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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 11, 2002 to December 02, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals
Number: 88 Sprague-Dawley rats (44 males and 44 females) were received at CIT on 19 November 2002.
Strain and Sanitary status: Crl CD® (SD) IGS BR, Cesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
Breeder: Charles River Laboratories, l’Arbresle, France.
Age/Weight: on the first day of treatment, the animals were approximately 6 weeks old and had a mean body weight of 185 g (range: 142 g to 205 g) for the males and 153 g (range: 127 g to 172 g) for the females.
Receipt: on arrival, the animals were given a clinical examination to ensure that they were in good condition.
Acclimation: an 8-day acclimation period to the conditions of the study preceded the beginning of the treatment period. A larger number of animals than necessary was acclimated to permit selection and/or replacement of individuals.
Allocation to groups: during the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition and allocated to the groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar.
Identification: each animal was identified by an individual ear tattoo. At the beginning of the study, each animal received a unique CIT identity number.

Environmental conditions
From arrival at CIT, the animals were housed in a barriered rodent unit, under specific pathogen free SPF) standard laboratory conditions.
The animal room conditions are set as follows:
. temperature : 22 ± 2°C
. relative humidity : 50 ± 20%
. light/dark cycle : 12h/12h (7:00 - 19:00)
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously and the records checked daily and filed. The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- Type: Oral via diet in feed
- Vehicle: Plain diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of food showed dosages were within 10% of nominal concentrations and mean achieved dosages of test substance were 0, 42, 84 or 175 mg a.i./kg bw/day for the males and 49, 96 or 201 mg a.i./kg bw/day for the females.
Duration of treatment / exposure:
13 week
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations: 0, 1500, 3000 and 6000 ppm of test material in diet corresponds to active substance DDAC (males/females): 1500 ppm: 42 /49 mg/kg/d; 3000 ppm : 84/96 mg/kg/d; 6000 ppm : 175 / 201 mg/kg/d
Basis: actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, plain diet
Details on study design:
A total of 80 (40 males and 40 females) Sprague-Dawley rats was assigned to one control and three treated groups, each comprising of 10 males and 10 females. One control group received untreated diet and three treated groups received the test item mixed with the diet at constant concentration of 1500, 3000 or 6000 ppm of test substance (corresponding to 644, 1287 or 2574 ppm of DDAC). The animals were checked twice daily for mortality and clinical signs were observed once a day. All animals were subjected to a detailed clinical observation on a weekly basis.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
The body weight of each animal was recorded once before allocation of the animals to 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): Yes
The quantity of food consumed by the animals of each cage was recorded once a week (over a 7-day period) during the study. Food consumption was calculated per animal and per day.

Ophthalmology: Yes
Ophthalmological examinations were performed before the beginning of the treatment period on all animals and in week 12 (control and high-dose groups). The pupils of the animals were dilated with tropicamide (Mydriaticum®, Merck Sharp & Dohme - Chibret, Paris, France). After assessment of the corneal reflex (by instillation of tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy.

HAEMATOLOGY: Yes
- Number of animals: all animals
- Time points: end of study
- Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, prothrombin time

CLINICAL CHEMISTRY: Yes
- Number of animals: all animals
- Time points: end of study
- Parameters: sodium, potassium, chloride, calcium, Inorg. Phosphorous, glucose, urea, total bilirubin, creatinine, total protein, albumin, total cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lipids.

URINALYSIS: No
Sacrifice and pathology:
Sacrifice
On completion of the treatment period, after at least 14 hours fasting, all animals were asphyxiated by carbon dioxide and killed by exsanguination.

Organ weights
The body weight of all animals killed at the end of the treatment period was recorded before sacrifice, and the following organs were weighed wet as soon as possible after dissection: Adrenals, Aorta, Brain (including medulla/pons, cerebellar and cerebral cortex), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes with Harderian glands, Femoral bone with articulation, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs with bronchi, Lymph nodes (mandibular and mesenteric), Mammary glands/area, Ovaries (with oviducts), Pancreas, Pituitary gland, Prostate (dorso-lateral and ventral), Rectum, Salivary glands (sublingual and submandibular), Sciatic nerve, Seminal vesicles (including coagulation gland), Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with bone marrow, Stomach with forestomach, Testes, Thymus, Thyroids with parathyroids, Tongue, Trachea, Urinary bladder, Uterus (horns and cervix), Vagina. Additionally, mesenteric lymph nodes of the low- and intermediate-dose groups were examined.

The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Preservation of tissues
For all study animals, the tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes and Harderian glands which were fixed in Davidson's fixative, and the testes and epididymides which were preserved in Bouin's fluid).

Preparation of histological slides
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin. This work was performed at Novaxia (Yvoy le Marron, France) under the responsibility of CIT.

Microscopic examination
A microscopic examination was performed on:
. all tissues listed in the Tissue Procedure Table for animals of the control and high-dose groups (groups 1 and 4) killed at the end of the treatment period,
. mesenteric lymph nodes of the low- and intermediate-dose groups,
. all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) killed on completion of the treatment period.
Other examinations:
-Other examinations: Functional observation battery (FOB)
All animals were evaluated once at the end of the treatment period. This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity. The animals were randomized. All animals were observed in the cage, in the hand and in the standard arena. Then, the following parameter measurements, reflexes and responses were recorded:
. touch response,
. forelimb grip strength,
. papillary reflex,
. visual stimulus response,
. auditory startle reflex,
. tail pinch response,
. righting reflex,
. landing foot splay,
. at the end of observation: rectal temperature.
Motor activity of all animals was measured by automated infra-red sensor equipment over a 15-minute period in week 12.
Statistics:
Depending on normal distribution (Kolmogorov–Lilliefors test, possibly after a Logarithmic transformation of the values – except for organ weights) and following testing of homogeneity of variances between groups: Bartlett test (3 or more groups) or Fisher test (2 groups). If Homogeneous: Student test (2 groups) or Dunnett test (3 or more groups), Else: Dunn test (3 or more groups) or Mann–Whitney/Wilcoxon test (2 groups)
If not normal distribution: Dunn test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During week 2, most of the animals treated at 6000 ppm showed emaciated appearance. This finding was consecutive to the body weight loss recorded during the first week of treatment. Soft feces were generally confined to week 2 observation only, except for 7/10 females in which this finding lasted during the whole treatment period. Piloerection was noted during week 2 in a few males and in the majority of females treated at the highest concentration. All these findings were ascribed to the test-treatment. The few other clinical signs observed were not considered to be of toxicological importance as they are commonly recorded in rat housed in laboratory (e.g. alopecia) or they were noted at very low incidence (e.g. round back in 1/10 females of group 4). Additionally, these signs were observed during a part of the study period only, and generally resolved again.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During week 1, animals treated at 6000 ppm lost weight (males: -9 g, p<0.01 ; females: -14 g, p<0.01) and the body weight gain of animals treated at 3000 ppm was lower than that of controls (males: 39 g vs. 64 g, p<0.01; females: 21 g vs. 30 g, p<0.05). Afterwards, the body weight gain of the animals of these groups was still affected, reaching statistical significance in the high-dose group. Throughout the dosing period, the body weights of animals treated at 3000 and 6000 ppm were significantly lower than those of controls. Decrease in body weight gain, noted in treated males and females at 1500 ppm during the first week of the study was considered to be related to palatability of the compound in the diet, resulting in a slightly lower food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
When compared to controls, food consumption of animals treated at 6000 and 3000 ppm was affected during the whole treatment period, with a more marked effect at the highest concentration and during the first week of the study. At 1500 ppm, the decreased food consumption was considered to be related to palatability of the compound when mixed with the diet, as this difference was only noted during week 1.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ocular findings were observed in week 12.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with control values, a decrease in WBC (white blood cell) count was recorded in treated males and in females treated at 6000 ppm. When looking to the relative fractions of lymphocytes and neutrophils, these were not very much changing over the groups, and not deviating to the CIT historical data. Although the statistical significance noted in all treated male groups and in the top dose female group could be explained by the elevated mean WBC values compared to the mean CIT historical data, a relationship to treatment with the test substance could not be ruled out. None of the other differences were considered to be relevant since they were slight and all the individual values were within the range of CIT historical background data (e.g. increase in Mean Cell Volume in treated males of groups 3 and 4, increase in Mean Cell hemoglobin in treated males of group 4, increase in Red Blood Cell count and Packed Cell Volume in males and females of group 4) or did not show any dose-relationship (decreased Mean Cell hemoglobin and Mean cell Hemoglobin Concentration in treated females of group 2). This decrease in prothrombin time noted in treated males at 6000 ppm was not considered to be of toxicological importance since it was very slight and all individual values were within the range of CIT historical control data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test substance at 1500 ppm did not cause any changes of biological relevance in blood biochemical parameters. Compared with controls, the following changes in blood biochemical parameters were considered to be treatment-related:
. increase in sodium, chloride and inorganic phosphorus plasma levels at 6000 ppm,
. a tendency for decrease in glucose levels in males and females at 6000 ppm and in females at 3000 ppm,
. a tendency of an increased urea levels in treated males and in treated females at 6000 ppm (although no statistical significance was reached in females). Decrease in cholesterol level in treated females, although there was no dose-relationship between treated groups 2 and 3,
. decrease in triglyceride level in treated males at 3000 and 6000 ppm (when the abnormal high value of male A29704 was excluded, the mean concentration of triglyceride recorded for the lowest group was comparable to that of controls),
. decrease in protein concentration in the 6000 ppm group,
. increase in A/G ratio in males treated at 3000 and 6000 ppm and in females treated at 6000 ppm. This difference was related to a globulin decrease in males. In females, an albumin decrease was noted at 6000 ppm,
. increase in transaminase activities in females treated at 6000 ppm, with a more marked increase in Alkaline phosphatase activity.
It is to be noted that most of these changes were not accompanied with histopathological findings, for instance in the liver and/or in the kidneys. All other differences were not considered to be relevant since they were very slight, all the individual values were within the range of CIT historical background data (e.g. decrease in calcium in treated females at 6000 ppm), and/or no dose-relationship could be noted (e.g. increased in creatine levels in treated males).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Observation Battery (FOB)

Detailed clinical observation and reactivity to manipulation or to different stimuli
There was no evidence of disturbance of either autonomic or physiological functions at any dose-level.

Motor activity
There were no differences in the measured motor activity that could be attributed to treatment with the test item.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute mean weights of the kidneys and liver were lower in the treated animals than the lowest control. Taking into consideration the body weight loss in the treated animals, the differences in the relative weights of these two organs were minor and their absolute and relative individual weights were out of the range of the control group only in few treated animals. Moreover, no histopathological abnormality correlated these lower organ weights. They were thus considered to be without toxicological significance. Some other differences were observed in the mean weights of other organs. These differences were not dose-related or minor and/or without the same trend in the two sexes. Moreover the individual values of organ weights in the treated groups were for the most part in the range of the control group. The differences of the mean weights were thus considered to be of no toxicological importance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Relevant necropsy findings were observed in the cecum, colon and mesenteric lymph node:
. distension with feces of the cecum and/or colon, among animals given 3000 ppm or 6000 ppm of test substance, expressed a fecal stasis. Although this was not associated with relevant histopathological abnormalities, a relationship to treatment with the test substance cannot be ruled out. This finding and could be due to an effect on the functional activity of the large intestine,
. brownish, reddish or greenish color of the mesenteric lymph node, among animals given 3000 ppm or 6000 ppm of test substance. This was correlated with higher severity of histiocytosis at microscopic examination. A relationship to treatment cannot be ruled out.
The few other macroscopic post-mortem observations noted were not dose-related and/or were those commonly recorded spontaneously in the untreated laboratory rat of this strain and age. They were thus considered to be without toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In animals given 1500 ppm, 3000 ppm or 6000 ppm of test substance were noted:
. higher mean severity of histiocytosis (minimal to moderate), often with brownish-yellowish intracytoplasmic pigment and sometimes associated with sinusal hemorrhage (minimal to slight), accompanied by colour changes observed at necropsy, was seen at the two highest dose group,
. there seems a dose related increase of mastocytosis from minimal mastocytosis involving less than half of the animals in controls, to slight mastocytosis involving almost all animals in the high dose.
A relationship of the marginally higher mean severity of these findings in the animals given 3000 ppm or 6000 ppm to the treatment cannot be ruled out.
All the other microscopic observations encountered were minor and/or not dose-related and were those which are commonly recorded spontaneously in the untreated laboratory rat of this strain and age. They were thus considered to be without toxicological importance.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body weight; food consumption; gross pathology; histopathology;
Remarks on result:
other: equivalent to 644 ppm a.i.
Remarks:
equivalent to 42 mg a.i./kg bw/day for the males and 49 mg a.i./kg bw/day for the females
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body weight; food consumption; gross pathology; histopathology;
Remarks on result:
other: equivalent to 1287 ppm a.i.
Remarks:
equivalent to 84 mg a.i./kg bw/day for the males and 96 mg a.i./kg bw/day for the females
Key result
Critical effects observed:
not specified

0, 1500, 3000 and 6000 ppm of test substance, containing 42.9% of active ingredient DDAC. (0, 644, 1287 and 2574 ppm based on a.i.)

At 6000 ppm, main treatment-related findings were: soft feces, body weight loss (week 1), lower body weight gain and food consumption, perturbation of hematological and blood biochemical parameters, distension of the cecum/colon with feces in all animals, histiocytosis, mastocytosis and sinusal hemorrhage in the mesenteric lymph node, consistent with a continued action of a mild irritant.

At 3000 ppm, body weight gain and food consumption were affected, changes at clinical pathology were noted, the cecum was distended with feces in about third of the animals, histiocytosis and mastocytosis in the mesenteric lymph node were seen at histopathology.

At 1500 ppm, changes in hematological and blood biochemical parameters were recorded and possibly a marginal increase in histiocytosis and mastocytosis in the mesenteric lymph node which all do not immediately seem to be of toxicological significance.

The most obvious effect is the lowering of the food intake caused by the palatability of the compound, which at lower levels improves after some time. Some effects seen in haematology and blood chemistry at the higher dose levels could very well result from a poor alimentary status as opposed to a specific toxic mechanism. At the highest dose level, the finding of distended cecum and/or colon becomes noteworthy, and possibly a higher severity of histiocytosis at microscopic examination, although this aspect is also already observed in controls.


Conclusions:
Based on the results of the read across study, the NOAEL of the test substance is considered to be 1500 ppm (equivalent to 42 mg a.i./kg bw/day for the males and 49 mg a.i./kg bw/day for the females) in rats.
Executive summary:

A study was conducted to evaluate the repeated dose dietary toxicity in rats of the read across substance, DDAC (42.9% active), using the method according to the OECD Guideline 408, in compliance with GLP. The read across substance was given by dietary admixture to Sprague-Dawley rats for 13 weeks at concentration of 0, 1500, 3000 and 6000 ppm (equivalent to 0, 644, 1287 and 2574 a.i. ppm). Analysis of food showed dosages were within 10% of nominal concentrations and mean achieved dosages of test substance were 0, 42, 84 or 175 mg a.i./kg bw/day for the males and 49, 96 or 201 mg a.i./kg bw/day for the females. The animals were checked twice daily for mortality and clinical signs were observed once a day. All animals were subjected to a detailed clinical observation on a weekly basis. Neurotoxicity was assessed by a functional observation battery (including detailed clinical observation and reactivity to manipulation or to different stimuli) and by measurement of motor activity at the end of the treatment period. Body weight and food consumption were recorded before the beginning of the study and then once a week. The eyes of animals from control and high-dose groups were examined at the end of the treatment period. Hematological and blood biochemical parameters were determined at the end of the study. At scheduled necropsy, the animals were sacrificed, designated organs and tissues were weighed and preserved. A macroscopic post-mortem examination was performed on all animals. A microscopic examination was carried out for animals of the control and high-dose groups, on macroscopic lesions from all groups, and on mesenteric lymph nodes of the low- and intermediate-dose groups. At 6000 ppm, main treatment-related findings were: soft feces, body weight loss (at wk 1), lower body weight gain and food consumption, perturbation of hematological and blood biochemical parameters, distension of the cecum/colon with feces in all animals, histiocytosis, mastocytosis and sinusal hemorrhage in the mesenteric lymph node, consistent with a continued action of a mild irritant. At 3000 ppm, body weight gain and food consumption were affected, changes at clinical pathology were noted, the cecum was distended with feces in about third of the animals, histiocytosis and mastocytosis in the mesenteric lymph node were seen at histopathology. At 1500 ppm, changes in hematological and blood biochemical parameters were recorded and possibly a marginal increase in histiocytosis and mastocytosis in the mesenteric lymph node which all do not immediately seem to be of toxicological significance (Chevalier, 2004). Based on the results of the read across study, the NOAEL of the test substance is considered to be 1500 ppm (equivalent to 42 mg a.i./kg bw/day for the males and 49 mg a.i./kg bw/day for the females) in rats.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion

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