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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 February 2013 - 25 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted Oct. 2008
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted May 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lucirin TPO-L
- Physical state: Liquid, viscous /yellow, clear
- Analytical purity: >97.8%
- Lot/batch No.: Mischung 120204
- Expiration date of the lot/batch: May 29th, 2015
- Storage condition of test material: Room temperature; under light exclusion
- Stability under test conditions: analysis confirmed stability for 96h of the test substance preparations at room temperature
Specific details on test material used for the study:
Purity: 95.7 g/100g (by quanitative H-NMR; study No. 13L00016)
Homogeneity: given
Stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Physical state/ appearance: liquid, viscous/ yellow, clear
Storage conditions: ambient (room temperature); under light exclusion

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species.
Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 41-43 days
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: groups of 5 in polysulfonate cages (floor area 2065cm²)
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, meal ad libitum
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance emulsions were prepared at least twice a week by stirring the appropriate amount in water containing 1% CMC. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer.

- Concentration in vehicle: 0.5, 1,5, 5.0g/100mL
- Volume used: 10ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was verified in the highest and lowest concentration. Correctness of concentration was confirmed in all concentrations at the beginning of the study. All measured values were in the range of 95 - 105%.

The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The studies were carried out in compliance with the Principles of Good Laboratory Practice.
The stability of Lucirin TPO-L in water at room temperature for a period of 96 hours was demonstrated (project No. 01Y0459/038023; see PART III, Supplement).
Homogeneity of Lucirin TPO-L was verified in the highest and lowest concentration. Additionally, concentration control was verified in all concentrations at the beginning of the study.

See attached background material for more detail
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELCTION RATIONALE

In a test study 3 male and 3 female Wistar rats were treated with 1000mg/kg b.w. (BASF project No. 10C0459/03S008). The animals showed clear signs of systemic toxicity like poor general condition, piloerection, hunched posture and impaired body weight parameters. 2 females were sacrificed moribund on day 6. At scheduled necropsy on day 14, increased liver weights (absolute: males: 184%; females: 233% and relative: males: 221%; females: 223%), increased absolute (males: 111%; females: 140%) and relative (males: 135%; females: 134%) kidney weights, and increased adrenal gland and decreased spleen weights were observed in both genders.

Another set of male animals treated for 7 days with 500 mg/kg bw/d showed no clinical signs of concern, but liver weights were also increased, i.e. absolute (127%) and relative (121%) and spleen weights were decreased.
Based on these findings, 500mg/kg was chosen as the maximum dose level in the main study.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily before administration as well as 2-5h after administration
- The following observations were included: mortality, clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals starting on day 0 prior to administration
- The following observations were recorded in a standard arena: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, appearance/consistency of feces, urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: weekly starting on day 0

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes (at weekly intervals over a period of one day)

WATER CONSUMPTION: Yes
- Time schedule for examinations: monitored daily by visual inspections of water bottles

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (day 29)
- Animals fasted: Yes (at least for 16h, water available ad lib.)
- How many animals: all
- Parameters examined: Leucocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy (day 29)
- Animals fasted: Yes (at least for 16h, water available ad lib.)
- How many animals: all
- Parameters examined: ALT, AST, ALP, GGT, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: on day 23
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food or water provided)
- Parameters examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period (days 25/26)
- Dose groups that were examined: all
- Battery of functions tested: home cage observations in single animal cages, open field observations, sensory motor tests / reflexes, motor activity

OTHER:
- Oestrus cycle determination: vaginal smears were prepared in the morning for at least 3 weeks and at necropsy
- Sperm parameters: sperm motility, sperm morphology, sperm head count (cauda epididymis and testis)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Organ weights: anesthetized animals, adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix

histopathologic examinations:
all animals: adrenal glands, left epididymis, kidneys, liver, spleen, stomach, testis

all high dose and control animals: bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, heart, ileum, jejunum, lung, lymph nodes, ovaries, peyer's patches, pituitary glands, prostate, rectum, sciatic nerve, seminal vesicles, skeletal muscle, spinal cord, sternum, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Gross lesions were examined in all animals affected.
Other examinations:
All observations made reported in attached background material
Statistics:
BODY WEIGHT, BODY WEIGHT CHANGE
A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
* for p ≤ 0.05
** for p ≤ 0.01
DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096-1121
DUNNETT, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, Vol. 20, 482-491

Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, umber of cycles, cycle length
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians
* for p ≤ 0.05
** for p ≤ 0.01
SIEGEL, S. (1956): Non-parametric statistics for the behavioural sciences. McGraw-Hill New York

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in all high dose animals and most mid dose female animals shortly after treatment on several days throughout the study, starting on day 1. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.

(Tables IIA- 1 - IIA-24)
Mortality:
no mortality observed
Description (incidence):
No animals died prematurely.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related changes in body weight or body weight gain were observed for male and female animal of test groups 1-3 (50, 150 and 500 mg/kg bw/d) when compared to control groups.

The significantly decreased body weight change value in female animals of test group 2 (150 mg/kg bw/d) on study day 7 was assessed as being incidental and not related to treatment.

(Tables IIA-27 - IIA-28)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (50, 150 and 500 mg/kg bw/d).

(Tables IIA-25 - IIA-26)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume. No overt changes in volume were observed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.

Regarding red blood cell parameters, in male animals of test group 3 (500 mg/kg bw/d) mean corpuscular volume (MCV) was lower, and in females of the same test group relative reticulocyte counts were higher compared to controls. However, both parameters were within historical control ranges (males: MCV 48.1-53.7 fL, females: relative reticulocyte counts 1.0-4.9%; PART III, Supplement). Therefore, these alterations were regarded as incidental and not treatment-related.

In male animals of test groups 1 and 3 (50 and 500 mg/kg bw/d) absolute monocyte counts were decreased, although not dose-dependently, and in males of test group 3 (500 mg/kg bw/d) relative monocyte counts were lower compared to controls. However, all medians were within historical control ranges (absolute monocyte counts 0.05-0.19 Giga/L; relative monocyte counts 0.9-2.5%; PART III, Supplement). Therefore, both changes were regarded as incidental and not treatment-related.

(Tables IB 1 – IB 2 Red blood cell and coagulation parameters)
(Tables IB 3 – IB 4 White blood cell parameters)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were
observed.
At the end of the administration period, in male animals of test group 1 (50 mg/kg bw/d) inorganic phosphate levels were increased. In female animals of test group 1 and 2 (50 and 150 mg/kg bw/d) creatinine levels were increased. However, both parameters were not changed dose-dependently and therefore the alterations were regarded as incidental and not treatment-related.


In males of test group 3 (500 mg/kg bw/d) cholesterol concentrations were decreased and in females of the same test group triglyceride and inorganic phosphate levels were increased. The inorganic phosphate levels in females were within the historical control range (phosphate 1.46-2.24 mmol/L; PART III, Supplement). The decreased cholesterol levels in males and increased triglyceride levels in females of test group 3 (500 mg/kg bw/d) were isolated in either sex and, therefore, they were regarded as treatment-related but not adverse (ECETOC Technical Report No 85, 2002).

(Tables IB 5 – IB 6 Enzymes)
(Tables IB 7 – IB 8 Substrates)
(Tables IB 9 – IB 10 Electrolytes + minerals)
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed. Higher urobilinogen levels in the urine were measured in high dose rats of both sexes and, additionally, in mid dose males. Urobilinogen is synthesized in the intestine by bacteria and absorbed by the gut into the blood. With the blood circulation it normally enters the liver and is re-excreted via bile into the intestine. When a higher amount of urobilinogen is produced because of a greater breakdown of hemoglobin during hemolytic anemia, or a liver damage and cholestasis is preventing the biliary excretion of urobilinogen, higher amounts of the molecule are excreted via the kidneys.

However, in this study there was neither any indication of an anemia nor any sign of a liver cell damage or cholestasis. It is highly probable that the indicator field of the urobilinogen test strip is falsely affected by renal excreted compound or its metabolites coloring the urine light pastel orange. Therefore, because it was an isolated finding not accompanied by any red blood cell or liver cell alteration this change was regarded as treatment-related but not adverse (ECETOC Technical Report No 85, 2002).

(Tables IB 11 – IB 14)
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean absolute and relative liver and kidney weights (in females only the relative kidney weight was increased) were significantly increased in high dose animals above current and historical control data (app. 140% for liver, 120% for kidney)

The liver and kidneys weight increases in males and the liver weight increase in females of test group 3 (500 mg/kg bw/d) were regarded as treatment-related. The liver weight increase in males (7.698 g) and females (4.864 g) of test group 2 (150 mg/kg bw/d) was within the range of the historical control data (males: 6.486-8.468 g; females: 4.194-5.426 g; PART III, Supplement). Therefore, the latter change was not considered to be treatment-related.

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

The liver weight increases in males and females of test group 3 (500 mg/kg bw/d) correlated with histopathological findings and were regarded as treatment-related. Although the kidney weight increase of males and females in test group 3 (500 mg/kg bw/d) had no histopathological correlate, it was regarded as treatment-related but not adverse. The liver weight increase in males (2.893%) and females (2.868%) of test group 2 (150 mg/kg bw/d) showed no histopathological correlate and were within the range of the historical controldata (males: 2.505-3.223%; females: 2.472-3.208%) and, therefore, were not considered to be treatment-related. All other mean relative weight parameters showed no significant differences when compared to the control group 0.

(Tables IC 1 – IC 8)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In test group 3 (500 mg/kg bw/d), 2 out of 5 males and 3 out of 5 females showed dark discoloration of the liver. However, no histopathological correlate was observed for this macroscopic finding. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

(Tables IC 9)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Correlating to the increased liver weights, minimal to slight centrilobular liver hypertrophy was observed in males (grade 2) and females (grade 1) of the high dose group. This finding was considered treatment related, but adaptive and not adverse. No histopathological correlate was found for the absolute and/or relative kidneys weight increase in males and females of this test group. However, this change was not considered to be adverse and most likely reflects an adaptive effect.

(Tables IC 10 – IC 13)
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Length and number of estrous cycle was unaffected.

(Table IA-45)

Sperm motility, sperm head counts, and incidence of abnormal sperms were unaffected by treatment.

(Tables IB 15)
Details on results:
Lucirin TPO-L was administered orally by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg bw/d (test group 0), 50 mg/kg bw/d (test group 1), 150 mg/kg bw/d (test group 2) and 500 mg/kg bw/d (test group 3) over a period of 4 weeks.

Clinical examinations did not reveal treatment-related, adverse effects up to a dose level of 500 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of estrous cycles were obtained.
Salivation after treatment was seen in all animals of test group 3 (500 mg/kg bw/d) and sporadically in a few female animals of test group 2 (150 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 500 mg/kg bw/d.

Regarding pathology, the absolute and/or relative weight increases observed in the liver and the kidneys of male and female animals of test group 3 (500 mg/kg bw/d) were regarded as treatment-related. In the liver, the weight increase correlated with centrilobular hepatocellular hypertrophy, affecting 5 out of 5 males (slight) and 4 out of 5 females (minimal). As the hepatocellular hypertrophy occurred without histopathological or clinical pathology alterations indicative of liver toxicity, the liver findings were considered to be treatment-related but adaptive and were therefore regarded as non-adverse (Hall et al., 2012). No histopathological correlate was found for the absolute and/or relative kidneys weight increase in males and females of test group 3 (500 mg/kg bw/d). However, this change was not considered to be adverse and most likely reflects an adaptive effect.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance by gavage over a period of 4 weeks did not reveal signs of systemic toxicity in male and female Wistar rats up to a dose level of 500 mg/kg bw/d. In addition, no changes with regard to the reproductive organs were observed. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 500 mg/kg bw/d for male and female Wistar rats.
Executive summary:

METHODS

Lucirin TPO-L was administered orally by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 50 mg/kg bw/d (test group 1), 150 mg/kg bw/d (test group 2) and 500 mg/kg bw/d (test group 3) over a period of 4 weeks.

 

OBSERVATIONS

Food consumption and body weight were determined weekly. The animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out towards the end of the administration period.

Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations. In addition to the required examinations, special attention was given to the reproductive organs of male and female animals, i.e. for at least 3 weeks an estrous cycle determination was performed and sperm parameters were examined after necropsy.

 

RESULTS

Analytics

The various analyses confirmed the stability of the test-substance preparations for a period of 96 hours at room temperature the homogeneous distribution of the test article in the vehicle, the correctness of the prepared concentrations.

 

Findings

The following test substance-related, relevant findings were noted:

 

Test group 3: 500 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology4

No treatment-related, adverse effects were observed.

 

Test group 2: 150 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology

No treatment-related, adverse effects were observed.

 

Test group 1: 50 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology

No treatment-related, adverse effects were observed.

 

CONCLUSION

The oral administration of Lucirin TPO-L by gavage over a period of 4 weeks did not reveal signs of systemic toxicity in male and female Wistar rats up to a dose level of 500 mg/kg bw/d. In addition, no changes with regard to the reproductive organs were observed. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 500 mg/kg bw/d for male and female Wistar rats.