Reaction mass of hentriacontan-16-one and pentatriacontan-18-one and tritriacontan-16-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-09-21 to 2017-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of hentriacontan-16-one and pentatriacontan-18-one and tritriacontan-16-one

EC Number:

946-252-3

Molecular formula:

(C35H70O).(C33H66O).(C31H62O)

IUPAC Name:

Reaction mass of hentriacontan-16-one and pentatriacontan-18-one and tritriacontan-16-one

Test material form:

solid

Specific details on test material used for the study:

Batch No: FK650468
Purity: not speific

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Condition: Inbred, SPF-Quality
- Age at study initiation: Young adult animals (approximately 11 weeks old)
- Weight at study initiation: 20.9 to 23.9 g.
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material equipped with water bottles
- Diet (e.g. ad libitum): Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap-water, freely available
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 45 to 71%
- Air changes (per hr): Ten or greater air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- IN-LIFE DATES: From: 2017-09-27 To: 2017-10-24

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

5%, 10%, 20%

No. of animals per dose:

Five females per group.

Details on study design:

PRE-SCREEN TESTS:
- Irritation: Not observed
- Systemic toxicity: Not observed
- Ear thickness measurements: Variations in ear thickness during the observation period were less than 25% from Day 1 predose values.
- Erythema scores: 0

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer

TREATMENT PREPARATION AND ADMINISTRATION:
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) with the test item, at approximately the same time on each day. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity - Day 6: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter. Counting time was to a statistical precision of ±0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.2

Test group / Remarks:

at 5% concentrations

Results 2

Parameter:

SI

Value:

0.9

Test group / Remarks:

at 10% concentrations

Results 3

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

at 20% concentrations

Cellular proliferation data / Observations:

EC3 CALCULATION
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 20%, the test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.
CLINICAL OBSERVATIONS:
- Skin Reactions / Irritation: No irritation was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all test item treated animals between Days 1 and 5, which did not hamper scoring of the skin reactions.
- Systemic Toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopic Examination of the Lymph Nodes and Surrounding Area: All auricular lymph nodes of the animals of the experimental and control groups were
considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity Measurements: Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 20% were 150, 114 and 158 DPM, respectively. The mean DPM/animal value for the vehicle control group was 126 DPM.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 20%, the test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.
Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Executive summary:

The objective of this study was to evaluate whether test item induces skin sensitization in mice after three epidermal exposures of the animals based on OECD No.429 (2010).

Test item concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 20% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (methylethylketone). Three days after the last exposure, all animals were injected with 3Hmethyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation was observed in any of the animals.

The mean body weight gain shown by the animals over the study period was considered to be normal.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 20% were 150, 114 and 158 DPM, respectively. The mean DPM/animal value for the vehicle control group was 126 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.2, 0.9 and 1.3, respectively.

Since there was no indication that the test item elicits a SI≥3 when tested up to 20%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.

Based on these results, test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.