Morpholinium, 4-[2-[2-[(2-hydroxyphenyl)methylene]hydrazinyl]-2-oxoethyl]-4-methyl-, chloride (1:1)

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japan MAFF 8147

GLP compliance:

yes (incl. QA statement)

Remarks:

Bioassay Labor für biologische Analytik GmbH, Im Neuenheimerfeld 515/519, 69120 Heidelberg

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

EC Number:

700-714-9

Cas Number:

1254469-57-2

Molecular formula:

C14H20ClN3O3

IUPAC Name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

Details on test material:

- Name of test item: Tinocat ES 96000
- Test item No.: 11/0013-2
- Batch identification: OP 2/2011
- Purity: 98.4 g/100 g determined by 1H-NMR-spectroscopy (for details see analytical report No.: 12L00295)
- Homogeneity: The test item was homogeneous by visual inspection.
- Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Expiry date: Nov. 2013
- Storage conditions: Ambient (RT)
- Physical state/ color: Solid / white

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: approx. 9 weeks; females: approx. 13 weeks. The female animals were nulliparous and non-pregnant
- Weight at study initiation: males: 265 - 290 g; females 220 - 238 g, Animals of comparable weight (± 20% of the mean weight)
- Housing: Makrolon cage, type III, Single housing
- Bedding: H 15005-29; Ssniff, Spezialitäten GmbH (Experimental Animal Diets Inc., 59494 Soest, Germany). The bedding and enrichment were regula rly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
- Enrichment: NGM E-022; ABEDD® LAB & VET Service GmbH, Hasnerstraße 84/6; 1160 Wien – Austria
- Diet : ad libitum,VRF1(P); SDS Special Diets Services, 67122 Altrip, Germany. The feed used in the study was assayed for chemical and microbial contaminants by the manufacturer in quarterly intervals
- Water: Tap water ad libitum. The drinking water was regularly assayed for contaminants by the municipal authorities of Heidelberg. The German Drinking Water Regulation of Dec. 5, 1990 served as the guideline for maximum tolerable contaminants
- Acclimation period: at least 5 days before the beginning of the experimental phase; during the acclimatization period, the animals were accustomed to the environmental conditions of the study and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): Approx. 10
- Day / night rhythm: 12 h / 12 h

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: Deionized water

Details on dermal exposure:

EXPERIMENTAL PROCEDURE
- Clipping of the fur: About 24 hours before administration
- Route of application: Single application to the clipped epidermis (dorsal and dorsolateral parts of the trunk);
- Area of exposure: about 40 cm2
- % coverage: at least 10%
- Type of wrap: air-permeable dressing (4 layers of absorbent gauze (Ph. Eur. supplied by Lohmann GmbH & Co., KG) and stretch bandage (Fixomull® Stretch (adhesive fleece) supplied by Beiersdorf AG).

Test Item preparation:
The test item preparation was produced for each application group shortly before application by stirring with a high speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test item during application was provided by stirring with a magnetic stirrer

REMOVAL OF TEST SUBSTANCE
- Washing: warm water
- Time after start of exposure: 24 hours

VEHICLE
- Amount(s) applied (volume or weight with unit): 3.33 mL/kg bw
- Concentration (if solution): 60 g/ 100 mL

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5/sex/dose

Control animals:

no

Details on study design:

EXPERIMENTAL PROCEDURE
Duration of observation period following administration: 14 days

Observations and weighing:
Body weight determination: individual body weights shortly before administration (day 0), weekly thereafter and on the last day of observation;
Clinical observations: recording of clinical signs several times on the day of administration, and at least once daily thereafter each workday for the individual animals.
Scoring of skin findings: individual readings 30 – 60 minutes after removal of the semi-occlusive dressing (day 1), several times until the animals were free of findings, weekly thereafter and on the last day of observation.
Mortality: A check for any dead or moribund animals was made at least once each workday.

Pathology:
Necropsy with gross-pathology examination on the last day of the observation period after sacrifice with CO2 in a chamber with increasing concentrations over time.

Assessment of skin reactions:
The evaluation of skin reactions was performed according to Draize, J.H. (1959): Appraisal of the safety of chemicals in foods, drugs and cosmetics. The association of food and drug officials of the United States Austin, Texas:

Erythema and eschar formation:
Grading
0 No erythema
1 Very slight erythema (barely perceptible)
2 Well- defined erythema
3 Moderate to severe erythema
4 Severe erythema (beet redness) to eschar formation preventing grading of erythema

Edema formation:
Grading
0 No edema
1 Very slight edema (barely perceptible)
2 Slight edema (edges of area well- defined by definite raising)
3 Moderate edema (raised approx. 1 mm)
4 Severe edema (raised more than 1 mm and extending beyond area of exposure)

Descriptions of any dermal findings not covered by this scale were recorded.

ANALYSES OF THE TEST ITEM:
Samples of the first concentration of the test item formulation were sent to the sponsor for analysis (BASF SE, Z470, 67056 Ludwigshafen, Germany).Analytical work will be done under the responsibility of the sponsor under GLP. The analytical report is not part of the study report.
The stability of the test item in the vehicle will be determined indirectly by concentration control analysis. For this purpose, the samples taken were stored at room temperature over the maximum duration of the administration period, subsequently deep-frozen and sent to the sponsor for analysis.
The homogeneity of the test item preparation will be determined indirectly by concentration control analysis. A concentration control analysis of the test item preparation and archiving of the respective data will be done by the sponsor.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Delayed mortality occurred in the male dose group. The animal with the number R008 was found dead at study day 6. The mortality was most likely incidental and not substance-related. Renal failure with following ascites is rarely but sometimes observed with this rat strain used.

Clinical signs:

other: Male animal clinical signs: - No systemic clinical signs were observed during clinical examination. - Very slight erythema (grade 1) was noted in one animal from study day 1 up to study day 3. Moderate to severe erythema (grade 3 to 4) and very slight ed

Gross pathology:

The following macroscopic pathologic findings were observed in the male animal that died:
- Swollen kidneys
- Ascites with erythroid liquid
No macroscopic pathologic abnormalities were noted in the other animals (4 males and 5 females) sacrificed on the last day of observation.