Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to official OECD TG method and GLP with certificate
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Info specific to Dose Range Finding Animals:
Species and strain: CRL: NMRI BR mice
Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the test: Good conventional

Justification of strain: The aim of the preliminary screen was to assess the ability of the test item causing irritation and/or systemic toxicity (not the sensitizing effect), CRL: NMRI BR mice were used in the pre-screen test for a better visibility of erythema and oedema.

Number of animals: 4 animals (2 animals/group);
Sex: Female, nulliparous, non pregnant
Age of animals: Young adult mice, 6-7 weeks old

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Info specific to Main Test Animals:
Species and strain: CBA/Ca mice
Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional

Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.

Number of animals: 4 animals/treatment group; 20 animals/main test
Sex: Female, nulliparous, non pregnant
Age of animals: Young adult mice, 10-12 weeks old (at start of the experiment)
Body weight range at starting: 19.1-22.9 g; The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
Acclimatization time: 7 and 14 days

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General info:

Husbandry:
Animal health: Only healthy animals were used
Housing during acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.

Food and Water Supply:
Animals received ssniff (R) SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, and tap water from municipal supply, as for human consumption, from a bottle ad libitum. For contents of standard diet for rats and mice and actual batch number of the diet see Appendix III.

Bedding:
Lignocel (R) Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberger (Germany) Holzmühle 1) was available to animals during the study.

Identification of Animals:
The individual identification of the animals was performed by numbers on the tail. The cages were marked with identification cards, with information about study number, sex, dose group and individual animal numbers.

Randomization:
The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software according to the actual body weights verifying the homogeneity and deviations between the groups.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test item formulations were prepared with Acetone: Olive oil 4:1 (v/v) mixture (AOO), except at concentration of 100 % where the undiluted test item was used. Homogenous formulation was achieved with the vehicle.

AOO was used as a vehicle control (0 % w/v diethyl carbonate) both for the test item and the positive control (PC) groups. Concentration of 25 % of the PC substance was tested as positive control group (25 % w/v). The test item was administered in three different concentrations according to the results of the preliminary test (25 %, 50 %, 100 % (w/v)).
No. of animals per dose:
4 animals/treatment group (3 diethyl carbonate test groups, 1 positive control test group, 1 negative/vehicle control group)
5 treatment groups
20 animals/main test
Details on study design:
According to the preliminary test results (dose range finding test), test item concentration of 100 % (the undiluted test item) was selected as the highest test concentration in the main test.

Main Test:

In vivo Treatment:
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (positive control group) and the vehicle (negative control group) using a pipette, on the dorsal surface of each ear. After the treatments animals returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation Assay:
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technically failed treatment was observed during the test. Hence no animals were excluded from the evaluation (all animals treated were processed).

Methodology for Observations:

Clinical Observations:
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring (during the whole test) and recorded for each animal individually.

Measurement of Body Weight:
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Evaluation of the Results:
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPN (DPM divided by the number of pooled lymph nodes). The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

Interpretation of the Results:
The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Assay acceptance criteria:
- Lymph nodes from all 4 animals per dose groups were pooled for a valid experiment.
- Skin sensitizing effect was observed at the applied concentration of the positive control (the stimulation indexes were greater than 3).
According to this, the assay acceptance criteria given in the Study Plan was fulfilled, hence the test was valid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Key result
Parameter:
SI
Value:
0.3
Test group / Remarks:
100 % test item
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
50 % test item in acetone/olive oil (4:1 v/v)
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
25 % test item in acetone/olive oil (4:1 v/v)

Detailed information on the results of the dose range finding test (skin irritation test) can be found under 7.3.1 Skin irritation / corrosion (7.3.1.002).

Main test results:

Body Weight Measurement:

Loss of body weights was observed in all test groups (including controls) in case of some animals. The effect was not treatment related. The mean body weights did not decrease significantly (or did not decrease at all) at the end of the test, hence the effect was considered to be not significant.

Clinical Observations:

No mortality or symptoms of systemic toxicity were observed during the study. No sign of irritation was observed in any treatment groups.

Proliferation Assay:

Larger than normal lymph nodes were observed (by visual observation) in the positive control and in the 50 % test item treated groups. Appearance of the lymph nodes was normal in the negative (vehicle) control group and in the other test item treated groups (100 % and 25 %). Inspite of the increased lymph nodes observed visually at the 50 % dose group no significant lymphoproliferative(SI ≥ 3) response was noted for Diethyl carbonate at the tested concentrations. The observed stimulation index values were 0.3, 0.6 and 1.5 at test item concentrations of 100 %, 50 % and 25 %, respectively. No biologically relevant dose-related response was observed.

Interpretation of observations:

Larger lymph nodes was observed in the 25 % test item treated group but the proliferation value was less than the control (the SI value was 0.6). Although the increased lymph node size is in a good correlation with an increased lymphocyte proliferation rate and the observation can help to evaluate the proliferation results obtained, visual evaluation of the lymph node size is very subjective. Hence this inconsistency is considered to be not significant and has no any effect on the reliability and validity of the results.

According to evaluation criteria of the OECD Guideline 429, since no proliferation value above 3 was observed either at the maximum achievable concentration of 100 % (the test item itself) or in an appropriate vehicle and the lack of a biologically relevant dose-related response are considered to be good evidence that Diethyl carbonate is not a sensitizer.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: based on the results and the expert statement of the laboratory that performed the study
Conclusions:
According to the results of the Local Lymph Node Assay in mice, the test substance diethyl carbonate was shown to have no sensitising potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Justification for selection of skin sensitisation endpoint:
Study performed according to official OECD TG method and GLP with certificate

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the results of the Local Lymph Node Assay in mice, the test substance diethyl carbonate was shown to have no skin-sensitising potential (see endpoint record 7.4.1.001, study report, Novasol, key data).