Registration Dossier

Administrative data

Description of key information

In order to study a possible skin sensitising potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 1, 2.5, and 5% (w/w) in ethanol/water (3+7, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could 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 (ethanol/water (3+7, v/v)) 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. A statistically significant increase in ear weight was not observed.

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 1.18, 0.74, and 1.09 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in ethanol/water (3+7, v/v).

The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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:
guideline study
Remarks:
Guideline-conform study under GLP without deviations
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 18.7 - 22.3 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), 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 (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Vehicle:
other: rthanol/water (30/70 v/v)
Concentration:
1, 2.5, and 5% (w/w)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
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 10% (corresponds to 5.66% tri-sodium tri-oxalato-ferrate) solution in ethanol/water (3+7, v/v). Vortexing and warming to 37°C was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved by the use of other vehicles.
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 5 and 10% 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 systemic toxicity. On day 4, the animal treated with 10% test item concentration showed a very slight erythema of the ear skin (Score 1). Additionally, the threshold (recommended by OECD 429) for the increase in ear weight (compared to historical vehicle values) was slightly exceeded in the animal treated with 10% of the test item. The animal treated with 5% test item concentration did not show any signs of local skin irritation.
Thus, the test item in the main study was assayed at 1, 2.5, and 5% (w/w). 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 1, 2.5, and 5% (w/w) in ethanol/water (3+7, v/v). 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 20.2 µCi of 3H-methyl thymidine (equivalent to 80.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

INTERPRETATION OF RAW DATA
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser 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.


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.

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.
Positive control results:
Experiment performed in April 2015 (Harlan study number 1690900) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.93, 2.65, and 9.48, respectively.
The EC3 value calculated was 10.8 % (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.
Parameter:
other: disintegrations per minute (DPM) per node
Value:
>= 393.7 - <= 628.3
Test group / Remarks:
all
Remarks on result:
other: see depicted table below
Key result
Parameter:
SI
Value:
>= 0.74 - <= 1.18
Test group / Remarks:
all

Calculation and results of individual data

Vehicle: ethanol/water (3+7, v/v)

 

Test item concentration %
(w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

16

---

---

---

---

---

BG II

21

---

---

---

---

0

1

4265

4246.5

8

530.8

1.00

1

2

5045

5026.5

8

628.3

1.18

2.5

3

3168

3149.5

8

393.7

0.74

5

4

4659

4640.5

8

580.1

1.09

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

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 symtoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

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 or biologically relevant increase in ear weights was not observed.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item was not a skin sensitiser under the test conditions of this LLNA study in mice.
Executive summary:

In order to study a possible skin sensitising potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 1, 2.5, and 5% (w/w) in ethanol/water (3+7, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could 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 (ethanol/water (3+7, v/v)) 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. A statistically significant increase in ear weight was not observed.

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 1.18, 0.74, and 1.09 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in ethanol/water (3+7, v/v).

The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

no classification

in an LLNA in mice no signs of sensitisation could be detected.