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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, equivalent to OECD TG 407.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: E.E.C. Recommendation No. 84/449/E.E.C., Annex VB, 19th September 1984
GLP compliance:
yes
Remarks:
Centre International de Toxicologie, France
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,6 DCPTFMA
- Physical state: solid
- Storage condition of test material: room temperature
- Analytical purity: 89,7 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France (76410 Saint-Aubin-lès-Elbeuf, France)
- Age at study initiation: about 6 weeks old
- Weight at study initiation: mean 206 g for the males and 180 g for the females
- Housing: 3 rats of the same sex and group
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in the vehicle and homogenized using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance preparations were made on a weekly basis by the C.LT. Pharmacy.
- Storage temperature of food: Each preparation was sampled the day of preparation and after 4 and 7 days. Storage at +4°C.

VEHICLE
- Lot/batch no. (if required): The vehicle was corn oil provided by C.P.F. (77000 Melun, France), batch No. A 24899 91552.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suspension was stirred with a magnetic stirrer. A 1 ml-volume of suspension was sampled into a test tube and diluted 10 times with isopropanol using a Microlab 1000 Hamilton dilutor. After vortex-mixing, serial dilutions were carried out with acetonitrile in accordance with a target concentration of 20 µg/ml. A 20 µl-aliquot was analysed by High Performance Liquid Chromatography with Ultra-Violet detection.
Duration of treatment / exposure:
29 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 160, 500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were determined in agreement with the Sponsor following a previous 2-week toxicity study by oral route in rats, conducted at doses of 125, 250, 500 and 750 mg/kg/day. In the previous study, ptyalism was observed at all dose levels, decrease in body weight was noted at 500 and 750 mg/kg/day and signs of poor physical condition were noted at 750 mg/kg/day.
Moreover, a decrease in thymus weight and liver hypertrophy were observed at all dose levels, associated at 500 and 750 mg/kg/day with blood biochemical changes. Thus, for the present study, 500 mg/kg/day was selected as the highest dose and 50 mg/kg/day as the lowest dose. The intemediate dose was 160 mg/kg/day, which represents the geometric mean between 50 and 500 mg/kg/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice a day for mortality and signs of morbidity, including weekends.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were observed for each animal at least once a day, at the same approximate daily time.


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


FOOD CONSUMPTION AND COMPOUND INTAKE:
The quantity of food consumed by the animals of each cage was recorded once a week (over a 7-day period) until the end of the study.
Food intake per animal and per day was calculated using the amount of food given and left in each feeder, divided by 3.


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: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on week 4
- Anaesthetic used for blood collection: Yes (identity): ether
- Animals fasted: Yes
- How many animals: all animals


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on week 4
- Animals fasted: Yes
- How many animals: all animals



URINALYSIS: Yes
- Time schedule for collection of urine: on week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following sequence was used for the statistical analysis of body weight, food consumption, haematology, blood biochemistry, urinalysis and organ weight data: The normality of the distribution of the values in each group was checked by Kolmogorov-Smirnov's test (1948).
If the distribution was normal, the homogeneity of variances between the groups was assessed by Baden's test (1937) (more than 2 groups) or Fisher's test (1934) (2 groups).
If no significant heterogeneity of the variances was established, the comparison between treated and control groups was performed by Dunnett's test (1955).
If the variances were 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 was not normal, the analysis was repeated after logarithmic transformation of the values (except for organ weights).
If this logarithimic transformation failed to normalise the distribution of the values, comparison of treated and control groups was perfomed by Dunn's test using original values.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY: Ptyalism was observed in all animals given 160 or 500 mg/kg/day, from week 1 or 2 to the end of the treatment period, and in almost all the animals given 50 mg/kg/day (4/6 males and 3/6 females) from week 3 or 4 to the end of the treatment period. This clinical sign was considered to be treatment-related, and the latency was dose-related. Regurgitation was observed in one male of the control group, and pallor of extremities, cold to the touch, hypokinesia, abdominal breathing and right eye damaged were observed in one male given 500 mg/kg/day, after the blood sampling. These clinical signs were considered to be accidental and not treatment-related.
No mortalities occurred during the study.


BODY WEIGHT AND WEIGHT GAIN: A decrease in mean body weight gain was observed in males given 500 mg/kg/day: -16% when compared to that of respective controls. Although this difference was not statistically significant, it was related to the treatment. The mean body weight gain of males given 50 or 160 mg/kg/day and all treated females was similar to that of respective controls.


FOOD CONSUMPTION: During the treatment period, the mean food consumption of all treated animals was similar to that of respective controls.



HAEMATOLOGY: A slightly lower (approximately -9%) erythrocyte count, haemoglobin concentration and packed cell volume was noted in females given 500 mg/kg/day when compared to respective controls. A slightly lower mean cell haemoglobin concentration (-3% to -4%) was noted in males given 160 mg/kg/day and in males and females given 500 mg/kg/day, when compared to respective controls. As these differences were minimal and the individual values were within the range of the background data, they were considered to be of no toxicological importance. No perceptible differences between treated and control animals were observed in the other haematological parameters.


CLINICAL CHEMISTRY: On week 4, when the values obtained in treated animals were compared with that of the respective controls, the following differences were observed:
- moderately higher total protein (+15% to +30%) with a slightly lower A/G ratio (-12% to -14%) in males and females given 500 mg/kg/day,
- moderatly higher total cholesterol (x 1.7 to x 2.7) in animals of both sexes given 500 mg/kg/day. This was associated with a moderately higher triglyceride (x 3) in females of the same group.
- moderately lower (approximately -40%) alkaline phosphatase activity (ALP) in the males and females of the 500 mg/kg/day group,
- moderately higher aminotransferases (ASAT and ALAT) activities in males and females given 500 mg/kg/day (x 1.3 to x 2).
All these differences were considered to be treatment related. The other minor differences noted between treated and control animals namely in inorganic phosphorous, glucose, urea and creatinine, were considered to be of no toxicological importance as they were minimal, not dose related and the individual values were within the range of the background data.


URINALYSIS: Increase in number of animals with high urine volume was noted in animals of both sexes given 500 mg/kg/day when compared to respective controls. However, in the absence of relevant qualitative or quantitative abnormalities in the other urine parameters and the blood biochemical
parameters of kidney function, the increase in urine volume was considered to be of no toxicological importance.



ORGAN WEIGHTS: A higher mean absolute and relative liver weight was noted in the treated animals when compared to respective control values (see table 1). These differences from controls were considered to be treatment-related.
A statistically significant higher statistically significant (but not dose-related) mean absolute and/or relative kidney weight was noted in the males given 160 or 500 mg/kg/day, when compared to control values (absolute weight: +18% and +4% (NS); relative weight: +14% and
+13%, respectively). Taking into account that these differences were minor, not dose-related and not well correlated with the treatment-related microscopic changes noted in the kidneys of treated males, they were considered to be of minor toxicological importance. A statistically significant higher mean absolute (+11%) and relative (+12%) kidney weight was noted in the females given 500 mg/kg/day. A statistically significant higher mean absolute or relative testis weight was noted in the males given 160 mg/kg/day (absolute weight: +10%) and in the males given 500 mg/kg/day (relative
weight: +12%). These differences from control values were minor, not associated with any relevant microscopic changes, and were considered to be not treatment-related. No other perceptible differences were noted in the organ weights of the treated animals when compared to controls.


GROSS PATHOLOGY: An enlarged liver was noted in 4/6 males and 1/6 females given 160 mg/kg/day and in all animals of both sexes given 500 mg/kg/day. This was associated to an accentuated lobular pattern in one of these males in the high dose group, and to many punctifom greyish/whitish foci in the left lateral lobe and median lobes of the liver in one of these females of the same group. All these macroscopic findings were considered to be treatment-related, and were well correlated with the microscopic changes noted in the liver.
An irregular or grey/green colour was observed in the kidneys of 1/6 males given 50 mg/kg/day, in 4/6 males and 1/6 females given 160 mg/kg/day and 1/6 males. These variations in colour, although not dose-related, could be related to the accumulation of acidophilic globules in cortical tubular epithelium seen at microscopic examination. No relevant microscopic findings could explain these macroscopic changes in colour noted in the females. Consequently, these macroscopic changes were considered to be of minor toxicological importance. Enlarged hepatic lymph nodes or pancreatic lymph nodes were noted in 1/6 males given 160 mg/kg/day and in 2/6 males given 500 mg/kg/day. These macroscopic findings can be found spontaneously in untreated laboratory rats of this strain. The incidence reported here was considered to be of no toxicological importance. The other macroscopic findings noted are those which are commonly recorded as spontenous in untreated laboratory rats of this strain. Accordingly, they were considered to bear no relationship to the treatment.

HISTOPATHOLOGY: A dose-related minimal to marked hepatocellular hypertrophy was noted in the liver of 3/6 males and 3/6 females given 50 mg/kg/day and of all animals of both sexes given 160 mg/kg/day or 500 mg/kg/day. The hepatocellular hypertrophy was accompanied by a minimal to moderate focal or multifocal hepatocellular degeneration/necrosis in 1/6 males given 50 mg/kg/day, 1/6 males given 160 mg/kg/day and in 4/6 males and 1/6 females given 500 mg/kg/day. All these above-mentioned findings were considered to be treatment-related.
A moderate zonal coagulative hepatocellular necrosis surrounded by a moderate fibrosis (with multifocal calcification and multinucleated giant cells) were noted in 1/6 males given 50 mg/kg/day and in 1/6 females given 160 mg/kg/day. The same type of fibrosis was also noted without coagulative hepatocellular necrosis in another 1/6 males given 50 mg/kg/day. A minimal or slight focal or multifocal interlobular fibrosis (often associated with yellowish/greenish pigment laden macrophages) was noted in 1/6 males given 50 mg/kg/day, in 3/6 males given 160 mg/kg/day and in 1/6 females given 500 mg/kg/day. An accumulation of acidophilic globules was noted in the cortical tubular epithelium of kidneys in 3/6 control males (minimal to slight severity), in all males given 50 mg/kg/day (minimal to moderate), 160 mg/kg/day (slight to moderate) or 500 mg/kg/day (slight to marked). In the males given 50 mg/kg/day, the severity of this finding was comparatively similar to that of controls. Consequently, the higher incidence reported in that group was considered to be of no toxicological importance. The higher incidence and severity noted in the males given 160 or 500 mg/kg/day were considered to be treatment-related. A moderate chronic thymitis and perithymitis, a moderate chronic inflammation of adventitia in aorta and adjacent tissue, a moderate chronic endocarditis together with a moderate chronic myocarditis in the right atrium of heart was noted in 1/6 males given 500 mg/kg/day. In addition, a slight focal subacute myocarditis was noted in the left ventricle of the heart in 1/6 females of the same treated group. As these findings can be found spontaneously in untreated laboratory rats of this strain the low frequency reported here was considered to bear no relationship to the treatment. The other microscopic findings noted were those which are commonly recorded as spontaneous in laboratory rats of this strain. Furthermore, their incidence severity and morphological characteristics were similar between treated and control groups. Thus, they were considered to be not treatment-related.


Effect levels

Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: hepatocellular hypertrophy accompanied by focal or multifocal hepatocellular degeneration/necrosis

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Liver weights:

Dose

Sex

Differences (%) from controls in mean

mg/kg/day

 

Absolute liver weight

Relative liver weight

50

Male

+14.5 (ns)

+12 (ns)

Female

+14 (ns)

+17 *

160

Male

+43 **

+37 **

Female

+43 **

+51 **

500

Male

+87 **

+106 **

female

+137 **

+140 **

ns: not statistically significant

*/**: p < 0.05 or 0.01, Dunnett's test

Applicant's summary and conclusion