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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-10-25 to 2001-06-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Version / remarks:

(1992)

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): FAT80'220/B (DCPP)
- Source: Ciba Specialty Chemicals Inc, CH-4002, Basel, Switzerland
- Physical state: powder, beige, off white
- Analytical purity: >99%(w/w)
- Lot/batch No.: 2130B9AA
- Stability under test conditions: warranted until August 2009
- Storage condition of test material: ambient temperature, in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park, Macclesfield, Cheshire, UK
- Age at study initiation: approximately 5 weeks old on delivery
- Weight at study initiation: 201-227 g (males) and 197-230 g (females)
- Diet: diet (RMl) supplied by Special Diet Services Limited, Witham, Essex, UK, ad libitum
- Water: water supplied by an automatic system, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22+3°C
- Humidity: 30-70%
- Air changes: at least 15 changes/hour
- Photoperiod: 12 hours light/12 hours dark

Administration / exposure

Type of coverage:

occlusive

Vehicle:

polyethylene glycol

Details on exposure:

- % coverage: 10 % of the surface area
- Washing: the application site was washed after removal of the dressing using clean swabs of cotton wool soaked in warm water, and then dried gently with clean tissue paper
- Time after start of exposure: 6 hours
- Amount of test item solution applied: 2 mL/ kg bw
- Concentration of test item in vehicle: 1.5, 5.0 and 15 mg/mL
- Following each application, there was a rest period of up to 18 hours. Dosing was sequential in group order and performed at approximately the same time each day. On non-dosing days the animals were bandaged (to maintain the procedural routine) although no dose was given. Control animals were treated in the same manner as test animals except that only vehicle was applied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples of dosing preparations were analysed prior to dosing to verify the achieved concentration of the test substance in propylene glycol. As the preparations were solutions, samples were not taken for the determination of homogeneity of the test item in propylene glycol
The chemical stability of the test substance in propylene glycol, stored at room temperature, was determined prior to the start of the study at concentrations of 0.05 and 50mg/mL which spanned the range of concentrations used in this study (study number LR0590).

Duration of treatment / exposure:

28 days of treatment
14 days of recovery (control and high dose group)

Frequency of treatment:

20 times/ 28 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 3, 10, 30 mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5 rats/sex in each of the main study groups
5 rats /sex in each of the 2 recovery groups (0 and 30 mg/kg bw/d)

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Clinical observations
Prior to the start of the study, all rats were examined to ensure that they were physically normal and exhibited normal activity. Detailed clinical observations, including the finding of no abnormalities detected, were recorded daily, where appropriate, at the same time that the bodyweights were recorded, immediately prior to dose application and after decontamination, or at a similar time of day on the days when the animals were not dosed on day 29 and on days 36 and 43 for the recovery animals.

Bodyweights
The bodyweight of each rat was recorded daily, immediately prior to dose application and at a similar time on days when the animals were not dosed (these measurements were made in group order because the animals were dosed in sequence).

Food consumption
Food consumption was recorded continuously throughout the study for each cage of rats and calculated, at weekly intervals, as a mean value (g food/rat/day) for each cage.

Haematology and clinical chemistry
At scheduled termination, (day 29 or 43, as appropriate) all surviving rats were bled by cardiac puncture and samples were submitted to, and analysed by the Clinical Pathology Unit, CTL. Urine samples were also taken in the week prior to termination and submitted as above for analysis.
Haematology parameters:
red blood cell count haemoglobin
total white cell count haematocrit
platelet count mean cell volume
mean cell haemoglobin concentration mean cell haemoglobin
differential white cell count
blood cell morphology including differential white cell count.

Clinical chemistry 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
Individual urine samples were collected from all rats in groups 1, 3, 4 and 5 during week 4 and from all rats in groups 2 and 6 during week 6. Samples were collected over a period of 16-18 hours during which the rats were housed individually in metabolism cages and denied access to food and water. Parameters:
colour
volume
appearance
specific gravity
pH
glucose
ketones
bilirubin
protein
blood

Sacrifice and pathology:

At test and recovery ending, the animals were sacrificed (halothane Ph. Eur. vapour) and then exsanguinated by cardiac puncture.

Organ weights:
The following organs were removed from all animals surviving to scheduled termination, trimmed free of extraneous tissue and weighed (paired organs were weighed together): adrenal glands, liver, kidneys, testes

Macroscopic examination:
All animals were subjected to a full examination post mortem. This involved an external observation and an internal examination of all organs and structures. The following tissues were examined in situ, removed, examined and fixed in an appropriate fixative: abnormal tissue, adrenal gland, kidney, liver, skin (treated), skin (untreated), testis, epididymis, spleen, heart.

Histopathology:
Except for the epididymis which was stored, tissue samples were routinely processed, embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. All tissues processed from the control and high dose group in the main study (30 mg/kg bw/d) were examined by light microscopy.

Statistics:

Bodyweights were considered by analysis of covariance on initial (day 1) bodyweight, separately for males and females.
Weekly food consumption was considered by analysis of variance, separately for males and females.
Haematology and blood clinical chemistry and urine clinical chemistry were considered by analysis of variance. Male and female data were analysed together and the results examined to determine whether any differences between control and treated groups were consistent between the sexes.Organ weights were considered by analysis of variance and analysis of covariance on final bodyweight, separately for males and females. Summary data are presented for organ to bodyweight ratios but these were not analysed statistically as the analysis of covariance provides a better method of allowing for differences in terminal bodyweights.
All analyses were carried out separately for the main study and recovery groups.
Analyses of variance and covariance for main study groups allowed for the replicate structure of the study design and were carried out using the MIXED procedure in SAS (1996). Least-squares means for each group were calculated using the LSMEAN option in SAS PROC MIXED. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least-squares mean. Differences from control were tested statistically by comparing each treatment group least-squares mean with the control group least-squares mean using a two-sided Student’s t-test, based on the error mean square in the analysis.

Results and discussion

Results of examinations

Details on results:

ANALYTICAL MONITORING
The mean achieved concentrations of the test item were within 5% of the nominal concentration.
The chemical stability of the test item in the dosing preparations determined at concentrations of 0.05 and 50mg/mL was shown to be satisfactory for at least 14 days which covered the period of administration in the present study.
MORTALITY AND CLINICAL SYMPTOMS
One male given 30 mg/kg bw/d and one female given 3 mg/kg bw/d were found dead on day 4 and 5, respectively; there was not indication that the mortalities were treatment-related.
Treatment-related clinical changes, e.g. edema and skin sensitive to touch, were seen sporadically in most treated male groups and in one female of the high dose group towards the end of the dosing period. These changes did not persist into the recovery phase and were considered to be of no toxicological significance. Further clinical observations, e.g. thickening, desquamation, chromodacryorrhea, erythema, small scattered scabs and scabs at the edge of the application area, consistent with those commonly seen in dermal toxicity studies as a consequence of bandaging, were seen in control and treated animals at a similar incidence, and/or showed no evidence of a dose-response; thus, these findings were not treatment-related.
BODY WEIGHT
Group mean body weights in males given 30 mg/kg bw/d were slightly below control values but the difference seldom achieved statistical significance (main study 7% and recovery group 12% below concurrent control values). However the body weight gains in both main study and recovery animals at this dose level were generally statistically significantly lower than the control group values (85 and 57% of control values, respectively) with the difference being more marked in the recovery group animals. It was noted that the effect was variable with some animals more markedly affected than others. During recovery, the body weight gain for the treated animals was identical to control.
Body weights and body weight gains in males given 10 or 3 mg/kg bw/d and in females at all dose levels were similar to controls throughout the study. The slight, transient, dip in body weight noted in all main study groups during week 4 of the study was a consequence of withdrawal of food and water during urine collection.
FOOD CONSUMPTION
Food consumption in all groups was generally similar to control values. However individual males in the 30 mg/kg/day main and recovery showed some reduction in food consumption.
HEMATOLOGY AND CLINICAL CHEMISTRY
There were statistically significant differences from control values in some hematological parameters. These were slight, were present in the recovery group (30 mg/kg bw/d) only and/or showed no evidence of a dose-response relationship and therefore were considered to be unrelated to treatment.
URINALYSIS
There were statistically significant differences from control values in some urine clinical chemistry parameters. These were slight and were present in the recovery group only, and therefore are considered to be unrelated to treatment with the test substance.
NECROPSY
Organ weights in all treated groups were similar to control values.
There were no compound-related macroscopic changes.
There were no compound-related microscopic changes.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

LOAEL

Effect level:

> 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Only a NOEL but no NOAEL was derived by the study author. However, the NOAEL of 30 mg/kg bw/d can be derived based on the following rational: As there were no other compound-related effects, in males, at this dose level or in either sex at any dose level and moreover no compound related effect that could be related to the decrease in the body weight gain, the transient effect was not considered to be an adverse effect and a NOAEL of 30 mg/kg/bw/d was determined, despite the slightly statistically difference between the weight gain of the test group treated with 30 mg/kg bw/d and the control group.