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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
other information
Study period:
From March 23, 2009 to 05 August, 2009
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) was used to determine the cell proliferation.
Principles of method if other than guideline:
The present LLNA test used the non-radioactive modification, which utilised non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) instead of 3H-methyl thymidine, and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation and the total number of LNC. This is a fully valid and accepted procedure.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Cas Number:
9001-62-1
Molecular formula:
Not applicable, see remarks
IUPAC Name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Substance type: UVCB
- Physical state: translucent tan liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Lab., Bar Harbor, ME.
- Age at study initiation: approx. 8-12 weeks old
- Weight at study initiation: 18 - 23 g (main study)
- Housing: Individually in suspended wire cages
- Diet (e.g. ad libitum): ad libitum PMI Rodent Chow (Diet #5001)
- Water (e.g. ad libitum): Ad libitum mains tap water
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Animal room was temp controlled
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2009-03-26 To: 2009-03-31

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
50 %, 25% and 10% of the test material in PG.
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Propylene Glycol (PG) was found to be suitable for dosing.
- Irritation: The test material, 25 µL of 10%, 25% and 50% in PG, was applied to the dorsal surface of both ears of two mice per group for three consecutive days. Any signs of toxicity or local irritation was recorded. No irritation was noted at any of the concentrations tested.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA) according to OECD 429
- Criteria used to consider a positive response: The stimulation index (SI) for each group was calculated by dividing the proliferative response (i.e. the BrdU+ lymph node cells, LNC) of each test article treated animal with the mean proliferative response of the corresponding vehicle control group. The mean SI and the standard deviation was calculated for each group. According to the OECD guideline, substances at a given concentration with a SI ≥ 3 should be classified as a skin contact sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
The treatment groups consisted of five animals each, dosed with the test material at concentrations of 10%, 25% and 50% in PG. The induction phase of each group comprised a daily topical application of 25 µL test material spread over the entire dorsal surface of both ear lobes for three consecutive days. A negative control group receiving the vehicle, PG, and a positive control group receiving 25% alpha-hexylcinnamaldehyde in PG were included. 5 days after initial application, each mouse received an intraperitoneal injection of 200 µL Dulbeccos phosphate buffered saline (DPBS) containing a dose of approx 150 mg 5-bromo-2’-deoxyuridine (BrdU) per kg bw. Five hours later, the mice were euthanized and the draining auricular lymph node of each ear was excised. A single cell suspension of lymph node cells was prepared from each mouse in fetal bovine serum and fixed with 85% ethanol.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Students' t-test was used to compare each group to the control group.

Results and discussion

Positive control results:
The positive control material, alpha-hexyl cinnamic aldehyde (HCA), 25% v/v in PG, gave a positive response, SI = 14.5, and the study was therefore accepted as valid.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: (see Table 1 below)

Any other information on results incl. tables

Table 1. The results of the LLNA, expressed as mean number of proliferating cells in the lymph nodes of each treatment group and the stimulation index (SI) for each group.

Test material

Lymphocyte proliferation

(# BrdU+)

SI

Vehicle, PG

10267

1.0

25% HCA in PG

148989

14.5

Lipase 3, 10% in PG

7242

0.7

Lipase 3, 25% in PG

14078

1.4

Lipase 3, 50% in PG

8921

0.9

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The enzyme Lipase is classified as a dermal non-sensitiser.
Executive summary:

The study was performed according to OECD test guideline 429, using the non-radioactive modification, which utilised non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) instead of the normally used tritiated methyl thymidine. The lymph node cell (LNC) suspensions were analyzed by flow cytometry for BrdU incorporation and the total number of LNC. The dermal irritation potential of the test article at 10%, 25% and 50% in Propylene glycol (PG) was determined first in three groups of two female CBA/J mice per group. No irritation was noted and concentrations of 10%, 25% and 50% in PG were selected for the main sensitization study.

In the sensitization study, the test article was applied at 10%, 25% or 50% in PG to the dorsum of each ear, once daily for three consecutive days to three groups of 5 female mice. Vehicle (PG) and positive control (25% HCA) were applied in the same manner to two groups of 5 female mice. Five days after the initial application, all animals received an intraperitoneal injection of BrdU. Five hours after the intraperitoneal injection, all mice were sacrificed, the auricular lymph nodes were isolated, single-cell suspensions of LNC were generated and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation. The amount of BrdU-positive LNC was determined as a measure of the proliferative response of the local lymph node. The stimulation index (SI) was calculated by dividing the proliferative response of each treated animal by the mean proliferative response of the vehicle control group. The mean SI was calculated for each group. A stimulation index (SI) equal or above 3 indicates a positive response.

The SI for the positive control (25% HCA) and vehicle control (PG) groups were 14.5 and 1.0, respectively, validating the sensitivity of this local lymph node assay. The group SI for the test article at 10%, 25% and 50% in PG were 0.7, 1.4 and 0.9 indicating that the test article is not a skin sensitizer.

 

GHS Classification: Not classified