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Description of key information

Both Local Lymph node assay and Guinea Pig Maximization test gave clear evidence of the test item's skin sensitizing potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2003-04-2 until 2003-05-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Remarks:
RCC Ltd
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15.3 g - 21.1 g
- Housing: groups of four in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba 3433, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: Under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 2, 2003 To: May 28, 2003
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8: in acetone:olive oil, 4:1, w/v); Groups 1 and 5 were treated with the vehicle (control groups)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- The highest non-irritant and technically applicable test item concentration was determined in a non-GLP pretest in two mice with concentrations of 1 %, 5%, 10% and 25 % (w/v). The test item in the main study was assayed at three consecutive concentrations. The top dose (10%) was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
- Name of test method: LLNA
- Criteria used to consider a positive response: regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8) (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. Two control groups of mice were treated with an equivalent volume of the relevant vehicle alone. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
- Administration of 3H-Methyl Thymidine: Five days after the first topical application, all mice were administered with 250 µl of 78.4µCi/ml 3HTdR (equal to 19.6 µCi 3HTdR) (for Groups 1-4) and 85.0 µCi/ml 3HTdR (equal to 21.3 µCi 3HTdR) (for Groups 5-8) by intravenous injection via a tail vein.
- Determination of incorporated 3HTDR: Approximately five hours after treatment with 3HTdR all mice were euthanized. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of “Ultima Gold” scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a P-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
- Interpretation of Data: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
1. 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 SI.
2. 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
Positive control results:
SI of 2.5, 3.7 and 9.7 were determined with the positive control at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v).
Parameter:
SI
Remarks on result:
other: In this study, SI of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). (See Table 1)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 1

- Viability / Mortality: No deaths occurred during the study period.

- Clinical signs: No test item-related clinical signs were observed. On the third application day, a slight ear erythema was observed at both dosing sites in all mice of Group 7 (5 %) and Group 8 (10 %). On the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 8 (10 %). These signs persisted for the remainder of the in-life phase of the study.

- Body weights: The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

- LLNA: A dose related increase (> 26 folds) in SI was observed. An exact EC3 value could not be calculated because for such calculation, data immediately above and below SI value of 3 is required (Table 1).

 

Table 1: Results of individual data

Test item concentration % (w/v)

 

Measurement DPM

Calculation

SI

DPM-BG (a)

No. of lymph nodes

DPM per lymph node (b)

--

BG I

3

--

--

--

--

--

BG II

1

--

--

--

--

--

CG 5 (c)

5497

5495

8

687

--

2.5

TG2

121387

121385

8

15173

22.1*

5

TG3

172678

172676

8

21585

31.4*

10

TG4

165859

165857

8

20732

30.2*

2.5

TG5

146953

146951

8

18369

26.7

5

TG6

190092

190090

8

23761

34.6

10

TG8

212233

212231

8

26529

38.6

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

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

(a) = The mean value was taken from the figures BG I and BG II

(b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

(c) = The date of vehicle control Group 5 was used to calculate S.I. The date of vehicle control Group 1 was not reported due to measurement problems occurred during evaluation

* The data was just as reference, because the S.I. value was calculated based on dpm/LN of vehicle control Group 5.

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
In this study STIMULATION INDICES of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). The test item was therefore found to be a strong sensitizer.
Executive summary:

In order to study a possible contact allergenic potential of the test substance, a GLP-compliant local lymph node assay according to OECD guideline 429 was performed. Six groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8), (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. Two control groups of each four mice were treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labeled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. 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 ^H-methyl thymidine measured in a β-scintillation counter. No test item-related clinical signs were observed in any animals of the control groups (Groups 1 and 5), Group 2 (2.5 %), Group 3 (5 %), Group 4 (10 %) or Group 6 (2.5 %). On the third application day, a slight ear erythema was observed at both dosing sites in all mice of Group 7 (5 %) and Group 8 (10 %). On the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 8 (10 %). These signs persisted for the remainder of the in-life phase of the study. All treated animals survived the scheduled study period. In this study STIMULATION INDICES of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). As a result, the test item was found to be a strong sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 13, 1986 to Jul. 18, 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Remarks:
CIBA-GEIGY LIMITED (GU 2 Toxicology)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Species:
guinea pig
Strain:
other: Pirbright White Strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY LTD. Tierfarm, 4334 Sisseln, Switzerland.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: 320-463 g
- Housing: Housed individually in Macrolon cages (Type 3)
- Diet: guinea pig pellets - NAFAG No.846, ad libitum
- Water: ad libitum, fresh water
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
Route:
intradermal
Vehicle:
other: sesame oil
Concentration / amount:
1%
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
Approximately 0.4 g paste of 10% test substance in vaseline
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
Approximately 0.2 g paste of 0.3% test substance in vaseline.
No. of animals per dose:
10/sex/dose
Details on study design:
RANGE FINDING TESTS:
The concentration of the test compound for the induction and challenge periods were determined on separate animals.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Test groups: 1) Three pairs of intradermal application (0.1 ml per injection): 1:1 mixture of adjuvant and saline, test compound in sesame oil, and test compound in the adjuvant saline mixture; 1% of the test compound in the sesame oil and adjuvant mixture. 2) Epicutaneous application: 1 week after intradermal application, with approximately 0.4 g of the 10 % test compound in vaseline for 48 h under occlusive condition, patch 2x4 cm
- Control group: Treated with adjuvant and the vehicle during induction period
- Site: Neck of the animals

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after the epicutaneous induction application
- Exposure period: 24 hours- Test groups: test compound in vaseline
- Control group: treated with the vehicle as well as with the test compound (at least 10 animals) to control the maximal subirritant concentration of the test compound in adjuvant treated.animals.
- Site: Flank of the animals
- Concentrations: Approximately 0.2 g paste of 0.3% test compound in vaseline
- Evaluation (hr after challenge): 24 and 48 hours after removing the dressings

OTHER:
Twenty four hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings. The sensitizing potential was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.
Challenge controls:
Adjuvant treated control group was challenged with the test compound and the vehicle.
Positive control results:
The sensitivity of the strain is checked every six months with paraphenylene-diamine or potassium-dichromate.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.2 g paste of 0.3% test compound
No. with + reactions:
15
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
vehicle reated
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.2 g paste of 0.3% test compound
No. with + reactions:
12
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
vehicle treated
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.2 g paste of 0.3% test compound
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle treated
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.2 g paste of 0.3% test compound
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle treated
No. with + reactions:
0
Total no. in group:
20

Number of positive animals per group after epicutaneous application:

 

Test group

(sensitized with test compound)

Control group

(adjuvant-treated)

After 24 h

After 48 h

After 24 h

After 48 h

Test compound

15/20

12/20

0/10

0/10

Vehicle control

0/20

0/20

0/20

0/20

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of this study and other the conditions employed, the test article is regarded as a skin sensitizer.
Executive summary:

In a guinea pig maximization test according to OECD guideline No. 406, 10 male and 10 female animals were first induced and then challenged with the test article to investigate its sensitization potential. Induction was a two-stage operation: First, three pairs of intradermal injections (1 % test substance in sesame oil) were made into the neck of the animals with a 1:1 mixture (v/v) of adjuvant/physiological saline, the test substance in sesame oil, and the test substance in a 1:1 mixture (v/v) of adjuvant/physiological saline. One week later, the test article was incorporated in vaseline at a concentration of 10% (approx. 0.4 g) and applied on a filter paper patch to the neck of the animals (occlusive administration for 48 hours). Two weeks after the epidermal induction application the animals were tested on the flank with 0.3 % test substance in Vaseline (approx. 0.2 g) and the vehicle alone (24 h occlusive application). Twenty four hours after removing the dressings the challenge reactions were graded according the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings. A control group (20 m/20 f) was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound to control the maximal subirritant concentration of the test compound in adjuvant treated animals. Under the experimental conditions employed, 60% to 75% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings. Therefore, under the experimental conditions of this study the test substance is  classified as a strong sensitizer when topically applied to albino guinea pigs.

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

In a Local Lymph Node Assay performed according to OECD guideline 429, CBA mice were treated by topical application with different test item concentrations of 2.5%, 5% and 10% in acetone:olive oil 4:1 once daily for three consecutive days. Five days after the first topical application, all mice were treated with ³H-Methyl Thymidine by intravenous injection. Approximately five hours after treatment the mice were sacrificed, the draining lymph nodes of each group were excised, pooled and the level of incorporated ³HTdR was measured. In this study, SI (stimulation index) of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v), respectively. As a result, the test substance is considered a strong skin sensitizer. An EC3 value could not be derived due to the severe reaction induced even at the lowest doses.

In a GLP-compliant dermal sensitization study with the test item in sesame oil Pirbright White guinea pigs were tested using the guinea pig maximization test according to OECD guideline 406. In this study, the test group animals received 1 % of the test item for intradermal induction as well as 10 % of the test item for epicutaneous induction one week later. For intradermal induction, three pairs of injections (adjuvant/saline; test item in vehicle; test item in vehicle plus adjuvant/saline) were given. Epidermal challenge was performed by occlusive application for 24 h two weeks later (0.3% in vaseline). In 60% - 75% of the animals of the test group skin reactions were observed 24 and 48 hours after the removal of the dressings. Control animals did not show any reactions. Based on this result and under the conditions of this study, the test item is considered a skin sensitizer.

In conclusion, the test item was found to be a strong sensitizer and has to be classified accordingly.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Both the LLNA study and the epicutaneous sensitisation study indicate a potential for strong skin sensitisation. Thererfore classification with Skin Sens Ca 1A is required according to the Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008