Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 30, 1990 to Aug. 29, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
Adopted : 12 May 1981
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to OECD principles of GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 1-methoxy-2-amino-4-(2'-hydroxyethyl)-amino-sulfat (Lehmannblausulfat; 2-amino-4-hydroxyethylaminoanisole sulfate)
- Substance type: Pure active substance
- Physical state: Grey powder
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature (protected from light)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: not specified
- Expiration date of the lot/batch: not specified
- Purity test date: 7/08/1990 and 28/08/1990

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (25°C) , protected from the light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test article was prepared fresh daily immediately before application. The 30% (w/v) of test item solution in water and the application volume of 1ml/kg represent technbical limits with regard to solubility and application volume respectively.

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Remarks:
SPF-Pirbright White (Sub strain BOR:DHPW)
Details on species / strain selection:
THe guinea pig is, as a rodent, the standart experimental animal of choice. The large amount of available physiological data render it highly suitable for dermato-toxicological investigations. The guinea pig is one of the recommanded species according the OECD Guideline 410, "repeated dose dermal toxicity : 21/28-day study"
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animals were obtained form Firma Winkelmann, GartenstraBe 27, W-4799 Borchen.
- On arrival, the health of animals was examined by a veterinarian and animals with only good health conditions were used for the study.
- Age at study initiation: Not reported
- Weight at study initiation range: 307-360 g (males) and 302-351 g (females)
- Housing: The animals were housed individually in a battery of lattice plate boxes.
- Diet: "Ssniff-G diet produced by Ssniff-Spezial Diaten GmbH, 4770 Soest/Westf; ad libitum. The diet was periodically analyzed with respect to the content of chlorinated hydrocarbons, aflatoxins, heavy metals, arsenic and antibiotic activity.
- Water: Aqua fontana (from automatic watering nipples); ad libitum. Samples of drinking water were analyzed twice yearly for chlorinated hydrocarbons, heavy metals and arsenic. Bacteriological testing was also performed.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.5 ± 3.5°C
- Relative humidity: 50 - 85% (measured by thermohygrometer)
- Air changes: 16 times per hour and air was filtered adequately
- Photoperiod: The room was illuminated by artificial light for 12 hours daily (from 7.00 am to 7.00 pm)/12 hours dark.
IN-LIFE DATES: From: Aug. 01, 1990 To: Aug. 29, 1990

Administration / exposure

Type of coverage:
open
Vehicle:
water
Details on exposure:
PREPARATION: The test substance solution was prepared freshly daily immediately before application. Doses were adapted weekly according to the weight development of the animals

METHOD OF APPLICATIONS: The test solution was applied once daily (7 days/week) for 28 days with a graduated pipette within 1 h post preparation.

TEST SITE
- Area of exposure: 3 x 4 cm skin area (clipped free of hairs) on the animal’s back.
- % coverage: About 10% of the total body surface
- Type of wrap if used: None; Range finding studies showed that it was not necessary to cover the test site to ensure that the animals could not ingest the test substance.
- Time intervals for shavings or clippings: Fur was clipped twice per week.

TEST MATERIAL
- Amount(s) applied: 1 mL/kg bw/day
- Concentration: 5, 15 and 30% w/v for 50, 150 and 300 mg/kg bw/day, respectively.
- Constant volume or concentration used: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample of the used batch were analyzed at the IBR’s Bioanalytical Centre, Hannover with respect to identity, stability and homogenity prior to study initiation and at termination.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily (7 days/week)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
5% (w/v) in water
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
10% (w/v) in water
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
30% (w/v) in water
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of the results of a range finding test. The intended human dose was 3% formulation of the test substance. 30% (w/v) solution of the test substance and the application of 1 mL/kg represented technical limits with regard to solubility and application volume, respectively.

- Rationale for animal assignment: The animals were randomly allocated to following four groups (3 treatments and 1 control), using a computer program on the basis of mean group body weight. The procedure was carried out separately for both the sexes.
Group I (Control): Vehicle control (tap water)
Group II (Low dose): 5% (w/v) (equivalent to 50 mg/kg bw/day)
Group III (Mid dose): 15% (w/v) (equivalent to 150 mg/kg bw/day)
Group IV (High dose): 300% (w/v) (equivalent to 300 mg/kg bw/day)

Examinations

Observations and examinations performed and frequency:
VIABILITY/MORTALITY: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Observations included: Sensory and motor behavior, skin, body orifices, urine and fecal excretion, general health status and dose responses.

DERMAL IRRITATION: Yes
- Time schedule for examinations: Daily (during early morning hours), prior to each new administration.
- Scoring of the skin (dermal) reactions was based on modified Draize scheme.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded individually in weekly intervals.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were withdrawn on the day before initiating the study (Day 0) and at termination of the study (Day 28).
- How many animals: All animals
- Parameters examined: Erythrocytes (RBC), leukocytes (WBC), thrombocytes, hemoglobin, hematocrit, reticulocytes, MCV, MCH, MCHC, differential blood count and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples were withdrawn on the day before initiating the study (Day 0) and at termination of the study (Day 28).
- How many animals: All animals
- Parameters examined: Following parameters were observed:
1) Substrates: Bilirubin (total), creatinine, glucose, urea nitrogen (BUN) and Uric acid.
2) Lipids: Triglycerides and cholesterol
3) Proteins: Total proteins and electrophoresis for albumin, alpha1 + alpha2 globulin, beta-globulin and gamma-globulin.
4) Electrolytes: Calcium, chloride, inorganic phosphorus, iron, potassium, sodium and NA/K ratio (by statistical evaluation).
5) Enzymes: Lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, creatine kinase (CK), gamma-glutamyl transferase and AST/ALT ratio (by statistical evaluation).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
The animals were sacrificed by the intravenous injection of 0.5 mL/kg T61 (Hoechst), blood was removed by cutting the A.carotis and complete autopsy was performed in all animals.

GROSS PATHOLOGY: Yes; The macropathological examination was performed which included inspection of the cranial, thoracic, abdominal and pelvic cavities.

HISTOPATHOLOGY: Yes; The hearts and the kidneys of the control and high dose animals were examined histopathologically. In addition, all gross lesions noted and the liver and the skin of all dose groups were examined microscopically.

All dissected organs were preserved in 8% buffered formalin. Samples from the tissues were trimmed, embedded in tissues wax and stained with hematoxylin and eosin. Frozen sections (stained with Sudan red) were prepared for heart, liver and kidney for assessment of fat content.
Other examinations:
ORGAN WEIGHT: At autopsy, terminal body weights of all animals were measured to calculate relative organs weights of liver, kidneys, heart, spleen, testes (with epididymides), ovary, uterus and adrenal glands.
Statistics:
Statistical analyses were performed separately for male and female animals.
- One- rep. two-factorial analysis of variance was performed for the evaluation of weight changes in the animals. Scheffe’s method was employed to compare group mean values.
- The organ weights were evaluated by analysis of co-variance. The comparison of the mean values was performed by Scheffe’s method for the analysis of co-variance.
- Values of clinical chemistry and hematology were analyzed as follows:
a) Analysis of variance for dose-effect curves with the factors group and time and the interaction group/time. The degrees of freedom for the factor time and the interaction group/time were corrected according to Greenhouse and Geisser (Epsilon-correction).
b) Mean values were compared according to the Scheffe’s method, after a preceding analysis of co-variance. The comparison (of the mean values) was carried out by correction with analysis of co-variance in such a manner as if the curves were originated from the same starting-point.
c) If there were available one point time values only, an analysis of variance with subsequent Scheffe’s test for analysis of variance was performed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
- No mortality was observed during the whole study period. The animals were sacrificed at the end of the study

CLINICAL SIGNS
- All animals of the control and dose groups displayed normal habits and behaviors during the entire study. No clinical signs of toxicity were observed in any animal.

DERMAL (SKIN) REACTIONS
- No signs of erythema or oedema formation were observed.

BODY WEIGHT AND WEIGHT GAIN
- The body weight was not affected by the treatment of the test substance.

HAEMATOLOGY
- Hematology parameters were within normal range during the entire study.

CLINICAL CHEMISTRY
- Clinical chemical values generally were within normal ranges.

ORGAN WEIGHTS
- Organ weights did not reveal any intergroup differences and no treatment related changes were observed.

GROSS PATHOLOGY
- There were no test substance related changes in the gross (macroscopic) findings. The findings on lung, liver, intestine and salivary glands were similar in control and all test groups.

HISTOPATHOLOGY
No test substance related changes were observed in heart, kidney and liver. In the skin an increased incidence of hyperplasia and hyperkeratosis in the epidermis of treated animals was observed; especially in the male high-dose group animals i.e. 300 mg kg/bw/day. This response was due to mechanical irritations caused by the clipping process and not considered to be treatment-related.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No indication of systemic toxic effects at any of the tested concentrations.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

ANALYTICAL VERIFICATION OF TEST SOLUTION: The stability and homogeneity of the test solutions were analytically verified and were in good agreement with the nominal doses, if analysis was done within 1 h after preparation. Thus, a sufficient stability and correctness of the dosing solutions can be assumed during the application period of 1 h.

Applicant's summary and conclusion

Conclusions:
Percutaneous application of 1 mL/kg bw/day Lehmannblausulfat (1-methoxy-2-amino-4-(2'-hydroxyethyl)-amino-sulfat) at concentrations of 5, 15 and 30% (w/v) (equivalent to 50, 150 and 300 mg/kg bw/day) for 28 days to guinea pigs revealed a NOAEL of 300 mg/kg bw/day.
Executive summary:

A repeated dose percutaneous toxicity study of Lehmannblausulfat (1-methoxy-2-amino-4-(2'-hydroxyethyl)-amino-sulfat) was performed following OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study).

SPF-Pirbright White (Sub strain BOR: DHPW) guinea pigs (weight range: 307-360 g (males) and 302-351 g (females)) were obtained from Firma Winkelmann, GartenstraBe 27, W-4799 Borchen. The animals were housed individually in a battery of lattice plate boxes under standard laboratory conditions (temperature: 19.5 ± 3.5°C; relative humidity: 50 – 85%; air changes: 16 times per week; photoperiod: 12 h artificial fluorescent light/12 h dark). The animals were acclimatized for 7 days under laboratory conditions before study initiation.

1 mL/kg bw Lehmannblausulfat (0, 5, 15 and 30 % in tap water; equivalent to 0, 50, 150 and 300 mg/kg bw) was applied dermally once daily for 28 days to the clipped area of skin on the animal’s back (3 x 4 cm; approximately 10 percent total body surface).. The test solution was applied within 1 h post preparation.

During the 28 day treatment period, mortality was checked twice daily, clinical signs were recorded once daily and individual body weights of animals were recorded weekly. Clinical laboratory investigations (haematology, blood/clinical biochemistry and urinalysis) were performed on Day 0 and on Day 28. All animals were subjected to a detailed necropsy and a number of organs (adrenals, heart, kidneys, liver, ovaries, spleen, testes, uterus) were weighed. The hearts and the kidneys of the control and high dose animals were examined histopathologically. In addition, all gross lesions noted and the liver and the skin of all dose groups were examined microscopically.

The daily skin evaluation revealed no indications of erythema or oedema formation. Hyperplasia and hyperkeratosis noted in the epidermis of treated animals were observed; especially in the male high-dose group i.e. 300 mg kg/bw/day. These effects were considered to be due to mechanical irritations caused by the clipping process and were not considered to be treatment-related. No mortalities or clinical signs of toxicity were noted. The body weight was not affected by the treatment. There were no treatment-related haematological or clinical biochemistry changes. At necropsy, no gross lesions were noted. Organ weights did not reveal any differences between the dose groups. No indications of systemic toxic effects were noted in the dermal 28-day toxicity study in guinea pigs.

In conclusion, percutaneous application of 1 mL/kg bw/day Lehmannblausulfat (1-methoxy-2-amino-4-(2'-hydroxyethyl)-amino-sulfat) at concentrations of 5, 15 and 30% (w/v) (equivalent to 50, 150 and 300 mg/kg bw/day) for 28 days to guinea pigs revealed a NOAEL of 300 mg/kg bw/day

This sub chronic toxicity study is classified as acceptable, and satisfies the guideline requirements of OECD 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study).