Registration Dossier

Administrative data

Description of key information

(S.I.) > 3 --> Skin Sens. 1

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16. May. 2007 - 27. March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation Local Lymph Node Assay ( adopted 24 April 2002)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Test Item Preparation
The test item was placed into a volumetric flask on a tared balance and the vehicle (propylene glycol) was quantitatively added. The test item concentrations were prepared serially. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made freshly before each dosing occasion.
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest was performed in two mice on three consecutive days.
The data showed that the highest test item concentration, which could be technically used was 100% of the undiluted test item. After treatment with this concentration the treated mouse showed signs of irritation. At concentrations of 10, 25, and 50% the treated mice did not show any signs of irritation. After the second and third application of the high dose (100%) redness of the ear skin was observed. After the third application of 100% loss of
hair was also observed.
The test item in the main study was assayed at 10, 25, and 50%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
Concentrations were in terms of material as supplied.
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Test system: Mice, CBA/CaOlaHsd
- Rationale: Recognised as the recommended test system
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Number of animals for the pre-test (non-GLP): 2 females
- Number of animals for the main study: 16 females
- Number of animals per group: 4 females (nulliparous and non-pregnant)
- Number of test groups: 3
- Number of control (vehicle) groups: 1
- Age: 7 - 8 weeks (beginning of acclimatisation)
- Identification: Single caging. The animals were distributed into the test groups at random and identified by cage number.
- Acclimatisation: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top, (EHRET GmbH, D-79302 Emmendingen)
- Bedding: granulated soft wood bedding, (Harlan Winkelmann GmbH, D-33178 Borchen)
- Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Environment: temperature 22 + 3°C, relative humidity 30-74%, artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
propylene glycol
Concentration:
test item concentrations of 10, 25, 50 and 100%.. See more information on section 9.2 Allocation (page 13) of the study report.
No. of animals per dose:
Four groups of four animals. See more information on section 9.2 Allocation (page 13) of the study report.
Details on study design:
Outline of the Performed Study
In order to study a possible allergenic potential of Sergomil L-60, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight.
The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a -scintillation counter.

Experimental Design and Procedures
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25, and 50% (w/v) in propylene glycol. The application volume, 25 Ul, was spread over the entire dorsal surface (Ø~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of H-Methyl Thymidine
H-methyl thymidine (HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 Ul of 78.5 UCi/ml 3HTdR (corresponds to 19.6 UCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of HTdR incorporation was then measured on a -scintillation counter. Similarly,
background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
Parameter:
EC3
Value:
6.7
Parameter:
SI
Value:
2.43
Key result
Parameter:
SI
Value:
4.07
Key result
Parameter:
SI
Value:
4.88
Cellular proliferation data / Observations:
OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: once daily (week day) from experimental start to necropsy.
- Body weights prior to the first application and prior to treatment with HTdR.
- Clinical signs (local / systemic) once daily (week day). Especially the treatment sites were observed carefully.

RESULTS
- Viability / Mortality: No deaths occurred during the study period
- Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
- Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with HTdR, was within the range commonly recorded for animals of this strain and age.
The individual body weight values are included in Annex 1.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test item was found to be a skin sensitiser under the described conditions. According to the CLP criteria an stimulation index (S.I.) of 3 or above (extracted from an Lymph nodes study) is enough for classify the substance as Skin Sens. 1
Executive summary:

In the study the test item suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices of 5.81, 11.14, and 23.92 were determined with the test item at concentrations of 10, 25, and 50% in propylene glycol. The EC3 value could not be calculated, since all obtained SI´s were above 3.

The test item was found to be a skin sensitiser in this assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Although the physical state described on section 1.2 (composition) and 1.4 (Appearance/ physical state/ colour) for the substance Reaction mass of copper digluconate, sodium sulphate and copper sulphate is solid, the manufacturing method (described in section 3.5.1.) only allows substance synthetisation as a dissolution, containing at most a 50% of Reaction mass of copper digluconate, sodium sulphate and copper sulphate.

Therefore, studies for physical hazards determination, were developed using solid substance form, whereas for toxicology and ecotoxicology hazards, the dissolution (≤ 50% Reaction mass of copper digluconate, sodium sulphate and copper sulphate) form was used as stated on guidelines.

In consequence, the substance has been classified taking into account that it always will be in a dissolution in a maximum of 50% of concentration.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the CLP criteria an stimulation index (S.I.) of 3 or above (extracted from an Lymph nodes study) is enough for classify the substance as Skin Sens. 1