, mean: 1.46 mmol/L; 450 mg/kg bw/d: -11 %, mean: 1.54 mmol/L; control mean: 1.73 mmol/L) and for the lower mean concentrations of total bilirubin (TBIL) (-49 %, mean: 0.69 µmol/L, control mean: 1.35 µmol/L), albumin (-6 %, mean: 44.11 g/L, control mean: 46.96 g/L) and total protein (TPROT) (-7 %, mean: 61.67 g/L, control mean: 66.35 g/L) at 450 mg/kg bw/day. All mean values remained within the historical control ranges of the HCD-2018.

Recovery period
Statistical significance was observed at the slightly higher mean concentration of inorganic phosphorous in the male animals (8 %, mean: 1.9 mmol/L; recovery control mean: 1.76 mmol/L) and at the lower mean concentrations of total bilirubin (-23 %, mean: 1.3 µmol/L, recovery control mean: 1.68 µmol/L) and sodium (Na+) (-2 %, mean: 140.78 mmol/L; recovery control mean: 143.92 mmol/L) in female animals at 450 mg/kg bw/day group at the end of the recovery period.

The sporadic statistical differences detected at some parameters (ALP, CREA, TBIL, GLUC, Pi, Na+, ALB, TPROT and A/G ration) were considered to be of little or no biological relevance. All these changes were with minor degree and not related to doses (ALP, GLUC, Pi, ALB and A/G). Slight changes in CREA, TBIL, Na+, ALB and TPROT in animals at 450 mg/kg bw/day were judged to be toxicologically not significant as there were no supporting findings (hematology, histopathology, related biochemical parameters).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional observation battery did not demonstrate any treatment-related differences with respect to the controls in the behavior or in reactions to different type of stimuli at the end of the treatment period (male and female, 50, 150 or 450 mg/kg bw/day).
The behavior and reactions to different type of stimuli or manipulations of animals were judged to be normal in the control and all test item treated groups on Day 89.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related changes were not detected in the examined organ weights in male or female animals at 50, 150 or 450 mg/kg bw/day.

Treatment period
Statistical significant differences with respect to the control were noted for the slightly reduced mean thymus weights (absolute and relative to body and brain weights) in male animals in the 50 and 450 mg/kg bw/day dose groups at termination of the treatment period. In comparison to the concurrent control (mean: 0.43 g) the thymus weight was statistically significantly reduced in low (-19 %, mean: 0.35 g) and high dose group (-21 %, mean: 0.34 g) male animals, but neither in mid dose males nor in females of all dose groups. The thymus weights of all male dose groups were clearly within the historical control ranges (min. 0.25 g, max. 0.59 g) of the most recent historical control data, conducted in 2018, the year the study was performed (HCD-2018), and even within one standard deviation (SD) of the mean value of the HCD-2018 (HCD-2018: 0.40 g ± 0.08; low dose: 0.35 g, mid dose: 0.39 g, high dose: 0.34 g). This clearly underlines that the observed effects were not treatment related and that the seemingly reduced weights in the treatment groups were caused by the clustering of high values from individual animals of the concurrent control group. Furthermore, there was no dose response as thymus weights did not decrease with increasing dose levels and there were no histopathological findings supporting an adverse effect on the thymus.
In female animals, the weights of the examined organs were similar in the control and test item treated groups. There were no statistically significant differences between the control and 50, 150 or 450 mg/kg bw/day groups.

Recovery period
Statistical significances were revealed for the lower mean kidney weight (-10 %, mean: 2.33 g; recovery control mean: 2.59 g) and higher mean spleen weights relative to body (19 %, mean: 0.16 %; recovery control mean: 0.14 %) and brain weights (18 %, mean: 34.18 %; recovery control mean: 28.95 %) in male animals at 450 mg/kg bw/day at the end of the recovery period. Compared to the HCD-2018, all values remained within the mean value ± SD (kidney weight: 2.38 ± 0.26 g; spleen weight relative to body: 0.157 ± 0.018 %; spleen weight relative to brain weight: 31.91 ± 4.08 %)
In the female animals at 450 mg/kg bw/day, the mean liver weights (absolute and relative to body and brain weights) slightly exceeded that of the control group at the end of the post-treatment observation period. The mean liver weight was increased by 15 % (mean: 7.18 g; recovery control mean: 6.24 g), by 13 % for the weight relative to body weight (mean: 2.77 %; recovery control mean: 2.44 %) and by 16 % for the weight relative to brain weight (mean: 359.51 %; recovery control mean: 308.73 %). In addition, all mean values were within the historical control ranges of the HCD-2018.

The statistically significant differences noted during the treatment and recovery periods, with respect to the appropriate control, were of small degree and no related findings were detected at the histopathological examinations. Comparison with the most recent historical control values showed that all mean values remained within the historical control values. Therefore, these changes were not considered to be toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period.
Treatment period
Individual macroscopic changes were noted for some male animals in the control and 150 mg/kg bw/day dose groups: hard formation (1x2 cm in size) in the fatty tissue of abdominal cavity and smaller than normal seminal vesicle (right side) in one control animal (1/10), and smaller than normal seminal vesicle (right side; 1/10) and pale liver (1/10) at 150 mg/kg bw/day). These findings are common in untreated experimental rats and were not related to the test item.
In the female animals, slight, moderate or marked hydrometra in the uterus was observed as follows: 6/10 control, 4/10 at 50 mg/kg bw/day, 6/10 at 150 mg/kg bw/day, 4/10 at 450 mg/kg bw/day. Hydrometra is indicative of female sexual cycle. Histological findings were in accordance with macroscopic observation in the uterus. There were no pathological alterations in the uterus at the histopathological examination, therefore it was considered toxicologically not relevant.
Recovery period
In one control male animal, yellowish formation (1x2 cm in size) in the fatty tissue of abdominal cavity was detected at the necropsy after four weeks post-treatment observation period. There were no macroscopic changes in the male animals at 450 mg/kg bw/day.
In some female animals slight or moderate hydrometra was observed at the end of the recovery period in the control (2/5) and 450 mg/kg bw/day (2/5) groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histological examination did not reveal test item related adverse lesions in the organs or tissues of animals administered with 450 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.
The slight vacuolation of hepatocytes (hepatic lipidosis) observed in some female animals (3/10) at 450 mg/kg bw/day was considered to be a non-adverse and reversible test item related effect, caused by a disturbance of energy metabolism of affected hepatocytes.
Treatment period
In control group, alveolar emphysema in the lungs (1/10 male and 1/10 female), hyperplasia of bronchus associated lymphoid tissue (BALT; 1/10 male and 1/10 female), lipoma in the fatty tissue of abdominal cavity (1/10 male), decreased amount of secretion in the seminal vesicle (one side; 1/10 male), focal lymphocytic infiltration in the prostate (1/10 male) and hydrometra (6/10 female) were observed.
At 150 mg/kg bw/day, there was no alteration in the liver (1/1 male) subjected to histological evaluation on the basis of macroscopic findings (pale liver). Decreased amount of secretion was observed in the seminal vesicle (one side; 1/1 male) processed histologically.
At 450 mg/kg bw/day, pulmonary alveolar emphysema (1/10 male), hyperplasia of bronchus associated lymphoid tissue (1/10 male), vacuolation of hepatocytes (3/10 female) and dilatation of the uterus (4/10 female) were detected.
Recovery period
At the end of the recovery period, alveolar emphysema in the lungs (1/5 male and 1/5 female control), hyperplasia of bronchus associated lymphoid tissue (1/5 female control, 1/5 male at 450 mg/kg bw/day) and lipoma in the fatty tissue (1/5 control male) were observed at the histopathology examination. Dilatation of uterine horns was revealed in female animals at the end of the recovery period: 2/5 control and 2/5 at 450 mg/kg bw/day.

Vacuolation of hepatocytes in the centrilobular region – indicative of hepatic lipidosis – in some high dose treated female animals might be related to the test item as this finding was not observed in the control animals and was not detected in the recovery female animals at 450 mg/kg bw/day. Therefore the lesion in the female liver was judged to be a mild and reversible effect.
Alveolar emphysema in connection with the hypoxia, dyspnea and circulatory disturbance, developed during the exsanguination procedure occurred with similar incidence in the control and treated animals.
Hyperplasia of bronchus associated lymphoid tissue – a physiological immunomorphological phenomenon – was without toxicological significance and was with similar incidence in control and high dose male animals and was only present in control female but not in the high dose females.
Dilatation of uterine horn related to hydrometra is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrous phase – of the inner genital organs.
Morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the alimentary system, the pancreas, the cardiovascular system, immune system, the hemopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central and peripheral nervous system was not detected.
The structure and the cell morphology of the endocrine glands was the same in the control and high dose treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Examination of Estrous Cycle:
A test item effect on the estrous cycle was not detected at any dose level.
Treatment period
There were no significant differences between the control and test item treated groups in the percentage of female animals with regular or irregular cycles, mean number of cycles or mean cycle length.
The percentage of animals with irregular cycle was higher than normal (historical control) in each group including control. Based on the lack of related histological changes and observation of the same effect in the control group, this finding was considered to be incidental and not related to the test item.
The mean length of estrous cycle was slightly longer with respect to the control at 50 mg/kg bw/day without statistical significance. A similar finding was not detected for the mid and high dose treated animals, therefore the change in the low dose group was considered to be independent from the test item.
Recovery period
The examined parameters of estrous cycle were comparable in the control and 450 mg/kg bw/day groups during the last two weeks of the post-treatment observation period.

Serum Levels of Thyroid Hormones:
T4 and TSH levels were comparable in the control and test item treated groups (50, 150 and 450 mg/kg bw/day, male and female) at the end of the treatment and recovery periods.

Sperm Examinations:
The sperm count, morphology and motility of sperm cells were not affected after three months administration of 450 mg/kg bw/day of the test item.
The mean sperm count, percentage of motile cells and cells with normal morphology was similar in animals of the control and 450 mg/kg bw/day groups at the termination of dosing period.
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Critical effects observed:
no

Results:

Table 1: Summary of daily clinical observations

Observations

Control

50 mg/kg bw/day

150 mg/kg bw/day

450 mg/kg bw/day

Treatment period

Recovery period

Treatment period

Recovery period

male

No clinical signs

15/15

5/5

4/10

5/10

0/15

5/5

Nuzzling up the bedding material

0/15

0/5

6/10

5/10

15/15

0/5

Salivation

0/15

0/5

2/10

5/10

12/15

0/5

Salivation before the treatment

0/15

0/5

0/10

0/10

5/15

0/5

Noisy breathing

0/15

0/5

0/10

0/10

1/15

0/5

Porphyrin around the right eye

0/15

0/5

0/10

0/10

1/15

0/5

female

No clinical signs

15/15

5/5

10/10

7/10

4/15

5/5

Nuzzling up the bedding material

0/15

0/5

0/10

3/10

11/15

0/5

Salivation

0/15

0/5

0/10

2/10

11/15

0/5

Salivation before the treatment

0/15

0/5

0/10

1/10

8/15

0/5

Frequency of observations = number of animals with observations / number of animals examined.

 

Table 2: Summary of body weight

Group

Body weight (g) on days

0

7

14

21

28

35

42

49

56

63

70

77

84

89

97

104

111

117

male

Control (Recovery group day 97-117)

Mean

244.1

279.1

313.0

334.3

353.3

370.3

384.5

403.5

419.3

431.1

443.9

454.5

465.1

469.8

488.6

492.6

495.6

498.4

SD

10.42

15.28

20.46

23.6

23.89

30.09

33.62

33.62

35.89

39-82

38.44

40.66

43.64

44.01

22.18

21.82

21.76

19.35

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

50 mg/kg bw/day

Mean

244.6

279.1

311.1

333.1

350.5

365.8

381.9

39.2

410.8

421.8

433.3

440.8

452.7

462.1

SD

10.21

11.84

14.72

16.24

19.06

20.81

24.5

26.63

29.2

31.45

32.26

33.94

35.02

36.82

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

0

0

-1

0

-1

-1

-1

-2

-2

-2

-2

-3

-3

-2

150 mg/kg bw/day

Mean

245.9

280.5

310.3

332.7

351.3

366.5

382.5

398.9

411.7

423.7

431.8

441.1

451.1

460

SD

11.61

14.55

17.95

20.73

22.15

24.8

26.07

27.94

27.77

28.16

29.2

29.27

28.3

28.62

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

1

1

-1

0

-1

-1

-14

-1

-2

-2

-3

-3

-3

-2

450 mg/kg bw/day (Recovery group day 97-117)

Mean

246.2

277.6

308.5

331.3

348.5

366.2

382.7

401

418.3

429.5

437.7

448.4

458.3

464.3

466.8

475.2

482.4

486.8

SD

11.1

12.87

16.36

20.63

23.75

27.92

30.26

33.25

35.5

36.34

39.3

39.14

40.64

40.07

40.36

37.68

37.93

38.74

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

+/- %

1

-1

-1

-1

-1

-1

0

-1

0

0

-1

-1

-1

-1

-4

-4

-3

-2

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

female

Control (Recovery group day 97-117)

Mean

160.2

181.7

195.7

207.3

218.7

223.7

230.7

239.4

242.1

247.3

250.9

255.5

262.7

270.9

264

268

268.8

268.4

SD

9.28

10.73

10.9

11.7

13.96

14.08

15.47

14.13

17.05

16.64

18.31

18.68

19.54

22.24

17.85

13.78

12.74

14.26

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

50 mg/kg bw/day

Mean

158.4

179.9

19.32

205.7

216.3

220.6

230.8

238

243.3

245.2

249

253.9

265.6

279.3

SD

10.15

11.65

14.96

15.85

16.85

19.24

21.56

21.48

20.17

20.98

20.9

19.34

19.41

19.26

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-1

-1

-1

-1

-1

-1

0

-1

0

-1

-1

-1

1

3

150 mg/kg bw/day

Mean

157.0

176.6

189.3

200.5

209.2

216.2

222.5

231.2

234.6

239.9

241.3

244.8

254.8

266.2

SD

6.5

9.85

12.43

13.39

14.6

14.76

15.18

13.32

14.9

16.16

15.05

15.09

18.07

19.38

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-2

-3

-3

-3

-4

-3

-4

-3

-3

-3

-4

-4

-3

-2

450 mg/kg bw/day (Recovery group day 97-117)

Mean

162.7

183.1

196.9

211.7

220.7

226.3

233.3

242.4

247.7

251.7

254.8

257.4

267.5

273.6

267.4

270.2

271.4

276.2

SD

9.36

11.46

11.83

13.97

15.7

14.6

16.32

17.45

18.61

19.58

21.78

21.03

25.15

23.15

31.33

33.34

34.71

39.33

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

+/- %

2

1

1

2

1

1

1

1

2

2

2

1

2

1

1

1

1

3

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

+/- % = Percent Deviation Versus Control

NS = Not Significant

 

Table 3: Summary of body weight gain

Group

Body weight gain (g) between days

0-7

7-14

14-21

21-28

28-35

35-42

42-49

49-56

56-63

63-70

70-77

77-84

84-89

0-89

90-97

97-104

104-111

111-117

90-117

male

Control (Recovery group day 90-117)

Mean

35

33.9

21.3

19

17

14.1

19

15.8

11.9

12.8

10.6

10.5

4.7

225.7

4.4

4

3

2.8

14.2

SD

7.54

6.03

5.52

5.64

4.64

4.55

4.46

5.36

5.85

4.46

4.34

5.4

4.99

38.36

0.89

0.71

2.35

5.45

4.21

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

5

50 mg/kg bw/day

Mean

34.5

32.0

55

17.4

15.3

16.1

15.3

13.6

11

11.5

7.5

11.9

9.4*

217.5

SD

6.6

3.71

3.59

4.81

3.37

5.59

3.23

4.86

3.46

1.9

2.84

3.51

4.72

31.61

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

150 mg/kg bw/day

Mean

34.6

29.8

22.4

18.6

15.2

16

16.4

12.8

12

8.1**

9.3

10

8.9*

214.1

SD

4.88

4.49

5.17

3.78

4.61

3.74

4.22

4.59

2.67

2.77

2.41

2.62

2.56

20.71

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

450 mg/kg bw/day (Recovery group day 90-117)

Mean

31.4

30.9

22.7

17.2

17.7

16.5

18.3

17.3

11.3

8.1**

10.7

9.9

6

218.1

5

8.4

7.2

4.4

25

SD

6.03

4.61

7.11

5.85

5.51

6.19

5.16

5.05

4.46

4.49

4.17

3.97

4.9

34.21

3.54

4.16

3.83

2.41

9.3

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

5

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

NS

NS

U

NS

NS

DN

NS

NS

*

NS

NS

*

female

Control (Recovery group day 97-117)

Mean

21.5

14

11.5

11.4

5.1

7

8.7

2.7

5.2

3.5

4.6

7.3

8.1

110.7

-6

4

0.8

-0.4

-1.6

SD

6.29

4.66

5.18

5.77

4.64

5.59

3.58

2.69

4.09

4.24

3.54

5.12

4.94

18.27

4.06

5.79

4.32

3.78

3.78

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

5

50 mg/kg bw/day

Mean

21.5

13.3

12.5

10.6

4.3

10.2

7.2

5.3

1.9

3.8

4.9

11.7

13.7*

130.9

SD

5.56

5.58

3.5

4.53

4.6

6.78

5.14

4.35

4.86

5.33

5.15

7.3

6.31

15.3

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

150 mg/kg bw/day

Mean

19.6

12.7

11.2

8.7

7

6.3

8.7

3.4

5.3

1.4

3.5

10

11.4

109.2

SD

6.06

5.38

4.59

3.74

4.06

3.97

4.59

3.17

3.68

3.78

4.06

4.64

3.41

17.33

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

450 mg/kg bw/day (Recovery group day 90-117)

Mean

20.4

13.8

14.9

9

5.5

7

9.1

5.3*

3.9

3.1

2.6

10.1

6.1

110.9

-2.8

2.8

1.2

4.8

6

SD

4.48

4.63

5.14

4.38

4.26

4.52

3.54

2.97

6.25

6.25

4.75

6.15

5.67

17.31

4.76

3.35

5.26

7.73

12.43

n

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

5

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

DN

NS

NS

NS

NS

DN

NS

NS

NS

NS

NS

NS

+/- % = Percent Deviation Versus Control

NS = Not Significant

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

T - test Versus Control (for recovery)

 

Table 4: Summary of mean daily food consumption

Group

Daily mean food consumption (g/animal/day)

Week

1

2

3

4

5

6

7

8

9

10

11

12

13

R1

R2

R3

R4

Days

0-7

7-14

14-21

21-28

28-35

35-42

42-49

49-56

56-63

63-70

70-77

77-84

84-89

90-97

97-104

104-111

111-117

male

Control (Recovery group day 90-117)

Mean

20.9

21.5

20.8

19.5

19.6

19.8

19.1

19.7

20.1

19.4

19.1

18.4

19.3

20.6

21.1

20.7

21.6

SD

0.78

0.77

0.98

0.56

0.64

1.02

0.62

0.57

0.87

0.49

0.14

0.7

0.54

n (Number of cages)

3

3

3

3

3

3

3

3

3

3

3

3

3

1

1

1

1

50 mg/kg bw/day

Mean

21.4

21.2

21.2

19.3

19.9

19.4

18.2

18.4

18.5

18.1

17.7*

17.6

18.2

SD

0.24

0.61

0.85

0.1

0.06

0.06

0.28

0.63

0.38

0.53

0.16

0.34

0.06

n (Number of cages)

2

2

2

2

2

2

2

2

2

2

2

2

2

+/- %

2

-1

2

-1

-2

-2

-5

0.7

-8

-6

-7

-4

-5

150 mg/kg bw/day

Mean

21.2

20.8

20.8

19.5

19.5

19.5

18.8

19.1

19.5

18.4

17.9

17.6

18.3

SD

0.1

0.69

0.69

0.91

1.05

1.05

0.61

0.24

0

0.77

0.75

0.65

0.48

n

2

2

2

2

2

2

2

2

2

2

2

2

2

+/- %

1

0

0

0

-1

-1

-2

-3

-3

-5

-6

-4

-5

450 mg/kg bw/day (Recovery group day 90-117)

Mean

20.2

21.2

21.2

19.4

20.1

20.1

19.9

20.3

20

19.6

19.5

18.8

18.7

20.8

22.2

22.4

22.1

SD

0.85

0.91

0.91

0.26

0.94

0.94

0.74

0.86

1

1.46

0.88

1.06

0.89

n (Number of cages)

3

3

3

3

3

3

3

3

3

3

3

3

3

1

1

1

1

+/- %

-4

2

-1

-1

1

1

4

3

-1

1

2

2

-3

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

DN

NS

NS

female

Control (Recovery group day 90-117)

Mean

14.8

14.6

14.3

14.4

14.4

13.7

13.1

13.6

13.7

13.7

13.5

12.8

15

12.6

14.9

14.6

13.9

SD

0.62

0.29

0.48

0.34

0.4

0.75

0.59

0.96

0.76

0.76

0.74

0.71

0.73

n

3

3

3

3

3

3

3

3

3

3

3

3

3

1

1

1

1

50 mg/kg bw/day

Mean

15

14.8

14.3

14.3

14.7

14.2

13.3

13.7

13.9

13.7

13.7

13.9

16.9

SD

0

0.36

0.71

0.16

0.53

0.34

0.81

0.3

0.06

0.36

0.63

0.77

0.4

n (Number of cages)

2

2

2

2

2

2

2

2

2

2

2

2

2

+/- %

2

1

0

-1

2

4

1

1

1

0

1

9

13

150 mg/kg bw/day

Mean

14.2

13.8

13.6

13.6

13.9

13.3

13.5

13.2

13.5

12.8

12.5

12.8

15.4

SD

0.42

0.44

0.4

0.36

0.38

0.18

0.14

0.22

0.2

0.2

0.59

1.09

1.75

n (Number of cages)

2

2

2

2

2

2

2

2

2

2

2

2

2

+/- %

-4

-6

-5

-5

-3

-3

3

-3

-2

-6

-7

0

3

450 mg/kg bw/day (Recovery group day 90-117)

Mean

15

15.4

15

14.9

14.9

14.7

14.6*

14.8

14

14.2

14.2

14

16.3

14.4

15.1

16

17.7

SD

0.46

0.62

0.41

0.16

0.16

0.3

0.26

0.22

0.02

0.8

0.28

0.46

0.08

n (Number of cages)

3

3

3

3

3

3

3

3

3

3

3

3

3

1

1

1

1

+/- %

1

5

6

4

4

7

11

9

2

4

5

9

9

Statistical evaluation

NS

NS

NS

NS

NS

NS

DN

NS

NS

NS

NS

NS

NS

+/- % = Percent Deviation Versus Control

NS = Not Significant

* = p < 0.05

DN = Duncan's multiple range test

Please note that no statistical calculation for recovery group was done, as only one cage existed for each group.

 

Table 5: Summary of hematology

Group

WBC [x109/L]

NEU [%]

LYM [%]

MONO [%]

EOS [%]

BASO [%]

RBC [x1012/L]

HGB [g/L]

HCT [L/L]

MCV [fL]

MCH [pg]

MCHC [g/L]

PLT [x109/L]

RET [%]

PT [sec]

APTT [sec]

male

Control

Mean

7.76

21.52

74.54

2.45

1.1

0.05

8.87

162.3

0.46

51.99

18.29

351.8

792.7

1.88

10.48

13.2

SD

1.37

5.17

5.1

0.78

0.22

0.07

0.32

5.5

0.01

1.12

0.49

3.77

102.96

0.19

0.22

1.95

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

50 mg/kg bw/day

Mean

6.93

18.38

78.48

1.77*

0.86*

0.08

8.91

162

0.46

51.86

18.2

350.8

8.46.6

1.83

10.44

13.22

SD

1.48

3.77

4.01

0.45

0.15

0.04

0.34

4.99

0.01

1.13

0.44

5.73

73.88

0.31

0.24

1.17

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-11

-15

5

-28

-22

60

0

0

1

0

0

0

7

-2

0

0

150 mg/kg bw/day

Mean

7.29

21.41

74.98

2.04

11.11

0.06

7.85

161.5

0.45

51.96

18.47

355.5

826.8

2.05

10.25

13.07

SD

1.24

6.41

6.47

0.51

0.33

0.05

0.31

5.82

0.02

0.94

0.37

5.02

123.09

0.39

0.21

1.32

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-6

-1

1

-17

1

20

-1

0

-1

0

1

1

4

9

-2

-1

450 mg/kg bw/day

Mean

8.05

21.65

74.14

2.59

1.1

0.07

8.57

157.7

0.45

52.08

18.44

353.9

873.7

2.14

10.36

12.52

SD

1.64

6.76

7.71

0.71

0.41

0.05

0.6

8.22

0.02

1.88

0.96

7.43

88.74

0.65

0.3

2.17

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

4

-1

-1

6

0

40

-3

-3

-3

0

1

1

10

14

-1

-5

Statistical evaluation

NS

NS

NS

DN

U

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

male recovery

Control

Mean

7.98

19.06

75.54

2.9

2.02

0.08

9.81

171

0.51

51.66

17.42

337.6

941.2

1.58

10.6

14.76

SD

1.46

5.26

5.44

0.43

1.2

0.04

0.31

5.66

0.02

1.24

0.62

5.32

124.4

0.33

0.12

1.45

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

450 mg/kg bw/day

Mean

7.32

17.84

77.24

3.64

0.96

0.08

9.73

175

0.51

52.86

18.06

34.8

928.2

1.77

10.28

13.08

SD

0.77

2.13

3.83

1.64

0.28

4

0.68

4.53

0.02

1.67

0.86

6.3

152.76

0.22

0.27

0.54

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

+/- %

-8

-6

2

26

-52

0

-1

2

1

2

4

1

-1

12

-3

-11

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

female

Control

Mean

5.38

15.47

80.29

2.2

1.43

0.07

7.79

149

0.42

54.14

19.14

353.2

847.1

2.8

9.81

13.09

SD

1.63

3.61

4.05

0.6

0.39

0.07

0.23

5.58

0.01

1.23

0.58

5.96

94.08

0.64

0.19

0.82

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

50 mg/kg bw/day

Mean

4.97

14.16

81.69

2.32

1.27

0.05

7.97

153.7*

0.43

53.62

19.29

359.8

921.6

2.81

9.82

12.46

SD

1.09

3.65

4.02

0.66

0.68

0.05

0.26

2.79

0.01

1.06

0.58

9.37

85.72

0.36

0.23

0.71

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-8

-8

2

5

-11

-29

2

3

1

-1

1

2

9

0

0

-5

150 mg/kg bw/day

Mean

4.4

18.33

7.7

2.09

1.41

0.04

7.78

150.3

0.42

53.96

19.31

358.3

890

3.14

9.7

12.71

SD

1.67

4.01

4.29

0.54

0.58

0.05

0.2

3.77

0.01

0.99

0.21

6.83

97.43

0.78

0.19

0.54

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-18

18

-3

-5

-1

-43

0

1

0

0

1

1

5

12

-1

-3

450 mg/kg bw/day

Mean

4.42

16.86

78.62

2.68

1.4

0.04

7.86

151

0.42

53.33

19.22

360.2

862.8

2.76

9.67

12.1**7

SD

1.17

6.53

7.58

1.18

0.74

0.05

0.39

3.16

0.02

1.48

0.72

8.05

76.37

0.66

0.19

0.57

n

10

10

10

10

0

10

10

10

10

10

10

10

10

10

10

10

+/- %

-18

9

-2

22

-2

-43

1

1

0

-1

0

2

2

-1

-1

-7

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

NS

DN

NS

NS

NS

NS

NS

NS

NS

DN

female recovery

Control

Mean

4.68

15.08

79.16

3.44

1.8

0.12

8.8

165.4

0.48

54.14

18.8

347

1075.8

1.79

9.68

13.14

SD

0.91

1.11

0.97

0.83

0.19

0.04

0.43

7.33

0.02

1.01

0.48

8

145.15

0.75

0.15

1.24

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

450 mg/kg bw/day

Mean

5.36

17.44

75.82

3.3

2.96

0.04

8.77

161.4

0.47

53.92

18.46

342.2

961.8

2.48

9.84

12.86

SD

1.46

3.99

4.3

0.98

2.13

0.05

0.28

5.41

0.01

1.43

0.72

4.21

111.53

0.61

0.15

1.31

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

+/- %

15

16

-4

-4

64

-67

0

-2

0

0

-2

-1

-11

38

2

-2

Statistical evaluation

NS

NS

NS

NS

NS

NS

*

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

+/-% = Percent Deviation Versus Control

NS = Not Significant

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

T - test Versus Control (for recovery)

 

Table 6: Summary of clinical chemistry

Group

ALT [U/L]

AST [U/L]

GGT [U/L]

ALP [U/L]

TBIL [µmol/L]

CREA [µmol/L]

UREA [mmol/L]

GLUC [mmol/L]

CHOL [mmol/L]

Pi [mmol/L]

Ca++ [mmol/L]

Na+ [mmol/L]

K+ [mmol/L]

Cl- [mmol/L]

ALB [g/L]

TPROT [g/L]

A/G

male

Control

Mean

41.9

84.7

-

85.2

1.12

26.3

4.37

5.45

1.92

1.92

2.5

145.26

4.35

100.15

41.98

64.04

1.92

SD

8.35

10.8

-

16.22

0.3

1.49

0.68

0.41

0.45

0.12

0.09

2.14

0.1

1.99

1.32

2.46

0.21

n

10

10

-

10

10

10

10

10

10

10

10

10

10

10

10

10

10

50 mg/kg bw/day

Mean

45.9

84.1

-

122.1*

1.04

27.6

4.4

5.61

1.6

1.9

2.48

144.72

4.36

99.52

44.56**

64.63

2.28*

SD

9.33

10.08

-

39.48

0.28

2.46

0.61

0.76

0.28

0.14

0.08

2.34

0.18

1.65

1.77

2.18

0.35

n

10

10

-

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

10

-1

-

43

-7

5

1

3

-17

-1

-1

0

0

-1

6

1

19

150 mg/kg bw/day

Mean

41.7

82.10

-

89.6

1.04

27.3

4.62

5.94**

1.87

1.87

2.51

145.74

4.35

99.67

43.22

64.74

2.02

SD

9.59

7

-

20.8

0.46

2.36

0.69

0.3

0.39

0.09

0.09

1.26

0.26

1.58

1.89

1.11

0.23

n

10

10

-

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

0

-3

-

5

-7

4

6

9

-3

-2

-3

0

0

0

3

1

5

450 mg/kg bw/day

Mean

40.1

82.9

-

117.7

0.75

31

4.45

5.84

1.66

1.81

2.55

144.7

4.51

99.47

42.55

64.13

2.01

SD

12.87

14.19

-

43.04

0.5

2.62**

0.72

0.38

0.23

0.12

0.07

1.98

0.22

1.83

1.52

2.77

0.34

n

10

10

-

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-4

-2

-

38

-33

18

2

7

-14

-5

2

0

4

-1

1

0

5

Statistical evaluation

NS

NS

-

U

NS

DN

NS

U

NS

NS

NS

NS

NS

NS

DN

NS

DN

male recovery

Control

Mean

41.6

88.4

-

70.2

1.4

24

5.58

5.89

2.05

1.76

2.58

143.68

4.69

99.16

42.14

66.4

1.74

SD

5.68

5.08

-

11.99

0.26

2.35

0.72

0.34

0.38

0.1

0.04

1.8

0.23

0.64

1.37

1.03

0.13

n

5

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

5

450 mg/kg bw/day

Mean

43.6

92.6

-

79.2

1.4

26.2

5.62

6.33

1.96

1.9

2.53

143.18

4.6

98.98

42.48

64.34

2

SD

10.52

2.51

-

15.42

0.3

1.48

0.61

0.37

0.33

0.05

0.06

0.22

0.15

1.11

1.45

2.58

0.34

n

5

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

5

+/- %

5

5

-

13

0

9

1

8

-4

8

-2

0

-2

0

1

-3

15

Statistical evaluation

NS

NS

-

NS

NS

NS

NS

NS

NS

*

NS

NS

NS

NS

NS

NS

NS

female

Control

Mean

43.2

86.7

-

40.4

1.35

30.2

5.87

4.79

1.51

1.73

2.54

144.38

4.1

99.79

46.96

66.35

2.45

SD

18.45

16.66

-

10.95

0.52

2.94

1.27

0.69

0.41

0.22

0.1

1.65

0.14

2.09

2.82

2.8

0.47

n

10

10

-

10

10

10

10

10

10

10

10

10

10

10

10

10

10

50 mg/kg bw/day

Mean

52.4

90.8

-

47.32

1.26

32.1

6.08

4.68

1.65

1.73

2.5

143.98

4.08

99.58

45.85

65.37

2.37

SD

31.41

20.11

-

19.27

0.33

3.67

0.84

0.61

0.33

0.22

0.11

1.51

0.17

1.21

2.64

3.11

0.21

n

10

10

-

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

21

5

-

17

-7

6

4

-2

9

0

-2

0

0

0

-2

-1

-3

150 mg/kg bw/day

Mean

39.2

80.3

-

41.5

1.01

31.9

5.66

4.53

1.63

1.46**

2.51

143.67

4.05

99.73

47.38

65.48

2.64

SD

10.76

12.41

-

12.71

0.2

3.18

1.12

0.54

0.33

0.17

0.06

2.59

0.26

1.61

1.5

2.28

0.18

n

10

10

-

10

10

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-9

-7

-

3

-25

6

-4

-6

8

-16

-1

0

-1

0

1

-1

8

450 mg/kg bw/day

Mean

36.8

81.4

-

47.1

0.69**

33

5.54

4.73

1.81

1.54*

2.51

143.93

4.07

101.35

44.11*

61.67**

2.52

SD

6.61

8.88

-

13.64

0.45

2.36

0.9

0.6

0.54

0.18

0.11

1.9

0.42

1.92

3.03

3.7

0.21

n

10

10

-

10

0

10

10

10

10

10

10

10

10

10

10

10

10

+/- %

-15

-6

-

17

-49

9

-6

-1

20

-11

-1

0

-1

2

-6

-7

3

Statistical evaluation

NS

NS

-

NS

DN

NS

NS

NS

NS

DN

NS

NS

NS

NS

DN

DN

NS

female recovery

Control

Mean

43.6

95.6

-

22

1.68

35.8

7.5

5.06

1.9

1.44

2.62

143.92

4.3

99.72

47.48

70.32

2.14

SD

10.81

23.17

-

6.08

0.2

3.27

1.09

0.27

0.37

0.18

0.08

2.54

0.22

2.62

2.18

3.92

0.34

n

5

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

5

450 mg/kg bw/day

Mean

36.2

83.4

-

24.8

1.3

33.2

6.68

5.35

2.27

1.32

2.56

140.78

4.43

98.82

46.24

65.8

2.36

SD

7.16

11.65

-

3.11

0.25

1.79

0.61

0.41

0.23

0.22

0.07

0.74

0.3

0.67

1.64

3.02

0.19

n

5

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

5

+/- %

-17

-13

-

13

-23

-7

-11

6

19

-9

-2

-2

3

-1

-3

-6

10

Statistical evaluation

NS

NS

-

NS

*

NS

NS

NS

NS

NS

NS

*

NS

NS

NS

NS

NS

+/-% = Percent Deviation Versus Control

NS = Not Significant

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

T - test Versus Control (for recovery)

 

Table 7: Summary of necropsy findings

Organs

Observation

Control

50 mg/kg bw/day

150 mg/kg bw/day

450 mg/kg bw/day

Main group

Recovery group

Main group

Recovery group

male

No macroscopic findings

9/10

4/5

10/10

8/10

10/10

5/5

Abdominal cavity

Semi-hard formation (1x2 cm) in fatty tissue

1/10

0/5

0/10

0/10

0/10

0/5

Yellowish formation (0.5x1.2 cm)

0/10

1/5

0/10

0/10

0/10

0/5

Seminal vesicle

Smaller than normal - right side

1/10

0/5

0/10

1/10

0/10

0/5

Liver

Pale

0/10

0/5

0/10

1/10

0/10

0/5

female

No macroscopic findings

4/10

3/5

6/10

4/10

6/10

3/5

Uterus

Hydrometra

6/10

2/5

4/10

6/10

4/10

2/5

 

Table 8: Summary of Organ weight (g)

Group

Bodyweight

Brain

Liver

Kidney

Heart

Spleen

Thymus

Adrenal glands

Testes

Epididymides

Seminal vesicles, Prostate

Uterus

Overies

male

Control

Mean

451.8

2.7

10.54

2.35

1.13

0.65

0.43

0.069

3.55

1.48

2.38

n.a.

n.a.

SD

50.03

0.06

1.78

0.35

0.12

0.08

0.1

0.007

0.22

0.15

0.54

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

50 mg/kg bw/day

Mean

446.7

2.21

10.7

2.38

1.1

0.72

0.35*

0.074

3.55

1.61

2.34

n.a.

n.a.

SD

35.2

0.06

1.53

0.26

0.12

0.07

0.07

0.009

0.22

0.28

0.44

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

-1

2

2

1

-3

11

-19

7

0

9

-2

n.a.

n.a.

150 mg/kg bw/day

Mean

446

2.17

10.51

2.31

1.08

0.66

0.39

0.074

3.4

1.45

2.16

n.a.

n.a.

SD

27.49

0.07

0.72

0.22

0.09

0.08

0.05

0.008

0.19

0.13

0.46

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

-1

0

0

-2

-6

2

-9

8

-4

-2

-9

n.a.

n.a.

450 mg/kg bw/day

Mean

452.4

2.21

11.22

2.33

1.08

0.71

0.34*

0.07

3.56

1.48

2.32

n.a.

n.a.

SD

41.68

0.1

1.18

0.28

0.08

0.11

0.08

0.006

0.2

0.2

0.21

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

0

2

6

-1

-4

9

-21

1

0

0

-3

n.a.

n.a.

Statistical evaluation

NS

NS

NS

NS

NS

NS

DN

NS

NS

NS

NS

n.a.

n.a.

male recovery

Control

Mean

479.8

2.25

10.92

2.59

1.14

0.65

0.38

0.072

3.51

1.58

2.62

n.a.

n.a.

SD

19.68

0.05

0.97

0.22

0.08

0.08

0.08

0.004

0.25

0.08

0.31

n.a.

n.a.

n

5

5

5

5

5

5

5

5

5

5

5

n.a.

n.a.

450 mg/kg bw/day

Mean

466

2.19

11.32

2.33

1.1

0.75

0.3

0.071

3.28

1.57

2.73

n.a.

n.a.

SD

35.01

0.04

1.03

0.11

0.13

0.06

0.04

0.007

0.35

0.3

0.42

n.a.

n.a.

n

5

5

5

5

5

5

5

5

5

5

5

n.a.

n.a.

+/- %

-3

-2

4

-10

-4

15

-22

-1

-7

0

4

n.a.

n.a.

Statistical evaluation

NS

NS

NS

*

NS

NS

NS

NS

NS

NS

NS

n.a.

n.a.

female

Control

Mean

258.3

2.09

6.91

1.48

0.74

0.48

0.37

0.082

n.a.

n.a.

n.a.

0.76

0.1

SD

25.83

0.05

1.05

0.09

0.06

0.05

0.06

0.014

n.a.

n.a.

n.a.

0.22

0.012

n

10

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

50 mg/kg bw/day

Mean

262.7

2.06

9.73

1.51

0.73

0.45

0.36

0.089

n.a.

n.a.

n.a.

0.67

0.102

SD

18.14

0.05

0.55

0.12

0.07

0.03

0.06

0.01

n.a.

n.a.

n.a.

0.14

0.021

n

10

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

2

-2

-3

2

-1

-5

-1

9

n.a.

n.a.

n.a.

-12

2

150 mg/kg bw/day

Mean

250.3

2.04

6.77

1.51

0.72

0.46

0.37

0.07

n.a.

n.a.

n.a.

0.73

0.09

SD

19.08

0.04

0.94

0.13

0.06

0.08

0.08

0.013

n.a.

n.a.

n.a.

0.15

0.011

n

10

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

-3

-2

-2

1

-3

-5

1

-7

n.a.

n.a.

n.a.

-4

-10

450 mg/kg bw/day

Mean

260.3

2.03

7.31

1.58

0.74

0.48

0.36

0.08

n.a.

n.a.

n.a.

0.69

0.093

SD

17.75

0.09

0.55

0.13

0.05

0.05

0.07

0.01

n.a.

n.a.

n.a.

0.17

0.009

n

10

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

1

-3

6

6

0

-1

-2

-3

n.a.

n.a.

n.a.

-10

-7

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

NS

n.a.

n.a.

n.a.

NS

NS

female recovery

Control

Mean

255.6

2.02

6.24

1.53

0.72

0.45

0.27

0.073

n.a.

n.a.

n.a.

0.64

0.105

SD

11.55

0.05

0.29

0.09

0.02

0.04

0.05

0.009

n.a.

n.a.

n.a.

0.14

0.014

n

5

5

5

5

5

5

5

5

n.a.

n.a.

n.a.

5

5

450 mg/kg bw/day

Mean

261.8

2

7.18

1.51

0.74

0.48

0.33

0.079

n.a.

n.a.

n.a.

0.77

0.097

SD

37.31

0.11

0.59

0.14

0.1

0.05

0.1

0.009

n.a.

n.a.

n.a.

0.08

0.01

n

5

5

5

5

5

5

5

5

n.a.

n.a.

n.a.

5

5

+/- %

2

-1

15

-1

2

7

22

8

n.a.

n.a.

n.a.

21

-7

Statistical evaluation

NS

NS

*

NS

NS

NS

NS

NS

n.a.

n.a.

n.a.

NS

NS

Seminal vesicles with coagulating gland

+/-% = Percent Deviation Versus Control

NS = Not Significant

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

T - test Versus Control (for recovery)

 

Table 9: Summary of Organ weight relative to body weight (%)

Group

Brain

Liver

Kidney

Heart

Spleen

Thymus

Adrenal glands

Testes

Epididymides

Seminal vesicles, Prostate

Uterus

Overies

male

Control

Mean

0.486

2.326

0.522

0.25

0.144

0.095

0.0154

0.792

0.33

0.53

n.a.

n.a.

SD

00.057

0.191

0.035

0.01

0.015

0.017

0.0012

0.072

0.034

0.124

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

50 mg/kg bw/day

Mean

0.497

2.392

0.532

0.245

0.161

0.078*

0.0165

0.8

0.363

0.525

n.a.

n.a.

SD

0.043

0.257

0.052

0.013

0.013

0.015

0.002

0.082

0.068

0.104

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

2

3

2

-2

12

-18

8

1

10

-1

n.a.

n.a.

150 mg/kg bw/day

Mean

0.489

2.358

0.517

0.239

0.149

0.088

0.0167

0.764

0.327

0.486

n.a.

n.a.

SD

0.034

0.108

0.046

0.018

0.016

0.014

0.002

0.061

0.035

0.101

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

1

1

-1

-4

4

-7

9

-4

-1

-8

n.a.

n.a.

450 mg/kg bw/day

Mean

0.492

2.48

0.516

0.24

0.157

0.075*

0.0156

0.791

0.328

0.515

n.a.

n.a.

SD

0.041

0.138

0.036

0.014

0.023

0.017

0.0016

0.056

0.034

0.055

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

1

7

-1

-4

9

-21

1

0

-1

-3

n.a.

n.a.

Statistical evaluation

NS

NS

NS

NS

NS

DN

NS

NS

NS

NS

n.a.

n.a.

male recovery

Control

Mean

0.469

2.274

0.541

0.237

0.136

0.08

0.015

0.732

0.329

0.579

n.a.

n.a.

SD

0.016

0.134

0.038

0.01

0.015

0.018

0.001

0.049

0.011

0.083

n.a.

n.a.

n

5

5

5

5

5

5

5

5

5

5

n.a.

n.a.

450 mg/kg bw/day

Mean

0.473

2.429

0.502

0.235

0.161

0.064

0.015

0.702

0.338

0.588

n.a.

n.a.

SD

0.035

0.116

0.019

0.017

0.009

0.007

0.001

0.03

0.057

0.094

n.a.

n.a.

n

5

5

5

5

5

5

5

5

5

5

n.a.

n.a.

+/- %

1

7

-7

-1

19

-20

2

-4

3

7

n.a.

n.a.

Statistical evaluation

NS

NS

NS

NS

*

NS

NS

NS

NS

NS

n.a.

n.a.

female

Control

Mean

0.816

2.676

0.579

0.287

0.186

0.142

0.0321

n.a.

n.a.

n.a.

0.3

0.0391

SD

0.082

0.299

0.056

0.014

0.014

0.024

0.0057

n.a.

n.a.

n.a.

0.1

0.055

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

50 mg/kg bw/day

Mean

0.787

2.563

0.578

0.278

0.173

0.138

0.0341

n.a.

n.a.

n.a.

0.256

0.0389

SD

0.065

0.105

0.048

0.018

0.012

0.023

0.0035

n.a.

n.a.

n.a.

0.066

0.0076

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

-4

-4

0

-3

-7

-3

6

n.a.

n.a.

n.a.

-15

0

150 mg/kg bw/day

Mean

0.821

2.698

0.601

0.287

0.182

0.148

0.0307

n.a.

n.a.

n.a.

0.296

0.0361

SD

0.066

0.228

0.03

0.024

0.023

0.033

0.004

n.a.

n.a.

n.a.

0.074

0.0039

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

1

1

4

0

-2

4

-4

n.a.

n.a.

n.a.

-1

-8

450 mg/kg bw/day

Mean

0.783

2.813

0.606

0.285

0.183

0.139

0.0306

n.a.

n.a.

n.a.

0.263

0.357

SD

0.056

0.189

0.056

0.008

0.022

0.032

0.0034

n.a.

n.a.

n.a.

0.063

0.0029

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

-4

5

5

-1

-1

-2

-5

n.a.

n.a.

n.a.

-12

-9

Statistical evaluation

NS

NS

NS

NS

NS

NS

NS

n.a.

n.a.

n.a.

NS

NS

female recovery

Control

Mean

0.791

2.444

0.597

0.282

0.175

0.105

0.0285

n.a.

n.a.

n.a.

0.249

0.0409

SD

0.035

0.157

0.031

0.012

0.021

0.019

0.0028

n.a.

n.a.

n.a.

0.053

0.0052

n

5

5

5

5

5

5

5

n.a.

n.a.

n.a.

5

5

450 mg/kg bw/day

Mean

0.772

2.765

0.58

0.282

0.183

0.124

0.0305

n.a.

n.a.

n.a.

0.302

0.0377

SD

0.088

0.222

0.033

0.024

0.016

0.028

0.0036

n.a.

n.a.

n.a.

0.069

0.0056

n

5

5

5

5

5

5

5

n.a.

n.a.

n.a.

5

5

+/- %

-2

13

-3

0

5

18

7

n.a.

n.a.

n.a.

21

-8

Statistical evaluation

NS

*

NS

NS

NS

NS

NS

n.a.

n.a.

n.a.

NS

NS

Seminal vesicles with coagulating gland

+/-% = Percent Deviation Versus Control

NS = Not Significant

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

T - test Versus Control (for recovery)

 

Table 10: Summary of Organ weight relative to brain weight (%)

Group

Bodyweight

Liver

Kidney

Heart

Spleen

Thymus

Adrenal glands

Testes

Epididymides

Seminal vesicles, Prostate

Uterus

Overies

male

Control

Mean

20820.7

485.8

108.82

52.11

29.83

19.82

3.19

163.8

68.4

109.75

n.a.

n.a.

SD

2301.94

80.7

16.18

6.11

3.68

4.48

0.33

12.99

8

24.99

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

50 mg/kg bw/day

Mean

20238.5

485.43

107.62

49.72

32.45

15.79*

3.34

160.93

72.81

105.7

n.a.

n.a.

SD

1738.47

74.22

12.33

5.68

3.24

3.19

0.41

9.9

11.86

19.8

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

-3

0

-1

-5

9

-20

5

-2

6

-4

n.a.

n.a.

150 mg/kg bw/day

Mean

20554.3

484.15

95.8

49.12

30.55

18.07

3.43

156.45

66.98

99.68

n.a.

n.a.

SD

1466.27

34.11

35.46

4.26

3.39

2.25

0.38

9.89

5.92

21.1

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

-1

0

-12

-6

2

-9

8

-4

-2

-9

n.a.

n.a.

450 mg/kg bw/day

Mean

20450.2

507.03

105.37

49.0

31.85

15.34**

3.17

161.17

67.06

104.92

n.a.

n.a.

SD

1728.81

47.99

10.73

3.99

4.07

3.36

0.22

8.97

8.59

9.94

n.a.

n.a.

n

10

10

10

10

10

10

10

10

10

10

n.a.

n.a.

+/- %

-2

4

-3

-6

7

-23

-1

-2

-2

-4

n.a.

n.a.

Statistical evaluation

NS

NS

NS

NS

NS

DN

NS

NS

NS

NS

n.a.

n.a.

male recovery

Control

Mean

21323.5

485.06

115.24

50.55

28.95

17.12

3.19

156

70.23

n.a.

n.a.

SD

719.42

35.12

8.45

3

3.37

3.85

0.14

10.68

3.59

n.a.

n.a.

n

5

5

5

5

5

5

5

5

5

5

n.a.

n.a.

450 mg/kg bw/day

Mean

21241.3

515.74

106.42

50.01

34.17

16.69

3.24

149.42

71.78

n.a.

n.a.

SD

1584.64

44.73

5.74

5.4

2.66

2.17

0.32

16.11

13.58

n.a.

n.a.

n

5

5

5

5

5

5

5

5

5

5

n.a.

n.a.

+/- %

0

6

-8

-1

18

-20

1

-4

2

n.a.

n.a.

Statistical evaluation

NS

NS

NS

NS

*

NS

NS

NS

NS

NS

n.a.

n.a.

female

Control

Mean

12352.7

330.58

71.03

35.35

22.92

17.46

3.94

n.a.

n.a.

n.a.

36.47

4.79

SD

1130.38

48.8

4.58

2.82

2.27

3.01

0.66

n.a.

n.a.

n.a.

10.7

0.52

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

50 mg/kg bw/day

Mean

12774.2

327.3

73.69

35.57

22.02

17.59

4.35

n.a.

n.a.

n.a.

32.49

4.98

SD

1017.32

29.13

7.04

3.71

1.4

3.49

0.53

n.a.

n.a.

n.a.

7.53

1.13

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

3

-1

4

1

-4

1

10

n.a.

n.a.

n.a.

-11

4

150 mg/kg bw/day

Mean

12248.0

331.47

73.65

35.08

22.34

18.1

3.76

n.a.

n.a.

n.a.

35.83

4.41

SD

922.79

45.83

6.68

3.6

4

4.19

0.57

n.a.

n.a.

n.a.

7.35

0.51

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

-1

0

4

-1

-3

4

-5

n.a.

n.a.

n.a.

-2

-8

450 mg/kg bw/day

Mean

12837.5

360-81

77.73

36.55

23.51

17.71

3.93

n.a.

n.a.

n.a.

33.77

4.58

SD

9.27.98

31-39

7.16

2.86

3.07

3.54

0.54

n.a.

n.a.

n.a.

8.23

0.45

n

10

10

10

10

10

10

10

n.a.

n.a.

n.a.

10

10

+/- %

4

9

9

3

3

1

0

n.a.

n.a.

n.a.

-7

-4

Statistical evaluation

n.a.

n.a.

n.a.

female recovery

Control

Mean

12656.3

308.73

75.58

35.66

22.12

13.29

3.61

n.a.

n.a.

n.a.

31.57

5.17

SD

568.91

12.24

4.96

1.44

2.5

2.59

0.41

n.a.

n.a.

n.a.

6.87

0.6

n

5

5

5

5

5

5

5

n.a.

n.a.

n.a.

5

5

450 mg/kg bw/day

Mean

13074.8

359.51

75.59

36.87

23.87

16.29

3.97

n.a.

n.a.

n.a.

38.91

4.87

SD

1405.24

23.94

5.69

4.66

2.69

4.53

0.5

n.a.

n.a.

n.a.

6.22

0.43

n

5

5

5

5

5

5

5

n.a.

n.a.

n.a.

5

5

+/- %

3

16

0

3

8

23

10

n.a.

n.a.

n.a.

23

-6

Statistical evaluation

NS

**

NS

NS

NS

NS

NS

n.a.

n.a.

n.a.

NS

NS

Seminal vesicles with coagulating gland

+/-% = Percent Deviation Versus Control

NS = Not Significant

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control

DN = Duncan's multiple range test

T - test Versus Control (for recovery)

 

Table 11: Summary of histopathology findings

Organs

Observation

Control

50 mg/kg bw/day

150 mg/kg bw/day

450 mg/kg bw/day

Main group

Recovery group

Main group

Recovery group

male

Adrenal glands

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Aorta

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Bone marrow

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Brain

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Cecum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Colon

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Duodenum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Eyes + optic nerve

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Epididymides

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Esophagus

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Harderian glands

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Heart

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Ileum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Jejunum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Kidneys

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Lachrymal glands

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Liver

No lesion

10/10

5/5

n.e.

1/1

10/10

5/5

Lungs

Alveolar emphysema

1/10

1/5

n.e.

n.e.

1/10

0/5

Hyperplasia of BALT

1/10

0/5

n.e.

n.e.

1/10

1/5

Mesenteric lymph nodes

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Muscle (quadriceps)

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Pancreas

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Pituitary

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Prostate

Lymphocytic infiltration

1/10

0/5

n.e.

n.e.

0/10

0/5

Rectum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Salivary glands (submandibular)

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Sciatic nerve

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Seminal vesicle with coagulating gland

Decreased amount of secrete

1/10

0/5

n.e.

1/1

0/10

0/5

Skin

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Spinal cord

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Spleen

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Sternum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Stomach

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Submandibular lymph nodes

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Thymus

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Thyroid + parathyroid

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Trachea

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Urinary bladder

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Tissue formation

Lipoma

1/1

1/1

n.e.

n.e.

n.e.

n.e

female

Adrenal glands

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Aorta

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Bone marrow

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Brain

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Cecum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Colon

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Duodenum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Eyes + optic nerve

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Epididymides

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Esophagus

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Harderian glands

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Heart

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Ileum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Jejunum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Kidneys

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Lachrymal glands

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Liver

Vacuolation of hepatocytes

0/10

0/5

n.e.

n.e.

3/10

0/5

Lungs

Alveolar emphysema

1/10

1/5

n.e.

n.e.

0/10

0/5

Hyperplasia of BALT

1/10

1/5

n.e.

n.e.

0/10

0/5

Mammary gland

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Mesenteric lymph nodes

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Muscle (quadriceps)

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Ovaries

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Pancreas

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Pituitary

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Rectum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Salivary glands (submandibular)

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Sciatic nerve

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Skin

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Spinal cord

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Spleen

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Sternum

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Stomach

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Submandibular lymph nodes

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Thymus

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Thyroid + parathyroid

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Trachea

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Urinary bladder

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

Uterus

Dilatation

6/10

5/2

n.e.

n.e.

4/10

2/5

Vagina

No lesion

10/10

5/5

n.e.

n.e.

10/10

5/5

n.e. = not examined

BALT = Bronchus associated lymphoid tissue

 

Conclusions:
The test item did not cause adverse effects in male or female Han: WIST rats after consecutive 90/91-day oral (by gavage) administration at 50, 150 or 450 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) was determined to be 450 mg/kg bw/day for male and female rats
Executive summary:

The test item was examined for its possible health hazards after repeated exposure in a GLP compliant study according to OECD 408. A four weeks recovery period in the high dose and control dose animals was included in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects.

The test item was administered orally (by gavage) to male and female Han: WIST rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 50,150 and 450 mg/kg bw/day doses (dose volume of 5 mL/kg bw) for 90 or 91 days. Five animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations). Concentrations of the test item in the dosing formulations varied between ranges of 93 % and 108 % of nominal concentrations at each analytical occasion confirming proper dosing.

Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight was determined twice weekly for four weeks (weeks 1-4) then once weekly during the course of the treatment and recovery periods. Food consumption was measured and evaluated weekly. The estrous cycle of each female animal was examined for two weeks before necropsy and for two weeks before the end of the recovery period. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Additionally, liver and seminal vesicles in single male animals at 150 mg/kg bw/day were processed and evaluated histologically on the basis of necropsy observations.

No animals died during the course of the study and no signs of toxicity related to the test item were detected at any dose level at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. Test item related nuzzling up the bedding material and salivation were observed in male animals at 50, 150 and 450 mg/kg bw/day with different incidence and duration during the treatment period. Animals salivated shortly after the daily administration of the test item and it ceased shortly thereafter and was therefore judged to be not adverse. The body weight development was unaffected by the test item at 50, 150 and 450 mg/kg bw/day (male and female animals) during the 3-month treatment period. The mean body weight was similar to the control in male and female animals at 450 mg/kg bw/day during the recovery period. Slight changes in the mean body weight gain were detected in male recovery animals. As the mean body weight remained similar to the control group, the changes were considered to be toxicologically not relevant. The mean daily food consumption was comparable in animals of the control and test item treated groups and no abnormalities in the eyes of animals in the high dose group at termination of the treatment were observed. No test item related influence on the estrous cycle was detected and hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level. No pathologic test item effect was detected at the evaluation of clinical chemistry parameters and T4 and TSH levels were comparable in the control and test item treated groups at the end of the treatment and recovery periods. Test item induced macroscopic changes and changes in organ weights were not detected at termination of the treatment period or at the end of the recovery period. Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 450 mg/kg bw/day. Histological examination did not reveal severe or permanent test item related lesions in the organs or tissues of animals administered with 450 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period. Slight vacuolation of hepatocytes in the centrilobular region (hepatic lipidosis) in 3/10 female animals at 450 mg/kg bw/day was detected. As no such effect was observed in the recovery group, the observation was considered a mild, reversible and non-adverse test item related lesion.

Under the conditions of the present study, the test item did not cause adverse effects in male or female Han: WIST rats after consecutive 90/91-day oral (by gavage) administration at 50, 150 or 450 mg/kg bw/day. Vacuolation of hepatocytes in the centrilobular region (hepatic lipidosis) in some female animals at 450 mg/kg bw/day was assumed to be indicative of test item influence and was considered as a mild, reversible lesion in the female liver.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined to be 450 mg/kg bw/day for male and female Han:WIST rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data are available from a key sub-chronic study on the substance itself, this being a well described GLP compliant study conducted in accordance with recognised internatinal test guidelines.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The subchronic toxicity of 4-MHHPA has been investigated in the rat according to OECD/EU test methods. A four weeks recovery period in the high dose and control dose animals was included in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects. The substance was administered orally (by gavage) to male and female Han: WIST rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 50,150 and 450 mg/kg bw/day doses (dose volume of 5 mL/kg bw) for at least 90 days. Five animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations). Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery

was conducted in the last week of treatment. The body weight was determined twice weekly for four weeks (weeks 1-4) then once weekly during the course of the treatment and recovery periods. Food

consumption was measured and evaluated weekly. The estrous cycle of each female animal was examined for two weeks before necropsy and for two weeks before the end of the recovery period. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Additionally, liver and seminal vesicles in single male animals at 150 mg/kg bw/day were processed and evaluated histologically on the basis of necropsy observations. No animals died during the course of the study and no signs of toxicity related to the test item were detected at any dose level at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. Test item related nuzzling up the bedding material and salivation were observed in male animals at 50, 150 and 450 mg/kg bw/day with different incidence and duration during the treatment period. Animals salivated shortly after the daily administration of the test item and it ceased shortly thereafter and was therefore judged to be not adverse. The body weight development was unaffected by the test item at 50, 150 and 450 mg/kg bw/day (male and female animals) during the 3 -month treatment period. The mean body weight was similar to the control in male and female animals at 450 mg/kg bw/day during the recovery period. Slight changes in the mean body weight gain were detected in male recovery animals. As the mean body weight remained similar to the control group, the changes were considered to be toxicologically not relevant. The mean daily food consumption was comparable in animals of the control and test item treated groups and no abnormalities in the eyes of animals in the high dose group at termination of the treatment were observed. No test item related influence on the estrous cycle was detected and hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level. No pathologic test item effect was detected at the evaluation of clinical chemistry parameters and T4 and TSH levels were comparable in the control and test item treated groups at the end of the treatment and recovery periods. Test item induced macroscopic changes and changes in organ weights were not detected at termination of the treatment period or at the end of the recovery period. Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 450 mg/kg bw/day. Histological examination did not reveal severe or permanent test item related lesions in the organs or tissues of animals administered with 450 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period. Slight vacuolation of hepatocytes in the centrilobular region (hepatic lipidosis) in 3/10 female animals at 450 mg/kg bw/day was detected. As no such effect was observed in the recovery group, the observation was considered a mild, reversible and non-adverse test item related lesion. Under the conditions of this study it was concluded that the test item did not cause adverse effects in male or female Han: WIST rats after consecutive 90/91 -day oral (by gavage) administration at 50, 150 or 450 mg/kg bw/day. Vacuolation of hepatocytes in the centrilobular region (hepatic lipidosis) in some female animals at 450 mg/kg bw/day was assumed to be indicative of test item influence and was considered as a mild, reversible lesion in the female liver. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined to be 450 mg/kg bw/day for male and female Han:WIST rats.

Supporting study:

The sub-acute oral toxicity of 4-MHHPA was investigated in rats according to EU test methods and OECD guidelines. No signs of toxicity were observed during the "in-life" phase of the study. Post mortem

macroscopic observations, absolute and relative organ weights and microscopic examination did not show any changes indicating systemic toxicity of the substance. Thickening of the forestomach mucosa

(non-glandular region) was noted in the majority of animals treated with 450 mg/kg bw/day and in 1 female treated with 150 mg/kg bw/day. Microscopic examination of these lesions revealed a slight to

mild squamous hyperplasia of the forestomach mucosa (non-glandular region), often associated with submucosal oedema, inflammatory reaction or erosion of the gastric mucosa. These changes were no

longer present after 2 weeks of recovery. According to the results of the read across the NOAEL for MHHPA was considered 450 mg/kg bw/day and the NOEL for MHHPA was considered 50 mg/kg bw/

day.

Conclusion:

As an overall conclusion no adverse effects regarding repeated dose toxicity have been identified and the NOAEL was determined to be 450 mg/kg bw/day for 4-methylhexahydrophthalic anhydride (4-MHHPA).

Justification for classification or non-classification

No significant toxicity observed at a concentration of 450 mg/kg/day in a repeat dose study of 90 days duration with the substance itself.

 

According to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification and labelling is not indicated for repeated dose toxicity, as clear functional disturbances or morphological changes were not apparent, the effects that were observed being minor, probably adaptive, changes.