Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division. CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: Approximately 6 - 7 weeks
- Weight at week 1: 134 - 161 g (males), 113 - 143 g (females)
- Housing: Individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 % ± 10 %
- Air changes (per hr): 16 - 20/hour
- Photoperiod (hrs dark / hrs light): 12/12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % and 0.1 % Tween 80 in distilled water .
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

Suspensions of the test item in the vehicle at the appropriate concentrations were freshly prepared every day immediately prior to the dosing.
Volume applied: 10 ml/kg body weight

The test item was administered orally by gavage. The dosing volume was adjusted daily to individual body weights. The control animals were dosed with the vehicle only.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of the study, samples of the vehicle containing the test item at concentrations of 1, 5, 30 and 100 mg/ml were analysed for content, homogeneity, and stability.

During treatment, control analyses of the test item concentration in the vehicle were carried out at all dose levels on samples collected once per experimental week. The samples were collected on completion of dosing, immediately deep-frozen and sent to the analytical laboratories.
Duration of treatment / exposure:
The test item was administered to groups of 5 males and 5 females (Experimental Group I) orally by daily gavage, over a period of 4 weeks at doses of 0 (control), 10, 30, 100 and 1000 mg/kg. Additionally groups of 5 male and 5 female control and high dose rats (Experimental group II) were similarly treated, and were originally scheduled to be maintained for a 4-week recovery period at the end of treatment. The high dose was reduced to 500 mg/kg from study day 8 because of mortality and poor condition. Dosing of high dose animals was halted from study day 11 because of continued mortality and deterioration, and all animals (including those scheduled for the recovery phase) were sacrificed at the end of the treatment period.

Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 100, 300 and 1000 mg/kg
Basis:
nominal in water
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
A total of 70 animals (10 males and 10 females in the control and dose group 5; 5 males and 5 females in dose groups 2, 3 and 4) was used.
The general outline of the experiment is presented in Table 1.

The test item was administered to groups of 5 males and 5 females (Experimental Group I) orally by daily gavage, over a period of 4 weeks at doses of 0 (control), 10, 30, 100 and 1000 mg/kg. Additionally groups of 5 male and 5 female control and high dose rats (Experimental group II) were similarly treated, and were originally scheduled to be maintained for a 4-week recovery period at the end of treatment. The high dose was reduced to 500 mg/kg from study day 8 because of mortality and poor condition. Dosing of high dose animals was halted from study day 11 because of continued mortality and deterioration, and all animals (including those scheduled for the recovery phase) ere sacrificed at the end of the treatment period.

- Dose selection rational:
Based on study by Cantoreggi, S. (1999) Acute oral toxicity in the rat (Test Number 981116). Final Report released February 16, 1999, Novartis Crop Protection Toxicology, Stein, Switzerland. LD50 in rats of both sexes: greater than 2000 mg/kg body weight.

Examinations

Observations and examinations performed and frequency:
Parameter Time Point

Mortality: Twice daily

Clinical signs: Daily

Detailed clinical observations: During pre-test and once weekly thereafter

Body weight: Days -6, 1, 4, 8, 11, 15, 22, 28

Food consumption: Once per week

Water consumption: Once per week

Neurological examinations: Week 4

Laboratory investigations: Week 5

Necropsy: Week 5
Sacrifice and pathology:
GROSS PATHOLOGY:
The following treatment-related necropsy findings were present in animals receiving daily doses of 300 mg/kg and above:
• Entire animal discoloured (without correlating microscopic findings)
• Liver granulated/nodular, enlarged, yellowish (correlating with microscopic findings of necrosis, regenerative hyperplasis and
cholangiofibrosis)
• Spleen enlarged (correlating with hematopoiesis)


HISTOPATHOLOGY:
Primary treatment-related changes were confined to the liver.
Th efollowing
Decedents:
The following treatment-related histopathological changes were present in the liver of animals receiving the high dose of 1000 mg/kg (reduced to 500 mg/kg from day 8 , and discontinued from day 11):
• Periportal necrosis
• Cholangiofibrosis
• Deposition of pigment (in macrophages)

Unscheduled deaths of high dose animals were due to periportal necrosis in the liver.

Terminal sacrifice:
The following treatment-related changes were present in the liver of terminal sacrifice animals:
High dose (1000/500 mg/kg/day):
• Cholangiofibrosis
• Regenerative hyperplasia
• Deposition of pigment (in macrophages)
300 mg/kg/day:
• Cholangiofibrosis
• Periportal necrosis
• Deposition of pigment
• Periportal deposition of glycogen

The following treatment-related changes present in decedent high dose animals considered secondary to the stress of treatment:
• Increase in hematopoisesis in the spleen (correlating with increased weight at necropsy)
• Lymphoid atrophy in spleen, thymus , lymph nodes and Peyer’s patches
• Various degenerative changes in the gastro-intestinal and reproductive tract

Incidental fndings:
Periportal deposition of glycogen in the liver of one male receiving 100 mg/kg/day was considered to be within the normal range, and unrelated to treatment. A renal carcinoma in the kidney of male no. 19 (100 mg/kg/day) was an incidental finding. A variety of other minor degenerative and inflammatory lesions were also observed which fell within the expected range for rats of this age, and were considered unrelated to treatment.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Due to discontinued exposure motor activity was not measured in group 5 animals.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY:

Mortality:
One male of group 5 (1000/500 mg/kg) had to be sacrificed in moribund condition at study day 4. Additionally, two males each of group 5 were found dead at days 7 and 12, respectively, and one male of this group at day 10. One female each of group 5 was found dead at day 5 and day 10 respectively, and two females of this group died at day 11.

Clinical observations:
From a few days after treatment start, marked clinical signs of systemic toxicity such as piloerection, hunched posture, hypoactivity and distended abdomen were progressively recorded for animals of both sexes treated at 100/500 mg/kg. Besides these prominently observed signs, lacrimation, prostration, pale skin, dyspnea, salivation, genital discharge and diarrhea were noted for isolated animal before early study death.
Although the high dose was reduced to 500 mg/kg from day 8 and dosing was halted from day 11, 6 males and 4 females died or were sacrificed in moribund condition.

BODY WEIGHT AND WEIGHT GAIN:
The mean body weights of group 5 (1000/500 mg/kg) males were significantly decreased from day 4 with a maximum depression of 29 % at day 11. Thereafter, correlated with the discontinuation of dosing, the mean body weight for group 5 males gradually attained values close to control level (-8 % below the control level at day 28). In females of group 5, the mean body weights were decreased by 9 % and 13 % at study days 8 and 11, and thereafter values comparable to those of the control group were reached at the end of the treatment period.
The presentation of mean body weight changes illustrates the negative (week 1) and strongly decreased (week 2) mean body weight gain of male group 5 and the markedly decreased mean body weight gain of female group 5 during week 1, and on the other hand, the compensatory increase in mean body weight gains during week 3 and to a minor degree during week 4 (for males only).
The body weight development of the other treated groups (10, 100 and 300 mg/kg) was not affected by treatment.

FOOD CONSUMPTION:
The mean food consumption of animals of group 5 (1000/500 mg/kg) was significantly depressed during week 1(by -58 % in males and -44 % in females) and week (by -59 % in males and -40 % in females. Thereafter at weeks 3 and 4, mean food consumption values in both male and female group 5 resumed levels comparable to or even minimally above those of the respective control groups.
The mean food consumption of the other treated groups (10, 100 and 300 mg/kg) was not influenced by treatment.

WATER CONSUMPTION:
The mean water consumption of the male high dose group (1000/500 mg/kg) was decreased by-15 % and -16 % at treatment weeks 1 and 2 respectively. After discontinuation of dosing, the mean water consumption of the surviving males of group 5 was above that of the control males (by +20 % and +31 % at weeks 3 and 4). In females of group 5 (1000/500 mg/kg) there was no consistent treatment-related effect, except at week 3 where the mean water consumption was significantly above the control level (by +67 %).
The mean water consumption in groups 2, 3 and 4 (10, 100 and 300 mg/kg) was not affected by treatment.

HAEMATOLOGY:
At the end of the treatment period, one female each of group4 and 5 (300 and 1000/500 mg/kg) showed an anemia. In addition, females of groups 4 and 5 (300 and 1000/500 mg/kg) had lower values for MCHC and higher values for HDW pointing to anisochromia of erythrocytes. Lower MCHand higher RDW values observed in males and females of group 5 (1000/500 mg/kg) reflected a tendancy to hypochromia and anisocytosis of red blood cells. Platelet counts for males and females of group 5 (1000/500 mg/kg) were near the lower end of the normal range. Furthermore, males of group 4 (300 mg/kg) had slightly higher lymphocyte counts and a marginal increase in large unstained cells.
The significantly higher mean value for relative monocyte counts in males of group 5 was not attributed to the treatment since absolute counts did not differ appreciably from controls.

BLOOD CHEMISTRY:
At the end of the treatment period, males and females of group 5 (1000/500 mg/kg) had slightly higher mean values for bilirubin and lower values for protein and albumin with a lower albumin to globulin ratio recorded for the females. Females of group 4 (300 mg/kg) also had significantly lower mean values for albumin and albumin to globulin ratio. In addition, males and females of group 5 (1000/500 mg/kg) and females of group4 (300 mg/kg) had higher activities recorded for aspartate and alanine aminotransferase and alkaline phosphatase. Furthermore, males and females of group 5 (1000/500 mg/kg) and females of group 4 (300 mg/kg) had higher plasma cholesterol levels.

No relevance was attributed to the significantly higher mean glucose value recorded for males of group 3 as the finding did not form a dose-response relationship. Similarly, no relevance was attributed to the significantly lower mean urea value for females of group 5 as the change was in the opposite direction to that considered to be toxicologically relevant.

URINALYSIS:
At the end of the treatment period, males of group 5 (1000/500 mg/kg) excreted more acidic urine than the controls.
No relevance was attributed to the higher concentrations of erythrocytes in the urine excreted by one male in group 3, two males in group 4 and one male in group 5 or to the significantly higher leukocyte content of the urine excreted by females of group 3 as the changes did not form a dose relationship and lacked concomitant histopthological findings.

NEUROBEHAVIOUR:
Functional observational battery:
Due to compound-related toxicity 6 males and 4 females of group 5 died or had to be killed at weeks 1 or 2. Therefore, exposure was discontinued as of test day 11 and week 4 functional measurements and motor activity were not assessed.
In these animals hunched posture, piloerection, distended abdomen and reduced muscle tone were the most common signs observed that were considered to be non-specific signs of systemic toxicity,. In individual animals touch response or ease of handling was affected as well.
No treatment-related signs were observed in animals of other dose groups and all functional measurements were comparable to those of the controls.
Group comparisons using multiple t-tests showed no statistically significant differences between treated and control groups.

Motor activity:
Motor activity measurements conducted at the end of the treatment period did not reveal any compound-related effects in groups 2 to 4 as compared to controls. Due to discontinued exposure motor activity was not measured.

ORGAN WEIGHTS:

At treatment and the mean carcass weight of surviving males of group 5 (1000/500 mg/kg) was minimally blow the control level (by-8 %).

Absolute and relative mean liver weights were increased in group 4 (300 mg/kg) animals.
Compared to the control groups, absolute values were increased by 14 % and 24 % for males and females, respectively, and values relative to carcass or brain weights by 15 % (males) and 26 % /19 % /females).

Significantly increased absolute and relative mean kidney weights were recorded for surviving high dose males (1000/500 mg/kg) with values of +16 % (absolute weight) and +26 %/+20 % (relative to carcass/brain weight)above the control mean values.

Decreased absolute and relative mean thymus weights were recorded for male groups 4 and 5 (300 and 1000/500 mg/kg) with values below the control levels (by -12 % to -13 % in group 4 and -11% to -19 % in group 5).

Mean testes weights and testes to brain weight ratios were significantly decreased in group 5 with values of -16 % and -12 % below the control values. Equally, absolute and relative mean ovary weights in the high dose group were significantly below the control values by -30 % to -33 %.

Mean values of absolute and relative spleen weights were significantly increased in both male (by 59 5 – 74 %) and female (by 71 5 – 78 %) high dose groups (1000/500 mg/kg). Additionally, spleen weights and ratios were also above control values (by 32 % to 40 %) in female group 4 (300 mg/kg).

The weights and weight ratios of the other organs were not affected by treatment.

Although minimally above the range of historical control values, significantly higher mean heart to body weight ratio in male group 5 was considered not to be directly related to treatment, but to be a consequence of poor body weight development. This is supported by the absence of corroborative histopathologic findings. In the absence of dose relationship and corroborative histopathological changes, also no toxicological relevance was attributed to slightly increased kidney weight and/or ratios in male group 3 or female group 4 and to the significantly increased mean testes weight and testes to brain weight ratio in male group 3. Equally, in the absence of histopathological findings, no toxicological relevance was attributed to increased mean liver weight and liver to brain weight ratio in male group 3.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 100 other: mg/kg body weight per day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the results of this study a No-Observable-Adverse-Effect Level (NOAEL) of 100 mg/kg body weight per day was defined for animals of both sexes.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study for CA 2219 A (Intermediate of CGA 276854), the liver was identified as primary target organs of toxicity at dose levels of 300 mg/kg and above. Additionally, a number of secondary effects related to the condition of the animals were found at these doses. A No-Observable-Adverse-Effect Level (NOAEL) of 100 mg/kg body weight per day is defined for animals of both sexes.
Executive summary:

This study in rats was conducted to determine the oral toxicity of CA 2219 A (Intermediate of CGA 276854) when administered by gavage over a period of 28 consecutive days and to estimate a No-Observable-Adverse-Effect Level (NOAEL) of exposure. As dosing at the high dose level was discontinued from study day 11 onwards, there was no evaluation of reversibility, persistence of, or delayed occurrence of potential toxic effects. 

 

The test item, suspended in the vehicle, was administered by daily gavage at doses of 0 (control), 10, 100, 300 and 1000 mg/kg to a total of 70 albino rats (Hanlbm: WIST), using 5 animals per sex and dose group (Experimental Group 1) and, additionally at the control and high dose level, 5 animals per sex for the originally planned recovery evaluation (Experimental Group II). Because of the mortality and poor condition of animals at 1000 mg/kg, the high dose was reduced to 500 mg/kg from day 8 before dosing of the high dose group was halted from day 11. 

Clinical signs, body weight, food consumption, water consumption, and mortality were monitored throughout the study for all animals. Neurotoxicologic investigations were performed weekly (detailed clinical observations) and at week 4 (functional observational battery and motor activity; not assessed/performed with group 5 animals). Hematological, blood chemistry, and urine analyses were performed on all animals at the end of the treatment period. At final sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathologic evaluation. 

 

Conclusion:

Based on the results of this study, the liver was identified as primary target organs of toxicity at dose levels of 300 mg/kg and above. Additionally, a number of secondary effects related to the condition of the animals were found at these doses. A No-Observable-Adverse-Effect Level (NOAEL) of 100 mg/kg body weight per day is defined for animals of both sexes.