Registration Dossier

Administrative data

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: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17.3 - 20.3 g
- Housing: group caging
- Diet: 2018C Teklad Global 18% protein rodent diet, ad libidum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding

Study design: in vivo (LLNA)

Vehicle:
other: 1% aqueous Pluronic®
Concentration:
5, 10, and 20%
No. of animals per dose:
4
Details on study design:
Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 20% solution in 1% aqueous Pluronic®. Vortexing and brief ultrasonication will be used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 20% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. From day 2 to 5, redness of the ear skin could not be detected, due to the colour of the test item.
Thus, the test item in the main study was assayed at 5, 10, and 20%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.


MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 20% in 1% aqueous Pluronic®. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.6 µCi of 3H-methyl thymidine (equivalent to 78.5 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of Ear Weights
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.

Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.

Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).

Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.

Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

INTERPRETATION OF RAW DATA
The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-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.
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.

Results and discussion

Positive control results:
Experiment performed in October 2015 (Harlan study number 1728500) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.62, 4.23, and 17.56, respectively.
The EC3 value calculated was 7.6 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see depicted table below

Any other information on results incl. tables

Calculation and results of individual data

Vehicle: 1% aqueous Pluronic®

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

17

---

---

---

---

---

BG II

11

---

---

---

---

0

1

4107

8

4093

511.6

1

5

2

5570

8

5556

694.5

1.36

10

3

4116

8

4102

512.8

1

20

4

4077

8

4063

507.9

0.99

1    =  Control Group

2-4=  Test Group

a)   =  The mean value was taken from the figures BG I and BG II

b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Calculation of the EC3 value

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.From day 2 to 5, redness of the earskin could partly not be detected due to the colour of the test item.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

 Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase in ear weight was observed in the low and high dose groups.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Aluminium-Braun 1134 p was not a skin sensitiser under the test conditions of this study.
Executive summary:

In order to study a possible skin sensitising potential of Aluminium-Braun 1134 p, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 20% in 1% aqueous Pluronic®by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (1% aqueous Pluronic®) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. From day 2 to 5, redness of the earskin could partly not be detected due to the colour of the test item. A statistically significant increase in ear weight was observed in the low and high dose groups. This is not biological relevant, since the increase is below the recommended threshold of 25% as recommended by OECD 429 and all S.I. determined were below 3.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.36, 1.00, and 0.99 were determined with the test item at concentrations of 5, 10, and 20% in 1% aqueous Pluronic®. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.