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Administrative data

Description of key information

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:
29 July 2015 to 10 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse, CBA/J strain, inbred, SPF-Quality.
Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Janvier, Le Genest-Saint-Isle, France
Number of animals: 20 females (nulliparous and non-pregnant), five females per group (main study only).
Age and body weight: Young adult animals (approx. 11 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification: Tail mark with marker pen.
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.
Reliability check: The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in APPENDIX 2 of the report. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
An extensive data base is available with reliability checks performed at half year intervals during at least the past 9 years showing reproducible and consistent positive results.

Animal husbandry
Conditions: Environmental controls for the animal room are set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod is between 07:00 and 19:00 hrs daily. The light/dark cycle may be interrupted for study related activities. Any variations to these conditions will be evaluated and maintained in the raw data.
Accommodation: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap water.
Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.
Vehicle:
methyl ethyl ketone
Concentration:
test substance concentrations of 10, 25 or 50% w/w
No. of animals per dose:
Three experimental groups of five female CBA/J mice
Details on study design:
Test substance preparation
Vehicle: Methyl ethyl ketone (Merck, Darmstadt, Germany).
Rationale: The vehicle was selected on the basis of maximizing the solubility using the test substance data provided by the Sponsor and trial preparation results performed at WIL Research Europe. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide. There was no information available regarding the solubility or stability in vehicle.
Preparation: The test substance preparations (w/w) were prepared within 4 hours prior to each dosing. The test substance was ground to a powder using a mortar and pestle prior to weighing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions.
Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.

Weight of evidence analysis
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected.

Pre-screen test
A pre-screen test 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.
Two test substance concentrations were tested; a 50% and 25% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids).
The test system, procedures and techniques were identical as those used in the main study except that the animals were approximately 12 weeks (at initiation of treatment) that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). 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 substance concentration per group. The highest test substance concentration was selected from the pre-screen test.
One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day. 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 substance.

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).

Observations
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. In addition, a description of all other (local) effects was recorded.
Necropsy: No necropsy for gross macroscopic examination was performed according to protocol.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None specified in the study report
Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 3.0 and 9.1 respectively. An EC3 value of 10% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.5, 14.5, 13.4, 14.1, 17.3 and 9.8%.
Parameter:
SI
Value:
>= 1 - <= 1.4
Test group / Remarks:
10, 25 and 50%
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 514 - <= 757
Test group / Remarks:
test substance concentrations 10, 25 and 50%

PRE-SCREEN TEST

 

Body weights and skin reactions

TS1(%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw (g)2

Erythema3

Erythema

Erythema

Erythema

Erythema

Erythema

bw (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

25

1

2

22.2

22.4

0

0

0

0

0F

0F

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

0

0

0

0

0

0

21.7

22.5

504

3

4

23.2

23.8

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

0

0

0

0

0

0

22.7

23.1

1TS = test substance (% w/w)

2Body weight (grams)

3Grading erythema and eschar formation (Left = dorsal of left ear; right = dorsal of right ear): 0 = No erythema

4Applied using a pipette with the tip cut off

F = White staining of test substance remnants on the dorsal surface of the ears did not hamper scoring of erythema

 

Ear thickness measurements

TS1(%)

Animal

Day 1

Day 3

Day 6

Left

Right

Left

Right

Left

Right

(mm)

(mm)

(mm)

%2

(mm)

%2

(mm)

%2

(mm)

%2

25

1

2

0.225

0.225

0.225

0.225

0.245

0.235

9

4

0.245

0.235

9

4

0.225

0.225

0

0

0.225

0.225

0

0

50

3

4

0.225

0.225

0.220

0.225

0.245

0.245

9

9

0.240

0.235

9

4

0.220

0.225

-2

0

0.225

0.225

2

0

 Left (mm) = thickness of left ear in millimetres; right (mm) = thickness of right ear in millimetres

1TS = Test substance (% w/w)

2Present increase compared to Day 1 pre-dose value.

 

 

MAIN STUDY

 

Body weights and skin reactions

Group

TS1(%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw (g)2

Erythema3

Erythema

Erythema

Erythema

Erythema

Erythema

 

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

bw (g)

1

0

1

2

3

4

5

22.1

23.9

24.0

22.5

21.1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

21.7

23.1

24.2

22.2

20.9

2

10

6

7

8

9

10

22.6

24.7

24.2

25.1

22.8

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

21.3

23.8

22.9

24.3

22.4

3

25

11

12

13

14

15

23.2

20.4

23.4

20.0

22.0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

23.8

20.9

23.7

20.4

23.0

4

504

16

17

18

19

20

22.0

21.4

23.7

23.0

22.4

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

22.2

22.9

24.0

24.6

23.0

1TS = test substance (% w/w)

2Body weight (grams)

3Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear): 0 = No erythema

4Applied using a pipette with the tip cut off.

Note: white staining of test substance remnants on the dorsal surface of the ears of all experimental animals on Days 1, 2 and 3 did not hamper scoring for erythema.

 

Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

Group

TS1(%)

Animal

Size nodes2

DPM3/ animal

Mean

Mean

Left

Right

DPM ± SEM4

SI ± SEM

1

0

1

2

3

4

5

N

N

N

N

N

N

N

N

N

N

359

497

429

781

597

533 ± 73

1.0 ± 0.2

2

10

6

7

8

9

10

N

N

N

N

N

N

N

N

N

N

661

825

625

1031

460

720 ± 97

1.4 ± 0.3

3

25

11

12

13

14

15

N

N

N

N

N

N

N

N

N

N

492

400

279

712

685

514 ± 83

1.0 ± 0.2

4

50

16

17

18

19

20

N

N

N

N

N

N

N

N

N

N

1244

578

427

690

845

757 ± 140

1.4 ± 0.3

1TS = test substance (% w/w)

2Relative size auricular lymph nodes (-, --, or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).

3DPM = Disintegrations per minute

4SEM = Standard Error of the Mean

 

RELIABILITY CHECK

Group1

% Alpha-Hexylcinnamaldehyde, technical grade

Mean

 

DPM ± SEM

SI ± SEM

1

0% (Acetone:Olive oil (4:1 v/v))

468 ± 91

1.0 ± 0.3

2

5%

808 ± 265

1.7 ± 0.7

3

10%

1391 ± 473

3.0 ± 1.2

4

25%

4243 ± 281

9.1 ± 1.9

1Five females per group

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was not considered to be a skin sensitizer.
Executive summary:

Assessment of skin sensitization to 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] in the Mouse (Local Lymph Node Assay).

 

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2010),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

Test substance 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 substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

 

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.

 

No irritation and no signs of systemic toxicity were observed in any of the animals.

 

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 substance concentrations 10, 25 and 50% were 720, 514 and 757 DPM, respectively. The mean DPM/animal value for the vehicle control group was 533 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.0 and 1.4, respectively.

 

Since there was no indication that the test substance elicited a SI ≥ 3 when tested up to 50%, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was not considered to be a skin sensitizer.

 

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

 

Based on these results, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Assessment of skin sensitization to 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] in the Mouse (Local Lymph Node Assay).

Test substance 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 substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

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.

No irritation and no signs of systemic toxicity were observed in any of the animals.

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 substance concentrations 10, 25 and 50% were 720, 514 and 757 DPM, respectively. The mean DPM/animal value for the vehicle control group was 533 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.0 and 1.4, respectively.

Since there was no indication that the test substance elicited a SI ≥ 3 when tested up to 50%, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was not considered to be a skin sensitizer.

Migrated from Short description of key information:

Not sensitising in the mouse local lymph node assay (LLNA).

Justification for selection of skin sensitisation endpoint:

Endpoint conclusion derived from GLP laboratory study according to OECD Guideline 429, EU Method B.42 and US EPA Standard 870.2600.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin Sensitisation

Based on these results, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).