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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-06 to 2010-11-30
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl oxalate
EC Number:
EC Name:
Diethyl oxalate
Cas Number:
Molecular formula:
diethyl oxalate
Test material form:
other: colourless oily liquid
Details on test material:
- Name of test material: Diethyloxalate

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at study initiation: 8-10 weeks (at start of dosing)
- Weight at study initiation: 16.46 to 19.77 g (at start of dosing)
- Housing: group-wise maximum six in macrolon cages (35x20x15cm) with sterilized softwood shavings.
- Diet: Pelleted standard diet for experimental animals ad libitum. Microbiological control and content of nutrients was performed.
- Water: Drinking tap water ad libitum.
- Acclimation period: at least 5 days
- Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed.
- Cage identification: By cage number, study number and group specific colour.
- Animal identification: By felt tip marking (from 1 to 5 per each group and group specific colour).
- Random selection: Yes.

- Temperature: 22±3°C, permanently monitored
- Humidity: 30 – 70 %, permanently monitored
- Air changes: approximately 15 air changes per hour
- Photoperiod: 12-hour light/dark cycle

Animal arrival/ start of acclimatization: 2010-03-03
Pilot experiment: 2010-10-06 to 20120-10-09
First day of administration: 2010-11-24
End of treatment period: 2010-11-26
Application of radionuclide and necropsy: 2010-11-29
Experimental completion date: 2010-12-06

Study design: in vivo (LLNA)

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
The test substance was administered as suspensions in DAE 433.
Following concentrations were formulated:
30% (w/v); 300 mg/mL
3% (w/v); 30 mg/mL
0.3% (w/v); 3 mg/mL
No. of animals per dose:
5 females
Details on study design:
The highest concentration 30% was administered to three animals to assess a possible systemic toxicity. The route of administration was the same as in the main study. During the pilot experiment no clinical symptoms were observed in all three animals and no macroscopic changes (after necropsy) were detected.

Animals were subjected to a clinical examination (health check) shortly after arrival.
Study animals were randomly allocated to the dose groups manually and assigned animal numbers.

- Criteria used to consider a positive response:
The results of the LLNA were evaluated according to the following criteria.

Stimulation index:
The SI was obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group; this yields a mean SI.

Cell proliferation:
The response towards the test substance was considered positive, if the stimulation index (SI) was ≥ 3, and the response increased in dose-related manner (dose-response relationship).
The response was considered negative, if the stimulation index (SI) was < 3 without the dose–response relationship.
The response was considered ambiguous if the stimulation index was < 3, but the response increased in dose-related manner (dose–response relationship), and eventually statistical significance was observed.

Validity criteria:
The test was considered valid, if the positive control substance DNCB produced a positive LLNA response, and if the stimulation index SI was > 3 over the negative control group.

Ear weight – irritation effect:
If after treatment with the test substance a statistically significant increase of the ear weight together with a clear concentration dependence of the effect was recorded, the inflammatory effect was considered as irritation induced by the test substance.
A positive result in cell proliferation reveales that the test substance could be a contact allergen, but an irritation effect of the test substance (increased ear weight) did not rule out the possibility that it could be a false positive result.

- Dosage volume: 25μL / ear / animal
- Preparation for administration: All solutions were prepared by mixing an appropriate amount test substance and the vehicle to obtain the application form at concentrations of 30%, 3% or 0.3% (w/v). The solutions were prepared before the start of application by mixing on a magnetic stirrer and then were still mixed during application.
- Frequency of preparation: Each day before administration.
- Application: The volume of the application form was constant at all groups of animals - 25μL of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipettes to avoid losses caused by draining from the ear.
- In vivo examination:
Health and mortality: at least twice daily during the dosing period
Clinical observations: at least twice daily during the dosing period
Body weight: on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide
- Post mortem investigations:
Immediately after death, both ears of one animal were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (= 0.5 cm2) were excised using a disposable punch and weighed together on analytical scales.
- Lymph node cell counts:
Incorporation of 3H-methyl thymidine: The tissues of both of the lymph nodes were prepared by gentle mechanical disaggregation through 100 mm mesh nylon gauze with concomitant pooling in 1 mL PSB (phosphate buffered saline). The pairs of lymph nodes were analysed separately for each animal per group. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C for 18h. The pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine were measured by β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM).
Positive control substance(s):
other: dinitrochlorbenzene (DNCB), 0.5% (w/v)
For statistical calculations the software Statgraphic Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for all two-group comparisons.

Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Results and discussion

Positive control results:
Examination of cell proliferation in lymph nodes: In the positive control group, the SI was 9.19 (≥ 3) – therefore this LLNA test was valid.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
high dose level (30 %)
Test group / Remarks:
mid dose level (3 %)
Test group / Remarks:
low dose level (0.3 %)
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM for the test groups treated with the test substance was decreased in a dose-related manner (see Table 1).
Cellular proliferation data / Observations:
In the positive control group, the SI was > 3 (9 .19). At the highest dose level of the test article, the SI was 0.84, at the middle dose level the SI was 1.07, and at the lowest dose level, the mean SI was 1.01. The stimulation indexes of all the test groups were below the threshold.

Stimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from the test substance groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.

not apllicable

No symptoms of toxicity were observed in animah; from the negative control group and in all treated animals. All animals in the positive control group showed these symptoms caused by the application ofDNCB: decreased response on stimuli, hyperaemia ofskin and clonospasm.

There was no significant difference in body weight of treated groups in comparison to the vehicle control.

Any other information on results incl. tables

The animals exposed to the test substance at all concentrations showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment.

Table 1  Summary table

Group Radioisotope incorporation Ear weight
Mean DPM SI Mean (mg)
NC 187.12 1.00 23.42
PC 1720.05  9.19+  36.86*
30% 157.21 0.84 22.84
3% 199.94 1.07 22.82
0.3% 189.57 1.01 22.78
Figures with asterisk = values increased with statistical significance on probability level < 0.05 (Mann-Whitney test)
Figures with cross = values ≥ 3
NC – Negative control group
PC – Positive control group

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Under the given test conditions, the test substance elicited a negative response in the LLNA test.
Executive summary:

The test substance was tested for the assessment of skin sensitisation potentia with the murine local lymph node assay.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. Ll42, 2008.

In this study the contact allergenic potential of of the test substance was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day to test and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated on the basis of the use of radioactive labelling. The ratio of the proliferation in treated groups to that in vehicle controls, termed the Stimulation Index, was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and

test substance: 30%, 3%, 0.3% (w/v) in DAE 433.

The animals exposed to the test substance at all concentrations showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment.

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and Stimulation Index of cell proliferation 9.19, which was in congruence with its expected mode of action as a contact allergen.

The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance did not cause a significant increase in radioisotope incorporation into the DNA of proliferating lymphocytes.

In conclusion, at the given experimental conditions the result of the LLNA study with the test substance was negative.