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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 34 ± 1 day
- Weight at study initiation:
- Fasting period before study: No
- Housing: in groups of 5 animals per cage (Polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany; floor area about 2065 cm²)
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, ad libitum (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 pm - 6.00 am dark; 6.00 am - 6.00 pm light)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as an emulsion. The appropriate amount of test substance was weighed out depending on the desired concentration. Thereafter, the vehicle (drinking water containing 1% carboxymethyl cellulose) was filled up to the desired volume + Cremophor EL + one drop Hydrochloric Acid (1mol/L) and subsequently mixed using a magnetic stirrer. The test-substance preparations were produced daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration control analyses of the test-substance preparations in samples of all concentrations were performed at the start of the administration period and thereafter weekly.
Duration of treatment / exposure:
about three months
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
60, 120 and 360 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
Control and high dose groups: 15 animals per sex per group (thereof 5 animals per sex per group for 28-day recovery)
Low and mid dose groups: 10 animals per sex per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 28 days (5 rats/sex of control and high dose groups only)
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
MORTALITY
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS
All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS (DCO)
Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high).The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

FOOD CONSUMPTION
Group food consumption was determined weekly for each cage. The average food consumption/cage was used to estimate the mean food consumption in grams per animal per day.

FOOD EFFICIENCY
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption:
[(BWx - BWy)/FCy to x] x 100 = Food efficiency for day x,
with BWx = body weight on day x (g), BWy = body weight on day y (last weighing date before day x) (g), FCy to x = mean food consumption from day y to day x; calculated as mean daily food consumption on day x, multiplied with the number of days from day y to day x (g).

WATER CONSUMPTION
Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

BODY WEIGHT DATA
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FUNCTIONAL OBSERVATION BATTERY (FOB)
A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 am. At least one hour before the start of the FOB the animals were transferred singly to cages. Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touch¬ing the cage or rack, noise) were avoided during these examinations in order not to in¬fluence the behavior of the animals. Attention was paid to posture, tremor, convulsions, abnormal movements, impairment of gait, other findings
Open field observations: The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes.
Sensorimotor Tests/Reflexes: The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.

MOTOR ACTIVITY (MA) ASSESSMENT
MA measurements were carried out in all animals toward the end of the administration period. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. 18 beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals for 5 minutes in each case. The animals were randomly placed in the cages. Motor activity measurements were carried out from 2.00 pm onwards. On account of the measuring variant "staggered", the starting time varied by the time which is needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was given to the animals during measurements. After the transfer of the last animal in each case, the room of measurement was darkened. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

OPHTHALMOSCOPY
Prior to the administration period, the eyes of all animals were examined using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany). At the end of the administration period the eyes of the control (0 mg/kg bw/day) and high dose animals (360 mg/kg bw/day) were examined for any changes, again.

HAEMATOLOGY
- Time schedule for collection of blood: at the end of the administration period
- Anaesthetic used for blood collection: Yes (Isoba, Essex GmbH Munich, Germany)
- Animals fasted: Yes
- How many animals: All animals per test group and sex. At the end of the recovery period only clotting analyses were examined in the remaining animals of the control and high dose group.
- The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time (Hepato Quick’s test; HQT). The clotting analyses were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at the end of the administration period
- Anaesthetic used for blood collection: Yes (Isoba, Essex GmbH Munich, Germany)
- Animals fasted: Yes
- How many animals: All animals per test group and sex. At the end of the recovery period only blood chemistry parameters were examined in the remaining animals of the control and high dose group.
- An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. The activities of the following enzymes were determined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT). The following blood chemistry parameters were determined: sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), magnesium (MG).

URINALYSIS
- Time schedule for collection of urine: at the end of the administration period overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Investigated parameters: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume. With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur 9 test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).

Sacrifice and pathology:
NECROPSY
All animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

WEIGHT PARAMETERS
The following weight determinations were carried out on all animals: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus, thyroid glands.

HISTOPATHOLOGY
After the organs were fixed, processing, examination by light microscopy and evaluation of findings in the following tissues of all control and high-dose animals of the main groups were performed: salivary glands (mandibular and sublingual glands), esophagus, stomach (forestomach and glandular stomach), duodenum, ileum, jejunum (with Peyer’s plaques), cecum, colon and rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cords), eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, nose (nasal cavity/level III), aorta, heart, bone marrow (femur), lymph nodes (mesenteric and axillary lymph nodes), spleen, thymus, kidneys, urinary bladder, testes, ovaries, oviducts, uterus and vagina, prostate, seminal vesicle and epididymides, female mammary gland, skin.
Additionally, nose tissue (nasal cavity/level III) was examined in all low- and mid-dose animals of the main groups and in all control and high-dose animals of the recovery groups. Furthermore, all gross lesions were examined in all affected animals of all groups.
Statistics:
Statistics of clinical examinations: Means and standard deviations of each test group were calculated for several parameters. Further statistical analyses were DUNNETT's test (two-sided) for body weight and body weight change, and KRUSKAL-WALLIS test (two-sided)/Wilcoxon-test (two-sided) for feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, and motor activity.
Statistics of clinical pathology: Means, medians and standard deviations of each test group were calculated for several parameters. Further statistical analyses were KRUSKAL-WALLIS test (two-sided)/Wilcoxon-test (two-sided) for clinical pathology parameters, urine volume, and urine specific gravity, and FISHER's exact test for urinalysis, except color, turbidity, volume and specific gravity.
Statistics of pathology: KRUSKAL-WALLIS test (two-sided)/Wilcoxon-test (two-sided) for weight parameters.

Results and discussion

Results of examinations

Details on results:
ANALYSES
Stability analyses: The stability of the test substance in drinking water containing 1% Carboxymethylcellulose + Cremophor EL + one drop Hydrochloric Acid (1mol/L) was demonstrated over a period of 4 h at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.

Homogeneity control analyses: Considering the standard deviation in the homogeneity analysis, it can be concluded that n-Butyl Methacrylate was distributed homogeneously in 1% Carboxymethylcellulose + Cremophor EL under the addition of one drop Hydrochloric Acid (1 mol/L).

Concentration control analyses: Due to the physical-chemical properties of the test article (esp. volatility), concentration control analyses were performed weekly. Thus, 13 analytical results were obtained for each dose group throughout the entire study. Taken together these results of the low dose group, the measured concentration was 85.2% of the nominal value. For the mid dose group, the corresponding value was 87.0% and for the high dose group 91.8% of the nominal concentration were determined.
Independently from the obtained variations of the analytical series, these mean values are assessed to demonstrate the correctness of the test-substance preparations within the current study.

CLINICAL EXAMINATIONS
- Mortality: No animal died prematurely in the present study.
- Clinical examinations: During clinical examinations, only Microphthalmia left was observed in 1 female of test group 2 (120 mg/kg bw/day).
This isolated finding only observed in one mid dose female animal was assessed as spontaneous in nature and therefore not substance related.
- Food consumption: Food consumption was significantly increased in males of group 1 (60 mg/kg bw/day) on day 91. This finding, which occurred only once in low dose males, was assessed as incidental in nature and therefore not substance related.
- Water consumption: No substance-related overt changes in water consumption were observed.
- Body weight data: The body weight was significantly decreased in male animals of test group 3 (360 mg/kg bw/day) towards the end of the study (-7% on days 84 and 91). The corresponding body weight change was also significantly decreased in these animals from day 77 till the end of the administration period (-12.1% on day 84 and 91). This impairment of body weight data in high dose males was assessed as related to treatment with the test substance and indicates general systemic toxicity. During the recovery period, no significant influence on body weight data was measured anymore. The body weight of the female animals in all test groups was not significantly influenced by the test article during the administration period. However, in female animals of test group 3 (360 mg/kg bw/day) a significantly lower (-7.8%) weight was noted on day 98, at the beginning of the recovery period, only. This significant difference evolved from an apparent above-average "increase" (by 9 grams) of the mean control body weights in the recovery animals, right after cessation of the treatment. The control group rats designated at the start of the study for recovery, by chance, weighed more than the control group rats sacrificed at the end of the administration period. Because of this, the body weight of the test group 3 (360 mg/kg bw/day) recovery rats appeared to be significantly decreased compared to the control group recovery rats at the beginning of the recovery period. Thus, the apparent weight decrease in the high-dose recovery females is not related to the test article and is considered to be toxicologically irrelevant.
- Food efficiency: No substance-related findings were observed.
- Functional observational battery (FOB): Deviations from "zero values" were obtained in several animals. However, as all findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. Besides this, during functional observational battery the following examinations were carried out and have to be assessed individually: Home cage observations: No substance-related findings were observed. Open field observations: No substance-related findings were observed. Sensorimotor tests/reflexes: No substance-related findings were observed. Quantitative parameters: No substance-related findings were observed.
- Motor activity (MA) measurement: Regarding the overall motor activity, no substance-related findings were observed. Comparing the single intervals, the motor activity was significantly decreased in female animals of group 3 at interval 10, compared to the control value. This single occurrence only observed towards the end of the period of measurement was without any significant influence on the overall motor activity and therefore assessed as toxicologically irrelevant.
- Ophthalmological examinations: The observed corneal stipplings and remainders of the pupillary membrane were equally distributed between the dosed animals and the controls. Therefore, these findings were clearly not related to treatment and were without any toxicological relevance.

CLINICAL PATHOLOGY
- Hematology: At the end of the administration period no treatment-related adverse changes were found in the rats of both sexes concerning the complete blood cell counts. In the male rats of the 360 mg/kg bw/d dose group the relative reticulocyte counts were significantly lower compared to the controls. This change was not regarded as toxicological relevant, because no other red blood cell parameter were affected. After substance administration, the prothrombin time (Hepatoquick’s time) was significantly prolonged in male and female rats of the 360 mg/kg bw/d dose group. This change was no longer present after the 4 weeks recovery period.
- Clinical chemistry: At the end of the administration period in rats of both sexes of the 360 mg/kg bw/d dose group the inorganic phosphate levels were significantly increased and the calcium levels were significantly decreased. The changes were no longer present after the recovery period apart from weakly significantly increased inorganic phosphate levels in males. The urea levels were significantly increased in male and female rats of the 360 mg/kg bw/d dose group, and additionally in males of the 120 mg/kg bw/d dose group. This increased urea level was the only clinical pathology value affected in the mid dose group. Therefore, it was regarded as treatment-related but not adverse. After the recovery period, the urea level was still slightly significantly increased in female rats of the high dose group. After the last administration day, the triglyceride concentrations were significantly decreased in rats of both sexes of the 360 mg/kg bw/d dose group. Additionally, in rats of the high dose group the total bilirubin levels in males as well as the glucose levels in females were significantly increased and the globulin values were significantly decreased in females. All these changes were not present after the recovery period, anymore. But in male rats of the high dose group the glucose levels were weakly significantly decreased after the recovery period. These lower glucose levels were regarded as incidentally, because in these animals the glucose levels were not changed at the end of the administration period, and in female rats the glucose levels were increased during the substance administration. At the end of the administration period, the inorganic phosphate and urea levels were statistically significantly higher in females of the 60 mg/kg bw/d dose group. These were the only findings in this dose group, and they were not dose-dependent. Therefore, theses changes were regarded as incidentally rather than treatment-related. At the end of the administration period, the sodium levels were significantly lower in males of the 360 mg/kg bw/d dose group. This change was not accompanied by any other electrolyte level change, and was not present in females. Therefore, this deviation was regarded as incidentally rather than treatment-related.
- Urinalyses: At the end of the administration period no treatment-related adverse effects were found in the dosed rats regarding the urinalysis parameters. In the males of the 360 mg/kg bw/d dose group there were found less phosphate crystals in the urine sediment compared to the controls. Regarding the individual pH values of the urine samples, there appeared to be slightly lower pH values in the dosed rats. Therefore, it was assumed, that the phosphate crystals were dissolved in the urine of the dosed males. Nevertheless, this effect was regarded as treatment-related, but not adverse.

PATHOLOGY
- Absolute organ weights: The increased kidney weights (+ 11%) in female animals of the 360 mg/kg body weight group are regarded as treatment-related, although there was no histopathologic correlate which could explain this weight increase. The decrease in adrenal gland weights in males of the mid-dose (- 11%) and high-dose (- 10%) groups is regarded as a consequence of the decreased terminal body weights, although these decreases were not statistically significant. It is therefore not regarded as a treatment-related effect. All other mean absolute weight parameters did not show significant differences when compared to the control group. The significant increase in kidney weights of the male animals of the recovery group (+ 17%) is not treatment-related, as the kidney weights of the main group males of the 360 mg/kg dose group were within the range of the control group. All other mean absolute weight parameters did not show significant differences when compared to the control group.
- Relative organ weights: The increased relative brain, kidney, and liver weights in the 360 mg/kg body weight group of male animals are regarded as a consequence of the reduction in terminal body weight. In females, the significant increase in relative kidney and liver weights are regarded as treatment-related. Due to the fact that the terminal body weight was not changed in treated animals and the absolute liver weight was increased up to +9%, although not statistically significant, a substance relationship can not be excluded. This effect is not regarded to be adverse in nature as no histopathologic finding were observed. The decrease in the uterus weight of the 60 mg/kg body weight group is assessed as incidental, because there is no dose-response relationship and the uterus weights in the higher dose groups were greatly than that of the control level. All other mean relative weight parameters of the main groups and the recovery groups did not show significant differences when compared to the control groups.
- Gross lesions: All gross lesions occurred singly or they were biologically equally distributed between control and treatment groups.
- Histopathology: In males and females of the 360 mg/kg body weight group degeneration and regeneration of the olfactory epithelium was observed. The nasal cavities of animals of the recovery groups (control and 360 mg/kg body weight) were examined. There were no abnormalities detected. Mainly the nasal septum and the ethmoid turbinates were affected. In the more severe cases, there was extension to almost all parts of the nasal cavity. The finding was always observed at the transition of the respiratory to the olfactory epithelium at the nasal septum and extended upwards until up to 2/3 of the septum was affected. There was a mixture between degenerative findings (loss of epithelial cells, reduction in epithelium height, irregular architecture, apoptosis) and regenerative findings (hyperplasia/hypertrophy of basal cells, nest-like accumulation of basal cells, occasionally extending into the submucosa, mitotic figures). In animals diagnosed with grade 1, the hyperplasia/hypertrophy was often the most evident finding. All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them are considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects on the liver activity (increased liver weight, prolonged prothrombin time, lower serum globulin and triglyceride levels in males and/or females) and kidneys weight (increased absolute weight in females)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

ABSOLUTE ORGAN WEIGHTS:

Main groups:

 

Male animals

Female animals

Group (mg/kg bw)

60

120

360

60

120

360

Adrenal glands

- 4%

- 11%*

- 10%*

 

 

 

Kidneys

 

 

 

+ 4%

+ 3%

+ 11%**

*: p ≤ 0.05, **: p ≤ 0.01; Kruskal-Wallis H and Wilcoxon test, two-sided

 

Recovery groups:

 

Male animals

Group (mg/kg bw)

360

Kidneys

+ 17%*

*: p ≤ 0.05, **: p ≤ 0.01; Wilcoxon test, two-sided

 

 

RELATIVE ORGAN WEIGHTS:

Main groups:

 

Male animals

Female animals

Group (mg/kg bw)

60

120

360

60

120

360

Brain

+ 1%

+ 1%

+ 11%**

 

 

 

Kidneys

0%

- 2%

+ 13%**

+ 1%

+ 2%

+ 13%**

Liver

+ 4%

+ 7%

+ 9%**

- 3%

+ 4%

+ 11%**

Uterus

 

 

 

- 23%*

+ 3%

+ 9%

*: p ≤ 0.05, **: p ≤ 0.01; Kruskal-Wallis H and Wilcoxon test, two-sided

 

 

INCIDENCE AND GRADING OF THE OBSERVED DEGENERATION AND REGENERATION OF THE OLFACTORY EPITHELIUM:

 

Nasal cavity level III

Male animals

Female animals

Dosemg/kg bw

0

60

120

360

0

60

120

360

Organs examined

10

10

10

10

10

10

10

10

Degeneration/regen-eration olf. epithelium

0

0

4

5

0

0

2

7

Grade 1

 

 

3

3

 

 

 

2

Grade 2

 

 

 

1

 

 

1

3

Grade 3

 

 

1

 

 

 

1

2

Grade 4

 

 

 

1

 

 

 

 

 

Applicant's summary and conclusion