Registration Dossier

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:
22 July 2015 to 21 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.429, "Skin Sensitization: Local Lymph Node Assay", Paris Cedex, July 2010.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B42: "Skin Sensitization: Local Lymph Node Assay". Official Journal of the European Union No. L142, May 2008, including most recent amendments.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. “Skin Sensitization”, March 2003.
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test substance: 206774/A (formerly known as 202199)
Identification: Lowinox® AH25
Appearance: White to cream coloured powder
Batch: WAH4L0016/WAH8E0007
Purity/Composition: 95.1%/95.88%
Test substance storage: At room temperature
Stable under storage conditions until: 09 November 2018 (retest date)/07 May 2022 (retest date)
Purity/composition correction factor: No correction factor required
Test substance handling: No specific handling conditions required
Stability at higher temperatures: Not indicated
Chemical name (IUPAC), synonym or trade name: 2,5-di-tert-pentylhydroquinone
CAS Number: 79-74-3
Molecular formula: C16H26O2
Molecular weight: 250.38
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated

Vehicle information
Solubility in water: 1 g/L at 20°C
Stability in water: Not indicated
Specific details on test material used for the study:
No further details specified in the sudy report.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
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. 10 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..

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.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Initially, two test substance concentrations were tested; a 25% and 50% concentration.
Eight additional animals were treated with four lower concentrations (5%, 10%, 1% and 2%)
In the main study CBA/J mice were treated with test substance concentrations of 0.5, 1 or 2% w/w
No. of animals per dose:
In the main study, three experimental groups of five female CBA/J mice were treated with the test substance.
Details on study design:
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.
Initially, two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical as those used in the main study except that the animals were approximately 10-12 weeks (at initiation of treatment), the application method may have been different 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 (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.
Based on the results of the initially treated animals, eight additional animals were treated in a similar manner with four lower concentrations (5%, 10%, 1% and 2%) at a later stage.

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)

Results and discussion

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%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.6
Test group / Remarks:
0.5% w/w
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
1% w/w
Key result
Parameter:
SI
Value:
5.6
Test group / Remarks:
2% w/w
Cellular proliferation data / Observations:
Main study
Skin reactions / Irritation
No irritation and no signs of systemic toxicity were observed in any of the animals.

Systemic toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic examination of the auricular lymph nodes and surrounding area
The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals of the highest dose group which were considered enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity measurements and SI values
Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.5, 1 and 2% were 881, 1056 and 1865 DPM, respectively. The mean DPM/animal value for the vehicle control group was 335 DPM. The SI values calculated for the substance concentrations 0.5, 1 and 2% were 2.6, 3.2 and 5.6, respectively.

Any other information on results incl. tables

PRE-SCREEN TEST

 

Body weight 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

254

1

2

20.3

23.8

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0s

0s

0s

0s

20.2

25.1

505

3

4

20.7

28.9

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0s

0s

0s

0s

21.3

20.0

Additional pre-screen tests

5

5

6

21.2

22.3

0

0

0

0

1

1

1

1

1

1

1

1

0s

1s

1s

1s

0s

0s

0s

0s

0s

0s

0s

0s

21.0

22.6

10

7

8

24.2

20.4

0

0

0

0

1

1

1

1

1

1

1

1

1s

1s

1s

1s

0s

0s

0s

0s

0sk

0sk

0sk

0sk

23.7

20.1

1

9

10

22

25

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

25

2

11

12

23

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

23

21

s: Scaliness

k: scabs

1. TS = test substance (% w/w)

2. Body weight (grams)

3. Grading erythema and eschar formation (Left = dorsal surface of left ear; Right = dorsal surface of right ear).

               0 = No erythema

               1 = Very slight erythema (barely perceptible)

4. Applied using a pipette with the tip cut off (day 1 and 2).

5. Applied using a spatula, instead of using a pipette.

Note: Brown staining of the dorsal surface of the ears by test substance remnants was noted for both animals at 50% on Days 1 to 5. The staining did not hamper the scoring of the ears. Bald spots behind the ears were noted for the animals treated at 5% and 10% on Days 2 and 3.

 

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

0.220

0.220

0.225

0.220

0.215

0.230

-2

5

0.225

0.225

0

2

0.275

0.245

25

11

0.280

0.260

24

18

50

3

4

0.225

0.225

0.225

0.225

0.215

0.215

-4

-4

0.220

0.220

-2

-2

0.295

0.280

31

24

0.255

0.295

13

31

Additional pre-screen tests

5

5

6

0.225

0.220

0.225

0.220

0.245

0.320

9

45

0.255

0.340

13

55

0.260

0.380

16

73

0.280

0.360

24

64

10

7

8

0.225

0.220

0.225

0.225

0.315

0.305

40

39

0.315

0.335

40

49

0.350

0.375

56

70

0.410

0.390

82

73

1

9

10

0.220

0.230

0.220

0.230

0.240

0.245

9

7

0.245

0.245

11

7

0.240

0.240

9

4

0.240

0.245

9

7

2

11

12

0.230

0.220

0.225

0.220

0.240

0.235

4

7

0.235

0.235

4

7

0.240

0.235

4

7

0.240

0.245

7

11

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

1. TS = test substance (% w/w)

2. Percent 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

bw (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

1

0

1

2

3

4

5

21.0

21.5

20.3

20.7

23.6

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

21.2

21.2

20.7

21.5

24.7

2

0.5

6

7

8

9

10

25.2

22.1

23.8

21.7

23.4

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

24.2

21.7

23.4

23.5

23.4

3

1

11

12

13

14

15

19.9

22.0

21.8

22.1

25.3

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

20.9

23.2

22.0

22.4

23.0

4

2

16

17

18

19

20

22.3

19.9

21.7

19.9

18.7

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

21.9

21.0

22.5

21.2

20.0

1. TS = test substance (% w/w)

2. Body weight (grams)

3. Grading erythema and eschar formation (Left = dorsal surface of left ear; Right = dorsal surface of right ears):

               0 = No erythema

 

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

Group

TS1(%)

Animal

Size nodes2

DPM3/ animal

Mean

DPM ± SEM4

Mean

SI ± SEM

Left

Right

1

0

1

2

3

4

5

n

n

n

n

n

n

n

n

n

n

310

293

318

528

225

335 ± 51

1.0 ± 0.2

2

0.5

6

7

8

9

10

n

n

n

n

n

n

n

n

n

n

477

1085

994

872

976

881 ± 106

2.6 ± 0.5

3

1

11

12

13

14

15

n

n

n

n

n

n

n

n

n

n

230

2000

1587

700

761

1056 ± 322

3.2 ± 1.1

4

2

16

17

18

19

20

n

n

n

n

n

n

n

n

n

n

1210

1764

1552

2082

2715

1865 ± 256

5.6 ± 1.1

1. TS = test substance (% w/w)

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

3. DPM = Disintegrations per minute

4. SEM = Standard Error of the Mean

 

ASSESSMENT OF CONTACT HYPERSENSITIVITY TO ALPHA-HEXYLCINNAMALDEHYDE, TECHNICAL GRAD IN THE MOUSE (LOCAL LYMPH NODE ASSAY) A RELIABILITY CHECK

SUMMARY RELIABILITY CHECK

Group1

% Alpha-Hexylcinnamaldehyde, technical grade

Mean

DPM ± SEM

SI ± SEM

1

2

3

4

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

5%

10%

25%

468 ± 91

808 ± 265

1391 ± 473

4243 ± 281

1.0   ± 0.3

1.7 ± 0.7

3.0 ± 1.2

9.1 ± 1.9

1. Five females per group

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
The results show that the test substance elicits a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 0.8% was calculated.
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.
Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), Lowinox® AH25 would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), Lowinox® AH25 should be classified as skin sensitizer (Category 1A).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), Lowinox® AH25 should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

Assessment of skin sensitization to Lowinox® AH25 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 0.5, 1 or 2% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Propylene glycol).

 

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.

 

The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals of the highest dose group which were considered 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 test substance concentrations 0.5, 1 and 2% were 881, 1056 and 1865 DPM, respectively. The mean DPM/animal value for the vehicle control group was 335 DPM. The SI values calculated for the substance concentrations 0.5, 1 and 2% were 2.6, 3.2 and 5.6, respectively.

 

These results show that the test substance elicits a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 0.8% was calculated.

 

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.

 

Based on these results:

- according to the recommendations made in the test guidelines (including all amendments), Lowinox® AH25 would be regarded as skin sensitizer.

 

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), Lowinox® AH25 should be classified as skin sensitizer (Category 1A).

 

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), Lowinox® AH25 should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.