Glycerides, mixed C8-10 and succinyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given (comparable to guideline).

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

no bodyweights determined; individual DPM and individual SI values for each animal are missing; no SI values for the positive control stated

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

no bodyweights determined; individual DPM and individual SI values for each animal are missing; no SI values for the positive control stated

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

no bodyweights determined; individual DPM and individual SI values for each animal are missing; no SI values for the positive control stated

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Succinic acid

EC Number:

203-740-4

EC Name:

Succinic acid

Cas Number:

110-15-6

IUPAC Name:

succinic acid

Details on test material:

- Name of test material (as cited in study report): succinic acid
- Substance type: endogenous substance
- Analytical purity: 99%

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca01aHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS:
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 6-12 weeks
- Housing: animals were housed in groups in Macrolon cages on Altromin saw fiber bedding.
- Diet: Altromin 1324 maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

5, 10 and 25% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the test substance was soluble in DMSO.
- Irritation: 3 different concentrations were tested to detect the highest tolerable exposure concentration for the main assay. The test substance was applied daily to the ears of 3 animals. Treated animals were observed for systemic effects and local skin irritation, which was assessed by measuring the ear swelling.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by β-scintillation
- Criteria used to consider a positive response: a positive response was obtained, if SI values of treated animals were greater 3 (corresponds to a 3-fold higher ³H-methyl thymidine incorporation than in control animals) and if the data showed a conventional dose response relationship.

TREATMENT PREPARATION AND ADMINISTRATION: each mouse was topically treated with 25 μL of the appropriate formulations of the test substance, the positive control substance (1% (w/v) p-phenylenediamine in DMSO) and the vehicle (DMSO) on the dorsal surface of each ear. Topical applications were repeated once daily on Days 2 and 3. Local irritations were assessed on Day 5 (determination of ear swelling). On the following day, all mice received an intravenous injection of 20 µCi ³H-methyl thymidine. Approximately 5 h later, mice were sacrificed and the draining auricular lymph nodes were excised and weighed (the left and right lymph nodes). The nodes of each individual animal (two lymph nodes per animal) were pooled and collected in Petri dishes containing PBS. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). The gauze was washed with PBS and the cell suspension was pelleted by centrifugation. The supernatants were discarded and the pellets were washed twice with PBS. After washing, each pellet was suspended in approx. 1 mL 5% trichloroacetic acid and incubated at 4 °C overnight for precipitation of macromolecules. Each precipitate was washed again and suspended in 10 mL scintillation fluid, transferred into scintillation vials and stored at room temperature overnight. The ³H-methyl thymidine incorporation was measured in a scintillation counter.

Positive control substance(s):

other: 1% (w/v) p-phenylenediamine

Statistics:

The mean lymph node weights were calculated for each test group. Mean values and standard deviations for the stimulation index (SI) and the lymph node weight index (LNWI) were calculated for each test group. The one-sided Student’s t-test was used to identify statistically significant differences in the mean lymph node weights between treated and control animals.

Results and discussion

Positive control results:

No results (SI values) of the positive control group (1% (w/v) p-phenylenediamine) were described in the study.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.2

Test group / Remarks:

Test Concentration = 5 %

Remarks on result:

other: The stimulation index values of the test substance were 1.3, 1.2 and 1.2 at treatment concentrations of 25%, 10% and 5%, respectively. No significant dose-response relationship was observed. Based on the results, EC3 values were not calculated.

Results 2

Parameter:

SI

Value:

1.2

Test group / Remarks:

Test Concentration = 10%

Results 3

Parameter:

SI

Value:

1.3

Test group / Remarks:

Test Concentration = 25%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Lymph nodes from each individual animal were pooled and DPM values were measured from the pooled lymph node cell suspensions. The measured DPM values per group were corrected by subtracting the background DPM value (DPM values of 5% (w/v) TCA solutions). The individual DPM values for each animal were not presented in this study.

Any other information on results incl. tables

In addition, the lymph node weight index (LNWI) was calculated as the ratio of the arithmetic mean lymph node weight values of the test group and the vehicle control group. No statistically significant increases in the mean LNWI were observed after treatment with 5, 10 and 25% test substance.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified