Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
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)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Laromer LR 8887
- Physical state: clear, yellowish liquid
- Analytical purity: 78.1g/100g by 1H-NMR-spectroscopy
- Purity test date: 2011-11-03
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 2012-08-03
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9-10weeks
- Weight at study initiation: 16.6 - 21.6g
- Housing: group in makrolon type III cages
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45 - 65%
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1%, 2.5%, 5% + vehicle control
No. of animals per dose:
5 (1 dor the pre-tests)
Details on study design:
RANGE FINDING TESTS (three treatments on consecutive days):
1st pre-test:
100%: animal found dead on day 2, no reason given
50%: erythema (grade 1-2) and ear swelling from day 2 to 6

2nd pre-test:
25%: erythema grade 1-2 from day 3-6, ear swelling (increase in thickness and weight > 25%) and eschar formation on day 6, excitement before treatment on day three and salivation thereafter
10%: erythema grade 1 from day 3-6, ear swelling (increase in thickness and weight > 25%) and eschar formation on day 6

3rd pre-test:
2.5%, 5%: erythema grade 1 from day 3-5

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
First, that 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.
Second, that 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.
Excessive irritation was also considered.

TREATMENT PREPARATION AND ADMINISTRATION:
25µl were spread over the dorsal surface of each ear on three consecutive days. On day 6, 250µl of phosphate-buffered saline containing 20.0µCi of 3HTdR (equivalent to app. 79.9 µCi/mL 3HTdR) were injected into each mouse via the tail vein. App. 5 hours later all mice were euthanized by i.p. injection of Pentobarbital-Sodium. The draining lymph nodes were rapidle excised and pooled per animal.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers.

The EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
control: DPM: 460.5
5%: DPM: 621.1 (SI= 1.35)
10%: DPM: 1004.3 (SI=2.18)
25%: DPM: 3720.5 (SI=8.08)
EC3= 12.1%

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 1%: 1.18 2.5%: 2.49 5%: 6.18 EC3 (calculated) = 2.8%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: control: 1173.5 1%: 1386.3 2.5%: 2922.1 5%: 7248.3

Any other information on results incl. tables

No mortality or signs of systemic toxicity were observed.

Significant increase in lymph node weight in the highest dose group (5%).

Significant increas in lymph node cell count in high and mid-dose (2.5%, 5%). Cut-off value for lymph node cell count index Balb/c mice (1.55) exceeded in the high dose (2.39)

Significant increase in ear weight very slightly (by 0.01) exceeding Balb/c cut-off value, but not exceeding an increase of 25%, OECD guidance on excessive local irritation.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test item was found to be a skin sensitiser under the test conditions of this study. The EC3 value was calculated to be 2.8% (w/w).
Executive summary:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations of 1, 2.5, and 5% were prepared in the vehicle acetone:olive oil (4+1 v/v). The highest concentration tested was the highest concentration that could be applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by three pre-experiments).

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice (see Ref. 9) was slightly exceeded in the high dose group (index of 1.11). However, the obtained mean value of this group was still within the range of historical vehicle control data for the ear weight. Furthermore, the threshold value of 25% (increase in ear weight in comparison to the vehicle control group) for excessive local skin irritation mentioned in OECD guideline 429 was not exceeded in this group. Thus, this was not considered as biologically relevant.

Stimulation Indices (S.I.) of 1.18, 2.49, 6.18 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in acetone:olive oil (4+1 v/v), respectively. A clear dose response was observed. Based on the S.I.s obtained with 2.5 and 5% test item concentration, an EC3 value of 2.8% (w/w) was calculated.

A statistically significant and biologically relevant increase in DPM value and also in lymph node cell count was observed in the mid and high dose group in comparison to the vehicle control group (p<0.05). A statistically significant and biologically relevant increase in lymph node weight was only observed in the high dose group (5%) in comparison to the vehicle control group. Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice (see Ref. 8) was exceeded in the high dose group, and showed a borderline response in the mid dose group (indices of 2.39 and 1.51, respectively).