Black HM 2482

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:

From April 12th to May 10th, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

adopted May 12, 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive 84/449, Annex V, Part B, Methods for Determination of Toxicity "Sub-Acute Toxicity (dermal"

Version / remarks:

amended July 1983, doc. XI/450/83 D

GLP compliance:

not specified

Remarks:

The conduct of this study was subjected to periodic inspections and the report audited by the Quality Assurance Unit.

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-920-6

EC Name:

-

Cas Number:

89857-06-7

Molecular formula:

C50 H53 N11 O14 S

IUPAC Name:

5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

KFM-Han., outbred, SPF-quality

Details on species / strain selection:

Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: males: 233 - 261 ; females: 211 - 236 g
- Housing: individually in Makrolon type-3 cages with wire mesh lids. The animals had standard granulated soft wood bedding ("Lignocel" Schill AG , Switzerland).
- Diet: standard Kliba 343, Batch 15/85 and 18/85 rat maintenance diet ("KLIBA"-Futter, Klingentalmuehle AG, Switzerland) ad libitum.
- Water: community tap water from Itingen was available ad libitum
- Acclimation period: One week

DETAILS OF FOOD AND WATER QUALITY:
The feed batch was analyzed for contaminants.
The water was analyzed for chemical and bacteriological contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 55 +/- 10 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours of artificial fluorescent light each day

IN-LIFE DATES: From: To: From April 12th to May 10th, 1985

Administration / exposure

Type of coverage:

occlusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

2% solution

Details on exposure:

TEST SITE
- Area of exposure: shaved skin of the back
- % coverage: about 10 % of the total body surface
- Type of wrap if used: occlusive bandage of elastic adhesive dressing
- Time intervals for shavings or clipplings: once weekly throughout the study

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed of with luke-warm tap water
- Time after start of exposure: after termination of the daily treatment

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 ml/kg body weight

VEHICLE
- Amount(s) applied (volume or weight with unit): 4 ml/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days per week

Doses / concentrations

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

MORTALITY: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily prior to the following and after the application

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: end of application period
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 4 weeks of treatment
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 per dose; males and females
- Parameters checked: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet and reticulocyte count, nucleated erythrocytes, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin and partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 4 weeks of treatment
- Animals fasted: Yes
- How many animals: 10 per dose; males and females
- Parameters checked: glucose, urea, creatinine, bilirubin total, cholesterol total, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total

URINALYSIS: Yes
- Time schedule for collection of urine: at 4 weeks of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment,

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a pathologist. All animals were killed by intraperitoneal injection of sodium pentobarbital.

HISTOPATHOLOGY: Yes
The tissue examined included: adrenal glands, brain, heart, ileum, kidneys, liver, lungs with mainstem bronchi, ovaries, spleen, testes, treated skin, untreated skin, gross lesions.

Statistics:

The following statistical methods were used to analyze the bodycweights, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the t-test based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The U-test was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continous data and medians were calculated for discrete data (scores).
Individual values , means, standard deviations and statistcs were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled
variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
References:
C.W. Ounnett: A Multiple Comparison Procedure for Comparing several Treatments with a Control, J. Amer. Statist. Assoc. 50, 1096-121 ( 1955)
R.G. Miller: Simultaneous Statistical Inference, Soringer Verlag, New-York (1981) .
R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Slight to moderate crusts on the treated skin were observed in 7 out of 10 animals of the test article-treated group between day 4 to 28. The observed signs were of different intensity and duration in the individual animals. No other local or systemic symotoms were observed in the animals of the test article-treated or control group.

Mortality:

no mortality observed

Description (incidence):

No death occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences in body weight gain was observed between the animals of the test article-treated and control group during the test period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences in food consumption were observed between the animals of the test article-treated and control group during the test period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were observed in any animal of the test article-treated or control group.

Haematological findings:

no effects observed

Description (incidence and severity):

All statistical differences in the results of the hematological parameters were considered to be incidental and of normal bioogical variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All statistical differences in the results of the biochemical parameters were considered to be incidental and of normal bioogical variation.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

All statistical differences in the results of the urinalisis parameters were considered to be incidental and of normal bioogical variation.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The observed increased absolute kidney weights in female animals of group 2 were related to the significantly increased final body weights. Therefore it could be stated that the organ weights and organ to body weight ratios were comparable between the rats of the control and test article treated group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Slight focal inflammation was observed at the application site of two rats of group 2, only. In all other rats, the treated skin appeared to be normal.

Neuropathological findings:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

gross pathology

haematology

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based upon the results obtained, the no-observable-effect level of test substance is greater than 1000 mg/kg bw/day for male and female rats.

Executive summary:

The repeated dermal toxicity of the test item was evaluated following the guideline OECD Guideline 410. In this dermal toxicity study (limit-test), test item was applied to albino rats for 6 hours/day (7 days/week) for a total of 28 applications. The study was comprised of 2 groups, for a total of 20 rats.
The following dose of test item was applied: 0, 1000 mg/kg bw/day.
No death occurred during the study. The assessment of hematology, clinical biochemistry and urinalysis data indicated no changes of toxicological significance at termination of the treatment. Only local findings, described as slight to moderate crusts and slight focal inflammation of the skin were observed in the animals of the 1000 mg/kg group.
Based upon the results obtained, the no-observable-effect level of test substance is greater than 1000 mg/kg bw/day for male and female rats.