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Description of key information

Skin Sensitisation

Key value determined in a GLP accredited labatory study in the Mouse (Local Lymph Node Assay), performed in accordance with OECD Guideline 429, EU Method B.42 and US EPA Procedure OPPTS 870.2600.

Lowinox® 22IB46 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.

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:
15 April 2016 to 07 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study performed in accordance with OECD, EPA & EU test guidelines in compliance with GLP and reported with a valid GLP certificate.
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)
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 a 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. 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 were 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 was between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
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:
2, 5 or 10% w/w
No. of animals per dose:
20 females (nulliparous and non-pregnant), five females per group (main study only).
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 item concentrations were tested; a 25% 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, four additional animals were treated in a similar manner with two lower concentrations (5% and 10%) at a later stage. The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-12 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 labelled 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 item concentration per group. The highest test item concentration 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 with a pipet (25 μL/ear) with the test item, 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 item.
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, re-suspended 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.

Grading Irritation Reactions:
Erythema and eschar formation:
No erythema ..............................................................................………………………….. .... ...0
Very slight erythema (barely perceptible) ....................................................………………….. 1
Well-defined erythema ...................................................................………………………… ..... 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) … 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema ......…. 4

Necropsy: No necropsy for gross macroscopic examination was performed according to study plan.

Evaluation: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using 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. The results were evaluated 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 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not specified
Positive control results:
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:
>= 3

PRE-SCREEN TEST

Body weights and the 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

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0

0F

0F

0

0

0

0

0

21.3

21.8

504

3

4

23.2

23.8

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0

0F

21.2

23.3

Additional pre-screen test

 

 

 

 

 

 

 

 

 

 

 

 

 

5

5

6

26.6

21.4

0

0

0

0

0

0

0

0

0F

0F

0F

0F

0F

0F

0F

0F

0

0F

0F

0F

0

0

0

0

23.5

22.1

10

7

8

21.0

22.5

0

0

0

0

0

0

0

0

0F

0F

0F

0F

0F

0F

0F

0F

0

0F

0FS

0F

0

0

0S

0

21.3

2.8

 S. Scaliness

1. TS = test item (% 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

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

Note: one animal treated at 25% (no. 2) showed piloerection on Days 2 and 3. Both animals treated at 50% showed uncoordinated movements and tremors on Days 1, 2 and 3. Additionally, piloerection was noted for these animals 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

0.225

0.220

0.225

0.220

0.225

0.220

0

0

0.220

0.220

-2

0

0.230

0.235

2

7

0.240

0.235

7

7

50

3

4

0.220

0.225

0.225

0.225

0.225

0.220

2

-2

0.225

0.225

0

0

0.230

0.235

5

4

0.235

0.235

4

4

Additional pre-screen test

 

 

 

 

 

 

 

 

 

5

5

5

6

0.225

0.225

0.220

0.220

0.225

0.225

0

0

0.225

0.225

2

2

0.220

0.225

-2

0

0.220

0.225

0

2

10

10

7

8

0.220

0.220

0.220

0.220

0.230

0.230

5

5

0.230

0.230

5

5

0.230

0.225

5

2

0.230

0.225

5

2

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

1. TS = test item (% 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

 

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

bw (g)

1

0

1

2

3

4

5

21.9

22.2

22.2

20.0

23.4

0

0

0

0

0

0

0

0

0S

0

0S

0

0

0S

0

0S

0

0S

0S

0

0S

0S

0S

0S

0S

0S

0S

0S

0S

0S

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

22.9

22.4

22.2

20.0

22.3

2

2

6

7

8

9

10

22.4

19.7

21.6

22.2

23.4

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0S

0S

0

0

0

0S

0S

0S

0

0

0S

0S

0S

0

0

0

0

0

0

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

20.0

22.5

22.5

23.5

3

5

11

12

13

14

15

21.8

21.6

22.1

22.2

24.2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0S

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

22.5

21.5

22.8

21.8

25.6


4

10

16

17

18

19

20

21.1

21.1

21.1

21.1

23.0

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

21.5

22.1

21.8

21.0

21.8

S. Scaliness

1. TS = test item (% 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

F. White staining of test item remnants on the dorsal surface of the ears 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

525

635

959

399

930

690 ± 11

1.0 ± 0.2

2

2

6

7

8

9

10

N

N

N

N

N

N

N

N

N

N

455

775

781

316

2312

928 ± 358

1.3 ± 0.6

3

5

11

12

13

14

15

N

+

N

N

+

+

+

N

N

+

423

1371

522

1043

383

748 ± 196

1.5 ± 0.3

4

10

16

17

18

19

20

N

+

N

N

+

N

+

N

N

+

815

809

1633

1080

806

1029 ± 160

1.5 ± 0.3

 1. TS = test item (% 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

 

RELIABILITY CHECK

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

1Five females per group.

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, Lowinox® 22IB46 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.
Executive summary:

Assessment of skin sensitization to Lowinox® 22IB46 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 item 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 item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Methyl ethyl ketone).

 

Three days after the last exposure, all animals were injected with3H-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.

 

Results

No irritation was observed in any of the animals. The scaliness as shown by the control animals and some animals treated at 2 and 5% was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of the high dose animals but did not hamper scoring of the skin reactions.

 

All auricular lymph nodes of the animals control group and animals treated at 2% were considered normal in size. The nodes of some animals treated at 5 and 10% 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 item concentrations 2, 5 and 10% were 928, 748 and 1029 DPM, respectively. The mean DPM/animal value for the vehicle control group was 690 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 1.3, 1.5 and 1.5, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, Lowinox® 22IB46 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.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, Lowinox® 22IB46 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item 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)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin Sensitisation

Based on the results, Lowinox® 22IB46 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item 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).

Respiratory Sensistisation

 

Based on the results, Lowinox® 22IB46 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The substance is not anticipated to be a respiratory sensitiser.

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