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Description of key information

OECD TG 429 (GLP): not skin sensitizing;

OECD TG 406 (GLP): not skin sensitizing

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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
When this study was performed, the OECD guideline for the LLNA did not yet exist.
Specific details on test material used for the study:
- Physical state: blue powder
- Analytical purity: > 98 %
- Impurities (identity and concentrations): no data given
- Lot/batch No.: 29122
- Storage condition of test material: room temperature in the dark
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, UK
- Age at study initiation: four to seven weeks
- Weight at study initiation: 399 - 498 g
- Housing: 5 animals per suspended plastic cage with solid floor and sawdust bedding
- Diet: vitamin C enriched guinea pig diet (Harlan Teklad 9600 FD2 SQC), ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +- 3 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hrs dark / 12 hrs light
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D (coconut oil product)
Concentration / amount:
50 % (w/v)
Day(s)/duration:
48h
Adequacy of induction:
highest technically applicable concentration used
Route:
intradermal
Vehicle:
other: Alembicol D (coconut product)
Concentration / amount:
7.5%
Day(s)/duration:
Single injection
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D (coconut oil product)
Concentration / amount:
25 % (w/v)
Day(s)/duration:
24h and 48h
Adequacy of challenge:
other: 50% of the highest technically achievable concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D (coconut oil product)
Concentration / amount:
50%
Day(s)/duration:
24h and 48h
Adequacy of challenge:
other: highest technically achievable concentration
No. of animals per dose:
5 control animals and 10 test animals
Details on study design:
RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify the minimum irritant test substance concentration suitable for the induction phase and the maximum non-irritant concentration by the topical route of administration and a dilution of this for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- Site: 4 x 6 cm interscapular area
- Intradermal induction:
A clipped, 4 x 6 cm area of dorsal skin on the scapular region was prepared. 3 pairs of intradermal injections (0.1 ml/site) were conducted on day 1 into a 2 x 4 cm area within the clipped area. Injectables for the test animals were 1) Freund´s Complete Adjuvant, diluted with an equal volume of water for irrigation, 2) the test material 7.5 % (w/v) in Alembicol D and 3) the test material 7.5 % (w/v) in a 50 : 50 mixture of Freund´s Complete Adjuvant and Alembicol D.
- Epicutaneous induction:
On day 7 the same 4 x 6 cm area was clipped and the site was prepared by gentle rubbing with 0.5 ml per site of 7.5 % (w/w) sodium lauryl sulphate in petrolatum. On day 8, a 2 x 4 cm patch of Whatman No. 3 paper was saturated with ca. 0.4 ml of the test material (50 % in Alembicol D); the patch was placed over the injection sites and covered by an impermeable plastic adhesive tape which was secured by an elastic adhesive bandage, wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for ca. 48 hours.
- Test groups: During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from intradermal injections and topical applications.

B. CHALLENGE EXPOSURE
The control and test animals were challenged topically two weeks after the topical induction application (day 22), using the test material (25 and 50 % w/v in Alembicol D.
Hair were clipped on the left flank of each animal and a 2 x 2 cm patch of Whatman No. 3 paper was saturated with ca. 0.2 ml of the test material. TheWhatman papers containing 50 % w/v in Alembicol D were applied on the anterior site of the flank, the Whatman papers containing 20 % w/v in Alembicol D were applied in a similar manner on the posterior site. The patches were sealed on the flank for 24 hours under impermeable adhesive tape, secured by elastic adhesive bandage, wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The challenged sites were evaluated ca. 24 and 48 h after removal of the patches.

All animals were observed daily for signs of ill health or toxicity. The body weight of each animal was recorded on day 1 of the study and following completion of the study prior to termination.
The dermal reactions resulting from challenge reaction were assessed using a numerical scoring system (Draize scores).
On completion of the study the animals were sacrificed by cervical dislocation.
The sensitivity of the guinea pig strain is checked periodically by the testing laboratory with hexyl cinnamic aldehyde (HCA), a known moderate sensitizer.

Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde (HCA), periodically checkings
Positive control results:
The following concentrations of HCA were administered: Intradermal injection 7.5 % v/v in Alembicol D; topical application as supplied (neat); challenge application as supplied (neat) and 50 % v/v in Alembicol D.
Slight to well defined dermal reactions were observed for all test animals after challenge reaction compared to no dermal reactions in the controls. The reactions in the test animals represented hypersensitivity and therefore all test animals gave positive sensitization responses.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 % (w/v) in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation

No deaths and no signs of ill health or toxicity were observed. The body weight of the animals increased within the normal range over the period of the study.

During the induction period, necrosis was recorded at sites receiving FCA in test animals and also in control animals. No irritation was seen in test animals at sites receiving the test material in vehicle and in control animals receiving vehicle alone after intradermal injection as well as after topical application.

After challenge, no dermal reactions were seen in either animals, all tested animals gave negative responses.

Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Substance type: blue powder
- Analytical purity: >99%
- Lot/batch No.: SF20927
- Storage condition of test material: room temperature in the dark
Species:
mouse
Strain:
other: CBA/CaBkl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: Certified Rat and Mouse Diet, Code 5LF2 (BCM IPS Ltd., London, UK), ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5 %, 10 % or 25 % (w/w)
No. of animals per dose:
four animals per dose
Details on study design:
Mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for 3 consecutive days. The test material was administered by using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of 4 animals received the vehicle alone in the same manner.
Five days following the first topical application of the test material, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR, 80 µCi/ml, specific activity 2.0 µCi/mmol; Amersham Biosciences UK Ltd.), giving a total of 20 µCi to each mouse.
Animals were observed post dose on day 1, twice daily on days 2 and 3 and on a daily basis on days 4, 5 and 6. Any signs of toxicity or ill health were recorded. The body weight of the animals was determined on day 1 prior to dosing, and on day 6, prior to termination.
5 hours following the administration of 3HTdR all animals were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the mice were excised and pooled for each animal group. For each group, 1 ml of PBS was added to the pooled lymph nodes.
A single cell suspension of the pooled lymph node cells was prepared by gentle mechanic disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze and collected into a centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (ca. 190 g) for 10 min. The pellet was resuspended in PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspenden in 3 ml of 5 % Trichloroacetic acid (TCA).
After overnight incubation at 4 °C the precipitates were removed by centrifugation at 2100 rpm (ca. 450 g) for 10 min, resuspended in 1 ml TCA and transferred to 10 ml scintillation fluid (Optiphase, "Trisafe"). 3HTdR incorporation was measured by ß-scintillation counting. The number of radioactive disintegrations per minute was then measured using a Beckman LS6500 scintillation system.

The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). The test material will be regarded as sensitizer, if at least one of the tested concentrations results in threefold greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as non-sensitizer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Value:
1.32
Test group / Remarks:
5%
Parameter:
SI
Value:
1.65
Test group / Remarks:
10%
Parameter:
SI
Value:
1.49
Test group / Remarks:
25%
Cellular proliferation data / Observations:
DPM data:
Test group 1: vehicle (acetone/olive oil 4:1): 683.38 dpm/node
Test group 2: 5 % test substance in vehicle: 899.50 dpm/node
Test group 3: 10 % test substance in vehicle: 1131.00 dpm/node
Test group 4: 25 % test substance in vehicle: 1020.05 dpm/node

There were no deaths. No signs of systemic toxicity was noted in the test or in the control animals during the study. Blue-colored staining on the ears, face, feet and fur was noted in all test animals during the study.

The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period.

A stimulation index of less than 3 was recorded for the three concentrations of the test material.

The positive control alpha-hexylcinnamaldehyde provided positive results and was considered to be a sensitizer under the experimental conditions of the test.

Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
When this study was performed, the LLNA did not yet exist.
Specific details on test material used for the study:
- Lot INCU 002025
- blue powder
- Purity: >95%
- Homogeneity: homogeneous
- Storage conditions: Room temperature
Species:
guinea pig
Strain:
other: Mol:DH (Moellegaard)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: M & B A/S, DK-8680 Ry Denmark
- Microbiological status of animals, when known: no data
- Weight at study initiation: mean 318 g
- Housing: 5 animals per cage
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50 ± 20 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light / dark cycle
Route:
intradermal
Vehicle:
other: sesame oil
Concentration / amount:
5%
Day(s)/duration:
single
Adequacy of induction:
highest technically applicable concentration used
Route:
intradermal
Vehicle:
other: 50 % Freund 's Complete Adjuvant emulsion
Concentration / amount:
5%
Day(s)/duration:
single
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
25 %
Day(s)/duration:
2
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
25%
Day(s)/duration:
24h
Adequacy of challenge:
other: highest technically achievable concentration
No. of animals per dose:
10 (Treatment group)
5 (control group)
3 (range-finder for non-irritant concentration)
Details on study design:
DETERMINATION OF THE PRIMARY NON-IRRITATING CONCENTRATION
Prior to the determination of the primary non-irritation concentration in a dermal-occlusive test three animals each received 4 intradermal injections of a 50% Freund's Complete Adjuvant emulsion (4 x 0.1 mL) into the dorsal area, since Freund' s Complete Adjuvant may lower the threshold of primary irritation. Thereafter, the test substance was administered to the right or left flank of the guinea pigs according to the following procedure:

Table 2: Distribution of the test concentrations (dermal)

Animal No. Left flank Right flank
1 25 % in sesame oil 5 % in sesame oil
2 25 % in sesame oil 1 % in sesame oil
3 5 % in sesame oil 1 % in sesame oil

The hair on the flanks of the animals was removed mechanically. 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema

DETERMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 2 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders.

The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders

Table : Test concentrations
site appl. vol. conc. vehicle
1 2 x 0.1 ml 5.0 % sesame oil
2 2 x 0.1 ml 1.0 % sesame oil
3 2 X 0.1 ml 0.2 % sesame oil

24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.


Intradermal injections with Freund's Adjuvant (with and without test substance) caused clear edema as well as indurations. The administration sites treated with the test substance in sesame oil showed clear edema. Intradermal injections of the vehicle alone exhibited two animals showing slight erythema. Due to the blue color of the test substance the treated skin of the animals could not be assessed for erythema.

Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at day 7.
Challenge controls:
Animals with induction treatment of 50 % Freund's Adjuvants or sesame oil

Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamaldehyde
Positive control results:
After the challenge treatment 9 animals of the treatment group (90 %) showed a positive reaction during the observation period.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none

No skin reactions were observed in the control and the treatment group 24 and 48 hours after removal of the occlusive bandage. Additionally, the treated skin was discolored light blue which did not influence the assessment of skin reactions.

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitization:

A local lymph node assay was conducted according to OECD TG 429 and under GLP conditions (Safepharm 2004). Four female CBA/CaBkl mice per dose (5 %, 10 % or 25 % w/w of the test material in acetone/olive oil 4:1 v/v) were treated by daily application of 25 µl of the appropriate concentration of either the test material or with vehicle alone to the dorsal surface of each ear for 3 consecutive days. Five days following the first topical application, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 20 µCi 3H-methyl thymidine (3HTdR) to each mouse. Animals were observed post dose, any signs of toxicity or ill health were recorded. The body weight of the animals was determined prior to dosing and prior to termination. 5 hours following administration of 3HTdR all animals were killed. The draining auricular lymph nodes were excised, pooled for each group and a single cell suspension of the cells was prepared. 3HTdR incorporation was measured by ß-scintillation counting. The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). There were no early deaths during the study. No signs of systemic toxicity were noted in the test or in the control animals during the study. Blue-coloured staining on the ears, face, feet and fur was noted in all test animals during the study. The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period. A stimulation index of less than 3 was recorded for the three concentrations of the test material, indicating no sensitizing potential of the tested material.

An in vivo study according to OECD TG 406 (GLP) (Huntingdon 2001), with 10 female guinea pigs in the test group and 5 female guinea pigs as control group was conducted. The intradermal induction (6 injections in groups of two per animal) was conducted with FCA, the test material 7.5 % (w/v) in Alembicol D and the test material 7.5 % (w/v) in a mixture of FCA and Alembicol D. Epicutaneous induction (one week after intradermal induction) was conducted with 50 % of the test material in Alembicol D. The challenge (14 days after epicutaneous induction) was performed with 25 % and with 50 % of the test material in Alembicol D. No animal in the control group and no animal in the treated group exhibited positive reactions 24 or 48 hours after the challenge procedure.

The same outcome was observed in a further guinea pig maximisation test (according to OECD TG 406; Aventis 2002).

 


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.