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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:
19 November 2009 -08 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-compliant, GLP proprietary study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
OECD 429 and Regulation (EC)No 440/2008 method B.42 standard LLNA method
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
White flakes with pungent odour. Solubility in water of 50 g/L (20 C, hydrolised) and density of 1.2340. Melting point of 119.6 C. Stable at room temperature. - Name of test material (as cited in study report):succinic anhydride- Substance type: dihydro-2,5-furandion cyclic acid anhydride- Physical state: solid- Analytical purity: 99.5%- Purity test date: 22 October 2009- Lot/batch No.: LEBA5A3070- Expiration date of the lot/batch: 30 June 2010- Stability under test conditions: stable- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Mice were acclimated for 13 days and were approximately 8 weeks old at first administration and weighed 17.8 -22.2 g. Animals were housed singly in Makrolon cages type II with autoclaved aspen wood chips which were changed 1/week. Nesting material and a polycarbonate shelter was provided per cage. Average room temperature of 21.2 C and average relative humidity of 58.9%. Artificial light was provided from 6:00am to 6:00pm. Mice were given maintenance diet and tap water ad libitum.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25, and 31.3 % w/w succinic anhydride
No. of animals per dose:
5 animals/ group (including spare animals). The spare animal of the low dose group died and therefore, the spare animals of other groups were not used. The number of animals/group used for lymph node examination was 4.
Details on study design:
Solubility testing of the test substance showed the highest concentration suitable for application of the test substance can be achieved with DMF (31.3%, w/w). A range finding study was performed with 2 animals/concentration at 31.3 and 25% succinic anhydride. None of the animals showed overt systemic toxicity, excessive local skin irritation at the application sites or an important increase in ear thickness in the range finding study. Therefore, 31.3% was chosen as the highest test substance concentration and ear thickness measurement was not performed in the main study.Main study: Test solutions were prepared fresh on each day of administration and administered in 3 concentrations to the dorsal surfaces of the ears of mice. One negative control group and one positive control group were treated with DMF and HCA, respectively. Each animal was treated for 3 consecutive days. Five days after the first topical administration, each animal received 20 uCi 3HTdR by slow intravenous administration. Approximately 5 hours after 3HTdR injection, all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised. The proliferation of the lymphocytes of the draining lymph node was measured by the determination of the amounts of incorporated 3HTdR.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Stimulation Indices were calculated according to the standard method

Results and discussion

Positive control results:
Application of 25% HCA in acetone:olive oil (4:1 v/v) resulted in an SI of 7.3.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index of 9.2, 11.6, and 11.0 was calculated for the low, mid and high dose groups, respectively. The negative and positive control groups had a stimulation index of 1 and 7.3, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 43520, 54949, and 52036 dpm for the low, mid and high dose groups, respectively. The negative and positive control groups had a dpm of 4733 and 34509, respectively.

Any other information on results incl. tables

One animal of the low dose group died on day 6 near application of radiolabeled H-methyl thymidine. This animal showed no adverse effects and no body mass loss during the study and gross necropsy was without observation. Therefore, this death was assumed to be random and not associated with test substance administration.

Table 1. Body mass index of animals on days 1 and 6

 Negative control           Low dose                    Mid dose        High dose              Positive control
  No.  b.m. day 1  b.m. day 6  b.m. gain   No.  b.m.day 1 b.m.day 6   b.m.gain No.   b.m.day 1 b.m.day 6   b.mgain   No.  b.m.day 1 b.m.day 6  b.m.gain    No.  b.m.day 1 b.m.day 6  b.m.gain 
 1  19.8 21.2  1.4   6  19.1 20.1  1.0   11 21.5  22.8  1.3   16 22.2  23.2  1.0   21 19.8  20.9  1.1 
 2  19.0 19.5  0.5  7 17.8   19.4 1.6   12 19.2  20.3  1.1   17  20.0 20.7  0.7   22  20.2 21.2  1.0 
 3  20.7 21.6  0.9   8  20.6 21.6  1.0   13  18.9 20.3  1.4   18  18.3 20.0  1.7   23  19.0 20.7  1.7 
 4  19.1 20.3  1.2   9  20.8 21.6  0.8   14 19.6  20.9  1.3   19  20.2 20.9  0.7   24  19.3 20.9  1.6 
 Mean  19.7 20.7  1.0  Mean   19.6 20.7  1.1  Mean  19.8  21.1  1.3  Mean  20.2  21.2  1.0  Mean  19.6  20.9  1.4 
 SD  0.8 0.9  0.4  SD  1.4  1.1  0.3  SD  1.2  1.2  0.1  SD  1.6  1.4  0.5  SD  0.5  0.2  0.4 
 n  4  4  4  n
    No. = Animal number; b.m.= body mass in g                                                     

Table 2. General observations and observations of the application sites on days 1 -6

 Finding  Group  No. of the affected animals  Observation time (day)
 General behavior normal  Negative control  1, 2, 3, 4  1 -6
   Low dose  6, 7, 8, 9  1 -6
   Mid dose  11, 12, 13, 14  1 -6
   High dose  16, 17, 18, 19  1 -6
   Positive control  21, 22, 23, 24  1 -6
 Application sites normal  Negative control  1, 2, 3, 4  1 -6
  Low dose   6, 7, 8, 9  1 -6
   Mid dose  11, 12, 13, 14  1 -6
   High dose  16, 17, 18, 19  1 -6
 Slight erythma  Positive control  21, 22, 23, 24  3 -4

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
A stimulation index of 9.2, 11.6, and 11.0 was calculated for the low, mid and high dose groups, respectively, showing no clear dose response relationship. The potency for skin sensitisation according to ECHA guidance R8, appendix 8 -10, table R 8 -23, Succinic anhydride is assessed to be weak/moderate (EC3 (%) >2).
Executive summary:

In a GLP study conducted according to OECD guideline 429, succinic anhydride was dissolved in dimethylformamide and administered to three groups of 4 female CBA/Ca mice. Succinic anhydride was administered epicutaneously to the dorsal surface of both ears, once a day on three consecutive days, at concentrations of 10%, 25%, or 31.3% w/w. The volume administered was 25 uL per ear. Positive and negative control groups were tested concurrently under identical conditions. Five days after topical administration, 3H-methyl thymidine was administered intravenously to all mice via a tail vein. Approximately 5 hours later, the draining auricular lymph nodes were excised, incorporation of radiolabeled 3HTdR was determined and compared with negative controls, and the SI index was calculated.

No adverse effects were noted in any animal. The SIs of the test substance concentrations were 9.2 (low dose), 11.6 (mid dose), and 11.0 (high dose). Although no clear dose response was observed, the SIs of all test substance concentrations were greater than 3 and succinic anhydride is regarded as a (weak/moderate) sensitizer in the LLNA.