1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 28 - February 11, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study, conducted according to internationally accepted technical guidelines and in compliance with GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

of 1987

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

of 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Version / remarks:

of 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar rats, strain: Crl:WI (Han) (outbred, SPF-Quality), with appropriate range of bodyweight at study start.
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation (day of dosing): Approx. 8 weeks.
- Weight at study initiation( day of dosing): Mean (males): 243 g, minimum 235 g, maximum 249 g.
Mean (females): 168 g, minimum 159 g, maximum 180 g.
- Housing: Individual housing in M III type cages. (During acclimatization group housing).
- Diet (ad libitum): Commercially available rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days before treatment start under laboratory conditions.


ENVIRONMENTAL CONDITIONS

Animal housing and environmental conditions were appropriate for acute toxicity testing in the rat: Controlled environment with approximately 15 airchanges per hour, 12 hours artificial fluorescent light and 12 hours darkness per day and 19 – 21ºC. The relative humidity during the study period was 34 – 69%, except for three hours, during which the relative humidity dropped to a minimum of 18% due to a short term failure of the air humidifying system. This transient decline to low relative humidity did not compromise the quality, integrity or outcome of the study.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Preparation: One day before exposure an area of approximately 5x7 cm on the back of the animal was clipped. During health inspection of the animals prior to commencement of treatment, special attention was paid to the skin to be treated, which was intact and fre e from any abnormality.
- Area of treated skin: Approx. 25 cm2 in males, approx. 18 cm2 in females corresponding to approx. 10% of total body surface.
- Type of wrap used: Surgical gauze patch, successively covered with aluminium foil and Coban elastic bandage. For females additional fixation of the bandage with Micropore tape.

REMOVAL OF TEST SUBSTANCE
Topical treatment lasted 24 hours. Then the dressings (gauze, foil, bandages, tape) were removed and residual test substance washed off the skin with tap-water.


TEST MATERIAL AND DOSE PREPARATION
The dose listed in the present study is expressed as water- and minor impurity-free test substance. This was accounted for during dosage preparation by the application of factor 1.16 to all test substance concentrations and the test substance dose as follows: Test substance concentration and test substance dose administered = 1.16 times the test substance received at the testing laboratory. The formulation (w/w) was prepared within 4 hours prior to dosing. Homogeneity was accomplished to a visually acceptable level.

- Administered Dose (expressed as water- and minor impurity-free test substance): 2000 mg/kg bw (5 males +5 females).
- Vehicle: Water (Elix, Millipore S.A.S., Molsheim, France).
- Administration Volume: 10 ml/kg bw
- Justification for choice of vehicle:
Trial formulations at the testing laboratory and test substance data supplied by the sponsor led to the choice of water as a suitable vehicle. (The test substance is well soluble in water and stable for at least 96 hours in water and no adverse side effects of this vehicle on the animals are to be expected)

Duration of exposure:

24 hours

Doses:

Dose (expressed as water- and minor impurity-free test substance):
2000 mg/kg bw (5 males + 5 females)

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

Duration of the observation period following administration start (day 1) was 14 days during which mortality/survival and clinical signs were recorded at: 0, 2 and 4 hours after administration start on day 1 and twice daily (mortality/survival) or once daily (clinical signs) thereafter until day 15. Body weight was recorded on day 1 (prior to administration) for each animal and on days 8 and 15 for each survivor. The one premature death and all survivors killed at the end of the 14-day post-dosing observation period (day 15) were necropsied and macroscopic pathology findings recorded.

Statistics:

Statistical analysis is inappropriate for this study, as there was only one dose group.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

approximate LD50

Effect level:

> 2 000 mg/kg bw

Remarks on result:

other: Dose is expressed as water- and minor impurity-free test substance. 95% CL could not be determined, as only one dose was tested.

Mortality:

One female was found dead on Day 1, four hours after treatment start.

Clinical signs:

Piloerection in all surviving animals on Days 1 and 2;
Flat and/or hunched posture in all surviving animals on Days 1 to 3;
Ptosis in one animal on Day 1;
Chromodacryorrhoea (snout) in three animals on days 1, 2 and/or 3.
Scales and/or scabs and orange staining of the treated skin area in all surviving animals generally from bandage removal until termination of the 15-day observation period.
Clinical signs were not seen in the one decedent animal.

Body weight:

Body weight loss, marginal to slight in degree, was recorded in two female animals from Day 1 to 8 and was followed by slightly retarded body weight gain from Day 8 to 15. Body weight gain of males and the remaining female survivors was considered to be normal.

Gross pathology:

Necropsy of each animal did not reveal any macroscopic pathology findings.

Applicant's summary and conclusion

Interpretation of results:

relatively harmless

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The present study demonstrated that the LD50 for acute dermal toxicity of the test substance is higher than the limit dose of 2000 mg/kg b.w. Therefore, the test material is considered to be relatively harmless regarding acute dermal toxicity. It cannot be ruled out that the clinical signs seen in all survivors and the single death were caused by oral uptake (grooming behaviour) of residual test material after patch removal, since dermal absorption/toxicity of the test material seems rather unlikely, because of its high molecular weight.

According to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008] the results attained in this study do not necessitate any labelling regarding acute dermal toxicity.

Executive summary:

the test substance was tested for its acute dermal toxicity in male and female rats according to OECD Guideline 402 and the corresponding EC, EPA-OPPTS and JMAFF Technical Guidelines in compliance with GLP. Reliability grade 1 was assigned.

 

Over a period of 24 hours, five male and five female Wistar rats were exposed to the test substance at the limit dose of 2000 mg*/kg bw by occlusive administration onto clipped, intact skin on their back. The test substance was administered in aqueous solution (200 mg*/ml w/w) to approximately 10% of the total body surface (approx. 25 cm2 in males, approx. 18 cm2 in females). Treatment was followed by a 14-day observation period. Mortality/survival, clinical signs and gross necropsy findings were recorded.

 

One female animal was found dead on Day 1, four hours after treatment start. Clinical signs comprised flat and/or hunched posture and piloerection in all surviving animals, chromodacryorrhoea in three animals and ptosis in one animal on Days 1, 2 and/or 3. In addition, scales and/or scabs and orange staining of the treated skin area were seen in all surviving animals generally from bandage removal until termination of the 15-day observation period. Clinical signs were not seen in the one decedent animal. Body weight loss, marginal to slight in degree, was recorded in two female animals from Day 1 to 8 and was followed by slightly retarded body weight gain from Day 8 to 15. The body weight gain of males and the remaining female survivors was considered to be normal. Macroscopic pathology findings were not evident at necropsy.

 

According to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008] the results attained in this study do not necessitate any labelling regarding acute dermal toxicity.

____________________________________________________________________________________________________ 

* Expressed as water- and minor impurity-free test substance