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Description of key information

Skin sensitisation: skin sensitiser, LLNA study (OECD 429, GLP, K1).

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:
04-18 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD guideline 429 with minor deviations: temperature was sometimes outside of the target ranges
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
dated 22 July 2010
Deviations:
yes
Remarks:
temperature was sometimes outside of the target ranges
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
dated 30 May 2008
Deviations:
yes
Remarks:
temperature was sometimes outside of the target ranges
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
20 March 2009
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: VE00201963
- Expiration date of the lot/batch: 21 March 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature and protected from light
Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 20.9 ± 1.2 g
- Housing: Animals were housed in groups of five in Makrolon Type II / III cages, with wire mesh top.
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45-65% (>95% for several hours)
- Photoperiod: 12 hours dark / 12 hours light
Vehicle:
dimethylformamide
Concentration:
0, 10, 25 and 50% (w/w)
No. of animals per dose:
5 females per dose
Details on study design:
PRE-SCREEN TESTS:
- Vehicle selection: In the solubility experiment, the highest test item concentration, which could be technically used, was a 50% solution in dimethylsulfoxide. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonicating, warming to 37°C).
- Dose selection: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% (w/w) once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (on Day 1), on Day 3 and before sacrifice (on Day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (Day 6) at the apical area using a biopsy punch and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value >=3 was observed at any observation time and/or if an increase in ear thickness of >=25% was recorded on Day 3 or Day 6. At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Thus, the test item in the main study was assayed at 10, 25, and 50% (w/w). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations of 10, 25, and 50% (w/w) in dimethylformamide. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application (Day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.5 µCi of 3HTdR (equivalent to 3HTdR 78 µCi/mL) were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The level of 3HTdR incorporation, expressed as as the number of radioactive disintegrations per minute (DPM), was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). The proliferative response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For all statistical calculations SigmaStat for Windows (Version 2.0) was used.
A One-Way-Analysis-of-Variance (ANOVA) was conducted on the ear weights to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group. Furthermore, the ANOVA was conducted on the ear thickness values to assess whether a statistically significant increase in ear thickness could be observed when comparing the values measured on Day 1 prior to application with the values measured on Days 3 or 6 in the respective test item groups or within the vehicle control group. Statistical significance was set at the 5% level (p < 0.05).
The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003). However, both biological and statistical significance were considered together.
Positive control results:
In the historical study performed in April 2012 on positive control (Hexylcinnamaldehyde), S.I. values were 1.00, 0.88, 1.51 and 3.73 at the concentrations of 0, 5, 10 and 25% (w/v), and EC3 value was 20.1% (w/v). The study was therefore considered valid.
Key result
Parameter:
EC3
Remarks:
in % (w/w)
Value:
ca. 4.8
Remarks on result:
other: considered as a moderate skin sensitizer
Parameter:
SI
Value:
5.28
Test group / Remarks:
at the tested concentration of 10%
Parameter:
SI
Value:
8.22
Test group / Remarks:
at the tested concentration of 25%
Parameter:
SI
Value:
9.02
Test group / Remarks:
at the tested concentration of 50%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
In the proliferation assay, the observed SI values were 1.00, 5.28, 8.22 and 9.023 at 0, 10, 25 and 50%, respectively. A clear dose response was observed. Refer tables 7.4.1/1 and 7.4.1/2 for details.

EC3 CALCULATION
An estimated EC3 value was determined by means of extrapolation from the two lowest test item concentrations and their respective Stimulation Indices. This extrapolated EC3 value was calculated as 4.8% (w/w).

MORTALITY / CLINICAL OBSERVATIONS:
No clinical signs and no mortality were observed during the study.

BODY WEIGHTS
The body weight gain of the treated animals was within the normal range.

LOCAL IRRITATION
On Days 2-6, the animals treated with a test item concentration of 50% showed a very slight to well defined erythema of the ear skin (Days 2, 3, 4, 6: Score 1; Day 5: Score 2).

EAR THICKNESS /EAR WEIGHT
A relevant increase in ear thickness was not observed.

Table 7.4.1/1: Skin sensitization – results

 

Test item concentration

DPM values measured

DPM-BG per animal

(2 lymph nodes)a)

S.I.b)

% (w/w)

Group no.

Animal no.

---

---

BG I

17

---

---

---

---

BG II

16

---

---

0

1

1

484

467

---

0

1

2

1343

1326

---

0

1

3

907

890

---

0

1

4

1099

1082

---

0

1

5

441

424

---

10

2

6

6317

6300

7.5

10

2

7

5333

5316

6.3

10

2

8

4028

4011

4.8

10

2

9

3261

3244

3.9

10

2

10

3276

3259

3.9

25

3

11

8427

8410

10.0

25

3

12

8265

8248

9.8

25

3

13

5216

5199

6.2

25

3

14

5185

5168

6.2

25

3

15

7455

7438

8.9

50

4

16

7502

7485

8.9

50

4

17

9122

9105

10.9

50

4

18

9397

9380

11.2

50

4

19

6004

5987

7.1

50

4

20

5853

5836

7.0

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Groups

S.I. = Stimulation Index

a)= values corrected for mean background value (BGI and BGII).

b)= Stimulation Indices relative to the mean of the control group (Group 1)

 

Table 7.4.1/2: Calculation of Stimulation Indices per Dose Group

 

Test item concentration

Group Calculation

Mean DPM per animal (2 lymph nodes)a)

SDb)

S.I.

Vehicle (dimethylformamide)

838.3

390.3

1.00

10% Test item

4426.5

1344.9

5.28

25% Test item

6893.1

1603.1

8.22

50% Test item

7559.1

1669.7

9.02

a)Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

b)SD = stimulation index

 

Table 7.4.1/3: Calculation of the EC3 Value by Means of Extrapolation

 

S.I.

Concentration

25% (a)

8.2 (b)

Concentration

10% (c)

5.3 (d)

EC3 = 2(log2 (c) + (3-d) / (b-d) * (log2(a) - log2(c)))

EC3

4.8%

3.0

EC3 = Estimated concentration for a S.I. of 3.

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above the S.I. value of 3 on the LLNA dose response plot. The point with the higher S.I. is denoted as (a, b). The point with the lower S.I. is denoted as (c, d).

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under these test conditions, the test item was considered as a moderate skin sensitizer (i.e. EC3 >2%) and need to be classified as "Category 1 and sub-category 1B" according to the Regulation (EC) N° 1272/2008 and according to the Globally Harmonised System of classification and labelling of chemicals (GHS). The hazard statement "H317: may cause an allergic skin reaction" with the symbol “exclamation mark” and signal word "Warning" are required.
Executive summary:

In a Local Lymph Node Assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/CaOlaHsd mice (5 females/dose) were topically applied with 25 µL/ear/day of test item at concentrations of 10, 25 and 50% (w/w) in dimethylformamide to the dorsal surface of both ears for three consecutive days (Days 1, 2 and 3). One negative control group of five animals received the vehicle (dimethylformamide). Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On Days 2-6, the animals treated with a test item concentration of 50% showed an erythema of the ear skin (Days 2, 3, 4, 6: Score 1; day 5: Score 2). No statistically significant increases in ear thickness or ear weights were observed.

In this study, Stimulation Indices (S.I.) of 1.00, 5.28, 8.22 and 9.02 were determined with the test item at concentrations of 0, 10, 25, and 50% (w/w) in dimethylformamide. A clear dose response was observed. An estimated EC3 value was determined by means of extrapolation from the two lowest test item concentrations and their respective S.I. This extrapolated EC3 value was calculated as 4.8% (w/w).

In the historical study performed on positive control (Hexylcinnamaldehyde), S.I. values were 1.00, 0.88, 1.51 and 3.73 at the concentrations of 0, 5, 10 and 25% (w/v), and EC3 value was 20.1% (w/v). The study was therefore considered valid.

Under these test conditions, the test item was considered as a moderate skin sensitizer (i.e. EC3 >2%) and need to be classified as "Category 1 and sub-category 1B" according to the Regulation (EC) N° 1272/2008 and according to the Globally Harmonised System of classification and labelling of chemicals (GHS). The hazard statement "H317: may cause an allergic skin reaction" with the symbol “exclamation mark” and signal word "Warning" are required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a Local Lymph Node Assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/CaOlaHsd mice (5 females/dose) were topically applied with 25 µL/ear/day of test item at concentrations of 10, 25 and 50% (w/w) in dimethylformamide to the dorsal surface of both ears for three consecutive days (Days 1, 2 and 3). One negative control group of five animals received the vehicle (dimethylformamide). Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On Days 2-6, the animals treated with a test item concentration of 50% showed an erythema of the ear skin (Days 2, 3, 4, 6: Score 1; day 5: Score 2). No statistically significant increases in ear thickness or ear weights were observed.

In this study, Stimulation Indices (S.I.) of 1.00, 5.28, 8.22 and 9.02 were determined with the test item at concentrations of 0, 10, 25, and 50% (w/w) in dimethylformamide. A clear dose response was observed. An estimated EC3 value was determined by means of extrapolation from the two lowest test item concentrations and their respective S.I. This extrapolated EC3 value was calculated as 4.8% (w/w).

In the historical study performed on positive control (Hexylcinnamaldehyde), S.I. values were 1.00, 0.88, 1.51 and 3.73 at the concentrations of 0, 5, 10 and 25% (w/v), and EC3 value was 20.1% (w/v). The study was therefore considered valid.

Under these test conditions, the test item was considered as a moderate skin sensitizer (i.e. EC3 >2%) and need to be classified as "Category 1 and sub-category 1B" according to the Regulation (EC) N° 1272/2008 and according to the Globally Harmonised System of classification and labelling of chemicals (GHS). The hazard statement "H317: may cause an allergic skin reaction" with the symbol “exclamation mark” and signal word "Warning" are required.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Based on the available information the substance is classified as:

- Skin Sens. 1, H317 (May cause an allergic skin reaction) according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No information was available regarding respiratory sensitisation.