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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 February, 2015 - 02 March, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 429 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material in report: IFF 09-0035
- Physical state: Clear liquid
- Storage condition of test material: At room temperature protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
Female CBA/J strain mice were supplied by Janvier, Le Genest-Saint-Isle, France. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a tail mark with marker pen. At the start of the study the animals were in the weight range of 21 to 24 g, and were approx. ten weeks old.

The animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment. Free access to tap water and pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 18 to 24°C and 40 to 70 %, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least 10 changes per hour and the lighting was controlled to give twelve hours continuous light and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Undiluted test item and the test item at concentrations of 50 % and 25 % v/v in Acetone/Olive oil (4:1 v/v)
No. of animals per dose:
Groups of five mice were treated
Details on study design:
Preliminary Screening Test
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 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)).
Two test substance concentrations were tested; a 50% and 100% concentration. The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 12 weeks (at initiation of treatment) and 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. 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 Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50 % or 25 % v/v in acetone/olive oil 4:1. 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. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, 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.

The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, % Alpha- Hexylcinnamaldehyde,
technical grade, at a concentration of 5, 10 and 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
The vehicle control group and the positive control group served as common controls with Project number 507448 according to the summary of positive control data for the local lymph node assay.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were 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).

Observations
Mortality/Viability: Twice daily.
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. Furthermore, a
description of all other (local) effects was recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice 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.

Preparation of Single Cell Suspension: 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 stored in the refrigerator until the next day.

Determination of 3HTdR Incorporation: 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. Radioactive 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:
Not performed.

Results and discussion

Positive control results:
The positive control item, α-Hexylcinnamaldehyde, technical grade, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 1.8, 2.2 and 4.1, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 703, 846 and 1587 DPM, respectively. The mean DPM/animal value for the vehicle control group was 383 DPM.

Any other information on results incl. tables

Pre-screen test:

Very slight erythema was noted for the animals treated at 100% on Days 2 and 3. No irritation was observed on the animals treated at 50% and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for all animals.

Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

50

1, 2

24, 22

24, 21

0

0

0

0

0

0

0

0

0

 100  3,4  22, 24  21, 24  0  0

0=     No signs of systemic toxic

Local Skin Irritation – Preliminary Screening Test

Concentration

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

50%

1, 2

0

0

0

0

0

0

0

0

0

0

0

0

 100%  3,4  0  0  1  1  1  1  0  0  0  0  0  0

Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

50%

1

0.230

0.230

0.245

0.245

0.225

0.225

overall mean (mm)

0.230

0.245

0.225

overall mean
ear thickness change (%)

na

7

-2

 50%  2  0.225  0.225  0.240  0.245  0.225  0.225
    

overall mean (mm)

    0.225     0.243     0.225
     

overall mean
ear thickness change (%)

    na     8     0
 100%  3  0.220  0.220  0.245  0.250  0.230  0.230
     

overall mean (mm)

    0.220     0.248     0.230
     

overall mean
ear thickness change (%)

    na     13     5
 100%  4  0.220  0.240  0.240  0.245  0.230  0.225
     

overall mean (mm)

    0.230     0.243     0.228
     

overall mean
ear thickness change (%)

    na     9     2

na=     Not applicable

Individual Disintegrations per Minute and Stimulation Indices

Treatment Group

Animal Number

dpm/
Animala

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle
acetone/olive oil 4:1

1

422

383
(±66)

1.0

Negative

2

408

3

126

4

495

5

463

Test Item
25v/vin
acetone/olive oil 4:1

6

688

703
(±63)

1.8

Negative

7

630

8

890

9

782

10

525

Test Item
50v/vin
acetone/olive oil 4:1

11

655

846
(±60)

2.2

Negative

12

822

13

813

14

929

15

1010

Test Item
100%

16

1777

1587
(±150)

4.1

Positive

17

1187

18

1800

19

1909

20

1263

Positive Control Item

25% v/v in
acetone/olive oil4:1

4-1

no data

1994*
(±575)

3.7

Positive

4-2

 no data

4-3

 no data

4-4

no data

4-5

 no data

dpm=Disintegrations per minute

N/A=  Not applicable

a=     Total number of lymph nodes per animal is 2

b=     Stimulation Index of 3.0 or greater indicates a positive result

Summary of Positive Control Data for the Local Lymph Node Assay

Project Number

Start Date

Finish Date

Test Item

Concentration

Vehicle

Stimulation Indexa

Classificationb

507448

Nov2014

Nov2014

α‑Hexylcinnamaldehyde, technical grade

10% v/v

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

3.1

Positive

507448

Nov2014

Nov2014

 α‑Hexylcinnamaldehyde, technical grade

 25% v/v

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

3.7

Positive

a=      Ratio of test to control lymphocyte proliferation

b=      Stimulation index greater than 3.0 indicates a positive result

Individual Clinical Observations and Mortality Data

Treatment Group

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle
acetone/olive oil 4:1Å

1

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

Test Item
25v/vin
acetone/olive oil 4:1

6

0

0

0

0

0

0

0

0

0

7

0

0

0

0

0

0

0

0

0

8

0

0

0

0

0

0

0

0

0

9

0

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

0

Test Item
50v/vin
acetone/olive oil 4:1

11

0

0

0

0

0

0

0

0

0

12

0

0

0

0

0

0

0

0

0

13

0

0

0

0

0

0

0

0

0

14

0

0

0

0

0

0

0

0

0

15

0

0

0

0

0

0

0

0

0

Test Item
100%

16

0

0

0

0

0

0

0

0

0

17

0

0

0

0

0

0

0

0

0

18

0

0

0

0

0

0

0

0

0

19

0

0

0

0

0

0

0

0

0

20

0

0

0

0

0

0

0

0

0

0=     No signs of systemic toxicity

Calculation of EC3Value

These results show that the test substance elicits a SI ≥ 3. Although, the data do not permit the use of linear interpolation, calculation of EC3 values (the estimated test substance concentration that will give a SI =3) by loglinear extrapolation can provide a reliable estimation of sensitization potency class for use in risk assessment and can avoid the need for repeat animal testing. The data show a clear doseresponse and a slope ratio of 0.38 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value according to Ryan et al 2007. An estimated EC3 value of 23.9% was calculated.

Individual Bodyweights and Bodyweight Changes

Treatment Group

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle
acetone/olive oil 4:1

1

22

22

0

2

23

24

1

3

23

24

 1

4

22

22

0

5

21

21

0

Test Item
25v/vin
acetone/olive oil 4:1

6

23

23

0

7

22

25

3

8

22

25

3

9

24

23

-1

10

24

25

1

Test Item
50v/vin
acetone/olive oil 4:1

11

22

23

1

12

22

22

0

13

23

24

1

14

22

24

2

15

24

24

0

Test Item
100%

16

23

23

0

17

23

24

1

18

21

21

0

19

22

23

1

20

23

24

1

Clinical Observations and Mortality Data

No irritation of the ears was observed in any of the animals examined. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal at 100% which were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Bodyweight

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

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The SI values calculated for the substance concentrations 25, 50 and 100% were 1.8, 2.2 and 4.1, respectively. These results show that the test substance elicits a SI ≥ 3 and an estimated EC3 value of 71.1% was calculated.
The test item was considered to be a sensitiser under the conditions of the test.
Executive summary:

The skin sensitisation potential of FRET 09 -0035 has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 25, 50 and 100% the substance showed SI values of 1.8, 2.2 and 4.1, respectively. The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal at 100% which were considered enlarged. These results show that the test substance elicits a SI ≥ 3 and an estimated EC3 value of 71.1% was calculated. The test item was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction. A NOAEL of 12500 µg/cm2 is derived.