Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
long-term toxicity to birds
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
other: Tif: RAIf(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Further description: F3-hybrid of RII 1/Tif x RII 2/Tif
- Source: Ciba-Geigy Ltd. Tierfarm, 4334 Sisseln, Switzerland
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 173-195 (within ± 20% of mean value)
- Fasting period before study: overnight prior to dosing
- Housing: 5 per sex in Macrolon cage type 4 with standardized soft wood bedding (Societe Parisienne des sciures, Pantin).
- Diet: ad libitum; Rat food, NAFAG No. 890, NAFAG AG, Gossau, SG (Switzerland),
- Water: ad libitum
- Acclimation period: 6 days
- Rationale for choice: The rat has been selected for this test as being a standard species for the determination of an acute oral LD50

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: distilled water containing 0.5% carboxymethylcellulose and 0.1% polysorbate
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg bw
Doses:
5000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: mortality; daily; a.m. and p.m. on working days, a.m. on weekend days, clinical signs of toxicity; daily
- Frequency of weighing: on days 1, 7, and 14
- Necropsy of survivors performed: yes
Statistics:
From the body weights, the group means and their standard deviations were calculated.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Remarks on result:
other: No deaths occurred
Mortality:
No deaths occurred
Clinical signs:
other: Dyspnea, exophthalmos, ruffled fur, and curved body position were seen, beginning as early as 1 hour post dosing and lasting up to 10 day. The animals were fully recovered within 11 days.
Gross pathology:
No deviations from normal morphology were found.
Interpretation of results:
GHS criteria not met
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Centre Elevage Charles River (76410 Saint-Aubin-les-Elbeuf, France)
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: mean body weight was 198 g for the males and 151 g for the females.
- Housing: in suspended wire-mesh cages and each cage contained 2 rats from the same sex and the same group.
- Diet: free access to A04 C pelleted diet (U.A.R., 91360 Villemoisson-sur-Orge, France) distributed weekly.
- Water: free access to bottles containing filtered tap water.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 13 cycles/hour of filtered, not recycled air
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light
Route of administration:
oral: gavage
Vehicle:
other: the vehicle was a solution of water for injectable preparations with 0.5% carboxymethylcellulose and 0.1% Tween 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the test substance was diluted in the vehicle and homogenized by a magnetic stirrer.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
at least 92 days and at most 93 days
Frequency of treatment:
once a day, approximately at the same daily time (in the morning), 7 days per week
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 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:
0 and 1000 mg/kg bw: 20
10 and 100 mg/kg bw: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on the results of a previously conducted study: the test item was administered daily by gavage for 28 days at doses of 0, 10, 50, 250 and 1000 mg/kg/day.
- Post-exposure recovery period in satellite groups: 28 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during the study, clinical signs were recorded for each animal, at least once a day, at approximately the same daily time. All animals were checked at least twice a day for possible mortality.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: the bodyweight of each animal was recorded before the time of allocation of animals into groups, on the first day of treatment and then once a week until the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by the animals of each cage was recorded once a week for a period of 7 days until the end of the study.

FOOD EFFICIENCY:
- Efficiency of food utilization was calculated once a week for each sex and each group using bodyweight and food consumption means until the end of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of treatment and in week 13.
- Dose groups that were examined: all surviving males and females from the control and high dose level groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 13
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: No data
- How many animals: all surviving animals
- Parameters examined: Erythrocytes (RBC), Haemoglobin (HB), Mean Cell Volume (MCV), Packed Cell Volume (PCV), Mean Cell Haemoglobin Concentration (MCHC), Mean Cell Haemoglobin (MCH), Leucocytes (WBC), Thrombocytes (PLAT), Differential White Cell Count: neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M), Quick time (QT), Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13 and at the end of the recovery period (week 17)
- Animals fasted: No data
- How many animals: all surviving animals
- Parameters examined: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic Phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL), Total proteins (PROT), Cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT).

URINALYSIS: Yes
- Time schedule for collection of urine: in week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 18 h
- Parameters examined: volume (VOLUME), appearance (APP), pH (PH), specific gravity (SP.GRAV), urobilinogen (UROB), proteins (PROT), glucose (GLUC), ketones (CETO), bilirubin (BILI), blood (BLOOD), nitrites (NITR). Microscopy of deposit after centrifugation: investigation for leucocytes (WBC), erythrocytes (RBC), hyaline (HYAL) and granular (GRAN) cylinders, magnesium ammonium phosphate (MAM.PH.), calcium phosphate (CAL.PH) and calcium oxalate (CAL.OX) crystals, epithelial (EPITH.), bladder (BLAD) and kidney (KIDN) cells.

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights: For all animals, the following organs were weighed wet as soon as possible after dissection: brain, spleen, adrenals, testes, liver, kidneys, ovaries. The animals were weighed before necropsy. Paired organs were weighed separately.

Macroscopic examination: Aorta, caecum, brain including medulla/pons, cerebellar and cerebral cortex, heart, colon, duodenum, stomach, femoral bone with articulation (*), liver, mammary glands (*), submaxillary glands (*), sublingual glands (*), Harderian glands (*), pituitary, ileum, jejunum, spinal cord, cervical, thoracic and lumbar sections (*), sciatic nerve, skeletal muscle (*), lymph nodes, mandibular and mesenteric esophagus, ovaries, pancreas, skin (*), lungs with bronchia, prostate (*), spleen, rectum, kidneys, sternum with bone marrow, adrenals, testes and epididymides, thymus, thyroids and parathyroids, trachea, uterus, horns, and cervix, vagina (*), seminal vesicles (*), urinary bladder, eyes (*) with optic nerve and lacrymal glands.

HISTOPATHOLOGY: Yes
Microscopic examinations was performed on: all macroscopic lesions and tissues listed above (except those marked by (*)) in all animals from the high dose level and control groups sacrificed at the end of the treatment and recovery periods, in all animals that died. All macroscopic lesions and lungs, liver, kidneys for all animals from the low and intermediate dose level groups.
Statistics:
The following sequence was used for the statistical tests of the clinical parameters (bodyweight, food consumption and water consumption) and for the haematological and biochemical parameters, and for organ weights: A normal distribution of values in the samples was checked by Kolmogorov-Smirnov test (1948), In the case of an abnormal distribution, this test was performed after the logarithmic transformation of the values.
If a significant heterogeneity persists after the logarithmic transformation of the values, the analysis of variances was not performed, A comparison between the treated groups and the control group in order to prove a treatment-related difference was made by using Mann-Whitney's test (1947).

In the case of the normal distribution of values according to the normal law an analysis of variances was made by the Bartlett's test (1937) (more than 2 samples) or Fisher's test (1934) (2 samples),

A comparison between the treated and the control groups in order to prove a treatment-related difference was made by either: Dunnett's test (1955) if no significant heterogeneity of the variances was established, Mann-Whitney's test (1947) in the case of significant heterogeneity of the variance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hypersalivation after treatment was observed in 6/20 males and 6/20 females of the 1000 mg/kg/day group (observed daily from day 17 to day 22, except in one female, from day 31 to day 43). This clinical sign was considered treatment-related.
Additional clinical signs were noted, including:
- Area of hair loss on the head, neck or back of some animals of all groups, and swollen ear in 1 male of the control group, probably due the prehension of animals at test substance administration.
- Damaged eye in 1 male of the control group, due to a traumatism at blood sampling.
- Chromorhinorrhea (from day 7I to day 119) in 1 male of the 1000 mg/kg/day group, considered to be spontaneous without any treatment relationship,
- A palpable mass on 1 female of the 100 mg/kg/day group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male animal died in the control group on day 119, one male (after 11 days) and one female (after 36 days) died in the 100 mg/kg bw dose group. However, no clinical signs were observed preceding the death, and, at necropsy, no relevant macroscopically findings were found. The observed mortality either occurred accidentally during blood sampling or as consequence of test substance aspiration.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The bodyweight gain in all treated females and in males of the 10 and 100 mg/kg/day groups was similar to that of the control animals. A very slight increase in the bodyweight gain was observed in males of the 1000 mg/kg/day group, when compared with the control animals. The difference in bodyweight was in mean 6% and was not statistically significant. It was still noted at the end of the recovery period. Although this change was very slight, the relationship to the treatment cannot be ruled out.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption in all treated females and in males of the 10 and 100 mg/kg/day groups was comparable to that of the control animals. In males of the 1000 mg/kg/day group, it was very slightly increased throughout the treatment period. This change was in mean 3% and was occasionally statistically significant. This could be associated with the very slight increase in bodyweight gain noted in the same animals.
Throughout the recovery period, the food consumption was normal and similar to that of the control animals except in Week 17 when the mean value of food consumption in controls was lower than that recorded in the same animals on the previous weeks.
Food efficiency:
no effects observed
Description (incidence and severity):
No change of toxicological significance was observed.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The ophthalmological examination performed on animals of each sex from the control and high dose level groups revealed only a few minor changes often observed in the laboratory rat (cornea vacuolisation, persistence of the hyaloid vessel, diffuse opacity of the cornea and punctual opacity of the posterior cortical). Therefore, these changes were not considered to be treatment-related.
Haematological findings:
no effects observed
Description (incidence and severity):
No perceptible changes were found in the haematological parameters and the individual values were within the normal range of the background data for the animal species used.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The variations observed in some biochemical parameters (sodium, potassium, chloride, inorganic phosphorus, creatine, total protein, cholesterol, triglyceride, transaminases and proteinogram) were considered to be of no toxicological significance, although they were sometimes statistically significant (they were minor, not dose-related and the individual values still within the normal range of the background data of the testing laboratory).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related abnormalities were found.
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):
The statistical analysis of organ weights of the animals sacrificed at the end of the treatment period revealed the following changes:
- An increase in absolute {22%) and relative (14%) liver weight in the males at the high dose level (1000 mg/kg/day);
- An increase in relative liver weight in the females at the mid-dose (8%) and high dose (9%) levels.
The following statistical significant variations were found in the organ weights of the animals sacrificed at the end of the recovery period:
- An increase in absolute {22%) and relative (12%) liver weight in the test substance treated males;
- A decrease (11%) in relative testes weight in the test substance treated males,
No perceptible changes were found in the other organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic changes found at autopsy in the animals, at the end of the treatment and recovery periods, were recognized as those commonly recorded findings in the laboratory rat, therefore they were considered to be of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The microscopic findings encountered in all organs examined, at the end of the treatment and the recovery periods, were similar as those commonly recorded changes in the laboratory rat. Moreover, their incidence, severity and morphological characters were comparable between control and treated animals; therefore they bear no relationship to treatment. In the absence of any relevant histopathological findings in the liver and testes, the slight above mentioned variations in those organs weight were considered to be of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at this dose level
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At the high dose level, hypersalivation in some animals was recorded and a very slight increase in the bodyweight gain related to an increase of the same intensity in the food consumption in the males was reported.
Critical effects observed:
not specified
Conclusions:
In conclusion, the administration of the test substance by gavage to the rat for a 90-day period, at the dose levels of 10, 100 and 1000 mg/kg/day induced hypersalivation in some animals of the high dose level. A very slight increase in the bodyweight gain related to an increase of the same intensity in the food consumption was also noted in the males of the 1000 mg/kg/day dose level. Throughout the recovery period, the reversibility of the increase in food consumption was assessed but the difference in bodyweight was still noted. No other change with a toxicological significance was observed. Thus, the high dose level of 1000 mg/kg/day was well-tolerated.
Executive summary:

In a 90-day subchronic repeated dose toxicity study followed by a 28-day recovery period, three groups of Sprague-Dawley rats received the test substance by gavage at the 10, 100 and 1000 mg/kg/day dose levels. Each group contained 10 males and 10 females except the 1000 mg/kg/day group which contained 20 males and 20 females. An additional group of 20 males and 20 females received the vehicle only

and acted as control group. After a 91-day treatment period, the first 10 males and females of the control and high dose level groups were kept for a 28-day recovery period. The other animals were sacrificed after a 92 or 93-day treatment period according to the date of necropsy. Hypersalivation after treatment was observed in 6/20 males and 6/20 females of the 1000 mg/kg/day group. This clinical sign was considered treatment-related. The mortality was 1/20 males in the control group and 1/10 males and 1/10 females in the 100 mg/kg/day group. In the absence of dose-relationship, the mortality rate was not considered to be of toxicological significance. The bodyweight gain in all treated females and in males of the 10 and 100 mg/kg/day groups was similar to that of the control animals. A very slight increase in the bodyweight gain related to an increase of the same intensity in the food consumption was noted in the males of the 1000 mg/ kg/day group. Throughout the recovery period, the reversibility of the increase in food consumption was assessed but the difference in bodyweight was still observed. Although these changes were very slight, the relationship to the treatment cannot be ruled out. No drug-related abnormality was detected in ophthalmological examinations. No change of toxicological significance was observed in the haematological and blood biochemical parameters. Urinalysis did not reveal treatmentrelated abnormalities. A slight increase in the net and/or relative liver weight was found in the females of the 100 mg/kg/day group and in the animals of the 1000 mg/kg/day group. But in the absence of abnormalities at the macroscopic and microscopic examinations, this change was considered to be of no toxicological significance. No changes of a toxicological significance were observed at the macroscopic and microscopic examinations. In conclusion, the administration of the test substance by gavage to the rat for a 90-day period the high dose level of 1000 mg/kg/day was well-tolerated and can be regarded as a NOAEL.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Marshall Farms (North Rose, New York 14516, U.S.A.)
- Age at study initiation: 5 months
- Weight at study initiation: 7.8+/-0.5 kg for the males and to 7.4+/-0.7 kg for the females
- Housing: in individual stainless steel cages (0.75 x 1.00 x 0.82 m)
- Diet (ca. 300 g/d, 2 hrs after treatment): standard dry diet A SQC (Special Diets Services Ltd, Witham, Essex, U.K.)
- Water (ad libitum): tap water
- Acclimation period: one month

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19+/-3
- Humidity (%): 50+/-30
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 0.3% carboxymethylcellulose and 0.1% Tween 80 in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was diluted in the vehicle and homogenized by a
magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): the preparations were performed for 3, 4 or 5 days of treatment in accordance with the stability.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Infrared spectrophotometry
Duration of treatment / exposure:
91-93 d
Frequency of treatment:
daily
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 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:
control and high dose treatment: 6
low and mid dose treatment: 4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the doses were based on the results of a range-finding toxicity study where no systemic adverse effect was observed at 2000 mg/kg/day during 14 days. The dose of 1000 mg/kg/day corresponded with the requirement of the OECD Guideline 409. The doses of 10 and 100 mg/kg/day were the same as those used in a 3-month rodent study.
- Post-exposure recovery period in satellite groups: 4 weeks, only control and high dose treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/ DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all clinical signs were recorded once daily for each animal at approximately the same time. All animals were checked each day at approximately 8.00 a.m. and 4.00 p.m. for ill, moribund and dead animals. During weekends and Public Holidays, the final check was carried out at approximately midday.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each animal was recorded a week before the beginning of treatment, on the first day of treatment, and once weekly during treatment and recovery periods.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the beginning of treatment, and on week 13, 2 to 4 hours following drug administration, with an indirect ophthalmoscope; all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4, 8, 13 and at the end of the recovery period (week 17)
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
Erythrocytes (RBC)
Haemoglobin (HB)
Mean Cell Volume (MCV)
Packed Cell Volvune (PCV)
Mean Cell Haemoglobin Concentration (MCHC)
Mean Cell Haemoglobin (MCH)
Leucocytes (WBC)
Thrombocytes (PLAT)
Differential White Cell Count: neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M)
Quick time (QT)
Activated Partial Thromboplastin Time (APTT)
Fibrinogen (FIBRIN)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 4, 8, 13 and at the end of the recovery period (week 17)
- Animals fasted: No
- How many animals: all
- Parameters checked: Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic Phosphorus (I.PHOS)
Glucose (GLUC)
Urea (UREA)
Creatinine (CREAT)
Total bilirubin (TOT.BIL)
Total proteins (PROT)
Cholesterol (CHOL)
Triglycerides (TRIG)
Alkaline phosphatise (ALP)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Creatine kinase (CK)
Gamma Glutamyltransferase (GGT)
Protein electrophoresis: albumin (ALB), alpha-1 globulins, alpha-2 globulins, beta-globulins, gamma-globulins, albumin/globulins ratio

URINALYSIS: Yes
- Time schedule for collection of urine: before the beginning of treatment, on week 13 and at the end of the recovery period (week 17).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: . volume (VOLUME), appearance (APP), pH (PH), specific gravity (SP.GRAV), urobilinogen (UROB), proteins (PROT), glucose (GLUC), ketones (CETO), bilirubin (BILI), blood (BLOOD), nitrites (NITR) microscopy of deposit after centrifugation: investigation for leucocytes (WBC), erythrocytes (RBC), hyaline (HYAL) and granular (GRAN) cylinders, magnesium ammonium phosphate (MAM.PH.), calcium phosphate (CAL.PH) and calcium oxalate (CAL.OX) crystals, epithelial (EPITH.), bladder (BLAD) and kidney (KIDN) cells.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights
For all animals, the following organs were weighed wet as soon as possible after dissection: adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes, thyroids and parathyroids. The animals were weighed before necropsy. Paired organs were weighed separately.

Macroscopic examination
For all animals, samples of the macroscopic lesions and the following tissues were preserved in 10% buffered formalin, except the eyes and pituitary gland which were fixed in formol-sublimate, for the animals sacrificed at the end of treatment and recovery periods (the tissues marked by (1) were kept in a fixative in case of a possible microscopic examination): aorta, caecum, brain including medulla/pons, cerebellar and cerebral cortex, heart, colon, duodenum, stomach, femoral bone with articulation (1), liver, mammary glands, salivary glands (parotid and mandibular), pituitary gland, ileum, jejunum, spinal cord, cervical, thoracic and lumbar, skeletal muscle, sciatic nerve, lymph nodes, mandibular and mesenteric, esophagus, ovaries, pancreas, skin, lungs, prostate, spleen, rectum, adrenals, sternum (with bone marrow), testes, epididymides, thymus, thyroids with parathyroids, trachea, uterus with horns and cervix, vagina, gall bladder, urinary bladder, eyes, kidneys

HISTOPATHOLOGY: Yes
All the tissues required for the microscopic examination were embedded in paraplast, sectioned at approximately 4 microns in thickness and stained with hemalum-eosin. Microscopic examination was performed on all macroscopic lesions and tissues as listed above for all animals.
Statistics:
The following sequence was used for the statistical tests of the body weight, food consumption, haematological and biochemical parameters and organ weights: The normality of the distribution of the values in each group was checked by Kolmogorov-Smirnov's test (1948). If the distribution is normal, the homogeneity of variances between the groups was assessed by Bartiett's test (1937) (more than 2 groups) or Fisher's test (1934) (2 groups). If no significant heterogeneity of the variances is established the comparison between treated and control groups was performed by Dunnett's test (1955). If the variances are heterogeneous, the comparison between treated and control groups was performed by Dunn's test (1964) (more than 2 groups) or by Mann Whitney's test (1947) (2 groups). If the distribution of values in the groups is not normal, the analysis was repeated after logarithmic transformation of the values, except for the organ weights. If this logarithmic transformation fails to normalise the distribution of the values, comparison of treated and control groups was performed by Dunn's test (1964) using original values.
Clinical signs:
no effects observed
Description (incidence and severity):
Vomiting and liquid feces were sporadically observed in some treated animals. These symptoms were rare and had no relationship with the dose; moreover, they are commonly and spontaneously observed in dogs. Consequently, their absence in controls they were not considered as treatment-related.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Slight decrease in body weight (up to 11%) was observed in one male of the 1000 mg/kg/day group from week 1 to week 5. Thereafter, the body weight gain was normal for this animal. One male and one female of the 1000 mg/kg/day group showed a slight decrease in body weight from week 6 or 7 to week 10 (around 17 and 10%). Thereafter, their body weight was stable until the end of the recovery period. A relationship with the treatment cannot be ruled out. The body weight difference between the control and high dose group males, kept for the recovery period, is explained by a higher body weight of the control males throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was normal and similar in treated and control animals throughout the study.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
A few animals showed a hypopigmentation of the fundus. These variations from normal are commonly observed in dogs (Bellhorn, 1981; Rubin, 1974), and were not related to the treatment since they were, moreover, observed at predose and without change on week 13.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A slight increase in mean total white cell count was observed on weeks 4, 8 and 13 in the animals of both sexes of the 1000 mg/kg/day group. This increase was due to a moderate increase in neutrophils in a few individuals. This phenomenon was more prominent on week 4. Evidence of reversibility was found after the 4-week recovery period. Although this parameter showed a great variability, an increase in neutrophils was noted only in the animals of the high dose group; therefore the relationship to the treatment of this change cannot be ruled out. A slight decrease in Quick time was observed on week 13 in the animals of both sexes of the high dose group. The relationship of this phenomenon to the treatment with the test substance was considered unlikely, since the decrease was minor when compared with the lower limit of our background data (males: 5-9 s, females: 5-7 s).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 100 and 1000 mg/kg/day, a slight to moderate increase in aminotransferases (ASAT and/or ALAT) and/or alkaline phosphatase activities was observed in a few individuals on weeks 4, 8 or 13. These increases were more prominent at 1000 mg/kg/day, especially on week 13 for one female which showed a marked increase in ALP (factor of 3). Evidence of reversibility was found after 4 weeks of recovery. Although, in most cases, these increases had not progressed with time, they were considered to be related to the treatment with the test substance. The slight variations observed in the other biochemical parameters (glucose, cholesterol, triglycerides, alpha-2 globulins, creatine kinase) were considered to be of no toxicological significance, since they were minor and the individual values were within the normal range of our background data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No abnormalities that could be related to the treatment were noted.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, the analysis of organ weights revealed:
- a slight increase in the absolute and relative weights of the liver in the females of the 1000 mg/kg/day (+22%) and a slight decrease in relative liver weight (-24%) in the males of the 10 mg/kg/day. The contradictory variations, with respect to the liver weights as well as the absence of any changes in the liver weights of the males of the high dose group, did not suggest a treatment-relationship.
- a slight to moderate decrease in absolute and relative weights of the testes in the males of all treated groups and the ovaries in the females of the 100 and 1000 mg/kg/day groups. The variations in the testes weight were not dose-related and were without any corresponding histopathological changes. The decrease in the ovarian weights corresponded to the absence of sexual maturation in almost all animals except for 2 control females and one from the 10 mg/kg/day group which reached sexual maturity. Accordingly, thee above-mentioned variations in the weights of the gonads were considered to be of no toxicological significance.
In addition, slight increases were found in the absolute and/or relative weights of the spleen and thyroid gland in the females and slight decreases in relative weights of the spleen and adrenal glands in the males. These variations in organ weights were not considered to be treatment related since they were minor, not dose-related and without any corresponding histopathological changes. At the end of the recovery period, the minor to slight variations found in the organ weights were considered to be of no toxicological significance and there was no corresponding histopathological changes in these organs.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Colored areas or nodules (blackish, brownish, reddish, purplish and/or yellowish) sometimes together with granular consistency were found in the lungs of 1/4 males and 3/4 females of the 1000 mg/kg/day group at the end of the treatment period. Furthermore, similar changes (colored areas and foci) were found in the lung at the end of the recovery period in 1/2 males of the control group and 1/2 males and 1/2 females of the 1000 mg/kg/day group. The other macroscopic findings are commonly recorded in laboratory dogs. Furthermore, they were of similar incidence and morphological characters in control and treated animals. Therefore they were considered to be of no toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A slight to moderate biliary cell proliferation was found in the liver of 3/4 males and 3/4 females of the 100 mg/kg/day group and 3/4 males and 2/4 females of the 1000 mg/kg/day group sacrificed at the end of the treatment period. The severity of this change was not dose-related, however it was considered to be treatment-related effect. No evidence of reversibility was found since the same was found in 1/2 males and 2/2 females of the 1000 mg/kg/day group sacrificed at the end of the recovery period. Some pulmonary changes were found in the lungs of some control and treated animals. These included acute bronchopneumonia, histiocytosis and some other minor findings. These microscopic findings were in line with the macroscopic changes found at the macroscopic examination of the lungs. These pulmonary changes can be found spontaneously in untreated dogs. Furthermore, similar changes were found at the end of the recovery period in 1/2 males of the control group and 1/2 males and 1/2 females of the 1000 mg/kg/day group. Therefore they were not considered to be toxicologically meaningful.
The other microscopic changes are commonly recorded in laboratory dogs. Furthermore, their incidence, severity and morphological characters were comparable in control and treated animals. Therefore they were considered as not toxicologically significant.
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: slight to moderate biliary cell proliferation without evidence of dose-relationship or reversibility were observed at 100 and 1000 mg/kg bw. Slight increase of ALP
Critical effects observed:
not specified
Conclusions:
The administration of the test item by oral route (gavage) to Beagle dogs for a period of 13 weeks at the dose levels of 10, 100 and 1000 mg/kg/day, was clinically well-tolerated. At the two higher dose groups, observed effects were mostly not dose related and so weak that it is difficult to assess whether they are incidental or actually treatment-related. A slight decrease in body weight was noted at 1000 mg/kg/day. A moderate increase in neutrophils at the dose level of 1000 mg/kg/day At 100 and 1000 mg/kg/day, a slight to moderate increase in aminotransferases (ASAT and/or ALAT) and/or alkaline phosphatase activities was observed in a few individuals on weeks 4, 8 or 13. These increases were more prominent at 1000 mg/kg/day, especially on week 13 for one female which showed a marked increase in ALP (factor of 3). Evidence of reversibility was found after 4 weeks of recovery. In most cases, these increases had not progressed with time.
The histopathological examination showed a slight to moderate biliary cell proliferation in the 100 and 1000 mg/kg/day groups, without evidence of of dose-relationship or reversibility. Biliary cell proliferation reverses slowly and full reversibility could not be expected within four weeks. The proliferation is an adaptive rather than an adverse response. From the above data, the liver was considered as the target organ. Based on the liver enzyme activities, effects at 1000 mg/kg bw may be considered adverse, whereas those at 100 m/kg are difficult to judge. A clear no-observed effect level is identified at 10 mg/kg/day.
Executive summary:

In a subchronic toxicity study forty Beagle dogs (20 males and 20 females) were allocated into one control and one high dose group of 6 males and 6 females each, and 2 other treated groups of 4 males and 4 females each. The test substance was administered daily by oral route at the dose levels of 10, 100 and 1000 mg/kg/day in a total volume of 2 ml/kg. Two males and 2 females of the control and high dose groups were kept for a 4-week recovery period after the termination of the treatment. Examinations were performed daily and included observation of clinical signs and mortality, and estimation of food consumption. Body weight was recorded before the beginning of treatment and once a week during the study. Ophthalmological examinations were performed during the predosing period and on week 13. Haematological and blood biochemical examinations were performed during the predosing period on weeks 4, 8 and 13, and at the end of the recovery period. Urinalysis was performed during the predosing period, on week 13, and at the end of the recovery period. At the end of the treatment and recovery periods, the animals were sacrificed. Organ weights were recorded. The organs were subjected to a full macroscopic examination and tissue specimens were examined microscopically. No clinical signs related to the treatment were observed. and no deaths occurred throughout the study. Slight decreases in body weight were observed in 2 males and one female of the 1000 mg/kg/day group, from week 1 to week 5 for one male and from week 6 or 7 to week 10 for the other animals. The food consumption of the animals was normal. The ophthalmological examinations showed no abnormalities related to the treatment. A moderate increase in neutrophils was observed in a few males and females of the 1000 mg/kg/day on weeks 4, 8 and 13. Evidence of reversibility was found after the 4-week recovery period. A slight to moderate increase in aminotransferases and/or alkaline phosphatase activities was observed in a few males and females from the 100 and 1000 mg/kg/day groups on weeks 4, 8 and 13- These changes were dose-related and evidence of reversibility was found after the 4-week recovery period. No abnormalities related to the treatment were observed regarding the oragan weights. Moacroscopically, no abnormalities of toxicological significance were found. Microscopic examination revealed a slight to moderate biliary cell proliferation in 3 males and 3 females of the 100 mg/kg/day group and in 3 males and 2 females of the 1000 mg/kg/day group. This abnormality could probably be related to the increase in aminotransferases and alkaline phosphatase activities. No evidence of reversibility was found since the same abnormality was observed in 1/2 males and 2/2 females of the 1000 mg/kg/day group, sacrificed following the 4-week recovery period.

In conclusion, the administration of the test substance by oral route (gavage) to Beagle dogs for a period of 13 weeks at the dose levels of 10, 100 and 1000 mg/kg/day, was clinically well-tolerated. A slight decrease in body weight was noted at 1000 mg/kg/day. A moderate increase in neutrophils at the dose level of 1000 mg/kg/day, and a slight to moderate increase in aminotransferases and/or alkaline

phosphatase activities at the dose levels of 100 and 1000 mg/kg/day. were observed. Evidence of reversibility was found after the 4-week recovery period. The histopathological examination showed a slight to moderate biliary cell proliferation in the 100 and 1000 mg/kg/day groups, without evidence of reversibility. Consequently, the liver was considered as the target organ. In conclusion, the dose level of 10 mg/kg/day could be considered as the No Observable Effect Level under the conditions of the study.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
The study was set up as a two-generation-study (OECD 416). In the absence of findings, it was discontinued on day 21 of the F1 generation.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: supplied by Harlan Nossan S.r.l., San Pietro al Natisone (UD) Italy
- Age at study initiation: (P) 6-7 weeks and 8 to 9 weeks for males and females, respectively
- Weight at study initiation: the animals used; a total of 110 male and 110 female rats, were within a 25 gram weight range (P)
- Housing: (a) during the pre-mating period in clear polycarbonate cages measuring 59x39x20 cm with a stainless steel mesh lid and floor (Type 4: Techniplast Gazzada S.r.l., Buguggiate, Varese; each cage tray held absorbent material which was inspected daily and changed at least 3 times per week); (b) during the mating period on the basis of one male to one female in clear polycarbonate cages measuring 43x27x15 cm with a stainless steel mesh lid and floor (Type 3: Techniplast Gazzada S.r.l; each cage tray held absorbent material which was inspected and changed daily. The males were re-caged after mating as they were before mating. The mated females were caged in individual, solid bottomed, breeding cages (Type 3: Techniplast Gazzada S.r.l.).
- Diet (ad libitum): commercially available rodent diet (Altromin MT, Altromin-Rieper, Bolzano, Italy)
- Water (ad libitum): potable water
- Acclimation period: at leat 11 days; the male animals arrived on 8 October 1999 and were allocated to groups on 15 October 1999. The females arrived on 3 December 1999 and were allocated to groups on 10 December 1999. The first day of treatment for males was on 21 October 1999. The first day of treatment for females was on 16 December 1999 (the last sacrifice was performed on 3 April 2000).

ENVIRONMENTAL CONDITIONS
The animals were housed in the barriered rodent facility at RTC. Animal room controls were set
- Temperature (°C): 22±3
- Humidity (%): 30±70
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
peanut oil
Remarks:
a weighed amount of the test substance was suspended in the vehicle and brought to the final volume appropriate for each concentration
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test suspensions were prepared daily at room temperature
- Males: dose volumes were calculated according to individual body weights on the first day of treatment and adjusted according to individual body weights at weekly intervals thereafter.
- Females: dose volumes were calculated according to individual body weights at weekly intervals before pairing and on Days 0, 6, 10 and 15 post-coitum. Thereafter individual dose volumes remained constant

VEHICLE
- Concentration in vehicle: 0, 3, 12.5 or 40 mg/ml, respectively for the 0, 15, 50 and 200 mg/kg bw dose groups
- Amount of vehicle (if gavage): 5 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 4 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable. Samples of formulations taken during the first and last week of treatment were also analysed for concentration homogeneity and stability. All analytical results were in the acceptable range.
Duration of treatment / exposure:
- Males: a total of approx. 113 days (for 10 weeks prior to pairing, through the mating period and thereafter until the day prior to sacrifice)
- Females: a total of approx. 56 days (for 2 weeks prior to pairing, during the mating period and through to weaning of the offspring)
Frequency of treatment:
Daily
Details on study schedule:
- Selection of parents from F1 generation: since no signs of toxicity were observed, the Sponsor requested that the study be stopped at the first generation after weaning and selection of the F1 generation.
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: chosen because of the results of 28-day and 90-day toxicity studies, as well as the teratogenicity study performed in rats. In the 28-day oral toxicity study (gavage) the NOEL was 50 mg/kg body weight, with increased alkaline phosphatase and liver weight increase in the 250 and 1000 mg/kg bw dose groups. In the 90-day oral toxicity study in rats (gavage) the NOEL was 10 mg/kg bw, with increased liver weights at 100 and 1000 mg/kg bw. In the rat teratology study 150 mg/kg bw (gavage) represented the overall NOEL, with reduced body weight gain at 300 mg/kg bw of the pregnant dams.
Therefore, since animals were dosed for at least 100 days, 200 mg/kg was considered to yield a maximum tolerated dose, while 15 mg/kg was expected to yield a NOEL with 50 mg/kg to show intermediate toxicity.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS, DETAILED CLINICAL OBSERVATIONS: Yes
- Mortality: all cages were checked for dead or moribund animals twice daily throughout the study, once in the morning and again in the afternoon. A similar procedure was followed at weekends and Public Holidays except that the female check was carried out at approximately mid-day.
- Clinical signs: all signs were recorded for individual animals. During the treatment period, for both F0 males and females, examination of individual animals for signs of reaction to treatment was carried out daily prior to dosing, immediately after and at approximately 1 hour after dosing. All animals received a more thorough examination once weekly during the entire treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on the day of allocation, the day treatment commenced, and weekly thereafter and just prior to sacrifice. Females were weighed on the day of allocation, the day treatment commenced and weekly to pairing. During the post-coitum period females were weighed on Days 0, 6, 10, 15, and 20 post-coitum and also on Days 0/1, 4, 8, 12 and 21 post-partum.

FOOD CONSUMPTION:
Food consumption per cage of animals was measured weekly for males from allocation to pairing and for females from the day of allocation to pairing. Individual food consumption of females was measured over the periods of Days 0 to 5, 6 to 9, 10 to 14 and 15 to 19 post-coitum, and Days 0 to 3, 4 to 7, 8 to 11 and 12 to 20 postpartum.
Oestrous cyclicity (parental animals):
- Oestrous cycles and mating performance: vaginal smears were taken daily for 2 weeks prior to pairing starting from the first day of treatment until occurrence of mating. During the mating period each cage was checked each morning for the presence of a copulation plug and a vaginal smear was prepared from each female. This information was used to detect marked anomalies of the oestrus cycle and to determine the precoital interval (the number of nights paired before detection of mating).
-Duration of gestation: gestation periods were taken as the time between the day of successful mating and the commencement of birth (i.e. first detected presence of offspring).
Sperm parameters (parental animals):
(Only F0 males) Sperm count in one epididymis and evaluation of sperm viability and motility was performed in control and high-dose males and in one mid-dose male which did not induce pregnancy
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On Day 4 post-partum, all litters in excess of eight offspring were culled to eight pups (4 males and 4 females, where possible) by random selection, excluding those pups showing signs of ill health. Culled pups were sacrificed on Day 4 post-partum by subcutaneous injection of 'Tanax', necropsied and examined for external and internal abnormalities. Pup sex was confirmed at necropsy. One litter was inadvertently culled to seven pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The pups (live and dead) were counted, sexed, weighed and examined for external abnormalities as soon as possible after parturition (Day 0 or 1 post-partum). Live pups were identified individually within the litter by toe amputation. All litters were examined daily for dead or abnormal young. The pups were also weighed on Days 4, 8, 12 and 21 post-partum. All pups found dead were given a post-mortem examination.
- 24 males and 24 females were selected for further study. All pups were allocated to 4 groups and treated starting the day after weaning. Clinical observation, post dose observation, food consumption, body weight and post weaning tests were performed. Meanwhile the Sponsor decided to stop the study at the first generation, since no toxicity was observed.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF PRE-WEANING DEVELOPMENT
All pups in all litters were examined to determine the age at which the following developmental stages were attained:
a) Pinna unfolding, from Day 1 post-partum to 100% occurrence;
b) Hair growth, from Day 3 post-partum to 100% occurrence;
c) Incisor emption (upper), from Day 8 post-partum to 100% occurrence;
d) Startle response to sound, from Day 12 post-partum to 100% occurrence;
e) Eye opening (complete separation of eyelids), from Day 12 post-partum to 100% occurrence;
f) Air righting reflex, once on Day 20 post-partum;
g) Pupil reflex, once on Day 20 post-partum;
h) Testes descent (testes palpable in the scrotum), once on Day 21 post-partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: males were killed by carbon dioxide after the birth of the majority of litters and retained for external and internal examinations.
- Maternal animals: all parental animals were killed at weaning (PND 21) by carbon dioxide inhalation. Apparently non pregnant females were sacrificed after at least 25 days post-coitum.

GROSS NECROPSY
- Gross necropsy consisted of and examined for external and internal abnormalities
- The sex of each pup was determined by gonadal inspection and, for each dam, the number of visible implantation sites was recorded

HISTOPATHOLOGY / ORGAN WEIGHTS
- Females: uteri were immersed in a 10% solution of ammonium sulphide to reveal any evidence of implantations. The following tissues were preserved in 10% buffered formol saline: uterus with cervix, ovaries with oviduct, vagina and pituitary
- Males: the following tissues were preserved in 10% buffered formol saline (except testes and one epididymis which were fixed in Bouin's fluid and preserved in 70% alcohol): pituitary, testes, one epididymis, seminal vesicles, prostate, coagulating gland and any Abnormalities. Testes from males which did not induce pregnancy and from 5 concurrent controls were weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.
- All pups found dead in the cage were necropsied.
- Excess pups (i.e., those not selected for the Fl generation) were killed with carbon dioxide on or shortly after Day 21 post-partum
- All animals were killed with carbon dioxide and were subjected to necropsy

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Culled pups were sacrificed by intrascapular injection of Tanax on the day of culling and were subjected to a post-mortem examination. The sex of each pup was determined by gonadal inspection.
Statistics:
For the body weights, body weight gain and organ weights the significance of the differences amongst group means was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error of variance. The homogeneity of the data were assessed by Bartlett's Test before Dunnett's Test was performed. If the data were found to be inhomogeneous, a modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
The non-parametric Kruskal-Wallis analysis of variance was used for litter data.
Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams' test.
The criterion for statistical significance was p<0.05.
Reproductive indices:
mating index, copulation index, fertilitiy index
Offspring viability indices:
viability index, lactation index
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs seen during the observations performed at weekly intervals in F0 males and females were limited to common conditions of the skin and fur. No reaction to treatment was seen at the post-dose observations performed immediately and 1 hour after dosing during the entire treatment period except for a few occasions of salivation in the high-dose group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control male was found dead and another one was sacrificed for humane reasons during the course of the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean body weights and body weight gain in F0 males were comparable between the treated and the control groups. Statistically significant reductions in body weight were observed on day 8 of treatment in females receiving 15 and 50 mg/kg/day of the test substance when compared to controls. A statistically significant reduction in body weight gain was observed in females receiving the test substance at 50 mg/kg/day on day 8 of treatment. Body weight gain in females receiving the test substance at 15 and 50 mg/kg/day was statistically significantly higher compared to controls on day 15 of treatment. A statistically significant increase in body weight gain was observed on postpartum day 8 for animals of the low and mid-dose groups and on post-partum day 12 for the high-dose group compared to controls. A statistically significant reduction in body weight gain was observed in high-dose females on post-partum Day 0 and was considered to be related to the higher number of pups compared to controls. These occasional differences are not considered to be of toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of the F0 generation was unaffected by treatment
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
A total of three males, one in the control, one in the mid-dose and one in the high-dose group failed to induce pregnancy.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
All dams gave birth between days 21 or 22 post-coitum, with the exception of one low-dose female which gave birth prematurely on gestation day 18. This isolated case was considered incidental. The statistically significant reduction observed in gestation periods for high-dose females compared to controls was not considered to be of toxicological significance
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no differences in concentration (million sperm/gr epididymal cauda), viability, motility and morphology of the sperm between the control, the high-dose males and in males which failed to induce pregnancy.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no differences between the controls and the treated groups in litter size, litter weight, mean pup weight or pup loss throughout the whole lactation period. Lactation index was similar between the control and the treated groups.
Sex ratio of Fl pups: sex ratios of offspring at birth and on Day 21 post-partum did not show any differences between groups. The statistically significant difference in the number of male pups found dead during the weaning period was attributable to the low number of deaths in the high-dose litters compared to controls and was not considered to be a sex related mortality.
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity, fertility
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects up to this dose group
Clinical signs:
no effects observed
Description (incidence and severity):
The abnormalities observed in pups during the post-partum period were incidental with no relation to treatment.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no differences between the controls and the treated groups in litter size or pup loss throughout the whole lactation period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no differences between the controls and the treated groups in litter weight or mean pup weight throughout the whole lactation period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No signs of toxicological significance were observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related changes were seen in FI pups which died before weaning. No findings of toxicological significance were seen at the necropsy performed on F1 culled pups. There were no meaningful differences in the incidence of the abnormalities recorded at the necropsy of FI pups at weaning or in selected pups.
Description (incidence and severity):
Pre-weaning development of F1 pups: there were no significant differences between groups in the results obtained from the parameters used to monitor the pre-weaning physical and functional development. One pup in group 2 had bilateral anophthalmia (considered incidental) and therefore was not subjected to the pupil reflex test. Values of eye opening and pupil reflex for this litter were excluded from group mean calculation.
Dose descriptor:
NOAEL
Remarks:
Postnatal development (up to weaning at postnatal day 21)
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects up to this dose group
Reproductive effects observed:
not specified

Table 1: Reproduction parameters

Reproduction parameters

Test groups (mg/kg bw)

0

15

50

200

 

 

Females

Initial group size

24

24

24

24

Not pregnant

1

0

1

1

Total litter loss

0

0

1

0

With live pups at weaning

23

24

22

23

Pre-coital interval (days)

4.5

3.75

3.17

2.96

Copulation index (%)

100

100

100

100

Fertility index (%)

95.8

100

95.8

95.8

 

 

Males

Initial group size

24

24

24

24

Found dead

1

0

0

0

Humane kill

1

0

0

0

Failed to induce pregnancy

1

0

1

1

Copulation index (%)

100

100

100

100

Fertility index (%)

95.8

100

95.8

95.8

 

Table 2: Implantation and pre-birth loss data from F0 females(group mean data)

Treatment (mg/kg bw)

Total liter size

Implantations/litter

Pre-birth loss /litter

Gestation period (days)

N

Mean±SD

N

Mean±SD

Number

Percentage

N

Mean±SD

N

Mean±SD

N

Mean±SD

0

23

14.7±2.6

23

13.6±2.6

23

1.1±1.5

23

7.0±9.6

23

21.9±0.3

15

24

15.1±2.1

24

14.1±2.4

24

1.0±1.1

24

7.0±7.8

23

22.0±0.3

50

22

14.4±2.2

22

13.4±2.6

22

1.0±1.0

22

9.6±11.8

23

21.9±0.3

200

23

14.9±1.8

23

14.0±2.2

23

0.9±0.9

23

6.1±6.3

23

21.6±0.5*

SD: standard deviation; *: p<0.05

 

Conclusions:
Oral administration with the test item to parental F0 animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment. These results suggest that a dosage of 200 mg/kg/day can be considered the NOAEL.
Executive summary:

The effects of the test article on reproductive function and fertility were evaluated in male and female rats following oral administration by gavage at dosages of 15, 50 and 200 mg/kg/day. Male parental (F0) rats were treated for ten weeks prior to pairing until the day before sacrifice. Female parental (F0) rats were treated for two weeks prior to pairing, during gestation and lactation periods and up to the day before sacrifice, on or shortly after day 21 post-partum. A concurrent group received the vehicle (peanut oil) at the same dose volume and acted as a control. Each group was comprised of 24 males and 24 females.

There were no mortalities in the treated groups. Two control males died (one was found dead and another was humanely sacrificed) during the course of the study. A total of 3 males failed to induce pregnancy; one each in the control, mid and high-dose groups and consequently the females in the same groups were not pregnant. One mid-dose female had total litter loss on post-partum day 0 and one low-dose female gave birth prematurely on gestation day 18. No treatment-related effects were observed at the post-dose observations. There were no treatment-related clinical signs. There were no changes of toxicological significance in body weight or body weight gain in F0 males. The occasional differences observed in F0 females are not considered to be of toxicological significance. Food consumption of the F0 generation was unaffected by treatment. Oestrus cycle, mating performance, and pregnancy rate were unaffected by treatment. No effects on implantation, pre-birth loss data or gestation length were seen in F0 treated females. Total and live litter size, litter and mean pup weights and pup loss were similar between groups. Sex ratios and lactation index were not affected by treatment. No signs were observed during the post-partum period which could be considered related to maternal treatment. Pre-weaning development did not show any differences between the control and the treated groups. There were no toxicologically relevant necropsy findings in Fl pups which died during the lactation period or in Fl pups which were culled on Day 4 post-partum. No changes were seen at the necropsy of Fl pups sacrificed on or shortly after Day 21 post-partum. No findings of toxicological significance were seen at necropsy of Fl selected pups. No signs of toxicological significance were observed at necropsy of the F0 generation. There were no differences in sperm motility or sperm concentration between the control, the high-dose group or the mid-dose male which failed to induce pregnancy.

In conclusion, oral administration with the test item to parental F0 animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment. These results suggest that a dosage of 200 mg/kg/day can be considered the NOAEL.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with detailed information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Version / remarks:
MITI
GLP compliance:
not specified
Radiolabelling:
yes
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
- Chemical name of vehicle): Hydrogenated castor oil (HCO-30)
Test organisms (species):
Cyprinus sp.
Details on test organisms:
Before using the 1 year old carp, it was purchased on 11/17/1988 from Sankyou Suisan Inc. (1-1 Tanidachou, Shinjuku, Tokyo) and after fish farming in the said facility, it was fed once every day at 25±2°C (Kyorin made minipette) and naturalized for more than 1 week. (Lot.No.88-K-1117-1, No.B-3 water tank) No fish was found dead during the 1 week naturalization period before usage.
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
8 wk
Nominal and measured concentrations:
0.1 mg/L (nominal)
0.01 mg/L (nominal)
Type:
BCF
Value:
24 - 36 L/kg
Basis:
whole body w.w.
Remarks on result:
other: Conc.in environment / dose:0.1 mg/L
Type:
BCF
Value:
52 - 89 L/kg
Basis:
whole body w.w.
Remarks on result:
other: Conc.in environment / dose:0.01 mg/L

 Actual concentration  2 weeks  4 weeks  6 weeks    8 weeks
0.1 µg/ml  0.099  0.101  0.103  0.103
 0.01 µg/ml  0.01  0.01  0.01  0.0101

Bioconcentration factor  2 weeks  4 weeks  6 weeks    8 weeks
0.1 µg/ml  33 -24  36 -31  31 -29  34 -36
 0.01 µg/ml  69 -52  72 -89  78 -73 82 -81
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986-11-10 to 1986-11-18 (experimental phase)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study. Very high solvent concentration. Limit test. Nevertheless, result considered reliable as no indication of toxicity observed at the highest tested concentration (100 mg/L).
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
Highest vehicle concentration; 954 mg/l, Test performed as limit test with one concentration: 100 mg/l
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Samples for analysis were taken at 0h and 96h exposure immediately after intensively stirring.
- Extraction in dichloromethane (20 ml) with subsequent drying
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Vehicle: 950 mg DMF, 4 mg Alkylphenol-polyglycolether per L water in the concentration used for the highest test concentration
- Calculated amounts of test substance to produce the desired test concentrations were given into the water and homogeneously distributed
- Initially the test substance appeared homogeneously distributed in all test concentrations. Small oil droplets were observed on the water surface after 1h exposure.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra-Fish
- Source: West Aquarium, Bad Lauterberg, Germany
- Length at study initiation (length definition, mean, range and SD): 24 mm (22-25 mm)
- Weight at study initiation (mean and range, SD): 0.1 g (0.07-0.12 g)
- Feeding during test: None

ACCLIMATION
- Acclimation period: 61 days plus adaptation (24 hours without food prior to exposure)
- no treatment during acclimation
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
176 mg/L CaCO3
Test temperature:
23°C
pH:
8.0
Dissolved oxygen:
>95%
Nominal and measured concentrations:
100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass aquaria of 20 liters filled with 15 liters (36/22/25 cm)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.06 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water (carbon filter)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours daily
- Light intensity: Fluorescent
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Sublethal observations / clinical signs:

Controls: Mortalities blank 0 %

Mortalities vehicle: 0 %

Remarks: Values are based on nominal concentrations

 

Measured initial concentrations: 95-113 mg/L

Measured final concentrations: 150-113 mg/L

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion