Registration Dossier

Administrative data

Description of key information

Skin sensitisation: Not a skin sensitiser.

Respiratory sensitisation: No data available

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:
2016-05-09 to 2016-06-07
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AGC Chemicals Europe Ltd (Hillhouse International, Thornton Cleveleys, Lancashire, FY5 4QD, United Kingdom); Batch no. 41251
- Expiration date of the lot/batch: 01 March 2017 (nominal expiry date) (taken from label)
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until 01 March 2017 (nominal expiry date) (taken from label)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless liquid

OTHER SPECIFICS:

Purity: 100.00%
Molecular weight: 320
pH (1% in water, indicative range): 7.22 – 7.78 (determined by WIL Research Europe)
Specific gravity/density: 1.67 at 25°C
Species:
mouse
Strain:
CBA:J
Remarks:
CBA/J strain, inbred, SPF-Quality.
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: not specified
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean
- Housing: 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.
- 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: at least 5 days before the start of treatment
- Indication of any skin lesions: It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

- IN-LIFE DATES: From: 2016-05-09 To: 2016-06-07
Vehicle:
dimethylformamide
Remarks:
N,N-Dimethyl formamide (Merck, Darmstadt, Germany).
Concentration:
0, 25, 50, and 100 %w/w
No. of animals per dose:
5/concentration
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at WIL Research Europe. There was no information available regarding the
solubility or stability in vehicle.
- 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.
-Systemic toxicity: 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.
- Ear thickness measurements: 50% and 100% w/w test concentrations; measurements made on Day 1 (pre-dose value), Day 3, and Day 6.
- Erythema scores: See Table 1.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Main study - local lymph node assay (LLNA)
- 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 groupusing 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 (see Table 2.)

TREATMENT PREPARATION AND ADMINISTRATION:

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

Induction - Days 1, 2 and 3:

The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, 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 material.

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 numerical scoring system orovided in Table 1. In addition, a description of all other (local) effects was recorded.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by WIL Research Europe. In this study, performed in May 2016, females of the CBA/J mouse strain (Janvier, Le Genest-SaintIsle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approx. 10 weeks old at commencement of the study. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKBT2800V (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO).

The SI values calculated for the item concentrations 5, 10 and 25% were 1.4, 1.5 and 4.3 respectively. An EC3 value of 18.0% 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%.
Key result
Parameter:
SI
Value:
ca. 1
Variability:
± 0.2
Test group / Remarks:
Vehicle Control
Key result
Parameter:
SI
Value:
ca. 0.8
Variability:
± 0.2
Test group / Remarks:
25% w/w Test Material Concentration
Key result
Parameter:
SI
Value:
ca. 1
Variability:
± 0.2
Test group / Remarks:
50% w/w Test Material Concentration
Key result
Parameter:
SI
Value:
ca. 1.5
Variability:
± 0.3
Test group / Remarks:
100% w/w Test Material Concentration
Key result
Parameter:
SI
Value:
ca. 1.4
Variability:
± 0.3
Test group / Remarks:
5% v/v Positive Control
Key result
Parameter:
SI
Value:
ca. 1.5
Variability:
± 0.3
Test group / Remarks:
10% v/v Positive Control
Key result
Parameter:
SI
Value:
ca. 4.3
Variability:
± 1.0
Test group / Remarks:
25% v/v Positive Control
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

Main Study - All auricular lymph nodes of the control animals and animals treated with test material concentration of 25% and 50% were considered normal in size. Thenodes of the animals treated with100% test material were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION

Main Study - Mean DPM/animal values for the experimental groups treated with test itemconcentrations 25, 50 and 100% were 545, 720 and 1064 DPM, respectively. The mean DPM/animal value for the vehicle control group was 691 DPM. The SI values calculated for the test itemconcentrations 25, 50 and 100% were 0.8, 1.0 and 1.5, respectively.

EC3 CALCULATION

Reliability check for positive control Alpha- Hexylcinnamaldehyde - 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%.

CLINICAL OBSERVATIONS:

Pre-screen Test - No irritation and no signs of systemic toxicity were observed in any of the pre-screen animals. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White staining of the dorsal surface of the ears by test material remnants was noted. The staining did not hamper the scoring of the ears. Based on these results, the highest test material concentration selected for the main study was a 100% concentration.

Main Study - No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS

Main Study - Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Table 4. Main Study - Body Weights and Skin Reactions

 

Group

Test Material

Concn

(% w/w)

 

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Body wt (g)

Erythema1

Erythema

Erythema

Erythema

Erythema

Erythema

Body wt (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

 

 

1

 

 

0

1

21.7

0

0

0

0

0

0

0

0

0

0

0

0

23.4

2

22.3

0

0

0

0

0

0

0

0

0

0

0

0

24.1

3

21.8

0

0

0

0

0

0

0

0

0

0

0

0

22.0

4

21.0

0

0

0

0

0

0

0

0

0

0

0

0

23.1

5

20.8

0

0

0

0

0

0

0

0

0

0

0

0

22.9

 

 

 

2

 

 

25

1

21.2

0

0

0

0

0

0

0

0

0

0

0

0

22.5

2

21.6

0

0

0

0

0

0

0

0

0

0

0

0

21.4

3

20.6

0

0

0

0

0

0

0

0

0

0

0

0

22.3

4

20.9

0

0

0

0

0

0

0

0

0

0

0

0

21.0

5

20.5

0

0

0

0

0

0

0

0

0

0

0

0

23.0

 

 

 

3

 

 

50

1

21.9

0

0

0

0

0

0

0

0

0

0

0

0

22.2

2

20.0

0

0

0

0

0

0

0

0

0

0

0

0

21.3

3

20.3

0

0

0

0

0

0

0

0

0

0

0

0

20.8

4

21.5

0

0

0

0

0

0

0

0

0

0

0

0

23.3

5

20.8

0

0

0

0

0

0

0

0

0

0

0

0

21.8

 

 

 

4

 

 

100

1

21.2

0

0

0

0

0

0

0

0

0

0

0

0

22.1

2

22.6

0

0

0

0

0

0

0

0

0

0

0

0

22.8

3

23.3

0

0

0

0

0

0

0

0

0

0

0

0

23.6

4

21.8

0

0

0

0

0

0

0

0

0

0

0

0

23.5

5

22.4

0

0

0

0

0

0

0

0

0

0

0

0

23.2

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

 

Table 5. Main Study - Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

 

Group

Test Material

Concn

(% w/w)

 

Animal

 

Size nodes1

 

DPM2/animal

 

mean

DPM ± SEM3

 

mean

SI ± SEM

Left

Right

 

 

1

 

 

0

1

n

n

789

 

 

691 ± 88

 

 

1.0 ± 0.2

2

n

n

347

3

n

n

813

4

n

n

700

5

n

n

808

 

 

 

2

 

 

25

1

n

n

620

 

 

545 ± 84

 

 

0.8 ± 0.2

 

2

n

n

293

3

n

n

462

4

n

n

550

5

n

n

800

 

 

 

3

 

 

50

1

n

n

829

 

 

720 ± 119

 

 

 

 

1.0 ± 0.2

2

n

n

586

3

n

n

1137

4

n

n

498

5

n

n

552

 

 

 

4

 

 

100

1

+

+

1122

 

 

1064 ± 159

 

 

1.5 ± 0.3

2

+

+

745

3

+

+

758

4

+

+

1080

5

+

+

1616

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

2 - DPM = Disintegrations per minute

3 - SEM = Standard Error of the Mean

 

Table 6. Reliability Check Results for Alpha- Hexylcinnamaldehyde (HCA; CAS no. 101-86-0)

Group1

% HCA

mean

DPM ± SEM

SI ± SEM

1

0% (AcOO)

603 ± 123

1.0 ± 0.3

2

5%

868 ± 111

1.4 ± 0.3

3

10%

900 ± 97

1.5 ± 0.3

4

25%

2566 ± 300

4.3 ± 1.0

1 - Five females per group

AcOO - Acetone/Olive oil

Interpretation of results:
other: Not classified as a skin sensitizer
Remarks:
GHS Criteria
Conclusions:
Since there was no indication that the test material elicited a SI ≥ 3 when tested up to 100%, it was not considered to be a skin sensitizer.

Based on the results observed in the mouse local lymph node assay, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane(AsahiklinTM AC-2000) would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test material 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 (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments). 
Executive summary:

In a key in vivo guideline (OECD 429) study, the skin sensitisation potential of the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane; AsahiklinTM AC-2000) was evaluated in a Mouse Local Lymph Node Assay (LLNA).

 

Test material concentrations selected for the study were based on the results of a pre-screen test. In this LLNA study, three experimental groups of five female CBA/J mice were treated with the test material at concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-Dimethyl formamide).Three days post 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 was observed in any of the animals. All auricular lymph nodes of the control animals and animals treated with test material at concentrations of 25% and 50% were considered normal in size. The nodes of the animals treated with 100% test material were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Mean DPM/animal values for the experimental groups treated with the test material at concentrations of 25, 50 and 100% were 545, 720, and 1064 DPM, respectively. The mean DPM/animal value for the vehicle control group was 691 DPM. The SI values calculated for the test material at concentrations of 25, 50 and 100% were 0.8, 1.0, and 1.5, respectively.

 

Since there was no indication that the test material elicited a SI ≥ 3 when tested up to 100%, it 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 is an appropriate model for testing for contact hypersensitivity.

 

Based on these results,1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane(AsahiklinTM AC-2000) would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test material 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 (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments). 

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

In a key in vivo Guideline (OECD 429) study (WIL Research Europe B.V., 2016c), the skin sensitisation potential of the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) was evaluated in a Mouse Local Lymph Node Assay (LLNA).

 

Test material concentrations selected for the study were based on the results of a pre-screen test. In this LLNA study, three experimental groups of five female CBA/J mice were treated with the test material at concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-Dimethyl formamide).Three days post 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.

  

No irritation was observed in any of the animals. All auricular lymph nodes of the control animals and animals treated with test material at concentrations of 25% and 50% were considered normal in size. The nodes of the animals treated with 100% test material were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Mean DPM/animal values for the experimental groups treated with the test material at concentrations of 25, 50 and 100% were 545, 720, and 1064 DPM, respectively. The mean DPM/animal value for the vehicle control group was 691 DPM. The SI values calculated for the test material at concentrations of 25, 50 and 100% were 0.8, 1.0, and 1.5, respectively.

 

Since there was no indication that the test material elicited a SI ≥ 3 when tested up to 100%, it was not considered to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Not classified for skin sensitisation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).