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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:
2004-04-13 to 2004-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April 2002
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Succinimide
EC Number:
204-635-6
EC Name:
Succinimide
Cas Number:
123-56-8
Molecular formula:
C4H5NO2
IUPAC Name:
pyrrolidine-2,5-dione
Details on test material:
- Name of test material: Succinimide
- Chemical name: 2,5-Diketopyrrolidine
- Physical state: CWhite scalls
- Batch No.: SSGS 029
- Solubility: in water 330 g/L at 20 °C
- Melting point: 123 - 125 !C
- Storage condition of test material: Dark , usage under light possible.
- Stability at conditions of storage: Stable.
- Stability in solutions: Stable.
- Date of expiry: 30 June 2005.
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was solved in DMSO

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species, strain: Mice, CBA/CaOlaHsd
- Supplier: Harlan Winkelman GmbH, D-33176 Borchen
- Sex, specification: Females, healthy young adult nulliparous animals
- Age of the animals: About 8 weeks at the first administration
- Weight range of the animals at the first application: 16.2 - 22.9 g
- Spare animals: The 5th animal of each group served as spare animal. It was treated in the same way as the other animals of its group
Since all animals survived and all animals received the correct amount of 3HTdR all spare-animals were taken for the examination of the results.

ENVIRONMENTAL CONDITIONS
- Hygiene: Optimal hygienic conditions
- Room temperature: Average of 22.0 °C (continuous monitoring and recording)
- Relative humidity: Average of 44.4 % (continuous monitoring and recording)
- Light: Only artificial light from 6.00 a.m. to 6.00 p.m.
- Cages: Single caging. Makrolon cages type II, (22 cm x 16.5 cm ground area, 15 cm high)
- Feed: Altromin maintenance diet for rats and mice, item No. 1324forte, ad libitum
- Water: Tap water from Makrolon-bottles with stainless steel canules, ad libitum
- Bedding material: Aspen wood chips, ABEDD, Dominik Mayr KEG, A-8580 Köflach
Germ reduction by autoclaving
- Identification of animals: by shearing a defined for region on the back of the animal and by cage tag
- Acclimatisation: 6 days

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
•Group A (low dose): 10 % (w/w) in DMSO
•Group B (mid dose): 25 % (w/w) in DMSO
•Group C (high dose): 46.1 % (w/w) in DMSO
•Group P (positive control):     25% (v/v) solution of hexyl cinnamic aldehyde (technical grade, 85%) in acetone:olive oil (4:1, v/v)
•Group K (negative control):  DMSO
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A range finding study was performed with two animals/concentration with following concentrations: 46.1 % and 25 % (46.1 % is the maximal achievable concentration suitable for application). In this study the animals were treated in the same manner as in a main study with 25 µL test substance on the dorsum of each ear on three consecutive days. Ear thickness and body weight were measured on Day 1 before the first administration and on Day 4 about 24 hours after the last administration.
None of the animals showed overt systemic toxicity or excessive local skin irritation. The observed little increase in ear thickness is known from our experience within the normal range and does indicate slight irritation at the most. Therefore, 46.1% was chosen as highest test substance concentration and ear thickness measurement was not performed in the main study.


MAIN STUDY
Bias control
The individual animals were allocated to their groups by random numbers.

Test design
The test substance was administered in 3 concentrations to the dorsal surfaces of the ears of the animals of the test substance groups. In a manner identical to that of animals in the treatment groups animals of one negative control group and one positive control group were treated with DMSO and hexyl cinnamic aldehyde, respectively. Each animal was treated for 3 consecutive days. 3 days after the last administration the proliferation of the lymphocytes of the draining lymph nodes was measured by the determination of the amounts of incorporated 3HTdR.

Incorporation of 3H-methyl thymidine in vivo
5 days after the first topical administration, each animal received 20 µCi 3HTdR by slow intravenous administration. The injection solution containing nominal 80 µCi/mL 3HTdR was prepared by the dilution of 1.152 mL 3HTdR (amersham pharmacia biotech, Kat. Nr.: TRA 310, batch B314, specific activity 2.0 Ci/mmol, radioactive concentration 1 mCi/mL, radiochemical purity 99.0 %, thymine content 0.1 %) with 13.248 mL PBS. Several minutes prior to 3HTdR administration the animals were kept restrained in Plexiglas-tubes and the tail veins were visualised by placing the tails in warm water. Then 250 µL of the injection solution were intravenously administered to each animal.

Preparation of single cell suspensions and determination of incorporated 3H-methyl thymidine
Approximately 5 hours after 3HTdR injection all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised. The lymph nodes of each group were pooled in PBS. A single cell suspension (SCS) of lymph node cells (LNC) was prepared by gentle mechanical disaggregation of the pooled lymph nodes through a 70 µm cell strainer. The SCSs were then transferred into centrifuge tubes and LNC were pelleted by centrifugation (4 °C, max. 200 g, 10 min). Afterwards supernatants were removed by aspiration. Then the LNC were resuspended and washed twice with PBS. After the final washing the supernatants were removed leaving just a small volume (<0.5 mL) and macromolecules were precipitated by incubation with 5 % trichloroacetic acid (TCA) at 4°C overnight. Each precipitate was pelleted by centrifugation (4 °C, max. 200 g, 10 min) and resuspended in 1 mL TCA. This suspension was transferred into scintillation vials containing 10 mL scintillation cocktail (Packard Bioscience: Ultima Gold, order no. 6013329) and 3HTdR incorporation was determined with a beta-scintillation counter (TriCarb 2200CA, Packard Instrument Co., protocol 4: single label 3H, dpm, AEC-quenchcurve in Ultima Gold from 21 June 2002).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Application of 25% HCA in AOO resulted in an SI of 5.0.
This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.7
Test group / Remarks:
Group A (low dose) 10% w/w test substance, 5 females
Parameter:
SI
Value:
0.7
Test group / Remarks:
Group B (mid dose) 25% w/w test substance, 5 females
Parameter:
SI
Value:
0.4
Test group / Remarks:
Group C (high dose) 46.1% w/w test substance, 5 females
Parameter:
SI
Value:
5
Test group / Remarks:
Group P (positive control), 5 females
Parameter:
SI
Value:
1
Test group / Remarks:
Group K (negative control), 5 females
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: Results are presented as test/control ratio (Stimulation index), calculated as dpm test group/dpm negative control group.
(dpm = disintegrations per minute, corrected by the subtraction of the background)
A substance is regarded as a sensitiser in the LLNA if the test substance induces a 3-fold or greater increase in 3HTdR incorporation into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes, as indicated by the SI, together with the consideration of dose response.
The Sl of the test substance groups were between 0.4 and 0.7.

CLINICAL OBSERVATIONS:
No abnormal behaviour or clinical signs were detected during the experiment in the animals. No local irritations were observed at the application sites of all animals of all test substance groups and the negative control group throughout the whole study. On Days 2-5 all animals of the positive control group had moderate erythema on the application sites indicating slight irritative skin reactions.

BODY WEIGHTS:
Body masses and body mass gain of all animals were in the range to be expected from animals of the same strain, sex and age. Body weight loss was noted in 1/5 animals in group A and C. On Day 1 the body mass of one animal of group C (22.9g) exceeded a little bit over the 20%of the mean weight (mean body mass + 20%: 22.5g) which is not of relevance for the obtained results.

POSITIVE CONTROL: Application of 25% HCA in AOO resulted in an SI of 5.0. This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

Any other information on results incl. tables

Table 1. Disintegrations per minute and calculated SI

   dpm  SI
 Group K (negative control)  8861  1
 Group A (low dose)  6536  0.7
 Group B (mid dose)  6127  0.7
 Group C (high dose)  2242  0.4
 Group P (positive control)  44,360  5.0

dpm = disintegrations per minute

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
"Succinimide" is regarded as a non sensitiser in the LLNA according to OECD-Guideline 429, since the SI of all examined test substance concentrations were clearly smaller than 3.
Executive summary:

In a dermal skin sensitisation study conducted according to OECD guideline 429 (Local Lymph Node Assay) with succinimide dissolved in DMSO at concentrations of 10% (w/w), 25% (w/w) and 46.1% (w/w), young adult female CBA/CaOlaHsd mice were tested. The test substance was solved in DMSO and was administered to three groups of 5 female CBA/Ca mice. Administration was performed epicutaneously to the dorsal surface of both ears, once a day on three consecutive days. The volume administered was 25 µL per ear. A 25% (v/v) solution of hexyl cinnamic aldehyde in acetone:olive oil (4:1, v/v) was used a positive control. All animals survived till the end of the study. No adverse effects were noted in any animal. Body mass and body mass gain was in the range to be expected from animals of the same strain, sex and age. Moderate erythema was noted in all animals of the positive control group on Days 2-5, indicating slight local skin irritation. No skin irritating effects were observed in the test substance groups and the negative control group throughout the whole study. The SI of the tested test substance concentrations were 0.7 (low dose), 0.7 (mid dose) and 0.4 (high dose). The positive control substance led to a stimulation index of 5.0, thus demonstrating the validity of the experiment.

In this study, succinimide is not a dermal sensitiser according to CLP criteria.