Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May to June 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
454-800-3
EC Name:
-
Cas Number:
70258-18-3
Molecular formula:
C6H5Cl2N
IUPAC Name:
2-Chloro-5-chloromethylpyridine
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: not specified
- Weight at study initiation: 99-130 g (males), 95-115 g (females)
- Housing: 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55+/- 5
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
administration volume: 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical content checks of the test item in the vehicle performed twice in all concentrations during the study demonstrated that the test substance concentrations in the vehicle agreed to the target values within defined limits. The formulations to be used were homogeneous as the formulations were a clear solution.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
0.5 mg/kg bw/day (actual dose received)
Dose / conc.:
2.5 mg/kg bw/day (actual dose received)
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE:
Dose levels have been selected according to results obtained in a previous 2-week oral study (No. T3075632) with 0, 10, 50, 250 and 500 mg/kg body weight/day performed in rats. In this study 250 and 500 mg/kg were lethal. There was a very slight body weight depression in rats receiving 50 mg/kg. Necropsy revealed severe changes at the stomach of 250 and 500 mg/kg rats. Histopathology (done only in rats receiving 0, 10, or 50 mg/kg) revealed changes in
the stomach from 10 mg/kg onwards in males and females as follows: There was a squamous hyperplasia in two of three 10 mg/kg males and in all males and females receiving 50 mg/kg. Prominent basal cells were noted in all three males at 10 and 50 mg/kg as well as in two of three 10 mg/kg and all three 50 mg/kg females. In two of three 50 mg/kg males and all three 50 mg/kg females stomach edema were evident. Thus, the dose levels of 0, 0.5, 2.5 and 12.5 mg/kg were used in the present study.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
On working days the experimental animals were inspected twice a day for morbidity and mortality (once on weekends and public holidays). General clinical observations (in the home cage) were made daily about 30 minutes after administration. Once before the start of treatment and once weekly thereafter animals were clinically examined in detail including observations in a standard arena (open field) for behavioral observations. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed. Findings and abnormalities were recorded either using a coding system or uncoded.

FUNCTIONAL OBSERVATIONAL BATTERY:
Functional observations were performed once (not blind). The following observations/examinations were performed: body weights, home-cage observations (posture, piloerection, gait abnormalities, involuntary motor movements, vocalization, others), observations during handling (ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrimation, nasal discharge, salivation, stains, body temperature, others), open field observations (piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalization, arousal, rearing, defecation, urination, others), reflex/physiological observations (approach response, touch response, auditory response, tail pinch response, pupil size, pupil response, righting reflex, body temperature, grip strength, foot splay).

MOTOR ACTIVITY:
Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA). Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.

OPHTHALMOLOGICAL INVESTIGATION:
An ophthalmological investigation was done. The pupillary reflex of both eyes was first tested in a darkened room and the anterior regions of the eye were inspected. After dilating the pupils with Mydriaticum Stulln® drops, the
refractive elements of the eye as well as iris and fundus were examined using an indirect ophthalmoscope. In addition, the optical media were examined with a ZEISS photo-slit lamp.

BODY WEIGHTS:
The body weights of the animals were determined before the beginning of the study and weekly thereafter up to scheduled necropsy. The body weights were used to calculate the appropriate administration volumes. The corresponding administration volumes, which were recorded on-line, were filed together with the study raw data and will not be reported herein. Furthermore, body weights were also recorded immediately before scheduled necropsies for calculation of relative organ weights.

FOOD AND WATER INTAKE:
Food and water intake was determined individually at comparable periodical intervals (e.g. weekly). These primary data were then used to calculate the group means for each period of approximately 7 days. The weight of the food/water offered at the start of the measurement period minus the food/water at the end of the period is defined as the food/water consumption of the animal in g.

HEMATOLOGY:
The following hematological parameters were determined in peripheral blood (on all animals per group near termination):
Differential blood count, erythrocytes, hemoglobin, hematocrit, leucocytes, reticulocytes, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), thromboplastin time (Hepato-Quick), platelets.

CLINICAL CHEMISTRY:
The following parameters were determined in peripheral blood (on all animals per group near termination):
Alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, albumin, albumin globuline quotient, total bilirubin, cholesterol, creatinine, total protein, urea, glucose, triglycerides, potassium, sodium.

URINALYSES:
The following parameters were determined in urine (on all animals per group near termination):
Quantitatively: Volume, density, protein, protein per volume, creatinine, creatinine per volume, protein creatinine quotient as well as microscopy of sediment.
Semiquantitatively: pH, glucose, ketones, bilirubin, urobilinogen, blood.

Sacrifice and pathology:
NECROPSY:
All animals were killed as scheduled for terminal necropsy by exsanguination under deep diethyl ether anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. Changes were described in terms of localization, size, color and consistency whenever appropriate. Animals that died spontaneously during the study were necropsied at the earliest opportunity. From these animals the organs and tissues were handled as described above.

ORGAN WEIGHTS:
The following organs of the animals killed at the end of the treatment were weighed before fixation: brain, heart, liver, spleen, kidneys, thymus, adrenals, epididymides, testes and ovaries. The organ weights are specified in both absolute and relative terms. The relative weights were calculated in % of the terminal body weight.

HISTOPATHOLOGY:
The following organs/tissues were collected and fixed in 10 % neutral buffered formalin or Davidson's solution: Adrenals, aorta, brain (cerebrum, cerebellum, brain stem), epididymides, esophagus, eyes, eyelids, extraorbital lacrimal glands, femur, head (with skull cap), Harderian glands, heart, intestine (Peyer's patches, duodenum, jejunum, ileum, cecum, colon, rectum), kidneys, larynx, liver, lungs, lymph nodes (mandibular, mesenterics, popliteal), optical nerves, ovaries, oviducts, pancreas, pharynx, pituitary gland, prostate, salivary gland, sciatic nerve, skeletal muscle, skin (mammary region), spinal cord, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid glands with parathyroids, tongue, trachea, ureters, urethra, urinary bladder, uterus with cervix, vagina, Zymbal's glands and tissues with macroscopic findings. Histopathology was performed on all tissue specimens shown above at least in the control and high dose groups.
Other examinations:
none

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):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology revealed treatment-induced minimal to slight hyperplasia of the forestomach basal cells, which occurred in all males and 4 of 5 females at 12.5 mg/kg bw. Additionally, in the liver of females Kupffer cell activation was present at 12.5 mg/kg (incidence: 0-0-0-4) attributed to a minimal severity level.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
12.5 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
other: forestomach
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL: 2.5 mg/kg bw (target organs: forestomach, liver)
Executive summary:

2-Chlor-5-chlormethylpyridine was administered daily via gavage in polyethylene glycol 400 (PEG 400) to 5 male and 5 female Wistar rats per dose group, in doses of 0, 0.5, 2.5 and 12.5 mg/kg bw for a period of 4 weeks. Survival rate as well as behavior and appearance of the rats were not influenced by the treatment. There were no signs of neurotoxicity or oculotoxicity up to 12.5 mg/kg. Body weights were not affected up to 12.5 mg/kg. No toxicologically relevant change in food intake was seen up to 12.5 mg/kg. Hematology, clinical chemistry and urine parameters were not altered up to 12.5 mg/kg. Histopathology revealed treatment-induced minimal to slight hyperplasia of the forestomach basal cells, which occurred in all males and 4 of 5 females at 12.5 mg/kg. Additionally, in the liver of females Kupffer cell activation was present at 12.5 mg/kg (incidence: 0-0-0-4) attributed to a minimal severity level. Gross findings as well as organ weight measurements and histopathology in the other organs gave no indication of test substance-related effects up to 12.5 mg/kg. Under the conditions described the NOAEL (=NOEL) for 2-Chlor-5-chlormethylpyridine is established at 2.5 mg/kg body weight per day in male and female rats.

In a previous dose finding toxicity study for the main study groups of rats (3/sex/group) were given doses of 0, 10, 50, 250 and 500 mg/kg bw/d via gavage for a period of 14 consecutive days. Mortality occurred at 250 and 500 mg/kg bw/d. In rats receiving 50 mg/kg bw/d a slight body weight depression was noted. Necropsy revealed severe changes at the stomach in the died animals given 250 and 500 mg/kg bw/d. Microscopy of animals receiving 0, 10, or 50 mg/kg bw/d demonstrated changes in the stomach from 10 mg/kg bw/d onwards. There was a squamous hyperplasia in 2/3 males at 10 mg/kg bw/d and in all males and females at 50 mg/kg bw/d. Prominent basal cells were noted in 3/3 males at 10 and 50 mg/kg bw/d as well as in 2/3 females at 10 mg/kg bw/d and all three females at 50 mg/kg bw/d. Additionally at 50 mg/kg bw/d stoma edema was evident in 2/3 males and 3/3 females.

In summary, no test substance-related mortality occurred during the main study used dose levels up to 12.5 mg/kg bw/d. Relevant toxic effects were also noted in the forestomach in male and female rats. Microscopically, minimal to slight hyperplasia of the forestomach basal cells seen in the four week study at 12.5 mg/kg bw/d correlated with the observations in males and females given 10 and 50 mg/kg bw/d in the dose finding study.