Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul - 06 Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(octan-2-yl)benzene

EC Number:

950-576-0

Cas Number:

31047-57-1

Molecular formula:

C14H22

IUPAC Name:

(octan-2-yl)benzene

Test material form:

liquid

Details on test material:

Identification: SHR 1396
Physical Description: Clear colorless liquid
Storage Conditions: At room temperature

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 nulliparous and non-pregnant: yes.
- Microbiological status of animals: SPF-quality.
- Age at study initiation: 10 weeks old.
- Weight at study initiation: 19.6 to 23.0 grams.
- Housing: Animals were 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 (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days.
- Indication of any skin lesions: no.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The daily mean temperature during the study period was 22 to 23°C.
- Humidity (%): The daily mean relative humidity of 53 to 79%.
- Air changes (per hr): at least 10.
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From 17 Jul to 05 Aug.

Study design: in vivo (LLNA)

Vehicle:

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

No. of animals per dose:

5 animals

Details on study design:

PRE-SCREEN TESTS:
Two test item concentrations were tested; a 50% (w/w) and 100% (w/w) concentration.
Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

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.
Group 1 was treated with the vehicle (vehicle control group). Group 2, 3 and 4 were treated with 25%, 50% and 100% concentration, respectively.

TREATMENT PREPARATION AND ADMINISTRATION:

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing.
The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item.
- Induction: Test item (25 μL) was applied in the dorsal surface of both ears of each animal, for 3 consecutive days.
- Excision of the Nodes. In 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 euthanized 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: On day 6 and following the excision of the nodes, a single cell suspension of limph node cells was prepared in PBS. LNC were washed twice and DNA was precipitated using 5% trichloroacetic acid (TCA).
- Radioactivity measurements: On 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 (2910TR). 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).

ANALYSIS
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.

INTERPRETATION
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3) (see table 1 in 'any other information on material and methods')

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

2.5

Test group / Remarks:

100%

Cellular proliferation data / Observations:

PRE-SCREENING TEST:
- At a 50% and 100% test item concentration, no signs of systemic toxicity were noted and up to very slight erythema was observed and therefore the 100% concentration was selected as highest concentration for the main study.

MAIN STUDY

SKIN REACTIONS / IRRITATION:
The very slight erythema of the ears as shown by all test item treated animals on at 1 to 6 Days during the observation period was considered not to have a toxicologically significant effect on the activity of the nodes.

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

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

MACROSCOPIC EXAMINATION OF LyMPH NODES:
The majority of auricular lymph nodes were considered normal in size, except for the nodes in two animals treated at 100%, which were considered to be slightly enlarged.
No macroscopic abnormalities of the surrounding area were noted for the majority of animals. Pale discoloration of the surrounding area of the lymph nodes of the animals treated at 100% was noted, which was considered not to have affected the DPM values of these animals.

DETAILS ON STIMULATION INDEX CALCULATION : DPM (Disintegrations Per Minute) 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.

Applicant's summary and conclusion

Interpretation of results:

other: Not skin sensitising

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

In conclusion, since there is no indication that the test item elicited a SI ≥ 3 when tested up to 100%, SHR 1396 was considered not to be a skin sensitizer.

Executive summary:

A LLNA study was performed following OECD guidelines and GLP principles. Three experimental groups of five females were treated with test item concentrations of 15%, 50% and 100%. One control group of five females was treated with the vehicle (Acetone/olive oil 1:4 v/v). All animals were treated for 3 consecutive days. Three days after the last exposure all animals were injected with 3H-methyl thymidine for measuring radioactivity. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was calculated for each group. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity. No macroscopic abnormalities of the surrounding area were noted for the majority of animals. Pale discoloration of the surrounding area of the lymph nodes of the animals treated at 100% was noted, which was considered not to have affected the DPM values of these animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 717, 938 and 972 DPM, respectively. The mean DPM/animal value for the vehicle control group was 384 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 1.9, 2.4 and 2.5, respectively. Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, SHR 1396 was considered not to be a skin sensitizer. Based on these results, SHR 1396 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).