Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-08-09 to 2013-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented study according to OECD guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 45 - 94% instead of 45 to 65% for several hours. This deviation to the study plan, however, is not considered to affect the validity of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): FC-131
- Substance type: crystalline
- Physical state: solid
- Analytical purity: 99.8% (area HPLC)
- Lot/batch No.: 30610045
- Expiration date of the lot/batch: 2015-06-12
- Stability under test conditions: stability in used solvent not indicated by the sponsor
- Storage condition of test material: room temperature, moisture protected

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Netherlands

- Age at study initiation:
pre-test: 10-11 weeks (beginning of treatment)
final test: 8-9 weeks (beginning of treatment)

- Weight at study initiation:
pre-test: 24(animal 1, lower dose) and 23.8 g (animal 2, higher dose); only two animals tested in pre-test
final test: 20.9 ± 1.1 g, (16 animals)

- Housing: all animals belonging to the same treatment /control group were kept in one cage,
cage tape: Makrolon Type II (pre-test) / III (final test), with wire mesh top
bedding: granulated soft wood bedding

- Diet: ad libitum, 2018C Teklad Global 18% protein rodent diet (certified),
- Water: ad libitum tap water

- Acclimation period: at least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2 °C
- Humidity (%): approx. 45-94 %
- Photoperiod (hrs dark / hrs light): 12/12 (6a.m. to 6 p.m. artificial light)

-health examination of all animals prior beginning of experiment; only animals without any visible signs of illness used for the study

Study design: in vivo (non-LLNA)

Induction
Route:
other:
Vehicle:
other:
Challenge
Route:
other:
Vehicle:
other:

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
pre-test: 10 and 25 % FC-131 solution
final-test: 5, 10 and 25 % FC-131 solution
No. of animals per dose:
4 animals per tested concentration in the final-test, all in all 16 animals
Details on study design:
RANGE FINDING TESTS (Pre test):
- Compound solubility: highest FC-131 concentration which could be technically used was a 25% solution in acetone/olive oil (4+1, v/v)
- Irritation:
10% FC-131 solution: on day 4: erythema score 1
25% FC-131 solution: on day 2,3,5 and 6: erythema score 1 and on day 4: erythema score 2
--> no excessive irritation upto the highest tested concentration
- ear weights: increase of 9.2% (10% FC-131 solution) and 18 % (25% FC-131 solution) compared to vehicle controls (ear weights after necrospy)
- Lymph node proliferation response: not analysed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: pooled treatment group approach
- 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.


TREATMENT PREPARATION AND ADMINISTRATION:
- FC-131 solutions were made freshly before each dosing occasion, time between preparation and dose formulation: max. 30 min
- each test group of mice was treated by epidermal topical application to the dorsal surface of each ear in a volume of 25 µl/ear/day spread over the entire dorsal surface (approx. 8 mm diameter)
- the vehicle controls were treated with an equivalent volume of the vehicle alone
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations of body weights and were calculated.

Results and discussion

Positive control results:
EC3 = 12.6% (w/w)
(10%: S.I: 2.4
12%: S.I. 5.9)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: vehicle control: S.I. 1.00 5 % FC-131: S.I. 1.86 10% FC-131: S.I. 1.55 25% FC-131: S.I. 1.04
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: values background substracted vehicle control: 1578.5 5 % FC-131: 2941.5 10% FC-131:2450.5 25% FC-131: 1643.5

Any other information on results incl. tables

No deaths occurred during the study period and no symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were detected.

The local skin irritation observed in the pre-test could not be confirmed in the final test.

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

Table 1: DPM and S.I. values for the different test item concentrations (BG: background)

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

23

---

---

---

---

---

BG II

26

---

---

---

---

0

1

1603

1578.5

8

197.3

1.00

5

2

2966

2941.5

8

367.7

1.86

10

3

2475

2450.5

8

306.3

1.55

25

4

1668

1643.5

8

205.4

1.04

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to criteria metioned in the OECD guideline 429, a result is regarded as positive, when a stimulation index (S.I.) ≥ 3 is observed. In the present study, S.I. of 1.86, 1.55 and 1.04 were estimated after treatment with the test item at concentrations of 5, 10 and 25% in acetone/olive oil (4+1, v/v). A dose response was not observed and an EC3 could not be estimated as no S.I ≥3 was found. Hence, the test item FC-131 was not a skin sensitiser under the test conditions used in this study.

Executive summary:

The possible skin sensitising potential of FC-131 was studied using the Local Lymph Node Assay in mice according to the OECD guideline 429 and GLP. Four female mice per treatment group were treated once daily with the test item by topical application to the dorsum of each ear for three consecutive days. Three treatment groups of 5, 10 and 25% (w/w) FC-131 in acetone/olive oil (4+1, v/v) and one vehicle control group were included, all in all 16 animals. The highest concentration tested was the highest concentration that could be technically achieved as confirmed by a pre-experiment. Five days after the first topical application, the DPM was determined and the stimulation index (S.I.) calculated. S.I. of 1.86, 1.55 and 1.04 were estimated with the test item at concentrations of 5, 10 and 25% and thus no EC3 could be calculated. No dose-response reaction, no clinical signs and no mortality were observed at any time of the study. Hence, the test item FC-131 was not a skin sensitiser under the test conditions used in this study. The test ist considered valid.