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Description of key information

Under the conditions of the study, the test item 2 -hexyldecanoic acid produced by either the old or new manufacturing process is considered to be a weak skin sensitiser in mice.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April - July 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 17th July 1992
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Test started before LLNA method was adopted.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 323 - 456 g
- Housing: Housed during acclimatisation period in groups of up to 10 animals in stainless steel cages (87x71x24cm) with a grid floor. During the study housed in groups up to 5 animals in stainless steel cages (48x63x41cm) with a grid floor. Cages were suspended over metal trays which held an absorbent material, this was inspected daily and changed as necessary.
- Diet: commercially available laboratory diet (Altromin MSK, Altromin, Lage, Germany) ad libitum
- Water: drinking water ad libitum supplied to each cage via a water bottle
- Acclimation period: at least 5 days
- Indication of any skin lesions: nothing mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 2002-05-27 To: 2002-07-26
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
6 hours topical exposure, 3x at weekly intervalls (on days 1, 8-9, 15-16)
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
other: Acetone
Concentration / amount:
50%
Day(s)/duration:
for 6 hours on day 29
Adequacy of challenge:
other: A lower concentration than in the induction phase used was selected for use at challenge, being judged non-irritant
No. of animals per dose:
10 in the control group, 20 in the test group
Details on study design:
RANGE FINDING TESTS:
The flanks of five animals were clipped free of hair. Each animal was dosed with 2 concentrations of the test item, 1 on either flank. A total of 5 concentrations (100%, 50%, 20%, 10% and 5%) of the test item in the selected vehicle (80% ethanol/water) were each dosed in duplicate. A gauze patch (20x20 mm) was soaked with 0.2 ml of the selected concentration of the test item. This was placed onto the selected treatment site. When both sites of the animal had been treated, they were secured in position by wrapping the trunk with adhesive strapping. 24 and 48 hours after removal of dressings, the treated sites were examined for signs of reaction to treament following the below mentioned scoring scheme.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours
- Test groups: 0.4 ml undiluted test substance on a gauze patch (20x20 mm) was placed onto the selected skin site, secured in position by encircling the trunk of the animal with adhesive strapping, after patch removal treated sites were cleaned of remaining test item by washing with warm water
- Control group: similarly treated with the selected vehicle (80% ethanol/water)
- Site: left flank, clipped free of hair
- Frequency of applications: weekly intervalls
- Duration: Day 1 to 16
- Concentrations: 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 29
- Exposure period: 6 hours
- Test groups: 0.2 ml of the test item in acetone spread evenly over an absorbant patch (20x20 mm), placed onto the skin of the posterior region of the prepared site. A similar patch, containing the vehicle alone (acetone) was placed onto the anterior region of the prepared site. The patches were secured in position by encircling the trunk of the animal with adhesive strapping. After patch removal treated sites were cleaned of remaining test item by washing with warm water.
- Control group: Same treatment as test group animals, treated with both the test item and vehicle in the same manner
- Site: right flank (clipped free of hair)
- Concentrations: 50%
- Evaluation (hr after challenge): 24 hours and 48 hours after patch removal (21 hours after patch removal, the treated sites were again clipped free of hairs)

OTHER:
The degree of skin reaction was scored according to the following scheme:
No visible change: 0
Discrete or patchy erythema: 1
Moderate and confluent erythema: 2
Intense erythema and swelling: 3
Positive control substance(s):
yes
Remarks:
tested separately as reliability check in the laboratory (RTC study number: 8231-004INT)
Positive control results:
Positive control substance: α-Hexylcinnamalaldehyde
Results: 63% response in test group and 0% response in control group at second challenge (70% α-Hexylcinnamalaldehyde in DMSO at induction, 14% in acetone at challenge).
Dates of performance of positive control test: Inductions at 24 June 2002, 1 July 2002, and 8 July 2002. Challenge at 22 July 2002.
Interpretation of positive control result: Incidence at challenge acceptable, test system regarded as valid.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no other toxic effects observed, body weight changes similar in test and control goup animals
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
no other toxic effects observed, body weight changes similar in test and control goup animals
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no other toxic effects observed, body weight changes similar in test and control goup animals
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no other toxic effects observed, body weight changes similar in test and control goup animals
Remarks on result:
no indication of skin sensitisation

Preliminary screen: No reaction was apparent in any animals suggesting that the undiluted test item was reasonably tolerated. This concentration was selected for use during the induction phases of the main study. A lower concentration of 50% in acetone was selected for use at challenge, being judged non-irritant.

Induction: No response was seen to either the test item or the vehicle alone in animals of the test and control groups following 6 hours topical exposure.

Challenge: At challenge, a discrete erythema was observed in 5 of the 20 animals of the test group 48 hours following 6 hours topical exposure. No reaction to the test item was observed in test group animals 24 hours following topical exposure or in control group animals. No reaction was obsered to the vehicle alone. Results were summarised as follows:

 Group  Treatment  Incidence of response at challenge   
     24 Hours  48 Hours
 Control  Test item  0%  0%
   Vehicle  0%  0%
 Test  Test item  0%  25%
   Vehicle 0% 0% 
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The results of this study indicate that the test item may elicit a sensitisation response in the guinea pig, there being an evident reaction (25% of test group animals) at challenge following a period of induction exposure to the test item.
Executive summary:

The potential of the test item to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model. The procedures used were those of the Buehler test for skin sensitisation. These methods meet the requirements of OECD guidelin no. 406, adopted 17th July 1992. The study was performed in accordance with the principles of Good Laboratory Practice (GLP).

The concentrations of the test item used in the main study were determined by the results of a preliminary screening test. The main sensitisation test was undertaken using a test group of 20 animals and a control group of 10 animals. In an attempt to induce sensitisation, test animals were treated by topical application of the undiluted test item. This was repeated at weekly intervals for a total of 3 weeks. Animals of the control group were treated in the same manner but the vehicle alone (80% ethanol/water) was used in place of the test item. Two weeks after the third and final induction exposure, animals of the test and control groups were challenged by topical application of both the test item at 50% concentration in acetone and the vehicle alone. At challenge no response was observed to the test item in either test or control group animals at the 24 hour examination. Moderate reaction was observed in 5 of the 20 animals of the test group at 48 hours following 6 hours topical exposure. No reaction was observed to the vehicle alone. These results indicate that the test item may elicit a sensitisation response in the guinea pig, there being an evident reaction at challenge following a period of induction exposure to the substance.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP comnpliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
from April 2002
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 16 - 18 g
- Housing: singly (dose range only) or pairs in suspended cages (dimensions 36.5 x 20.7 x 14 cm) with stainless steel grid tops and solid bottoms, nesting material (Nestlets, supplied by Datesand Limited, UK) was provided, wood shavings were used as bedding (a certificate of the wood shavings used in the study is available in the study report)
- Diet: Rat and Mouse No. 1 Maintenance Diet, supplied by Special Diets Services Limited, UK, available ad libitum (each batch of diet was analysed for major nutritive constituents and significant contaminants, the certificate for the batch used is available in the study report)
- Water: Water from domestic mains supply available ad libitum (the water used is analysed by the local water authority for dissolved items, heavy metals, pesticide residues, pH, nitrates and nitrites, certificate for the analysis conducted most recently before commencement of the study is available in the study report)
- Acclimation period: at least 8 days
- Indication of any skin lesions: nothing mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 50 (mean value)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Prescreen test: 50, 100%
Main test: 25, 50 and 100%
No. of animals per dose:
Prescreen test: 1
Main test: 4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: soluble in vehicle
- Irritation: no findings at concentrations of 50% in acetone/olive oil (4:1 v/v) and in 100% (undiluted test substance)
- Systemic toxicity: no abnormalities detected
- Ear thickness measurements: not performed
- Erythema scores: no findings, no scores mentioned

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation index (SI) ≥ 3, together with consideration of dose-response and, where appropriate statistical significance

TREATMENT PREPARATION AND ADMINISTRATION:
Prescreen test: Dose levels were selected based on existing data from similar products from the sponsor previously tested at the test laboratory (Inveresk project Nos. 505909 and 505956). 4 animals were allocated and treated as follows: one animal each receiving 50% and 100% test item (new), and 50% and 100% test item (old). The animals received an open application for above 3 consecutive days (25 µl each ear), reactions were then recorded using the following scoring scheme:
No eythema: 0
Very slight erythema: 1
Well defined erythema: 2
Moderate to severe erythema: 3
severe erythema (beet redness) to slight eschar formation (injuries in depth): 4

Main test: 4 test animals per dose group received the following concentrations: 0 (actone/olive oil (4:1 v/v) only as vehicle control), 25% , 50%, 100% test items (old and new) each. All animals received open applications of 25 µl of the appropiate formulation for 3 consecutive days onto the dorsum of each ear. There were no treatment on days 4 and 5. On day 6 each animal received an intravenous injection of 250 µl of phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine into the lateral tail vein. Approxymately 5 h after intravenous administration the animals were killed by exposure to carbon dioxide and exsanguinated. Each pair of draining auricular lymph nodes was collected from each animal. A single cell suspension of lymph node cells from each group was prepared by gentle mechanical desegration through 200 µm mesh stainless steel gauze. The lymph node cells were washed twice in an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at approximately 4°C for 18 h. After removal of excess TCA the pellets were resuspended and solubilised in 200 µl soluene. Scintillation fluid (10 ml) was added and incorporation of tritiated thymidine measured by ß-scintillation counting as disintegrations per minute (DPM).
- Observations: All animals were checked for viability early in the morning and again as late as possible on each day.
- Clinical observations: All main study animals were examined for reaction to treatment (at least 3 times) on each day of dosing, and once daily thereafter. The onset, intensity and duration of any signs were recorded. Assessments of the application sites during the main test were not considered to be necessary.
- Body weights: The body weight of each individual animal was recorded immediately before dosing (day 1) and on day 4 (prescreen) or day 6 (main study).
- Calculation of results: Reported DPM were corrected for background radiation. Results were expressed as the Stimulation Index (SI), this was obtained by dividing the DPM within each test item group by the DPM of the vehicle control group. The SI for the vehicle control group is 1.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
Hexyl cinnamic aldehyde (HCA) was tested May and August 2003 in the test laboratory as validation of the Local Lymph Node Assay (Project Nos. 996459 and 996621). The studies were designed to be compliant with OECD Guideline No. 429, Skin Sensitisation: Local Lymph Node Assay, adopted April 2002. Four groups of 5 animals were used, 3 test groups and 1 control group in each study. During the induction exposure the test groups were exposed to 10, 20 or 40% HCA, and the control group was exposed only to the vehicle, acetone/olive oil (4:1 v/v). Stimulation Indices in the study completed in May 2003 were 1.9, 4.3, and 11.7 and in August 2003 were 1.7, 1.9, and 6.3. Under the conditions of these studies, HCA is considered to be a sensitiser in CBA/Ca mice, thus providing evidence that the procedure employed at the test laboratory were valid.
Parameter:
SI
Value:
1.1
Test group / Remarks:
25 % test substance (new)
Parameter:
SI
Value:
2.4
Test group / Remarks:
50 % test substance (new)
Key result
Parameter:
SI
Value:
6.4
Test group / Remarks:
100% test substance (new)
Key result
Parameter:
SI
Value:
7
Test group / Remarks:
100% test substance (old)
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION:
The Stimulation Indices, when compared with the vehicle control were calculated to be 1.1, 2.4, and 6.4 for 25%, 50% or 100% test item (new), respectively.
The Stimulation Indices, when compared with the vehicle control were calculated to be 1.8, 1.8, and 7.0 for 25%, 50% or 100% test item (old), respectively.

EC3 CALCULATION :
no EC3 value calculated

CLINICAL OBSERVATIONS:
no adverse clinical signs were noted during the observation period.

BODY WEIGHTS:
Body weight gain was considered satisfactory.

 Treatment  Dose level (%)  Disintegrations per minute (DPM)  Stimulation Indix (SI)
 Acetone/Olive oil (4:1 v/v)  0  2724  1
 Test item (new)  25  2902  1.1
   50  6403  2.4
   100  17515  6.4
 Test item (old)  25  4840  1.8
   50  4928  1.8
   100  19137  7.0
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the study, the test item produced by either the old or new manufacturing process is considered to be a weak skin sensitiser in mice.
Executive summary:

Using CBA/Ca mice this study investigated the delayed contact hypersensitivity potential of the test item, made using 2 separate production processes. The study was conducted based on OECD Guideline No. 429, Skin Sensitisation, Local Lymph Node Assay, April 2002 and in compliance with the principles of Good Laboratory Practice (GLP).

After dose ranging, 6 groups of 4 female mice received open applications of either test item (new) or test item (old) at concentrations of 25, 50 or 100%, another group of 4 females received only the vehicle, acetone/olive oil (4:1 v/v). Treatment was for 3 consecutive days. Three days later each animal received an intravenous injection of 3H methyl thymidine, and 5 h later the draining lymph nodes were collected and the incorporation of tritiated thymidine assessed by scintillation counting. The Stimulation Indices, when compared with the control group, were calculated to be 1.1, 2.4, and 6.4 for 25%, 50% , and 100% test item (new), respectively. The Stimulation Indices, when compared with the control group, were calculated to be 1.8, 1.8, and 7.0 for 25%, 50% , and 100% test item (old), respectively.

Under the conditions of the study, the test item produced by either the old or new manufacturing process is considered to be a weak skin sensitiser in mice.

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

Using CBA/Ca mice this study investigated the delayed contact hypersensitivity potential of the test item, made using 2 separate production processes. The study was conducted based on OECD Guideline No. 429, Skin Sensitisation, Local Lymph Node Assay, April 2002 and in compliance with the principles of Good Laboratory Practice (GLP).

After dose ranging, 6 groups of 4 female mice received open applications of either test item (new) or test item (old) at concentrations of 25, 50 or 100%, another group of 4 females received only the vehicle, acetone/olive oil (4:1 v/v). Treatment was for 3 consecutive days. Three days later each animal received an intravenous injection of 3H methyl thymidine, and 5 h later the draining lymph nodes were collected and the incorporation of tritiated thymidine assessed by scintillation counting. The Stimulation Indices, when compared with the control group, were calculated to be 1.1, 2.4, and 6.4 for 25%, 50% , and 100% test item (new), respectively. The Stimulation Indices, when compared with the control group, were calculated to be 1.8, 1.8, and 7.0 for 25%, 50% , and 100% test item (old), respectively.

Under the conditions of the study, the test item produced by either the old or new manufacturing process is considered to be a weak skin sensitiser in mice.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the classification criteria of regulation (EC) 1272/2008 2 -hexyldecanoic acid has to be classified as skin sensitizer (H317)..