5-chloro-2-(4-chlorophenoxy)phenol;[DCPP]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-01-27 to 2001-04-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1998)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1996)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-290-0

EC Name:

-

Cas Number:

3380-30-1

Molecular formula:

C12 H8 Cl2 O2

IUPAC Name:

5-chloro-2-(4-chlorophenoxy)phenol

Details on test material:

- Name of test material (as cited in study report): FAT 80'220/A (i.e. Diclosan)
- Physical state: solid, powder
- Analytical purity: >99%
- Lot/batch No.: GRU98
- Expiration date of the lot/batch: 2008-10-31
- Stability of test article: stable under storage conditions
- Storage conditions: at room temperature (20°C) away from direct sunlight

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
- Source: RCC Ltd., Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf/ Switzerland
- Age at delivery: 4 weeks
- Body weight range at acclimatization: Males: 65 - 92 g, Females: 53 - 67 g
- Diet: pelleted standard Provimi Kliba 3433 rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland), ad libitum. The feed batch was analyzed for contaminants.
- Water: community tap-water, ad libitum, in water bottles.
- Acclimatization: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 40-70 %
- Air changes (per hr): 10-15 air changes/ hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours light

REMARK
None of the contaminants analyzed in the water and diet is considered to have been present at a concentration which would have affected the validity of the results.

IN-LIFE DATES: 2000-01-27 to 2000-01-02

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

(PEG 300)

Details on oral exposure:

PREPARATION oF DOSING SOLUTIONS
The test item formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test article in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: standard vehicle for studies of this type
- Concentration in vehicle: 4, 20 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity and concentration of the test item in the dose preparations was determined in samples taken from the first preparation. Intercurrent samples for concentration and homogeneity analyses were taken at monthly intervals (ie. a total of 4 intervals).

Duration of treatment / exposure:

92 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 100 and 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

0 and 500 mg/kg bw/day: 15 males and 15 females per group; therefrom, 5/sex served for recovery study
20 and 100 mg/kg bw/day: 10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose level selection: based upon the results of a non-GLP five day range-finding study (RCC Project 712146) and a subsequent 28-day study (RCC Project 712135) in rats.
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

none

Examinations

Observations and examinations performed and frequency:

MORTALITY / VIABILITY
Time schedule: observations for mortality/viability were recorded twice daily

GENERAL CAGESIDE OBSERVATIONS (DAILY)
Time schedule: the animals were observed for clinical signs once before commencement of administration; once daily on days 1-91 and once daily during days 92-120 (recovery).

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. The observations were performed in random sequence (with the exception of week 13) once before commencement of administration and once weekly (weeks 1-13) thereafter.

GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed during week 13 using a push-pull strain gauge (Mecmesin, AGF 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). Animals were randomized and monitored during the last treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

FOOD CONSUMPTION
The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

WATER CONSUMPTION
The water consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

BODY WEIGHTS
Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATIONS
The ophthalmoscopic examinations of both eyes of all animals were performed after the application of a mydriatic solution using a Miroflex 2 Ophthalmoscope. A description of any abnormality was recorded. For unilateral findings unless otherwise indicated in the tables, the contralateral eye was without abnormalities.

HAEMATOLOGY:
- Time schedule for collection of blood: after 13 weeks and after 17 weeks
- Anaesthetic used for blood collection: Yes (anaesthetic not specified
- Animals fasted: Yes (18- hour fasting period)
- Parameters: red blood cell count, total white cell count, platelet count, mean cell haemoglobin concentration, differential white cell count, blood cell morphology including differential white cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: after 13 weeks and after 17 weeks
- Parameters:
glucose
urea
creatinine
albumin
total protein
albumin/globulin ratio
total bilimbin
alkaline phosphatase activity
alanine aminotransferase activity
aspartate aminotransferase activity
creatine kinase activity
gamma-glutamyl transferase activity
sodium
potassium
chloride
phosphorus (as phosphate)
calcium
cholesterol
triglycerides

URINALYSIS:
- Time schedule for collection of urine: after 13 after 17 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters:
colour
volume
appearance
specific gravity
pH
glucose
ketones
bilirubin
protein
blood

Sacrifice and pathology:

NECROPSY
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Samples of tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:
Brain, Thymus, Heart, Kidneys, Liver, Adrenals, Thyroids w/parathyroids, Uterus, Spleen, Testes, Epididymides, Ovaries
The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOPATHOLOGY
Slides of all organs and tissues which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. Because treatment-related morphologic changes were detected in the organs of high-dose animals, the pancreas (males only), esophagus, stomach, kidneys and liver from animals of the mid- and low-dose groupsalso were examined. The organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin for microscopical examination.

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, body weight, organ weight and all ratios, as well as clinical laboratory data and ophthalmoscopy:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Student's T-Test was applied to locomotor activity and grip strength.
- Fisher's exact-test was applied to ophthalmoscopy data, and macroscopic findings.
- Armitage/Cochran Trend Test for non-neoplastic lesions, if appropriate.

References:
- C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
- R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
- P. Armitage: Test for linear trends in proportions and frequencies. Biometrics 11, 375-386 (1955).

Results and discussion

Results of examinations

Details on results:

MORTALITY / VIABILITY
All animals survived until scheduled necropsy.

CLINICAL SIGNS
Increased incidences of breathing noises were noted during daily observations and weekly observations (performed weeks 1-12) in both sexes treated with 500 mg/kg bw/day, although more frequently in males. This finding was considered to be test article-related, although no morphological correlate was evident. All remaining clinical signs were considered to be unrelated to treatment. Breathing noises were not evident in the animals allocated to the recovery group.

FUNCTIONAL OBSERVATIONAL BATTERY
Breathing noises were noted during the functional observational battery (performed in week 13) in both sexes treated with 500 mg/kg bw/day, although more frequently in males. This finding was considered to be test article-related. All remaining clinical signs were considered to be unrelated to test article.

Grip Strength
No test article-related changes in the fore- or hindlimb grip strength were noted at any dose level.
Locomotor Activity
No test article-related differences were noted in the mean locomotor activity (measured over 60 minutes) of the control and test article-treated animals.

FOOD CONSUMPTION
Minor differences noted in the mean daily food consumption of test article-treated and control animals were considered unlikely to be related to the treatment with the test article.

WATER CONSUMPTION
A test article-related increase in water consumption was noted in males and females treated with 500 mg/kg bw/day. These changes often attained statistical significance: Although the differences were more clearly expressed in females during the treatment period, the mean water consumption remained slightly elevated for the first two weeks of recovery in males and for the first week of recovery for females. The mean water consumption values of rats treated with the test article at 20 mg/kg bw/day or 100 mg/kg bw/day were similar to those of the control rats during the treatment period.

BODY WEIGHT
Minor differences noted in the body weight development of test article-treated and control animals were considered unlikely to be related to the treatment with the test article.

OPHTHALMOSCOPY
No test article-related ophthalmologic findings were noted when compared with the control animals.

CLINICAL LABORATORY INVESTIGATIONS

Hematology
Findings noted in the hematology parameters of animals treated with 100 mg/kg bw/day or 500 mg/kg bw/day were not supported by commensurate changes in related parameters, or remained within the 95 % tolerance limits of the historical control data, and were therefore considered to be without toxicological significance.

Clinical Biochemistry
When compared with control values, creatinine levels were higher and chloride levels were lower in males and females treated with 500 mg/kg bw/day were considered to be indications of effects upon the kidneys, whereas higher levels of triglyceride, phospholipids and increased activity of alkaline phosphatase seen in these animals were related to hepatic effects. During recovery, higher creatinine levels persisted in the females previously treated with 500 mg/kg bw/day. All remaining differences reverted to control levels.

Urinalysis
Increased urine production, lower specific gravity and lower osmolality noted in males and females treated with 500 mg/kg bw/day when compared with controls, indicating test articlerelated changes considered to be associated with alterations in the kidneys' ability to concentrate urine. These changes were reversible after four weeks recovery.

ORGAN WEIGHTS
The mean absolute and relative liver weights of males and females treated with the test article for 13 weeks at 500 mg/kg bw/day were higher than those of the controls, and considered to be test article-related. This difference reverted to levels similar to that of the controls after four weeks' recovery. All other organ weights and ratios were unaffected.

MACROSCOPIC / MICROSCOPIC FINDINGS
No test article-related macroscopic findings were noted at any dose level. Morphologic differences from the control animals were noted in the esophagi, stomachs, kidneys, livers of males and females treated with 500 mg/kg bw/day, whereas changes in the pancreas were limited to males treated with 500 mg/kg bw/day. Most of these findings were reversible after the recovery period. The findings in the esophagus consisted of epithelial hyperplasia in some animals treated with 500 mg/kg bw/day for 13 weeks. Hyperkeratosis of the esophagus was increased in incidence in animals treated with 100 mg/kg bw/day or 500 mg/kg bw/day.
In the stomach of animals treated with 500 mg/kg bw/day for 13 weeks, the severity of hyaline droplets in the glandular stomach was slightly increased. Submucosal lymphoid follicles and inflammatory cell infiltrate was increased in incidence in these animals after treatment and after recovery. Forestomach hyperkeratosis and epithelial hyperplasia were noted in some animals treated with 100 mg/kg/day and all animals treated with 500 mg/kg bw/day. This finding persisted after recovery in some animals of the latter group. In the liver of animals treated for 13 weeks, hepatocellular hypertrophy was noted in ten males and three females treated with 100 mg/kg bw/day and ten males and three females treated with 500 mg/kg bw/day. This finding was reversible in the latter group after recovery. In the pancreas, increased vacuolation with dense bodies were recorded in three males treated with 500 mg/kg bw/day for 13 weeks.
The kidneys of males treated with 500 mg/kg bw/day showed a slightly higher grade of severity for hyaline droplets after 13 weeks. Multifocal tubular swelling was seen in several males treated with 500 mg/kg bw/day after 13 weeks, occasionally accompanied by tubular cell necrosis. Although these kidney findings were reversible after 13 weeks, the incidence of lymphoid cell foci increased after recovery in males treated with 500 mg/kg bw/day.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects were seen at the lowest tested dose level of 20 mg/kg bw/d.

2

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analysis of dose formulations:

the overall mean concentrations of the homogeneity samples were found to be 98.7 %, 99.9 % and 97.9 % of the nominal concentrations of dose group 2 84 mg(mL), dose group 3 (20 mg/mL9 and dose group 4 800 mg/mL), respectively. the individual concentrations varied in the range from -1 % to +1 % of the mean concentrations. Theregore, the test item was found to be homogeneously distributed in the vehicle.