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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 March 2016 - 04 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008, including most recent amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance: Brown coloured, brittle solid
- Storage condition of test material: At room temperature
- Chemical name: Zinc modified rosinate, hydrogenated zinc rosinate
- CAS No.: 68425-02-5
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in methyl ethyl ketone at least to the concentration of 50% (maximal concentration required by the guildeline)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was ground to a powder using a pestle and mortar prior to weighing and the test item preparations (w/w) were prepared within 4 hours prior to each dosing.

OTHER SPECIFICS:
pH (1% in water, indicative range) 6.3 – 6.8 (determined by Charles River Den Bosch)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: ca. 10 weeks
- Weight at study initiation: 20.3-23.7 g
- Housing: group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: the ears were intact and free from any abnormality

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7-21.9
- Humidity (%): 41-53
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 02-03-2016 To: 04-04-2016

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
Pre-screen: 5, 10, 25 and 50%
Main study: 0 (vehicle control), 1, 2 and 5%
No. of animals per dose:
Pre-test: 2 females/dose
Main study: 5 females/dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: soluble in methyl ethyl ketone at least up to 50% (maximal concentration required by the guideline)
- Irritation: Very slight to well-defined erythema was noted for all pre-screen animals. Additionally, scaliness, bald spots, dry skin and/or scabs were noted for the animals. Variations in ear thickness during the observation period were more than 25% from Day 1 pre-dose values for the animals treated at 25% and 50%.
- Systemic toxicity: Both animals treated at 10% and 25% showed piloerection on Day 3. The animals treated at 50% showed piloerection and hunched posture between Days 3 and 6.
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Variations in ear thickness during the observation period
were less than 25% for the animals treated at 5% and 10%. White staining of test item remnants on the dorsal surface of the ears of the animals treated at 25% and 50% (Days 2-6), did not hamper scoring of erythema.
- Erythema scores:
50%: Both ears 0 in both animals on day 1, both ears 1 in both animals on day 2, both ears 2 in one animal and left ear 2, right ear 1 in another animal on day 3, both ears 2 in both animals on days 4, 5 and 6
25%: Both ears 0 in both animals on days 1 and 2, both ears 1 in both animals on day 3, both ears 2 in both animals on days 4 and 5, both ears 1 in both animals on day 6
10%: Both ears 0 in both animals on days 1 and 2, both ears 1 in both animals on days 3 and 4, both ears 0 in both animals on days 5 and 6
5%: Both ears 0 in both animals on days 1 and 2, both ears 1 in both animals on day 3, both ears 0 in both aniamls on days 4, 5 and 6

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was ground to a powder using a pestle and mortar prior to weighing and the test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was
assessed by visual inspection of the solutions.
Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
On day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamaldehyde performed at Charles River Laboratories Den Bosch B.V. is available and indicates that the Local Lymph Node Assay as performed at Charles River Laboratories Den Bosch B.V is an appropriate model for testing for contact hypersensitivity

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.8
Variability:
± 0.2
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
2.8
Variability:
± 0.4
Test group / Remarks:
2%
Key result
Parameter:
SI
Value:
5.7
Variability:
± 0.6
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1
Variability:
± 0.1
Test group / Remarks:
0% (vehicle controls)
Key result
Parameter:
SI
Value:
4.4
Variability:
± 0.9
Test group / Remarks:
25% alpha-hexylcinnamaldehyde (positive control, reliability check)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 1287, 1927 and 4005 DPM, respectively. The mean DPM/animal value for the vehicle control group was 697 DPM. The mean DPM/amimal value for 25% alpha-hexylcinnamaldehyde in the reliability check was 4551 ± 643.

DETAILS ON STIMULATION INDEX CALCULATION
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 2.2% was calculated.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Very slight erythema was noted for the animals treated at 5% between Days 2 and 4. Scaliness was noted for three control animals, one animal treated at a concentration of 1% and all animals treated at 2% and 5%. White staining of test item remnants on the dorsal surface of the ears of the animals treated at 2% and 5% (Days 2-3), did not hamper scoring of erythema.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Any other information on results incl. tables

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In a GLP-compliant guideline study, the test item induced the Stimulation Indices of 1.8, 2.8 and 5.7 when tested as 1, 2 and 5% in methyl ethyl ketone, respectively. Based on the results of the study, the test item is considered to be sentising to skin.
Executive summary:

In a GLP-compliant OECD guideline 429 study (LLNA assay), the test item was tested as 1%, 2% and 5% solution in methyl ethyl ketone in female CBA/J mice (5/group). The test concentrations were chosen based on the results of the pre-screen test in which 4 groups of two animals were treated with 5, 10, 25 and 50% solution of the test substance. Both animals treated at 10% and 25% showed piloerection on Day 3, while the animals treated at 50% showed piloerection and hunched posture between Days 3 and 6. Variations in ear thickness during the observation period were less than 25% for the animals treated at 5% and 10%. Based on the systemic tolerability of the test substance, 5% was chosen as the highest concentration for the main study. In the main study there were no mortalities or clinical signs of systemic toxicity. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Very slight erythema was noted for the animals treated at 5% between Days 2 and 4. Scaliness was noted for three control animals, one animal treated at a concentration of 1% and all animals treated at 2% and 5%. White staining of test item remnants on the dorsal surface of the ears of the animals treated at 2% and 5% (Days 2-3), did not hamper scoring of erythema. The mean DPM/animal values in the main study were 1287, 1927 and 4005 DPM, respectively, vs. 697 DPM in vehicle controls and 4551 ± 643 DPM in 25% solution alpha-hjexylcinnamaldehyde (positive control). The calculated Stimulation Indices were 1.8, 2.8 and 5.7 for 1%, 2% and 5% solution of the test item, respectively. The EC3 value was determined by linear extrapolation and was 2.2%.The validity of the study was confirmed by the reliability check with alpha-hexylcinnamaldehyde, performed not more than 6 months previously at the test facility (SI 4.4 ± 0.9 at 25% alpha-hexylcinnamaldehyde). Based on these results, the test substance is considered to be sensitizing to skin under the conditions of the study and should be classified as Skin. Sens.1, H317 according to Regulation 1272/2008/EC.