Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 625-Triketone
- Substance No.: 95/225-1
- Physical state: Powder, beige
- Analytical purity: 99.4 %
- Lot/batch No.: LJ 27881/1
- Date of manufacturing: 18-27 Jul 1995
- Stability under test conditions: verified by reanalysis
- Storage condition of test material: refrigerator at 4° C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach, FRG
- Strain/Quality: WISTAR / CHBB: THOM (SPF)
- Age at study initiation: young adult animals, 42 days old
- Weight at study initiation: males: 177 - 195 g (group mean: 183 g); females: 132 - 155 g (group mean: 144 g)
- Fasting period before study: none
- Housing: single housing
- Diet: KLIBA-Labordiaet 343, Klingenthalmuehle AG Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 1 week


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

Analysis of drinking water
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the technical services of the BASF AG as well as for the presence of germs by a contract laboratory.

Analysis of feed:
The feed used in the study was assayed for chemical and microbial contaminates.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare the suspensions, the test substance was weighed in depending on the dose group. Then olive oil was filled up to the desired volume. The suspension were mixed with a Ultra-Turrax. During the administration to the animals the test substance preparations were kept homogeneous with a magnetic stirrer. The test substance preparations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test substance is hardly soluble in water and water based solvents
- Concentration in vehicle: dose dependent
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance and its preparations were carried out at the Analytical Department of BASF AG.
Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentration drawn from bottom, middle and top at the start of the administration period. These samples also served for concentration control analyses. Additional concentration control analyses were performed with samples from the mid concentrations drawn at the start of the administration period.
The stability of the test substance in the vehicle was tested over a period of 4 hours at room temperature.

Concentration control analyses
The concentration control analyses at the high and mid dose level confirmed the correctness of the concentrations (recovery rates of 92 – 100 %). At the low concentration, the analytically found values were only 67 – 73 % of the target concentration. The analyses of the reserve samples also revealed only 70 % of the target concentration. This low recovery was therefore taken into consideration for the calculation of the actual dose level, which was reduced from 15 to 10.5 mg/kg body weight (i.e. 70 %).

Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10.5, 60, 180 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
control group: 5 males and 5 females, additional 5 males and 5 females for a 2-week recovery period
10.5 mg/kg dose group: 5 males and 5 females
60 mg/kg dose group: 5 males and 5 females
180 mg/kg dose group: 5 males and 5 females, additional 5 males and 5 females for a 2-week recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
SELECTION OF DOSES
The LD50 (oral) in rats is >200 and <2,000 mg/kg body weight [BASF 1994]. The repeated administration by gavage (as suspension in olive oil) caused lethalities at dose levels > 250 mg/kg body weight in a range finding study. Macroscopically, ulcerations in the forestomach were observed. 5 repeated administrations of 150 mg/kg body weight did not cause any lethalities or abnormal clinical or macroscopic findings.

Based upon the above mentioned findings, following nominal dose levels were selected for the present study:
180 mg/kg bw as high dose with expected toxicological effects
60 mg/kg bw as mid dose
15 mg/kg bw as low dose

RATIONAL FOR SELECTING SATELLITE GROUPS
Animals of the highest dose and control group underwent a recovery period of two weeks in order to prove reversibility of possible effects.

Examinations

Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS
Clinical observations
The animals were examined for evident signs of toxicity or mortality twice a day from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. In the recovery period the animals were subjected to a comprehensive clinical examination once each working day.

Food consumption
Food consumption was determined weekly over a period of 7 days.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the study, the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals.

Food efficiency
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption:

((BWx – BWy) / (FCy to x) x100 = Food efficiency for day x

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]

CLINICAL PATHOLOGY

Blood was taken from the retroorbital venous plexus in the morning from non-fasted, unanesthetized animals.
The following examinations were carried out in all animals at the end of the administration period and in 5 animals of the control and highest dose group at the end of the recovery period.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter:
leukocytes, erythrocytes , hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count
Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.

Clinical chemistry
An automatic analyzer was used to examine the clinicochemical parameters. Chloride was measured with a chloride analyzer. The following parameters were determined:
alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

Urinalysis
For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight.
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips and a reflection photometer. The specific gravity was determined using an urine refractometer. The sediment was evaluated microscopically. The following examinations were carried out:
volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment
Sacrifice and pathology:
PATHOLOGY

Necropsy
The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals which died intercurrently were necropsied as soon as possible after death and assessed by gross pathology.

Organ weights
The following weight parameters of all animals sacrificed at scheduled study termination were determined:
anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, ovaries, brain, thymus, heart, spleen

Histopathology
The following organs or tissues were fixed in 4 % formaldehyde solution:
all gross lesions, brain, pituitary gland, thyroid glands and parathyroid glands, thymus, trachea, lungs, heart, liver, spleen, kidneys, adrenal glands, testes/ovaries, uterus/vagina, epididymides, prostate, seminal vesicle, stomach (glandular and non-glandular), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mandibular and mesenteric), sciatic nerve, bone marrow (femur), eyes, spinal cord (cervical, thoracic and lumbar)
After the organs were fixed, histotechnical processing and examination by light microscopy were performed.
Statistics:
Food consumption, body weight, body weight change, food efficacy:
non-parametric one-way analysis using the Kruskal-Wallis Test (two sided), pairwise comparison of each dose group with the control group was performed using Mann-Whitney U-test (two-sided) for equal means.

Clinical pathology parameters, except some parameters of the differential blood count:
Comparison of the dose groups with the control group using Mann-Whitney U-test (two-sided); non-parametric one-way analysis using the Kruskal-Wallis test ( two-sided); pairwise comparison of each dose group with the control group using the Mann-Whitney U-test (two-sided)

Urinalysis, except volume, color and turbidity:
Pairwise comparison of each dose group with the control group using Fisher's exact test

Weight parameters:
Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided); pairwise comparison of each dose group with the control group using the Wilcoxon test

Results and discussion

Results of examinations

Details on results:
CLINICAL EXAMINATIONS
Mortality
no test substance-related mortalities

Clinical observations
No substance-related effects were observed.

Food consumption
No substance-related effects were observed. All values were within the biological range of variation.

Body weight data
180 mg/kg bw: Impaired body weight and body weight changes in males
60 mg/kg bw: Impaired body weight and body weight changes in males
The effects on body weight in males of test group 2 and 3 were assessed as being substance-related. During the treatment-free recovery
period of test group 3, a slight increase in body weight was observed.

Food efficiency
180 ng/kg bw: Food efficiency was statistically significantly impaired in males of test group 3 on single days.
60 mg/kg bw: Food efficiency was statistically significantly impaired in males of test group 2 on single days.
This was assessed as being substance-related.

CLINICAL PATHOLOGY
Hematology
No treatment-related effects were reported.

Clinical chemistry
180 mg/kg bw: slight increase in alkaline phosphatase (males), increased total bilirubin concentrations (males and females), marginally increased urea (males), slightly prolonged prothrombin times (males). All findings were normalized during the recovery period.
60 mg/kg bw: increase total bilirubin levels (males and females)

Urinalyses
180 mg/kg bw: increased amount of renal epithelial cells (males) After withdrawal of the test compound no differences were detected between control and high dose males.

PATHOLOGY

Weight parameters
Absolute weights
all dose groups: significant increased weight of epididymides (-7.5 % to 22.6 %)

Absolute organ weights
180 mg/kg bw: reduced testis weight in the R1 recovery group

Relative weights (related to terminal body weight)
180 mg/kg bw: The relative weights of epididymides were decreased (- 13.9 %).

Gross lesions
No test-substance related gross lesions were recorded.

Histopathology
Animals of the main groups (F1)
180 mg/kg bw: decreased number of sperms and an increase of debris (i.e. sloughed sperm cell and residual bodies), degenerating (vacuolated) Sertoli cells (males)
60 mg/kg bw: increase of debris (i.e. sloughed sperm cell and residual bodies) in the epididymides

Animals of the recovery-groups (R1)
180 mg/kg bw: reduced spermatogenesis, decreased number of sperms and an increase of debris (i.e. sloughed sperm cell and residual bodies) in the epididymides, tubular giant cells coinciding with tubular atrophy in the testis



Effect levels

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Dose descriptor:
NOAEL
Effect level:
10.5 mg/kg bw/day (actual dose received)
Sex:
female
Dose descriptor:
NOAEL
Effect level:
< 10.5 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: increased weight of epididymides

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion