[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Apr - 24 Apr 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see Remark

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

736150-63-3

EC Number:

616-005-1

Cas Number:

736150-63-3

Molecular formula:

C25H46 O6; C27H48O8

IUPAC Name:

736150-63-3

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA7CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 19.4 ± 0.9 g
- Housing: animals were housed individually in Makrolon Type I cages with wire mesh top (EHRET GmbH, Emmedingen, Germany) and granulated soft wood bedding (Harlan Winkelmann GmbH, Borchen, Germany)
- Diet: pelleted standard diet (Harlan Winkelmann GmbH, Borchen, Germany), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

10, 20 and 50% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Study design: 50% (Batch D) was tested in a non-GLP pre-experiment
- Compound solubility: insoluble in water, soluble in DMSO
- Lymph node proliferation response: DPM-Background (1 mL 5% trichloroacetic acid) per animal (2 lymph nodes) controls: 2707.1 (animal 1), 2357.9 (animal 2), 2199.6 (animal 3), 2336.3 (animal 4) and 3184.5 (animal 5); DPM-Background (1 mL 5% trichloroacetic acid) per animal (2 lymph nodes) test group: 6794.2 (animal 1), 5977.4 (animal 2), 6981.8 (animal 3), 7739.5 (animal 4), 6305.7 (animal 5)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by β-scintillation and γ-counting, respectively
- Criteria used to consider a positive response: A test item was regarded as a sensitiser in the LLNA if the following criteria were fulfilled: The exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. The data were compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. The decision to select a stimulation index (SI) of 3 as an arbitrary indication of sensitising activity was made on the basis of investigations performed with a wide range of chemicals.

TREATMENT PREPARATION AND ADMINISTRATION: Each test group was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume, 25 µL, was spread over the entire dorsal surface of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application, all mice were administered with 250 µL of 80.6 µCi/mL ³HTdR (corresponds to 20.2 µCi ³HTdR per mouse) by intravenous injection via a tail vein. Five hours later, all mice were euthanized by intraperitoneal injection of Na-thiopental. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions in PBS of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with PBS the lymph node cells were resuspended in 5% trichloroacetic acid and incubated at approximately 4 °C for at least 18 h for precipitation of macromolecules. The precipitates were resuspended in 5% trichloroacetic acid and transferred to plastic scintillation vials.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables. An ANOVA (Kruskal Wallis) followed by a Dunnett-test or t-test (Mann-Whitney), when applicable, were conducted to assess whether the difference of DPM per animal was statistically significant between test item groups or the positive control (RCA) group and the negative control (vehicle) group. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The positve control substance (25% hexyl cinnamic aldehyde in acetone:olive oil (4+1)) induced positive reactions in 2/5 animals (40%).The SI-values of the positive control were 2.3, 1.6, 2.6, 4.2 and 4.8, respectively.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

10% formulation

Results 2

Key result

Parameter:

SI

Value:

1.53

Test group / Remarks:

20% formulation

Results 3

Key result

Parameter:

SI

Value:

1.86

Test group / Remarks:

50% formulation

Any other information on results incl. tables

In the study, stimulation indices of at the maximum 0.97 in the 10% groups, 1.53 in the 25% groups and 1.86 in the 50% groups were observed. At a concentration of 50% of Batch A a statistically significant difference of DPM per animal was observed (p ? 0.05), however, the stimulation index (S.I.) was 1.71. EC3 values of the test groups could not be calculated, since none of the tested concentrations induced a S.I. greater than 3.

VEHICLE CONTROL DATA:

DPM values per lymph node obtained for the vehicle control were relatively high when compared to the historical control data. The results for the individual animals showed low variability and the results are nonetheless considered as reliable. Differences in vehicle control data when compared to other studies are related to the use of different batches of 3H-Thymidine. Experiments performed with the same batch of 3H-Thymidine as used in the present study have shown DPM values per lymph node, which were considerably higher than the results obtained with earlier and later batches. It is assumed that incorporation of 3H-Thymidine is more efficient with the present batch even though evidence for this hypothesis cannot be provided. Since all animals used for this study were labeled with the same 3H-Thymidine-solution this increase is supposed to be higher for all test groups and does neither affect the sensitivity of the assay nor the calculation of S.l. values.

Table 1. Results of the LLNA.

Test item concentration % (w/v)	Group	Calculation	DPM per lymph node	Result
		number of lymph nodes		S.I.
-	BG_I		37.1*
-	BG_II		17.89*
-	Control group	10	2027.9	1.0
25	Positive control Group	10	6312.4**	3.11
10	Test group Batch A	10	1528.3	0.75
25	Test group Batch A	10	2088.3	1.03
50	Test group Batch A	10	3468.7**	1.71
10	Test group Batch B	10	1964.4	0.97
25	Test group Batch B	10	3107.2	1.53
50	Test group Batch B	10	3768.4	1.86
10	Test group Batch C	10	1211.2	0.60
25	Test group Batch C	10	2295.3	1.13
50	Test group Batch C	10	2213.7	1.09
10	Test group Batch D	10	1225.7	0.60
25	Test group Batch D	10	1688.8	0.83
50	Test group Batch D	10	2868.6	1.41

* Measurement of DPM only.

** Mean DPM value for the group was according to the Dunnett test significanlty higher than the corresponding control value. The p value for the analysis was <0.05

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

S.I. = Stimulation Index

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

CLP: not classified