Reactionmass of octan-2-yl prop-2-enoate and octan-3-yl prop-2-enoate and octan-4-yl prop-2-enoate

Endpoint summary

Description of key information

MTDID 44428 was tested in an in vivo skin sensitization study. The result of the study was:

Category 1B sensitizer (EC3 = 34.8%) when tested in the Local Lymph Node Assay (LLNA) according to OECD 429.

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Remarks:

No deviations ocurred that negatively impacted the integrity of the study results.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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
- Microbiological status of animals, when known: No data
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 19.7-23.4 g
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany)
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: 5 days
- Indication of any skin lesions: Prior to dosing all ears were inspected to ensure that they were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 02 December, 2015 To: 21 December, 2015

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 50, and 100%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Homogenity of the test article was confirmed via visual inspection of the solution at preparation.
- Irritation: Irritation (max. grade 1 or 2) was observed for all test items. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for all test items.
- Systemic toxicity: No signs of systemic toxicity were noted for the pre-screen animals treated with MTDID 44428.
- Ear thickness measurements: See 'irritation' field.
- Erythema scores: See 'irritation' field.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:

DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:

The test article was prepared at 10, 50 and 100% in acetone/olive oil (4:1 v/v)

Induction - Days 1, 2 and 3:

The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately priorto dosing.
The vehicle and positive control animals were treated in the same way as the experimental animals, except that the vehicle and/or positive control item 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) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). 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 approximately 3 mL 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 (diameter: 125 μm). 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) (Merck, Darmstadt, Germany) 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 (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). 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).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first.

Positive control results:

A six month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed by the laboratory is an appropriate model for testing contact hypersensitivity.

Key result

Parameter:

EC3

Value:

34.8

Test group / Remarks:

Mean

Parameter:

SI

Value:

1.2

Variability:

+/- 0.3

Test group / Remarks:

10% Test Article

Parameter:

SI

Value:

4.1

Variability:

+/- 1.1

Test group / Remarks:

50% Test Article

Parameter:

SI

Value:

5.8

Variability:

+/- 1.5

Test group / Remarks:

100% Test Article

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
All auricular lymph nodes of the animals were considered normal in size.

DETAILS ON STIMULATION INDEX CALCULATION AND EC3 CALCULATION: Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 50 and 100% were 788, 2789 and 3916 DPM, respectively. The SI values calculated for the test item concentrations 10, 50 and 100% were 1.2, 4.1 and 5.8, respectively.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals treated with the test article.

BODY WEIGHTS : Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SIGNS OF TOXICITY: The very slight irritation (10% Days 2-4; 50% and 100% Days 1-4) of the ears was considered not to have a toxicologically significant effect on the activity of the nodes.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results of the study (EC3 = 34.8%), MTDID 44428 is a GHS Category 1B sensitizer.

Executive summary:

The skin sensitization potential of MTDID 44428 was evaluated in mice in the Local Lymph Node Assay (LLNA). The study was conducted according to OECD 429 in compliance with OECD GLP. Based on the results of a pre-screen test, test article concentrations of 10, 50, and 100% were tested in the main test (vehicle: acetone:olive oil 4:1), along with a concurrent positive control (25% alpha-hexylcinnamaldehyde in acetone:olive oil). No mortality occurred and no clinical signs of systemic toxicity were observed in the animals treated with the test article. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The very slight irritation (10% Days 2-4; 50% and 100% Days 1-4) of the ears was considered not to have a toxicologically significant effect on the activity of the nodes. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 50 and 100% were 788, 2789 and 3916 DPM, respectively. The SI values calculated for the test item concentrations 10, 50 and 100% were 1.2, 4.1 and 5.8, respectively. Based on the results of the study (EC3 = 34.8%), MTDID 44428 is a GHS Category 1B sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

Based on the results of the study, MTDID 44428 is a Category 1B sensitizer according to GHS.