Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th December 2017 - 13th April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

None

Molecular formula:

C10-13H21-27-C6H4-SO2-O-CH2-C(H)OH-CH2-CO2-C9H19

Test material form:

liquid

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Test item storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

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, when known: SPF
- Age at study initiation: 11 weeks
- Weight at study initiation: 17.1-24.1 g ; all animals within ± 20% of the sex mean.
- Housing: group housed within treatmnet groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 40-43
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 15 January 2018 To: 04 March 2018

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0.5%, 1%, 2%

No. of animals per dose:

Five females

Details on study design:

A pre-screen was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied. Initially, two test item concentrations were tested; a 100% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines. Based on the results of the initially treated animals, eight additional animals were treated in a similar manner with four lower concentrations (10%, 5%, 2% and 1%) at a later stage. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.

In the main study, the dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day (Days 1, 2, 3). The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item. On 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®. 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. Following excision of the lymph nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (200 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) and then stored in the refrigerator until the next day. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed using a 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).

Positive control substance(s):

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

Statistics:

The EC3 value was calculated using linear interpolation.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

+/- 0.2

Test group / Remarks:

Vehicle control

Results 2

Key result

Parameter:

SI

Value:

0.5

Variability:

+/- 0.1

Test group / Remarks:

0.5%

Results 3

Key result

Parameter:

SI

Value:

1.3

Variability:

+/- 0.3

Test group / Remarks:

1.0%

Results 4

Key result

Parameter:

SI

Value:

1.5

Variability:

+/- 0.5

Test group / Remarks:

2.0%

Results 5

Key result

Parameter:

EC3

Remarks on result:

not determinable

Cellular proliferation data / Observations:

In the pre-screen, a 100% test item concentration caused mortality. At concentrations of 50%, 10% and 5%, variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values, and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At 2% and 1%, no signs of systemic toxicity were noted and no irritation was observed and therefore the 2% concentration was selected as highest concentration for the main study.

In the main study, no mortality occurred and no clinical signs of systemic toxicity were observed. Body weights and body weight gain of the animals treated at 2% remained in the same range as controls over the study period. Marked body weight loss was noted for all animals treated at 0.5% and a higher body weight gain compared to normal was noted for all animals treated at 1%. No erythema was observed in any of the animals. The scaliness shown by one animal treated at 1% on Day 4 was considered not to have a toxicologically significant effect on the activity of the nodes. 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 0.5, 1 and 2% were 324, 868 and 994 DPM, respectively. The mean DPM/animal value for the vehicle control group was 667 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 0.5, 1.3 and 1.5, respectively. The results of a separate reliability check performed with HCA confirm the sensitivity of the assay. An EC3 value of 19.2% for HCA was calculated using linear interpolation, and was within the acceptable range of 4.8 and 19.5%.

Any other information on results incl. tables

Summary of results

Group	Individual DPM	Mean DPM	Mean SI
Control	278, 1061, 1031, 338, 625	667 ± 166	1.0 ± 0.2
0.5%	306, 602, 457, 72, 181	324 ± 95	0.5 ± 0.1
1%	89, 1494, 1270, 437, 752	868 ± 222	1.3 ± 0.3
2%	435, 2092, 1189, 617, 639	994 ± 302	1.5 ± 0.5

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The SI values for the tested concentrations do not exceed 3; there is therefore no evidence that the test material IC-2 (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) has skin sensitising potential.

Executive summary:

The potential of IC-2 (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) to induce skin sensitisation was investigated in a LLNA performed according to GLP, OECD, EC and EPA OPPTS guidelines. Test item concentrations selected for the main study were based on the results of a pre-screen test. Due to mortality, signs of toxicity and variation in ear thickness seen in the pre-screen, concentrations of 0.5%, 1% and 2% were selected for the main study. In the main study, three groups of five female CBA/J mice were treated with test item concentrations of 0.5, 1 or 2% w/w on three consecutive days, by open application on the ears. Five control animals were similarly treated with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl 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. 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 0.5, 1 and 2% were 324, 868 and 994 DPM, respectively. The mean DPM/animal value for the vehicle control group was 667 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 0.5, 1.3 and 1.5, respectively. The SI values for the tested concentrations do not exceed 3; there is therefore no evidence that the test material IC-2 (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) has skin sensitising potential. The results of a separate reliability check performed with HCA confirm the sensitivity of the assay.