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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-04-16 till 2000-03-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform study under GLP without significant deviations (see "Deviations from the protocol" under section "Any other information on materials and methods"

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
This study has been performed in compliance with OECD Principles of Good Laboratory Practice (Council Decision 81/30, adopted on May 12, 1981, and the OECD Recommendation 89/87 concerning the 'Compliance with Principles of Good Laboratory Practice'
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MeNigu
- Physical state: solid, white
- Analytical purity: 98.0%
- Lot/batch No.: 601014
- Expiration date of the lot/batch: reanalysis date March 2003
- Stability under test conditions: not indicated by the sponsor
- Storage condition of test material: at room temperature
- Stability under test conditions: test item was homogeneously distributed in the vehicle and was stable at the target concentrations.

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, Fuellinsdorf, Switzerland
- Age at study initiation: no data
- Weight at study initiation: 142 to 186 g in males, 115 to 143 g in females
- Fasting period before study: not applicable
- Housing: The experiment was carried out under specified pathogen free (SPF) conditions. The animals were housed individually in macrolon cages type 3 (area: 900 square centimeters) with wire mesh tops and sterilized, granulated soft wood bedding. Neither insecticides nor chemicals were applied in the animal room with the exception of disinfectant Bradophen. Cages and water bottles were changed at one week intervals.
- Diet (e.g. ad libitum): Pelleted, certified standard diet was provided ad libitum (except for overnight fasting prior to blood collection)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: An acclimatization/quarantine period of 14 days was allowed between delivery and the start of the treatment.


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


IN-LIFE DATES: From: 1999-04-28 To: 1999-06-11 (group 1); 1999-07-09 (group 2)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
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.


DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: no data


VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given in the report
- Concentration in vehicle: test item concentrations 1, 10, 30, and 100 mg/ml, respectively
- Amount of vehicle (if gavage): 10 ml/kg body weight
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, 10, 30 and 100 mg/ml were analyzed for
content, homogeneity, and stability. During treatment, control analysis 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. Analyses results are given in the results and appendix sections of the study report.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily gavage
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10 mg/kg bw per day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw per day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw per day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw per day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose level selection was based on the results of the following study: Winkler, G. (1996). Acute oral toxicity in rats
(Test Number 962035), Novartis Crop Protection AG, Toxicology, Stein, Switzerland.
- Rationale for animal assignment (if not random): by means of computer-generated random numbers
- Rationale for selecting satellite groups: From the same batch of animals a small number was retained for possible replacement during the
acclimatization period of animals deemed not suitable for study. These animals were subjected to identical conditions during this period, and those
not used were removed at the start of the experiment.
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): random
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations explained in section 3.5 were included (results see Table 8.1 of the study report).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pretest and once weekly thereafter


BODY WEIGHT: Yes
- Time schedule for examinations:during pretest and once weekly thereafter


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, during pretest and weekly thereafter
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight
gain data: yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: pretest and once weekly thereafter


OPHTHALMOSCOPIC EXAMINATION: no data


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 5 (all animals), and week 9 (recovery animals)
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: week 5: 35 male and 35 female; week 9: 5 male and 5 female
- Parameters checked in table 3.8.1 were examined (results see Table 8.10 of the study report).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 5 (all animals), and week 9 (recovery animals)
- Animals fasted: Yes
- How many animals: week 5: 35 male and 35 female; week 9: 5 male and 5 female
- Parameters checked in table 3.8.3 were examined (results see Table 8.11 of the study report).


URINALYSIS: Yes
- Time schedule for collection of urine:bweek 5 (all animals), and week 9 (recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3.8.4 were examined (results see Table 8.12 of the study report).


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 (all animals), and week 8 (recovery animals)
- Dose groups that were examined: all groups
- Battery of functions tested: sensorimotor functions (approach, touch, vision, audition, pain, vestibular), autonomic functions (pupillary reflex,
body temperature), sensorimotor coordination (grip strength, landing foot splay)


OTHER:

MOTOR ACTIVITY:
Motor activity was measured in groups of 8 animals. Measurements were conducted between 8 a.m. and 3 p.m. in an illuminated and air conditioned
room with background noise provided by the PC's fan. An automated open field device was used that had been shown to allow
measurement of increased and decreased vertical and horizontal motor activity (FITZGERALD R.E., BERRES M., and SCHAEPPI U. 1988).

FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
An FOB was conducted towards the end of the treatment and recovery periods at about the same time each day. To control for variations in test
conditions and to make experimenters unaware of the animals' treatment, animals were randomized, the cage labels covered with the
corresponding FOB number and the animals tested in FOB-number order. Observation of animals was conducted in a structured way and included
1) observation of animals in the home cage, 2) observation and gait evaluation in a standard arena, and 3) observation during handling and were
followed by a neurological examination. Observations covered the functional domains of CNS activity, CNS overexcitation, sensorimotor, autonomic,
and physiological functions.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (in study report see table 8.13 organ weights, see table 8.14 macroscopial and microscopial findings )
HISTOPATHOLOGY: Yes (in study report see table 8.14 macroscopial and microscopial findings)
Statistics:
Statistical analyses of in-life and organ weight data (with the exception of FOB data and motor activity data) were carried out using the statistical
routines contained in the NOVATOX System (Novartis Crop Protection AG).
Quantitative data such as body and organ weights were analyzed either using parametric or non-parametric statistical tests following a pre-test for
uniformity of the within group variances based upon the Bartlett's test of homogeneity of variances. In the case of a non-significant Bartlett's test
(p>0.05) a one-way analysis of variance (ANOVA) was carried out. If the overall test of differences between the groups was significant (p≤0.05),
comparisons were made between the control group and each of the treatmentgroups using Dunnett's multiple comparison test. In cases where
Bartlett's test was significant (p≤0.05) the Kruskal-Wallis non-parametric test of the differences between the groups was carried out. If this test was
significant (p≤0.05), comparisons were made between the control group and eachof the treatment groups using Dunn's multiple comparison test.
Ordinal data such as urine components were analyzed using the non-parametric Kruskal-Wallis test. If this test was significant (p≤0.05),
comparisons were made between the control group and each of the treatment groups using Dunn's multiple comparison test.
FOB data and motor activity data were compared to the control group using multiple t tests. Raw and multiplicity-adjusted p-values based on
bootstrap resampling are given.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Mean water consumption of the male and female high dose group (1000 mg/kg) was significantly increased throughout the whole treatment period.
During the recovery period, water consumption declined in both sexes to normal values.

CLINICAL CHEMISTRY
Treatment resulted in higher plasma urea levels at 100, 300 and 1000 mg/kg with higher creatinine levels at 1000 mg/kg in males and females. In
addition, a disturbance of the electrolyte balance was in evidence with lower values for sodium recorded for males and females at 100, 300 and
1000 mg/kg, and lower values for chloride recorded for males and females at 1000 mg/kg and for males at 100 and 300 mg/kg. Males at 1000
mg/kg also had minimally lower values for calcium and females at 300 and 1000 mg/kg had minimally lower potassium levels. Following a 4-week
recovery period, normal values were recorded for the above parameters except for calcium that remained at a lower level in males.

URINALYSIS
At the end of the treatment period, males at 1000 mg/kg excreted a larger urine volume. Following the 4-week recovery period, the urine excreted
by previously treated rats was similar to that excreted by the respective controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically, hepatocellular hypertrophy was increased in dose groups 4 and 5 of either sex, but not after 4-week recovery.

All other investigated parameters no treatment-related findings.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: clinical chemistry, urinalysis
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: clinical chemistry, urinalysis

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Tab. 2: Blood chemistry (mean data of males): Ca in mmol/l

 Group 1 Control (vehicle only) Group 2 (10 mg/kg) Group 3(30 mg/kg)  Group 4(100 mg/kg) Group 5 (1000 mg/kg) 
 Week 5  Mean  2.695  2.628  2.622  2.662 2.574 
   SD  0.074  0.024 0.052   0.072  0.076
   N  10 5  5  5  10
 Week 9  Mean  2.654        2.578
   SD  0.034       0.056 
   N  5        5

Applicant's summary and conclusion

Conclusions:
Oral administration of CA 2342 A to rats by daily gavage was well tolerated at all dose levels tested. In the absence of histopathologic correlates as
well as supportive findings from organ weight analysis, the changes seen on some blood chemistry parameters (at dose levels of 100 mg/kg bw per day and above) point to influencing the renal function. In addition, adaptive changes of the liver were observed. Reversibility was demonstrated for all
changes within a 4-week treatment free period except for calcium concentration in blood of males. Due to the irreversibility in males, a No-
Observable-Adverse-Effect Level (NOAEL) of 300 mg/kg body weight per day is defined for males and 1000 mg/kg body weight per day for females.
Executive summary:

This study in rats was conducted to determine the oral toxicity of the test item when administered by gavage over a period of 4 weeks, to estimate a No-Observable-Adverse-Effect level (NOAEL), and to evaluate reversibility, persistence of, or delayed occurrence of potential toxic effects after a 4-week recovery period.

The test item CA 2342 A (Intermediate of CGA 293343), suspended in the vehicle, was administered by daily gavage at dose levels of 0, 10, 100, 300 and 1000 mg/kg body weight (groups 1, 2, 3, 4, 5 and 6, respectively), to albino rats (Hanlbm:WIST), 5 animals per sex and dose group (experimental group A) and, additionally at the control and high dose level, 5 animals per sex for recovery evaluation (experimental group B). 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 weeks 4 and 8 (functional observational battery, motor activity). Hematological, blood chemistry, and urine analyses were performed at the end of the treatment period on all animals, and at the end of the recovery period on animals of experimental group B. At sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation.

In life observations, body weight, food consumption, hematology, organ weights and necropsy

None of the animals died. Clinical observations. body weight development, food consumption, hematology profile, and organ weights were not affected by treatment. No treatment-related necropsy findings were observed.

Neurotoxicological endpoints

Observations and functional tests conducted as part of the Careful Clinical Observations and Functional Observational Battery, motor activity measurements, as well as, histological examination of peripheral and central nervous system tissues, eyes and muscles evidenced absence of a neurotoxic potential of the test item.

Water consumption

Mean water consumption of the male and female high dose group (1000 mg/kg bw per day) was significantly increased throughout the whole treatment period. During the recovery period, water consumption declined in both sexes to normal values.

Blood chemistry

Treatment resulted in higher plasma urea levels at 100, 300 and 1000 mg/kg bw per day with higher creatinine levels at 1000 mg/kg bw per day in males and females. In addition, a disturbance of the electrolyte balance was in evidence with lower values for sodium recorded for males and females at 100, 300 and 1000 mg/kg, and lower values for chloride recorded for males and females at 1000 mg/kg and for males at 100 and 300 mg/kg. Males at 1000 mg/kg also had minimally lower values for calcium and females at 300 and 1000 mg/kg had minimally lower potassium levels. Following a 4-week recovery period, normal values were recorded for the above parameters except for calcium that remained at a lower level in males.

Urine analysis

At the end of the treatment period, males at 1000 mg/kg excreted a larger urine volume. Following the 4-week recovery period, the urine excreted by previously treated rats was similar to that excreted by the respective controls.

Microscopical findings

Microscopically, hepatocellular hypertrophy was increased in dose groups 4 and 5 of either sex, but not after 4-week recovery.

Conclusion

The changes seen on some blood chemistry parameters (at dose levels of CA 2342 A of 100 mg/kg and above) point to influencing the renal function. In addition, adaptive changes of the liver were observed. Reversibility was demonstrated for all changes within a 4-week treatment free period except for calcium blood levels in males. Due to the irreversibility in males, a No-Observable-Adverse-

Effect Level (NOAEL) of 300 mg/kg body weight per day is defined for males and 1000 mg/kg body weight per day for females.

Remarks on GHS classification

For a classification as Category 2 STOT-RTD (specific target organ toxicity, repeated dose toxicity), both UN-GHS and EU-GHS (CLP) are using as guidance values for the 28 days oral chronic toxicity test a cut-off value of 300 mg/l (daily gavage) as observed effect level. Since the single observed irreversible health effect, the calcium blood level in males, occured at a higher dose only, GHS classification does not apply for chronic oral toxicity of CA 2342 A. This is also supported by section 3.9.2.8 in the GHS and CLP documents, where it is stated that small changes in blood chemistry would not justify classification.