Registration Dossier

Administrative data

Description of key information

In a GLP OECD 429 local lymph node assay, the test item Harpin-ab Fermentation Extract showed no sensitising properties and is therefore not regarded as a dermal sensitiser.

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
Study period:
2nd May 2018 - 9th May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Batch no: lot~R134-2
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17.2 - 20.9 g
- Housing: Full barrier in an air-conditioned room
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark
Vehicle:
propylene glycol
Concentration:
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
25% (v/v), 50% (v/v), each diluted with PG and 100% (undiluted test item)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item was found to be 100%. The undiluted test item sufficiently wetted the application sites and did not run off

- Irritation: order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.

- Systemic toxicity: Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal.

- Ear thickness measurements: Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6


MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 μL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 μCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.

A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The stimulation index of the positive control (25% Hexylcinnamaldehyd in PG) was 9.8 and therefore the test is considered to be valid
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
Test concentration 25%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
Test concentration 50%
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
Test concentration 100%
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study

WEIGHT OF LYMPH NODES:
The mean weight of the lymph nodes
for the 25% test group was 2.4 mg
for the 50% test group was 2.4 mg
for the 100% test group was 2.2 mg
for the negative control group was 2.1 mg
for the positive control group was 4.3 mg
Interpretation of results:
GHS criteria not met
Conclusions:
According to OECD 429 the test item is expected to have no sensitising properties and therefore should not be regarded as a skin sensitiser.
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

The skin sensitisation study performed in accordance with OECD Guideline 429 showed that Harpin-ab Fermentation Extract is not sensitising. Therefore, in accordance with Regulation (EC) No. 1272/2008, Harpin-ab Fermentation Extract does not require classification as a skin sensitiser.