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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
based on EPA-TSCA Guideline "Functional Observational Battery" & EPA-TSCA Guideline " Neuropathology" (Federal Register Vol. 50, No. 188)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lucirin LR 8728
- Purity test date: 94.8%
- Lot/batch No.: 49/0193
- Storage condition of test material: protect from light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr Karl Thomae GmbH, Biberach/Riss, FRG
- Age at study initiation: app. 5 weeks
- Weight at study initiation: 191 (177 - 210) g for males, 156 (143 - 217) g for females
- Housing: single in DK III stainless steel wire cages. The animal cages were placed on the racks in a manner such that uniform test conditions (air inflow/air outflow/light) were guaranteed
- Diet: ground Kliba rat/mouse/hamster maintenance laboratory diet, GLP 343 meal, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; ad libitum
- Water: drinking water; ad libitum
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparations were made prior to each administration.

VEHICLE
- Concentration in vehicle:
_________________________________
Dose Dose Concentration
group (mg/kg bw) (g/100 ml)
---------------------------------
0 0 -
1 100 1.0
3 300 3.0
4 1000 10
_______________________________
- Amount of vehicle (if gavage): 10 ml/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance in the vehicle was verified for a period of 24h.
The concentrations from the beginning and the end of the study were verified via UV spectroscopy at 379 nm and found to be within 10% of the target concentration.
Duration of treatment / exposure:
3 months (total of 64 applications)
Frequency of treatment:
once daily on workdays (5days/week, excepted for 3 working days over the whole experimental period)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the results of a test study where a dose of 1000 mg/kg bw/day was applied 10 times in 14 days and did not lead to marked toxicity besides a minimal reduction in body weight (6 %) in male but not in female animals if compared to controls.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for any evident signs of toxicity / mortality; once weekly for exact clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, once a week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning on days 33 and 87 after the beginning of administration
- Animals fasted: No
- How many animals: all
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets and thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above, HAEMATOLOGY
- Parameters examined: 1) enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase: 2) blood chemistry (sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once prior to the start of the study (Oct 16 1987), at 1, 6, and 24 hours after the first administration and before the daily exposure on days 7 (Oct 26 1987) and 14 (Nov 2 1987) and then monthly to day 91 (Nov 30 1987; Dec 21 1987 and Jan 18 1988)
- Dose groups that were examined: all animals
- Battery of functions tested:
- observation of present/absent:
tremors, convulsions, lacrimation/secretion of pigmented tears, salivation, piloerection, pupil size (constriction/dilatation), diarrhea, vocalization, paresis, paralysis and staxia
- Appearance of impairments refering to:
general appearance, posture, skin colour, locomotor activity, respiration, urination (volume, odour, colour), pupillary reflex, winking reflex, righting reflex, behaviour, grip strength, body tone, vision, olfaction, audition, sensitivity of the body surface, pain perception (hot plate), tail Pinch, toe Pinch, visual placing response.
Sacrifice and pathology:
1) Perfusion fixation
Organs of the first three animals per group and sex were fixed by perfusion using 2.5% glutaraldehyde and 4% formaldehyde.

GROSS PATHOLOGY: Yes: brain, gasserian ganglia of both sides, dorsal root ganglia (C3-C6 and L1-L4 ), proximal sciatic nerve (mid thigh and sciatic notch ), gastrocnemius muscle, kidneys, adrenal glands, skin, medulla oblongata, spinal cord at cervical ( C3-C6) and lumbar swelling (L1-L4 ), dorsal and ventral root fibers (C3-C6 and L1-L4 ), sural and tibial nerve (knee region), liver, spleen, testes, all gross lesions

HISTOPATHOLOGY: Yes
all groups: gastrocnemius muscle, skin ( auricle), skin ( paw region), liver, kidneys, testes, spleen, all gross lesions (if observed), gasserian ganglia, dorsal root ganglia, dorsal and ventral root fibres, proximal sciatic nerve, sural and tibial nerve
high dose and control: brain (forebrain, cerebrum, midbrain, cerebellum, pons), medulla oblongata, spinal cord at C3-C6 and at L1-L4


2) Immersion fixation
Organs of the remaining 7 animals per group and sex were necropsied and assessed by gross pathology prior to fixation in 4% formaldehyde solution.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
all groups: liver, kidney, spleen, adrenals, testes, left hind extermity with all skeletal muscles and sciativ nerve roots, all gross lesions
high dose and control: gasserian ganglia of both sides, spinal cord at cervical and lumbar swelling (C3-C6 and L1-L4), dorsal root ganglia (C3-C6 and L1-L4 ), dorsal and ventral root fibers (C3-C6 and L1-L4 ), proximal sciatic nerve (mid thigh and sciatic notch), sural and tibial nerve (knee region), brain, medulla oblongata,
Statistics:
The statistical assessement of the findings was based on the analysis of variance (ANOVA) followed by a Dunnett´s test (Dunnett CW, J. Amer. Statist. Assoc., 50, 1096-1121, 1955,´Dunnett CW, Biometrics, 20, 482-491, 1964.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The females of the 1000 mg/kg group showed a reduced general state of health. Lesions on the hairless skin of the extremities and reddening and scale formation on the ears were reported for males and females of the 1000 mg/kg group. In males of both, the 300 and the 1000 mg/kg group, the testes were reduced in size, which was palpable from week 6 onwards.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females of the 1000 mg/kg group died during the experimental period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For males and females of the 1000 mg/kg group body weight was reduced (23% in males, 8% in females). At 300 mg/kg, body weight reduction only concerned the males (10%). Body weight gain was also reduced by 38% (high dose males), 16% (mid dose males), and 16% (high dose females).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
An increase in food consumption was reported for the females of the 1000 mg/kg.

Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the females of the 1000 mg/kg group, erythrocytes, hemoglobin, hematocrit and thromboplastin time (Hepato Quick test) were decreased. Hemoglobin and hematocrit values were also found to be reduced in females exposed to 300 mg/kg. In contrast, leucocytes, platelets, eosinophilic granulocytes, neutrophilic polymorphonuclears, and calcium levels were increased in mid and high dose females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum alkaline phosphatase and gamma-glutamyltransferase activity were significantly increased in both sexes of the high dose group (1000mg/kg). In addition, alanine aminotransferase activity was significantly increased in high dose males. Triglyceride levels were decreased in both sexes of the high dose group, while cholestrol levels were increased and clotting time was decreased in females only. These findings are attributed to liver dysfunction.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Necropsy revealed increased absolute and relative kidney and liver weights in the females and increased relative kidney and liver weight in males of the 1000 mg/kg group and females of the 300mg/kg group. In all males in the mid and high dose group, the absolute and relative testes weights were decreased, on average by about 50%.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Neither functional defects nor any other signs of neurotoxicity were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Despite altered blood and urine parameters and increased liver and kidney weight, histopathology revealed no damage on these organs. In the testes of mid and high dose males marked diffuse atrophy of the testicular parenchyma and a slight to moderate interstitial oedema was detected. No further substance related effects were found. Especially, no damage was detected in any samples taken from nervous tissues.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body ans testes weight development; results of testes patology:

Dose group/finding

0

mg/kg bw/day

100

mg/kg bw/day

300

mg/kg bw/day

1000

mg/kg bw/day

Body weight

day 0

190.4 ± 7.9

190.1 ± 7.4

190.9 ± 5.1

192.4 ± 5.5

day 91

498.8 ± 44

493.5 ± 29.8

451.3 ± 37

384.0 ± 27.6

Testes

10

10

10

10

absolute weights

3.563 ± 0.193 g

3.68 ± 0.353 g

1.691 ± 0.328 g

1.693 ± 0.369 g

relative weights

0.78 ± 0.062 g

0.818 ± 0.094 g

0.421 ± 0.1 g

0.477 ± 0.1 g

diffuse atrophy

--

--

10/10

10/10

edema

--

--

10/10

10/10

focal atrophy

2/10

1/10

--

--

vacuolar degeneration

7/10

10/10

--

--

reduced spermiogenesis

--

1/10

--

--

Incidence and grading of microscopic findings:

Dose group

Grading

0

mg/kg bw/day

100

mg/kg bw/day

300

mg/kg bw/day

1000

mg/kg bw/day

Testes

10

10

10

10

diffuse atrophy

--

--

10

10

1 (minimal)

--

--

--

--

2 (slight)

--

--

--

--

3 (moderate)

--

--

1

--

4 (marked)

--

--

9

10

5 (severe)

--

--

--

--

edema

--

--

10

10

1 (minimal)

--

--

--

--

2 (slight)

--

--

3

7

3 (moderate)

--

--

7

3

4 (marked)

--

--

--

--

5 (severe)

--

--

--

--

focal atrophy

2

1

--

--

1 (minimal)

--

--

--

--

2 (slight)

--

--

--

--

3 (moderate)

1

--

--

--

4 (marked)

1

1

--

--

5 (severe)

--

--

--

--

vacuolar degeneration

7

10

--

--

1 (minimal)

3

3

--

--

2 (slight)

1

6

--

--

3 (moderate)

3

1

--

--

4 (marked)

--

--

--

--

5 (severe)

--

--

--

--

reduced spermiogenesis

--

--

1

--

--

1 (minimal)

--

--

--

--

2 (slight)

--

--

--

--

3 (moderate)

--

1

--

--

4 (marked)

--

--

--

--

5 (severe)

--

--

--

--

Applicant's summary and conclusion