Chlorphenesin

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August to September 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

reproductive toxicity, other

Remarks:

Data regarding reproductive toxicity obtained from the oral repeated dose toxicity study (see section 7.5.1 of this IUCLID dossier)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August to September 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: Data regarding reproductive toxicity obtained from the oral repeated dose toxicity study conducted similar to OECD407 (see section 7.5.1 of this IUCLID dossier)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: the test substance was stored in the original container under ambient conditions in non-continuous artificial light.

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 28 ± 1 days old
- Weight at study initiation: 74 - 103 g.
- Housing: the cages (each containing four rats) constituting each group were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments. Each cage was identified by a coloured label according to group. Each label displayed the study schedule number, cage number, sex, individual animal numbers and the initials of the Study Director and Home Office Licensee.
- Diet (e.g. ad libitum): free access to Biosure LAD 1 Diet. Biosure LAD was a closed formula diet suitable for normal health, growth and reproduction of laboratory rats and mice. The standards of production adopted by the manufacturers have been approved by the Quality Assurance Department. Analyses were made of all batches of diet for most nutrients and for specified substances and micro-organisms likely to be present in feed ingredients or the finished diet and which, if in excess of specified amounts, might have had an undesirable effect on the test system. Although occasional slight deviation may have been permitted, batches of diet conformed with the acceptable standards agreed by the Study Director and Quality Assurance.
- Water (e.g. ad libitum): free access to tap water. Results of the routine physical and chemical examination of drinking water at source as conducted usually weekly by the supplier. Additionally, levels of specified substances known to be present from time to time in local water and which, if in excess of the maxima recommended (for humans) might have had an undesirable effect on the test system, were determined in the tap water at approximately 6-monthly intervals.
- Acclimation period: 19 days was allowed between delivery of the animals and start of treatment.

DETAILS OF FOOD AND WATER QUALITY: analyses were made on all batches of diet used to establish levels of basic nutrients and of specified substances and micro-organisms likely to be present in feed components and which, if in excess of specified amounts, might have had an undesirable effect on the test system. Results of the routine physical and chemical examination of drinking water at source as conducted usually weekly by the supplier. Additionally, levels of specified substances known to be present from time to time in local water and which, if in excess of the maxima recommended (for humans) might have had an undesirable effect on the test system, were determined in the tap water at approximately 6-monthly intervals. Quarterly summary analyses of source water normally included levels of nitrites, nitrates, Ca, Mg, Na, K, P, CI, Si, Fe. Six-monthly analyses of HRC tap water included levels of As, Se, Ba, Ag, Sb, organophosphorus, organochlorine and other pesticides, haloforms, chlorophenols, polychlorinated biphenyls and polycyclic aromatic hydrocarbons.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): animal room temperature was controlled at a mean minimum of 21.0 °C and a mean maximum of 21.6 °C.
- Humidity (%): relative humidity was controlled at a mean minimum of 50.7 % and a mean maximum of 58.8 %.
- Air changes (per hr): air exchange was maintained at a rate of approximately 20 air changes per hour.
- Photoperiod (hrs dark / hrs light): lighting was controlled to give twelve hours artificial light in each twenty-four hour period.

Route of administration:

oral: gavage

Vehicle:

other: aqueous methylcellulose (1 % MC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the high dosage concentration of the test substance was prepared freshly each day as a 10.0% w/v suspension in 1% aqueous methylcellulose (1% MC). The intermediate (1.0% w/v) and low (0.1% w/v) dosage concentrations were prepared by direct dilution of the test substance with the vehicle. Prior to dosing, the test substance suspensions were mixed initially by inversion and sequently using a magnetic stirrer for a period of 5 to 15 minutes. Dosing was completed within one hour of the commencement of stirring.

VEHICLE
- Concentration in vehicle: 10.0%, 1.0% and 0.1% w/v suspension in methylcellulose (1% MC) corresponds respectively to dosage levels of 1000, 1000 and 10 mg/kg/day

Details on mating procedure:

The animals were chaged in groups of four of the same sex and no mating was planned during the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical verification of dose and concentration was performed by High performance liquid chromatograph (HPLC) using ultra-violet detection. A representative sub-sample (1 ml) of test formulation was accurately weighed and dissolved in a suitable volume of mobile phase. The solution obtained was further diluted with mobile phase to provide an expected concentration of Chlorphenesin between 4 and 8 mcg/mI. The final solution was filtered (Whatman GF/F) prior to quantification.

Typical chromatographic conditions
- Analytical column: Apex ODS, 5 mcm, 250 x 4.6 mm ID, Jones Chromatography Ltd.
- Guard column: RP 300 aquapore octyl, 7 mcm, 30 x 4.6 mm ID, Browniee Laboratodes Inc.
- Mobile phase: Methano/water (55/45 v/v).
- Detector: wavelength UV, 279 nm.
- Flow rate: 1.0 ml/minute.
- Injection volume 65 mcl.
- Integrator attenuation 32.
- Retention volume 8 ml

Determination of concentrations of Chlorphenesin in dose formulations prepared for Day 1 of the study
Representative samples (approximately 20 ml) of freshly prepared dose formulation, received from the Department of Formulation, were thoroughly mixed by shaking and duplicate sub-samples (1 ml) were analysed.

- Chemical stability
Freshly-prepared batches of test formulations, at nominal concentrations of 1 mg/ml and 100 mg/ml, were each sampled (6 replicates of approximately 1 ml) into pre-weighed volumetric flasks. Two samples were analysed immediately and the remaining four samples were stored in the dark at ambient temperature for 4 hours and at +40 °C for 24 hours before analysis.
At each time-point, two samples from each formulation were analysed.

- Physical stability
After sampling for chemical stability, the remainder of each test formulation was thoroughly mixed by repeated inversion and magnetically stirred for 5 minutes. Samples (approximately 1 ml) were removed from points approximately one-quarter, one-half and three-quarters the depth (representing the top, middle and bottom) of the formulation and analysed. The formulation was magnetically stirred for a further 1 hour and sampled, as above, at time-points corresponding to 0.5 hour and 1-hour storage.
The magnetic stirring was then discontinued and the remainder of each formulation was stored in the dark at -ambient temperature for a further 3 hours and subsequently at +4°C overnight. After storage for 4 and 24 hours, the suspension was re-mixed and sampled, as above, for analysis.
The three samples removed from each formulation, at each time-point, were analysed.

Calculation
The response for Chlorphenesin in each calibration chromatogram was measured and calibration curves were constructed by linear regression of standard response versus standard concentration. The response of the peak observed at the characteristic retention volume for Chlorphenesin in sample and procedural recovery chromatograms was measured and the concentration of Chlorphenesin calculated using the equation below.
Concentration, mg/ml = (Y-I)/S x (V/W) x D x 10-3
Where:
Y = Peak response in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution volume of sample (mi)
W = Weight of sample (g)
D = Density (g/ml)
Results were corrected for the appropriate cumulative mean procedural recovery value at analysis.

Limit of detection
The limit of detection, defined as the concentration of Chlorphenesin in control matrix producing a peak response equivalent to 3 x baseline noise, was determined as 0.075 mg/ml.

Results regarding verification of doses
The mean concentrations of Chlorphenesin in dose formulations analysed during the study and the deviation of mean results from nominal values are measured:mean results are all within 5% of nominal concentrations. Based on the data obtained during the study Chlorphenesin forms a homogeneous suspension in the 1% MC formulation. The data confirm that homogeneity can be maintained by magnetic stirring for 1 hour and that the formulations can be successfully re-suspended 4 and 24 hours after preparation. The chemical stability of Chlorphenesin in 1% MC formulations, was confirmed for a period of 24 hours .
The data confirm that the analytical method described is both precise and accurate for Chlorphenesin in 1% MC formulations. A mean recovery of 101.1% ± 1.38% SD (n=8) was obtained at 1 mg/ml and 103.4% ± 1.85% SD (n=8) at 100 mg/ml. Results are corrected for the appropriate cumulative mean procedural recovery value at analysis.

Duration of treatment / exposure:

Twenty-eight consecutive days (Dosing started on 29 August 1989 and ended on 25th September 1989).

Frequency of treatment:

Animals were treated once daily for twenty-eight consecutive days. Treatment of animaIs not scheduled for sacrifice on Day 29 continued until the day prior to being kilied.

Details on study schedule:

Animals were treated once daily for twenty-eight consecutive days. The animals were chaged in groups of four of the same sex and no mating was planned during the study. Treatment of animals not scheduled for sacrifice on Day 29 continued until the day prior to being killed.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per dose (5 males and 5 females)+ 6 animals per dose as satellite subgruops (3 males and 3 females). Total animals effective used in the test: 64 animals. One animal of satellite group was killed during the test.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: dosage levels of 10, 100 and 1000 mg/kg/day were selected on the basis of acute oral toxicity data available from the Sponsor and a 7-day preliminary oral toxicity study in the rat.
- Rationale for animal assignment (if not random): before the start of treatment (Week -1) each animal was weighed and sixty-four rats were randomly allocated to four groups, each consisting of eight males and eight females. This allocation was carried out using a computer program. Following this random allocation five male and five female rats from each group were selected for allocation to the main study (rats with the lowest study numbers were chosen).
- Rationale for selecting satellite groups: the remaining rats within each group (three males and three females) were allocated to satellite sub-groups. These satellite groups were used for terminal immunological investigations
- Post-exposure recovery period in satellite groups: after 28 days of treatment (Day 29) all animals from the main study were randomly killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The macroscopic appearance of the tissues was recorded. All surviving rats in the satellite groups were similarly killed on Days 29 to 32 (five or six animals per day).

Positive control:

Not present.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: all animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post-mortem examination to be undertaken during the working part of that day. At weekends a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was isolated, and if found to be in extremis was killed.

MORTALITIES
Any rats killed for humane reasons, were subjected to detailed macroscopic examination in an attempt to define the cause of death. Any abnormal tissues and some tissues were subjected to histological examination.

BODY WEIGHT:
- Time schedule for examinations: all rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE :
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.
The substance was administrated by oral gavage and not in food.

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations:
Daily monitoring by visual appraisal was maintained throughout the dosing period. After sixteen days of treatment, this assessment indicated possible differences between control and treatment groups. Daily gravimetric measurement of water consumption was therefore initiated during Week 4 of the study.
The substance was administrated by oral gavage and not in drinking water.

OPHTHALMOSCOPIC EXAMINATION: included in clinical findings

HAEMATOLOGY:
- Time schedule for collection of blood: prior to termination (during Week 4) blood was withdrawn under light ether anaesthesia from the orbital sinus of ail rats allocated to the main study.
- Anaesthetic used for blood collection: yes
- Animals fasted: food was withdrawn overnight prior to collection of samples.
- How many animals: all animals allocated to the main studies.
- Parameters checked:
Packed cell volume (PCV) (unit:%)
Haemoglobin (Hb) (unit: g/dl)
Red blood cell count (RBC) (unit: x10^6/mm3)
Platelet count (Pits) (unit: x10^3/mm3)
Mean corpuscular haemoglobin concentration (MCHC) (unit:%): calculated: Hb (g/dl) x 100 + PCV (%)
Mean corpuscular volume (MCV) (unit:fl): calculated: PCV (%) x 10 + RBC (x10^6/mm3)
Total white blood cell count (WBC) (unit: x10^3/mm3)
Differential white blood cell count (Diff) - by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B)and Monocytes (M) (unit for all: x10^3/mm3). The percentage distribution of each cell type was determined by microscopy. These values were then converted to absolute values by computer. This inevitably involved a "rounding off" in a proportion of the results and for this reason the measured total WBC may differ slightly from the total of the different cell types.
Cell morphology : no definitely abnormal cells were observed when examining the stained slides.
Thrombotest (TT) - Method of Owren, P.A. (Lancet, 1959, ii, 754) (unit:s)

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: prior to termination (during Week 4) blood was withdrawn under light ether anaesthesia from the orbital sinus of ail rats allocated to the main study.
- Animals fasted: food was withdrawn overnight prior to collection of samples.
- How many animals: all animals allocated to the main studies.
- Parameters checked:
Glucose - using BCL Test Kit (Hexokinase mediated) (unit:mg/dl)
Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase' - using BCL Test Kit , reaction temperature 30°C, (unit: mU/ml)
Glutamic-oxaloacetic transaminase (GOT), also. known as 'aspartate aminotransferase' - using BCL Test Kit, reaction temperature 30°C.
Alkaline phosphatase (AP), reaction temperature 30°C (unit: mU/ml)
Total bilirubin (unit:mg/dl)
Cholesterol (Choi) (unit:mg/dl)
Urea nitrogen (Urea Nitr) (unit:mg/dl)
Total protein (unit: g/dl)
Albumin (Alb)
Globulin (Glob) - subtraction:
Total protein (g/dl) minus Albumin (g/dl)
Albumin/Globulin ratio (A/G) - by calculation from Total Protein and Albumin concentrations
Sodium (Na) (unit: mEq/l)
Potassium (K) (unit: mEq/l)
Calcium (Ca) (unit: mEq/l)
Chloride (CI) (unit: mEq/l)
Inorganic phosphorus (P) (unit: mEq/l)
Creatinine (unit:mg/dl)

URINALYSIS: not performed.

NEUROBEHAVIOURAL EXAMINATION: not performed.

IMMUNOLOGY:
- Time schedule for examinations: blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to the study initiation (Week -1) and from all rats allocated to the main study prior to termination (Day 29).
Blood samples were also withdrawn from all remaining rats of the satellite groups immediately prior to sacrifice (Days 29 to 32). On this occasion, sampling was carried out by cardiac puncture under ether anaesthesia
- How many animals: all rats allocated to the main study and all remaining rats of the satellite groups immediately prior to sacrifice .
- Dose groups that were examined: all
- Parameters checked:
The following immunological investigations were carried out using the above blood samples:
Clotted samples (pre-dose 1.0 ml clotted and terminals samples 1.0 ml clotted): the determination of IgG, IgM and IgA levels in sera
EDTA samples (only for satellite sub-groups 0.5 ml EDTA anticoagulant): the determination of the differential and total white blood cell counts in peripheral blood
Heparin samples (only for satellite sub-groups 3.0 ml heparin anticoagulant): the assessment of the numbers of B and T lymphocytes in peripheral blood
-Method:
#Determination of IgG, IgM and IgA levels in rat sera
Serum levels of IgG, IgM and IgA were determined using radial immunodiffusion test kits supplied by Serotec. Each kit consisted of ready-prepared immunodiffusion plates containing monospecific antiserum in buffered agarose, and reference standards. Each test was carried out according to the manufacturer's instructions supplied with each kit using procedure 2, "accurate analysis". A standard volume (5µI) of each 1 g reference solution was pipetted into the wells on the appropriate plates for each assay. The test sera were diluted 1 : 10 in sheep serum for the IgG assay and used undiluted for the IgM and IgA assays. A 5 gl aliquot of test serum or dilution was pipetted into the remaining test wells. The plates were resealed in their foil pouches and left at room temperature for 72 hours (IgG), 96 hours (IgA) or 120 hours (IgM).
For each assay the diameter of the precipitin rings was then measured for all test sera and reference standards. A standard curve was constructed by plotting the square of the ring diameters of the standards against the known concentrations. The concentration of immunoglobulin in each serum sample was determined by reading the square of the ring diameter against the standard curve. The values were recorded in mg immunoglobulin/litre serum.
This test was carried out on sera obtained pre-dose and at termination from all animals in the main study and the satellite sub-groups.
#Assessment of B and T lymphocytes in peripheral blood
Each blood sample was diluted in RPMI 1640 medium w/o serum, layered onto the surface of a density gradient separation medium and centrifuged at 400 x g for 30 minutes at room temperature.
The lymphocyte layer was pipetted off from the interface and the cells washed in Hepes-buffered RPMI 1640 medium and resuspended in that medium. A viable count was then carried out on a trypan blue-stained preparation using an Improved Neubauer Haemacytometer and the cell suspension was washed and resuspended in Azide-serum buffer (ASB) and adjusted to contain a maximum 2 x 10' viable cells/mI in ASB.
50 µl volumes of each cell suspension were placed in each of 2 wells in a 96-well plate. One of each pair of wells received 25 µl anti-rat T lymphocyte monoclonal antibody (Serotec) conjugated to fluorescein isothiocyanate (FITC) at the pre-determined optimal dilution. The remaining well received 50 µl polyvalent anti-rat Ig (IgA, IgM, IgG H and L chains) FITC conjugated. The plate was then stood at 4°C for 30 minutes. It was then centrifuged and the cell deposits washed twice in ASB and resuspended in 25 p1 ASB for fluorescent microscopy. Wet preparations were then counted and the number of B and T lymphocytes noted, the total number of lymphocytes/field was also recorded using tungsten light. The percentage of B and T lymphocytes present in each preparation was calculated and the relative number of B and T lymphocytes in each sample of rat blood determined using the total lymphocyte count.
#Assessment of total WBC, differential arid B and T lymphocyte count in the spleen
Each spleen removed from rats of the satellite groups was placed in a petri-dish and dissected free of fat and connective tissue and weighed (wet wt.). The spleen was chopped into small pieces and the pieces were washed into a nylon sieve using Hopes-buffered medium and gently pushed through the sieve, disrupting the tissue cells. The resultant suspension was passed through a Nybolt cloth filter to remove cell debris. The cell suspension was then pipetted into a fresh bottle and centrifuged, the cell deposit was washed twice in Hepes-buffered medium and subjected to hypotonic lysis if necessary to remove excess contaminating red cells. The cell suspension was then treated as follows:
(a) 1 ml suspension was centrifuged and the deposit mixed with 1 ml foetal calf serum and sent to Haematology for total WBC count and differential counts.
(b) a viable count was performed.
An appropriate aliquot was then removed and the cells washed in Azide-serum buffer (ASB) and resuspended in ASB to give a maximum of 2 x 10' cells/ml followed by staining and counting for numbers of B and T lymphocytes as described previously for the blood samples

Oestrous cyclicity (parental animals):

Not performed but the weight of the ovaries was performed for all the animals treated for 28 days at three different dosage levels (10, 100, 1000 mg/kg bw/day) and compared with untreated animals.

Sperm parameters (parental animals):

Not performed but the weight of the testes was performed for all the animals treated for 28 days at three different dosage levels (10, 100, 1000 mg/kg bw/day) and compared with untreated animals.

Litter observations:

Not performed.

Postmortem examinations (parental animals):

After 28 days of treatment (Day 29) all animals from the main study were randomly killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The macroscopic appearance of the tissues was recorded.
All surviving rats in the satellite groups were similarly killed on Days 29 to 32 (five or six animais per day)

GROSS PATHOLOGY:
The following organs from each animal of the main study killed after four weeks were dissected free of fat and weighed:
adrenals
liver
ovaries
kidneys
thymus
spleen
testes (with epididymides).

Samples of the following tissues from all rats in the main study were preserved in 10% buffered formalin:
adrenals
liver
skeletal muscles
aorta
lungs
spleen
brain (medullary, cerebellar and cortical sections)
lymph nodes (cervical and mesenteric)
sternum (for bone and marrow mesenteric)
mammary gland
stomach
caecum
oesophagus
testes (including epididymis)
colon
ovaries
duodenum
pancreas
thymus (where present)
eyes (Davidson's fluid as fixative)
Peyers patches
thyroid (with parathyroid)
pharynx
tongue
heart
pituitary
trachea
ileum
prostate
urinary bladder
jejunum
salivary gland
uterus
kidneys
sciatic nerve
vagina
larynx
seminal vesicles


HISTOPATHOLOGY:
The hystopatological examination was performed only on abnormal tissues:
adrenals
liver
spleen
lymph nodes (cervical and mesenteric)
sternum (for bone and marrow mesenteric)
thymus (where present)
Peyers patches
heart
kidneys
Fixed tissue samples required for microscopic examination were embedded in paraffin wax (m.p. 56°C), sections cut at 4 µm and stained with haematoxylin and eosin.
Microscopic examinations were carried out for:
(a) any animals that died during the study in an attempt to ascertain the course of death.
(b)all rats in Group 1 (control) and Group 4 (high dosage group) killed on Day 29.

Postmortem examinations (offspring):

Not performed.

Statistics:

See other information on method.

Reproductive indices:

Not performed.

Offspring viability indices:

Not performed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Intermittent increased salivation was noted for all male and female rats in the high dosage group (1000 mg/kg/day) during the treatment period. Following several days of dosing, this was accompanied by persistent hunched posture, abnormal gait (waddling) and pallor and by occasional lethargy and ptosis. All animals appeared badly groomed from Day 21 until termination and instances of noisy respiration were noted for five surviving male rats and all eight female rats. Pilo-erection was also recorded for two surviving male rats during Week 4.
The right eye of one male in this high dosage group (no. 26) appeared enlarged from Day 2 to Day 4, and appeared dull and darkened from Day 2 to Day 10. From Day 16 to termination the right eye appeared sunken. These observations were considered likely to be related to some mechanical damage and not to the oral administration of the test substance.
For male and female rats in the intermediate dosage group (100 mg/kg/day), increased salivation was noted intermittently throughout the treatment period. All rats from this intermediate group and also all rats from the low dosage group (10 mg/kg/day) appeared to be badly groomed during the last week of treatment.
No other clinical findings were noted for animals in the control, low or intermediate dosage groups that were considered to be of toxicological importance.
On Day 28 pallor was noted for most animals in the control, low and intermediate dosage groups allocated to the main study. This was considered to be related to the blood sampling procedure carried out for these animals on Day 28.
The macroscopic examination performed at termination revealed general brown staining of the fur in all five female rats examined in the high dosage group compared to none in the control group.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male rat (no. 30) from the high dosage group (1000 mg/kg/day) was found to be in extremis and was sacrificed on humane grounds on Day 25. The clinical signs observed in this animal were similar to other male rats in the high dosage group and included hunched posture, abnormal gait (waddling), increased salivation, a pale appearance, lethargy, ptosis and a badly groomed appearance. Noisy respiration, pilo-erection, cold extremities and hair loss along both sides of the abdomen were also noted on the day it was sacrificed (Day 25).
A post modem revealed general patchy alopecia and moist fur around the genital region with blood in the thoracic cavity, congestion of the adrenal glands and enlargement and pallor of the kidneys. Watery distension of the stomach with multiple haemorrhagic depressions in the corpus mucosa and dark contents in the ileum and large intestine were also noted.
Microscopic examination revealed renal tubular dilatation and necrosis of the papillary tip that was considered to be related to treatment with Chlorphenesin. Lymphoid depletion in the thymus and increased apoptosis in the lymphoid organs were also noted but were considered to be possible secondary to the renal lesions.
The poor health status of this animal was considered to be related to treatment with Chlorphenesin.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower bodyweight gains were recorded for male rats of the high dosage group (1000 mg/kg/day), achieving significance (P<0.01) during Weeks 1 to 4 when compared with control rats. A similar trend to lower bodyweight gains during Weeks 2 to 4 was observed for the corresponding females, with statistical significance being achieved (P<0.01) during Week 4 when compared to control rats. Instances of bodyweight losses were noted during Weeks 2 to 4 for female rats in the high dosage group.
Bodyweight gains for rats in the intermediate and low dosage groups were similar to those for control rats.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption was recorded for male rats from the high dosage group during Weeks 1 to 4 when compared with controls. Slightly lower food consumption was also recorded for female rats in the high dosage group during Weeks 3 and 4 when compared with controls.
These changes corresponded to the decreased bodyweight gains recorded for the same rats during the study period.
Food consumption for rats from the intermediate and low dosage groups were similar to that for control rats.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Following sixteen days of treatment, the appraisal of water consumption by visual assessment indicated possible differences between the control and treatment groups. Daily gravimetric measurement of water consumption was therefore initiated during Week 4 of the study.
Water consumption was higher for both male and female rats from the high dosage group compared to control rats. Water consumption for male rats in the intermediate dosage group was also slightly higher than that of the controls.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigations were carried out during Week 4 (Day 28) for all rats allocated to the main study.
Haemoglobin levels were significantly (P<0.05) lower in male and female rats from the high dosage group and in male rats from the intermediate dosage group when compared to control animals. The change in haemoglobin levels appeared to be associated with a lower red blood cell count and lower packed cell volume, although statistical significance was not achieved in these instances.
All other haematological parameters for rats treated with the test substance were similar to controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical investigations were also carried out during Week 4 (Day 28) for all rats allocated to the main study.
Statistically significantly (P<0.01) raised glutamic-pyruvic transaminase (GPT) levels were recorded for male and female rats from the high dosage group when compared with control rats. Alkaline phosphatase levels for male rats in the high dosage group were also slightly higher than controls although statistical significance was not achieved.
Total protein, albumin and globulin levels were lower in male rats treated at 1000 mg/kg/day, achieving significance (P<0.05) for total protein and albumin levels. Significantly lower (P<0.05) albumin levels were also recorded for male rats from the intermediate dosage group (100 mg/kg/day) In comparison with controls.
Potassium and calcium ion concentrations were significantly (P<0.05) lower in female rats from the high dosage group than in control rats. This change was not observed in the corresponding male rats.
Lower glucose levels were recorded for male rats in the high dosage group. This change was statistically significant (P<0.05) when compared to control rats, but was not observed in female rats of this group. This effect was considered unlikely to be related to treatment with Chlorphenesin.

Urinalysis findings:

not examined

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

One of the five male rats from the main group receiving Chlorphenesin, 1000 mg/kg/day, and killed at termination, showed a focus of degeneration in the renal papilla and minimal focal degeneration of urothelium of the papillary tip. The possibility that these changes are related to treatment with Chlorphenesin must be considered.
No microscopic changes were seen in the female rats or remaining male rats from the main group receiving Chlorphenesin, 1000 mg/kg/day, and killed at termination, to account for the statistically significant changes in haematology, biochemistry and organ weights.

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

The estrous cycle was not examined during the study. The animals were chaged in groups of four of the same sex and no mating was planned during the study. However the weight of ovaries was performed for all female rats treated for 28 days with Chlorphenesin and no statistically significantly changes in ovaries weight was observed between control animals and treated animals. The mean ovaries weight was 102.5 mg, 95.6 mg, 105.9 mg and 91.2 mg respectively for control, 10 mg/kg bw/day, 100 mg/kg bw/day and 1000 mg/kg bw/day groups.

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

The sperm measure was not performed during the study. The animals were chaged in groups of four of the same sex and no mating was planned during the study. However the weight of testes was performed for all male rats treated for 28 days with Chlorphenesin and no statistically significantly changes in testes weight was observed between control animals and treated animals. The mean testes weight was 4.30 g, 4.51 mg, 4.33 g and 3.93 g respectively for control, 10 mg/kg bw/day, 100 mg/kg bw/day and 1000 mg/kg bw/day groups.

Reproductive performance:

not examined

Description (incidence and severity):

The animals were chaged in groups of four of the same sex and no mating was planned during the study.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: changes in reproductive organs weight

Remarks on result:

other: See remarks

Remarks:

Based on the result indicated in this study NOAEL Chlorphenesin (repeated oral toxicity rat 28 days) = 10 mg/kg bw/day. At 10 mg/kg bw/day the only effect observed is a badly groomed appearance and at following level 100 mg/kg bw/day no specific effects on target organ (included ovaries and testes) that could compromise the animal health were registered.The NOAEL should be reasonable increased at 100 mg/kg bw/day. The NOAEL defined for repeated dose toxicity (100 mg/kg bw/day) should be applied also for reproductive toxicity.

Critical effects observed:

no

Remarks on result:

other: See remarks

Remarks:

The animals were chaged in groups of four of the same sex and no mating was planned during the study. The effect on F1 generation can not be evaluated with this study.

Key result

Reproductive effects observed:

no

Conclusions:

Based on the result indicated in this study NOAEL Chlorphenesin (repeated oral toxicity rat 28 days) = 10 mg/kg bw/day. At 10 mg/kg bw/day the only effect observed is a badly groomed appearance and at following level 100 mg/kg bw/day no specific effects on target organ (included ovaries and testes) that could compromise the animal health were registered.The NOAEL should be reasonable increased at 100 mg/kg bw/day. The NOAEL defined for repeated dose toxicity (100 mg/kg bw/day) should be applied also for reproductive toxicity.

Executive summary:

Chlorphensin has been tested for repeated oral toxicity during an administration period of 28 days.

Chlorphenesin was formulated daily as 10.0 %, 1.0 % and 0.1 % w/v suspensions in 1 % aqueous methylcellulose (1% MC) and administered orally to rats at dosage levels of 1000, 100 and 10 mg/kg/day for a minimum of 28 consecutive days. Control animals received 1% MC alone.

Following administration of Chlorphenesin, 100 or 1000 mg/kg/day, changes were noted in the parameters and tissues examined that were considered to be related to treatment.

For rats in the low dosage group receiving Chlorphenesin, 10 mg/kg/day, a badly groomed appearance was noted during the last week of treatment only. This finding was not considered to be of toxicological importance and, in the absence of any other changes at this dosage level, the no adverse effect level of Chlorphenesin can be considered as 10 mg/kg/day. At 10 mg/kg/day the only effect observed is a badly groomed appearance and at following level 100 mg/kg/day  no specific effects on target organ that could compromise the animal health were registered. In fact the only effects observed at 100 mg/kg/day were increase salivation, lower level of haemoglobin in male rats  and lower albumin level in male rats if compared with control group. The NOAEL should be reasonable set at 100 mg/kg bw/day. The animals were chaged in groups of four of the same sex and no mating was planned during the study. However the weight of testes/ovaries was performed for all male/female rats treated for 28 days with Chlorphenesin and no statistically significantly changes in reproductive organs weight was observed between control animals and treated animals. The NOAEL defined for repeated dose toxicity (100 mg/kg bw/day) should be applied also for reproductive toxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorphenesin

EC Number:

203-192-6

EC Name:

Chlorphenesin

Cas Number:

104-29-0

Molecular formula:

C9H11ClO3

IUPAC Name:

3-(4-chlorophenoxy)propane-1,2-diol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: the test substance was stored in the original container under ambient conditions in non-continuous artificial light.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 28 ± 1 days old
- Weight at study initiation: 74 - 103 g.
- Housing: the cages (each containing four rats) constituting each group were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments. Each cage was identified by a coloured label according to group. Each label displayed the study schedule number, cage number, sex, individual animal numbers and the initials of the Study Director and Home Office Licensee.
- Diet (e.g. ad libitum): free access to Biosure LAD 1 Diet. Biosure LAD was a closed formula diet suitable for normal health, growth and reproduction of laboratory rats and mice. The standards of production adopted by the manufacturers have been approved by the HRC Quality Assurance Department. Analyses were made of all batches of diet for most nutrients and for specified substances and micro-organisms likely to be present in feed ingredients or the finished diet and which, if in excess of specified amounts, might have had an undesirable effect on the test system. Although occasional slight deviation may have been permitted, batches of diet conformed with the acceptable standards agreed by the Study Director and Quality Assurance.
- Water (e.g. ad libitum): free access to tap water. Results of the routine physical and chemical examination of drinking water at source as conducted usually weekly by the supplier. Additionally, levels of specified substances known to be present from time to time in local water and which, if in excess of the maxima recommended (for humans) might have had an undesirable effect on the test system, were determined in the tap water at approximately 6-monthly intervals.
- Acclimation period: 19 days was allowed between delivery of the animals and start of treatment.

DETAILS OF FOOD AND WATER QUALITY: analyses were made on all batches of diet used to establish levels of basic nutrients and of specified substances and micro-organisms likely to be present in feed components and which, if in excess of specified amounts, might have had an undesirable effect on the test system. Results of the routine physical and chemical examination of drinking water at source as conducted usually weekly by the supplier. Additionally, levels of specified substances known to be present from time to time in local water and which, if in excess of the maxima recommended (for humans) might have had an undesirable effect on the test system, were determined in the tap water at approximately 6-monthly intervals.Quarterly summary analyses of source water normally included levels of nitrites, nitrates, Ca, Mg, Na, K, P, CI, Si, Fe. Six-monthly analyses of HRC tap water included levels of As, Se, Ba, Ag, Sb, organophosphorus, organochlorine and other pesticides, haloforms, chlorophenols, polychlorinated biphenyls and polycyclic aromatic hydrocarbons.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): animal room temperature was controlled at a mean minimum of 21.0 °C and a mean maximum of 21.6 °C.
- Humidity (%): relative humidity was controlled at a mean minimum of 50.7 % and a mean maximum of 58.8 %.
- Air changes (per hr): air exchange was maintained at a rate of approximately 20 air changes per hour.
- Photoperiod (hrs dark / hrs light): lighting was controlled to give twelve hours artificial light in each twenty-four hour period

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered by oral gavage to rats inclusive using a syringe and rubber catheter at a dosage volume of 10.0 ml/kg/day.
Each animal received a constant dosage level based on its most recently recorded bodyweight.

Vehicle:

methylcellulose

Remarks:

aqueous methylcellulose (1 % MC)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: the high dosage concentration of the test substance was prepared freshly each day as a 10.0% w/v suspension in 1% aqueous methylcellulose (1% MC). The intermediate (1.0% w/v) and low (0.1% w/v) dosage concentrations were prepared by direct dilution of the test substance with the vehicle. Prior to dosing, the test substance suspensions were mixed initially by inversion and sequently using a magnetic stirrer for a period of 5 to 15 minutes. Dosing was completed within one hour of the commencement of stirring.

VEHICLE
- Concentration in vehicle: 10.0%, 1.0% and 0.1% w/v suspension in methylcellulose (1% MC) corresponds respectively to dosage levels of 1000, 100 and 10 mg/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical verification of dose and concentration was performed by High performance liquid chromatograph (HPLC) using ultra-violet detection. A representative sub-sample (1 ml) of test formulation was accurately weighed and dissolved in a suitable volume of mobile phase. The solution obtained was further diluted with mobile phase to provide an expected concentration of Chlorphenesin between 4 and 8 mcg/mI. The final solution was filtered (Whatman GF/F) prior to quantification.

Typical chromatographic conditions
- Analytical column: Apex ODS, 5 mcm, 250 x 4.6 mm ID, Jones Chromatography Ltd.
- Guard column: RP 300 aquapore octyl, 7 mcm, 30 x 4.6 mm ID, Browniee Laboratodes Inc.
- Mobile phase: Methano/water (55/45 v/v).
- Detector: wavelength UV, 279 nm.
- Flow rate: 1.0 ml/minute.
- Injection volume 65 mcl.
- Integrator attenuation 32.
- Retention volume 8 ml

Determination of concentrations of Chlorphenesin in dose formulations prepared for Day 1 of the study
Representative samples (approximately 20 ml) of freshly prepared dose formulation, received from the Department of Formulation, were thoroughly mixed by shaking and duplicate sub-samples (1 ml) were analysed.

- Chemical stability
Freshly-prepared batches of test formulations, at nominal concentrations of 1 mg/ml and 100 mg/ml, were each sampled (6 replicates of approximately 1 ml) into pre-weighed volumetric flasks. Two samples were analysed immediately and the remaining four samples were stored in the dark at ambient temperature for 4 hours and at +40 °C for 24 hours before analysis.
At each time-point, two samples from each formulation were analysed.

- Physical stability
After sampling for chemical stability, the remainder of each test formulation was thoroughly mixed by repeated inversion and magnetically stirred for 5 minutes. Samples (approximately 1 ml) were removed from points approximately one-quarter, one-half and three-quarters the depth (representing the top, middle and bottom) of the formulation and analysed. The formulation was magnetically stirred for a further 1 hour and sampled, as above, at time-points corresponding to 0.5 hour and 1-hour storage.
The magnetic stirring was then discontinued and the remainder of each formulation was stored in the dark at -ambient temperature for a further 3 hours and subsequently at +4°C overnight. After storage for 4 and 24 hours, the suspension was re-mixed and sampled, as above, for analysis.
The three samples removed from each formulation, at each time-point, were analysed.

Calculation
The response for Chlorphenesin in each calibration chromatogram was measured and calibration curves were constructed by linear regression of standard response versus standard concentration. The response of the peak observed at the characteristic retention volume for Chlorphenesin in sample and procedural recovery chromatograms was measured and the concentration of Chlorphenesin calculated using the equation below.
Concentration, mg/ml = (Y-I)/S x (V/W) x D x 10-3
Where:
Y = Peak response in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution volume of sample (mi)
W = Weight of sample (g)
D = Density (g/ml)
Results were corrected for the appropriate cumulative mean procedural recovery value at analysis.

Limit of detection
The limit of detection, defined as the concentration of Chlorphenesin in control matrix producing a peak response equivalent to 3 x baseline noise, was determined as 0.075 mg/ml.

Results regarding verification of doses
The mean concentrations of Chlorphenesin in dose formulations analysed during the study and the deviation of mean results from nominal values are measured:mean results are all within 5% of nominal concentrations. Based on the data obtained during the study Chlorphenesin forms a homogeneous suspension in the 1% MC formulation. The data confirm that homogeneity can be maintained by magnetic stirring for 1 hour and that the formulations can be successfully re-suspended 4 and 24 hours after preparation. The chemical stability of Chlorphenesin in 1% MC formulations, was confirmed for a period of 24 hours
The data confirm that the analytical method described is both precise and accurate for Chlorphenesin in 1% MC formulations. A mean recovery of 101.1% ± 1.38% SD (n=8) was obtained at 1 mg/ml and 103.4% ± 1.85% SD (n=8) at 100 mg/ml. Results are corrected for the appropriate cumulative mean procedural recovery value at analysis.

Duration of treatment / exposure:

Twenty-eight consecutive days (Dosing started on 29 August 1989 and ended on 25th September 1989).

Frequency of treatment:

Animals were treated once daily for twenty-eight consecutive days. Treatment of animaIs not scheduled for sacrifice on Day 29 continued until the day prior to being killed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per dose (5 males and 5 females)+ 6 animals per dose as satellite subgruops (3 males and 3 females). Total animals effective used in the test: 64 animals. One animal of satellite group was killed during the test.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dosage levels of 10, 100 and 1000 mg/kg/day were selected on the basis of acute oral toxicity data available from the Sponsor and a 7-day preliminary oral toxicity study in the rat.
- Rationale for animal assignment (if not random): before the start of treatment (Week -1) each animal was weighed and sixty-four rats were randomly allocated to four groups, each consisting of eight males and eight females. This allocation was carried out using a computer program. Following this random allocation five male and five female rats from each group were selected for allocation to the main study (rats with the lowest study numbers were chosen).
- Rationale for selecting satellite groups: the remaining rats within each group (three males and three females) were allocated to satellite sub-groups. These satellite groups were used for terminal immunological investigations
- Post-exposure recovery period in satellite groups: after 28 days of treatment (Day 29) all animals from the main study were randomly killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The macroscopic appearance of the tissues was recorded. All surviving rats in the satellite groups were similarly killed on Days 29 to 32 (five or six animals per day)

Positive control:

Not present.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: all animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post-mortem examination to be undertaken during the working part of that day. At weekends a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was isolated, and if found to be in extremis was killed.

MORTALITIES
Any rats killed for humane reasons, were subjected to detailed macroscopic examination in an attempt to define the cause of death. Any abnormal tissues and a lot of them were subjected to histological examination.

BODY WEIGHT:
- Time schedule for examinations: all rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE :
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.
The substance was administrated by oral gavage and not in food.

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations:
Daily monitoring by visual appraisal was maintained throughout the dosing period. After sixteen days of treatment, this assessment indicated possible differences between control and treatment groups. Daily gravimetric measurement of water consumption was therefore initiated during Week 4 of the study.

OPHTHALMOSCOPIC EXAMINATION: included in clinical findings

HAEMATOLOGY:
- Time schedule for collection of blood: prior to termination (during Week 4) blood was withdrawn under light ether anaesthesia from the orbital sinus of ail rats allocated to the main study.
- Anaesthetic used for blood collection: yes
- Animals fasted: food was withdrawn overnight prior to collection of samples.
- How many animals: all animals allocated to the main studies.
- Parameters checked:
Packed cell volume (PCV) (unit:%)
Haemoglobin (Hb) (unit: g/dl)
Red blood cell count (RBC) (unit: x10^6/mm3)
Platelet count (Pits) (unit: x10^3/mm3)
Mean corpuscular haemoglobin concentration (MCHC) (unit:%): calculated: Hb (g/dl) x 100 + PCV (%)
Mean corpuscular volume (MCV) (unit:fl): calculated: PCV (%) x 10 + RBC (x10^6/mm3)
Total white blood cell count (WBC) (unit: x10^3/mm3)
Differential white blood cell count (Diff) - by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B)and Monocytes (M) (unit for all: x10^3/mm3). The percentage distribution of each cell type was determined by microscopy. These values were then converted to absolute values by computer. This inevitably involved a "rounding off" in a proportion of the results and for this reason the measured total WBC may differ slightly from the total of the different cell types.
Cell morphology : no definitely abnormal cells were observed when examining the stained slides.
Thrombotest (TT) - Method of Owren, P.A. (Lancet, 1959, ii, 754) (unit:s)

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: prior to termination (during Week 4) blood was withdrawn under light ether anaesthesia from the orbital sinus of ail rats allocated to the main study.
- Animals fasted: food was withdrawn overnight prior to collection of samples.
- How many animals: all animals allocated to the main studies.
- Parameters checked:
Glucose - using BCL Test Kit (Hexokinase mediated) (unit:mg/dl)
Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase' - using BCL Test Kit , reaction temperature 30°C, (unit: mU/ml)
Glutamic-oxaloacetic transaminase (GOT), also. known as 'aspartate aminotransferase' - using BCL Test Kit, reaction temperature 30°C.
Alkaline phosphatase (AP), reaction temperature 30°C (unit: mU/ml)
Total bilirubin (unit:mg/dl)
Cholesterol (Choi) (unit:mg/dl)
Urea nitrogen (Urea Nitr) (unit:mg/dl)
Total protein (unit: g/dl)
Albumin (Alb)
Globulin (Glob) - subtraction:
Total protein (g/dl) minus Albumin (g/dl)
Albumin/Globulin ratio (A/G) - by calculation from Total Protein and Albumin concentrations
Sodium (Na) (unit: mEq/l)
Potassium (K) (unit: mEq/l)
Calcium (Ca) (unit: mEq/l)
Chloride (CI) (unit: mEq/l)
Inorganic phosphorus (P) (unit: mEq/l)
Creatinine (unit:mg/dl)

URINALYSIS: not performed.

NEUROBEHAVIOURAL EXAMINATION: not performed.

IMMUNOLOGY:
- Time schedule for examinations: blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to the study initiation (Week -1) and from all rats allocated to the main study prior to termination (Day 29).
Blood samples were also withdrawn from all remaining rats of the satellite groups immediately prior to sacrifice (Days 29 to 32). On this occasion, sampling was carried out by cardiac puncture under ether anaesthesia
- How many animals: all rats allocated to the main study and all remaining rats of the satellite groups immediately prior to sacrifice .
- Dose groups that were examined: all
- Parameters checked:
The following immunological investigations were carried out using the above blood samples:
Clotted samples (pre-dose 1.0 ml clotted and terminals samples 1.0 ml clotted): the determination of IgG, IgM and IgA levels in sera
EDTA samples (only for satellite sub-groups 0.5 ml EDTA anticoagulant): the determination of the differential and total white blood cell counts in peripheral blood
Heparin samples (only for satellite sub-groups 3.0 ml heparin anticoagulant): the assessment of the numbers of B and T lymphocytes in peripheral blood
-Method:
#Determination of IgG, IgM and IgA levels in rat sera
Serum levels of IgG, IgM and IgA were determined using radial immunodiffusion test kits supplied by Serotec. Each kit consisted of ready-prepared immunodiffusion plates containing monospecific antiserum in buffered agarose, and reference standards. Each test was carried out according to the manufacturer's instructions supplied with each kit using procedure 2, "accurate analysis". A standard volume (5µI) of each 1 g reference solution was pipetted into the wells on the appropriate plates for each assay. The test sera were diluted 1 : 10 in sheep serum for the IgG assay and used undiluted for the IgM and IgA assays. A 5 gl aliquot of test serum or dilution was pipetted into the remaining test wells. The plates were resealed in their foil pouches and left at room temperature for 72 hours (IgG), 96 hours (IgA) or 120 hours (IgM).
For each assay the diameter of the precipitin rings was then measured for all test sera and reference standards. A standard curve was constructed by plotting the square of the ring diameters of the standards against the known concentrations. The concentration of immunoglobulin in each serum sample was determined by reading the square of the ring diameter against the standard curve. The values were recorded in mg immunoglobulin/litre serum.
This test was carried out on sera obtained pre-dose and at termination from all animals in the main study and the satellite sub-groups.
#Assessment of B and T lymphocytes in peripheral blood
Each blood sample was diluted in RPMI 1640 medium w/o serum, layered onto the surface of a density gradient separation medium and centrifuged at 400 x g for 30 minutes at room temperature.
The lymphocyte layer was pipetted off from the interface and the cells washed in Hepes-buffered RPMI 1640 medium and resuspended in that medium. A viable count was then carried out on a trypan blue-stained preparation using an Improved Neubauer Haemacytometer and the cell suspension was washed and resuspended in Azide-serum buffer (ASB) and adjusted to contain a maximum 2 x 10' viable cells/mI in ASB.
50 µl volumes of each cell suspension were placed in each of 2 wells in a 96-well plate. One of each pair of wells received 25 µl anti-rat T lymphocyte monoclonal antibody (Serotec) conjugated to fluorescein isothiocyanate (FITC) at the pre-determined optimal dilution. The remaining well received 50 µl polyvalent anti-rat Ig (IgA, IgM, IgG H and L chains) FITC conjugated. The plate was then stood at 4°C for 30 minutes. It was then centrifuged and the cell deposits washed twice in ASB and resuspended in 25 p1 ASB for fluorescent microscopy. Wet preparations were then counted and the number of B and T lymphocytes noted, the total number of lymphocytes/field was also recorded using tungsten light. The percentage of B and T lymphocytes present in each preparation was calculated and the relative number of B and T lymphocytes in each sample of rat blood determined using the total lymphocyte count.
#Assessment of total WBC, differential arid B and T lymphocyte count in the spleen
Each spleen removed from rats of the satellite groups was placed in a petri-dish and dissected free of fat and connective tissue and weighed (wet wt.). The spleen was chopped into small pieces and the pieces were washed into a nylon sieve using Hopes-buffered medium and gently pushed through the sieve, disrupting the tissue cells. The resultant suspension was passed through a Nybolt cloth filter to remove cell debris. The cell suspension was then pipetted into a fresh bottle and centrifuged, the cell deposit was washed twice in Hepes-buffered medium and subjected to hypotonic lysis if necessary to remove excess contaminating red cells. The cell suspension was then treated as follows:
(a) 1 ml suspension was centrifuged and the deposit mixed with 1 ml foetal calf serum and sent to Haematology for total WBC count and differential counts.
(b) a viable count was performed.
An appropriate aliquot was then removed and the cells washed in Azide-serum buffer (ASB) and resuspended in ASB to give a maximum of 2 x 10' cells/ml followed by staining and counting for numbers of B and T lymphocytes as described previously for the blood samples

Sacrifice and pathology:

GROSS PATHOLOGY:
The following organs from each animal of the main study killed after four weeks were dissected free of fat and weighed:
adrenals
liver
ovaries
kidneys
thymus
spleen
testes (with epididymides).

Samples of the following tissues from all rats in the main study were preserved in 10% buffered formalin:
adrenals
liver
skeletal muscles
aorta
lungs
spleen
brain (medullary, cerebellar and cortical sections)
lymph nodes (cervical and mesenteric)
sternum (for bone and marrow mesenteric)
mammary gland
stomach
caecum
oesophagus
testes (including epididymis)
colon
ovaries
duodenum
pancreas
thymus (where present)
eyes (Davidson's fluid as fixative)
Peyers patches
thyroid (with parathyroid)
pharynx
tongue
heart
pituitary
trachea
ileum
prostate
urinary bladder
jejunum
salivary gland
uterus
kidneys
sciatic nerve
vagina
larynx
seminal vesicles


HISTOPATHOLOGY:
The hystopatological examination was performed only on abnormal tissues:
adrenals
liver
spleen
lymph nodes (cervical and mesenteric)
sternum (for bone and marrow mesenteric)
thymus (where present)
Peyers patches
heart
kidneys
Fixed tissue samples required for microscopic examination were embedded in paraffin wax (m.p. 56°C), sections cut at 4 µm and stained with haematoxylin and eosin.
Microscopic examinations were carried out for:
(a) any animals that died during the study in an attempt to ascertain the course of death.
(b)all rats in Group 1 (control) and Group 4 (high dosage group) killed on Day 29.

Statistics:

See other information on method

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Intermittent increased salivation was noted for all male and female rats in the high dosage group (1000 mg/kg/day) during the treatment period. Following several days of dosing, this was accompanied by persistent hunched posture, abnormal gait (waddling) and pallor and by occasional lethargy and ptosis. All animals appeared badly groomed from Day 21 until termination and instances of noisy respiration were noted for five surviving male rats and all eight female rats. Pilo-erection was also recorded for two surviving male rats during Week 4.
The right eye of one male in this high dosage group (no. 26) appeared enlarged from Day 2 to Day 4, and appeared dull and darkened from Day 2 to Day 10. From Day 16 to termination the right eye appeared sunken. These observations were considered likely to be related to some mechanical damage and not to the oral administration of the test substance.
For male and female rats in the intermediate dosage group (100 mg/kg/day), increased salivation was noted intermittently throughout the treatment period. All rats from this intermediate group and also all rats from the low dosage group (10 mg/kg/day) appeared to be badly groomed during the last week of treatment.
No other clinical findings were noted for animals in the control, low or intermediate dosage groups that were considered to be of toxicological importance.
On Day 28 pallor was noted for most animals in the control, low and intermediate dosage groups allocated to the main study. This was considered to be related to the blood sampling procedure carried out for these animals on Day 28.
The macroscopic examination performed at termination revealed general brown staining of the fur in all five female rats examined in the high dosage group compared to none in the control group.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male rat (no. 30) from the high dosage group (1000 mg/kg/day) was found to be in extremis and was sacrificed on humane grounds on Day 25. The clinical signs observed in this animal were similar to other male rats in the high dosage group and included hunched posture, abnormal gait (waddling), increased salivation, a pale appearance, lethargy, ptosis and a badly groomed appearance. Noisy respiration, pilo-erection, cold extremities and hair loss along both sides of the abdomen were also noted on the day it was sacrificed (Day 25).
A post modem revealed general patchy alopecia and moist fur around the genital region with blood in the thoracic cavity, congestion of the adrenal glands and enlargement and pallor of the kidneys. Watery distension of the stomach with multiple haemorrhagic depressions in the corpus mucosa and dark contents in the ileum and large intestine were also noted.
Microscopic examination revealed renal tubular dilatation and necrosis of the papillary tip that was considered to be related to treatment with Chlorphenesin. Lymphoid depletion in the thymus and increased apoptosis in the lymphoid organs were also noted but were considered to be possible secondary to the renal lesions.
The poor health status of this animal was considered to be related to treatment with Chlorphenesin.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower bodyweight gains were recorded for male rats of the high dosage group (1000 mg/kg/day), achieving significance (P<0.01) during Weeks 1 to 4 when compared with control rats. A similar trend to lower bodyweight gains during Weeks 2 to 4 was observed for the corresponding females, with statistical significance being achieved (P<0.01) during Week 4 when compared to control rats. Instances of bodyweight losses were noted during Weeks 2 to 4 for female rats in the high dosage group.
Bodyweight gains for rats in the intermediate and low dosage groups were similar to those for control rats.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption was recorded for male rats from the high dosage group during Weeks 1 to 4 when compared with controls. Slightly lower food consumption was also recorded for female rats in the high dosage group during Weeks 3 and 4 when compared with controls.
These changes corresponded to the decreased bodyweight gains recorded for the same rats during the study period.
Food consumption for rats from the intermediate and low dosage groups were similar to that for control rats.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Following sixteen days of treatment, the appraisal of water consumption by visual assessment indicated possible differences between the control and treatment groups. Daily gravimetric measurement of water consumption was therefore initiated during Week 4 of the study.
Water consumption was higher for both male and female rats from the high dosage group compared to control rats. Water consumption for male rats in the intermediate dosage group was also slightly higher than that of the controls.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigations were carried out during Week 4 (Day 28) for all rats allocated to the main study.
Haemoglobin levels were significantly (P<0.05) lower in male and female rats from the high dosage group and in male rats from the intermediate dosage group when compared to control animals. The change in haemoglobin levels appeared to be associated with a lower red blood cell count and lower packed cell volume, although statistical significance was not achieved in these instances.
All other haematological parameters for rats treated with the test substance were similar to controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical investigations were also carried out during Week 4 (Day 28) for all rats allocated to the main study.
Statistically significantly (P<0.01) raised glutamic-pyruvic transaminase (GPT) levels were recorded for male and female rats from the high dosage group when compared with control rats. Alkaline phosphatase levels for male rats in the high dosage group were also slightly higher than controls although statistical significance was not achieved.
Total protein, albumin and globulin levels were lower in male rats treated at 1000 mg/kg/day, achieving significance (P<0.05) for total protein and albumin levels. Significantly lower (P<0.05) albumin levels were also recorded for male rats from the intermediate dosage group (100 mg/kg/day) In comparison with controls.
Potassium and calcium ion concentrations were significantly (P<0.05) lower in female rats from the high dosage group than in control rats. This change was not observed in the corresponding male rats.
Lower glucose levels were recorded for male rats in the high dosage group. This change was statistically significant (P<0.05) when compared to control rats, but was not observed in female rats of this group. This effect was considered unlikely to be related to treatment with Chlorphenesin.

Urinalysis findings:

not examined

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood was withdrawn from all animals prior to the start of the study and again at termination for determination of serum immunoglobulin levels. Blood was also withdrawn at termination from all animals from the satellite sub-groups for assessment of total WBC, differential and B and T lymphocyte counts in peripheral blood. The assessment of total WBC, differential and B and T lymphocyte count in spleen suspensions was also carried out for satellite animals at termination.

- Number of B and T lymphocytes in peripheral blood and spleen
No dosage-related effects were noted on the numbers of B and T lymphocytes present in the peripheral blood and spleen cell suspensions from male or female rats which had been treated with Chlorphenesin for 28 days in comparison with control rats.

- Serum immunoglobulin levels
A rise in serum IgG and IgM levels was noted for all control and treated animals between the pre-dose and terminal samples. This was probably due to maturation of the immune system during the four-week experimental period.
At termination no statistically significant changes in adjusted IgG, IgM or IgA levels were noted in the sera of male rats from the low, intermediate or high dosage groups. Adjusted serum IgM and IgA levels were very similar in control animals and treated animals at all dosage levels. Adjusted serum IgG levels were slightly higher than control values in animals treated with 10 or 100 mg/kg/day but were similar to control values in sera from the high dosage group.
At the end of the treatment period no differences in adjusted serum levels of IgA were found between control and treated female rats at all three dosage levels. In female rats in the low and intermediate dosage groups mean adjusted IgG and IgM serum levels were both higher than control values but were not dosage-related or statistically significant. For female rats from the high dosage group the adjusted levels of serum IgG and IgM were significantly higher than those of control animals (P>0.05 and P>0.01 respectively). Since there was no dosage-related increase, this is likely to be a reflection of the general haematological and biochemical changes produced by Chlorphenesin in rats from the high dosage group, rather than to a specific effect on the immune system. The total lymphocyte and B and T cell counts in spleen and peripheral blood were similar to control values in the high dosage group and no pathological changes were observed in spleen, lymph nodes or bone marrow.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute spleen weights were statistically significantly (P<0.01 or P<0.05) lower in male and female rats in the high dosage group than in control rats.
Adjusted liver and kidney weights were significantly (P<0.05 or P<0.01) higher in female rats treated at 1000 mg/kg/day than for control rats. For male rats in this high dosage group, adjusted liver and kidney weights were comparable to those of the controls.
Significantly lower (P<0.05) absolute thymus weights were recorded for male rats in the high dosage group than for control rats. This apparent change was not noted for the corresponding female rats.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All macroscopic abnormalities were considered incidental and not to be related to treatment with Chlorphenesin.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

One of the five male rats from the main group receiving Chlorphenesin, 1000 mg/kg/day, and killed at termination, showed a focus of degeneration in the renal papilla and minimal focal degeneration of urothelium of the papillary tip. The possibility that these changes are related to treatment with Chlorphenesin must be considered.
No microscopic changes were seen in the female rats or remaining male rats from the main group receiving Chlorphenesin, 1000 mg/kg/day, and killed at termination, to account for the statistically significant changes in haematology, biochemistry and organ weights.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Remarks on result:

other: See remarks

Remarks:

Based on the result indicated in this study NOAEL Chlorphenesin (repeated oral toxicity rat 28 days) = 10 mg/kg bw /day. At 10 mg/kg/day the only effect observed is a badly groomed appearance and at following level 100 mg/kg bw/day no specific effects on target organ that could compromise the animal health were registered.The NOAEL should be reasonable set at 100 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the result indicated in this study NOAEL Chlorphenesin (repeated oral toxicity rat 28 days) = 10 mg/kg bw /day. At 10 mg/kg/day the only effect observed is a badly groomed appearance and at following level 100 mg/kg bw/day no specific effects on target organ that could compromise the animal health were registered.The NOAEL should be reasonable set at 100 mg/kg bw/day.

Executive summary:

Chlorphensin has been tested for repeated oral toxicity during an administration period of 28 days.

Chlorphenesin was formulated daily as 10.0 %, 1.0 % and 0.1 % w/v suspensions in 1% aqueous methylcellulose (1% MC) and administered orally to rats at dosage levels of 1000, 100 and 10 mg/kg/day for a minimum of 28 consecutive days. Control animals received 1% MC alone.

The following comments are made in summary:

 

Mortalities

One male rat treated at 1000 mg/kg/day was killed on humane grounds during the fourth week of treatment. Several clinical and macroscopic findings were recorded for this animal and microscopic examination revealed renal tubular dilatation and necrosis of the papillary tip. The changes were considered to be related to treatment with Chlorphenesin.

 

Clinical findings

For rats in the intermediate and low dosage groups, a badly groomed appearance was noted during the last week of treatment. Increased salivation was also noted intermittently for rats in the intermediate dosage group.

Several clinical findings were recorded during the treatment period for rats in the high dosage group treated at 1000 mg/kg/day. These findings included hunched posture, abnormal gait, pallor, lethargy, ptosis, a badly groomed appearance, noisy respiration and pilo-erection.

 

Bodyweight changes

In comparison with controls, lower bodyweight gains were recorded for male and female rats in the high dosage group, achieving statistical significance (P<0.01) during Weeks 1 to 4 for male rats and during Week 4 for females rats. Instances of bodyweight losses were noted during Week 4 in female rats from this dosage group.

Food consumption

Lower food consumption was recorded for male rats in the high dosage group during Weeks 1 to 4 and for female rats in this group during Weeks 3 and 4 when compared to the controls.

 

Water consumption

Water consumption was higher for both male and female rats from the high dosage group when compared to control rats during Week 4. Slightly higher water consumption was also recorded for male rats of the intermediate dosage group.

 

Haematology

Haemoglobin levels were significantly (P<0.05) lower in male and female rats from the high dosage group and in male rats from the intermediate dosage group when compared to control animals. The change in haemoglobin levels appeared to be associated with a lower red blood cell count and lower packed cell volume although statistical significance was not achieved in these instances.

Biochemistry

Statistically significantly (P<0.01) raised glutamic pyruvic transaminase (GPT) levels were recorded for male and female rats from the high dosage group when compared with control rats. Alkaline phosphatase levels for male rats in the high dosage group were also slightly higher than controls although statistical significance was not achieved.

Total protein, albumin and globulin levels were lower in male rats treated at 1000 mg/kg/day, achieving significance (P<0.05) for total protein and albumin levels. Significantly lower albumin levels were also recorded for male rats from the intermediate dosage group (100 mg/kg/day) in comparison with controls.

Potassium and calcium ion concentrations were significantly (P<0.05) lower in female rats from the high dosage group than in control rats.

 

Organ weights

Absolute spleen weights for male and female rats and thymus weights for male rats in the high dosage groups were statistically significantly (P<0.01 or P<0.05) lower than control rats.

Adjusted liver and kidney weights were significantly (P<0.05 or P<0.01) higher in female rats treated at 1000 mg/kg/day than for control rats.

 

Macroscopic pathology

Macroscopic pathology revealed general brown staining of the fur in all five female rats examined in the high dosage group compared to none in the control group.

 

Microscopic pathology

A focus of degeneration in the renal papilla and minimal focal degeneration of urothelium of the papillary tip were noted in one of the five male rats from the main group receiving Chlorphenesin, 1000 mg/kg/day.

 

Immunological investigations

When adjusted for pre-dose levels, IgG and IgM serum levels in female rats from the high dosage group were significantly higher than those of the controls at termination. These changes were considered to be a reflection of the haematological and biochemical changes produced following treatment with Chlorphenesin and not to be a specific effect on the immune system.

 

CONCLUSION

Following administration of Chlorphenesin, 100 or 1000 mg/kg/day, changes were noted in the parameters and tissues examined that were considered to be related to treatment.

For rats in the low dosage group receiving Chlorphenesin, 10 mg/kg/day, a badly groomed appearance was noted during the last week of treatment only. This finding was not considered to be of toxicological importance and, in the absence of any other changes at this dosage level, the no adverse effect level of Chlorphenesin can be considered as 10 mg/kg bw/day. At 10 mg/kg bw/bw the only observed effect is a badly groomed appearance and at following level 100 mg/kg bw/day  no specific effects on target organ that could compromise the animal health were registered. In fact the only effects observed at 100 mg/kg bw/day were increase salivation, lower level of haemoglobin in male rats  and lower albumin level in male rats if compared with control group. The NOAEL should be reasonable set at 100 mg/kg bw/day.