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Description of key information

Skin sensitisation (OECD TG 406): sensitising

Skin sensitisation (OECD TG 429): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981, June 1 - 1981, July 8
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
The in vivo information is retrieved from a Buehler test which was conducted before the REACH regulation on requesting in vitro information, came into force (October, 2016)
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
yes
Remarks:
The test item is from IFF and therefore according to internal standards. Ten animals are used in the treatment group and 5 in the negative control group in view of the positive result this does not affect the outcome of the study.
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
LLNA method was not available yet by the time the study was conducted. An appropriate Buehler test is available which would not justify conducting an additional LLNA due to animal welfare.
Test material information:
Composition 1
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Perfection Breeders, Inc., Douglasville, Pennsylvania
- Age at Acclimatization Start: No data
- Weight at Acclimatization Start: 290 - 500g
- Housing: Fill: two per cage in stainless steel wire mesh cages
- Diet: free access to Wayne Guinea Pig diet
- Water: free access to tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 20 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: June 1, 1981 To: July 8, 1981
Route:
epicutaneous, occlusive
Vehicle:
other: Ethanol
Concentration / amount:
test substance: 0.4 mL 60%
Day(s)/duration:
3 times/week for 3 weeks (total 9 exposures), 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Ethanol
Concentration / amount:
Test substance: 0.4 mL 60%
Day(s)/duration:
17 days after last induction, 6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test animals: 10
Positive Control animals: 10
Negative Control animals: 5
Details on study design:
RANGE FINDING TESTS
Four unexposed animals were exposed to 0.4 mL 20%, 40%, 60% and 100% concentration of the test substance by patching technique as described for main study. Treated sites were scored at 24 hours. The highest non-irritating concentration was 60%.



MAIN STUDY
A. INDUCTION EXPOSURE
The dorsal area of each animal was clipped free of hair 24 hours pior to the 1st, 4th, 6th and challenge applications of the test material. The shaved area was approx. 5 x 10 cm, i.e. 10% of the body surface. The test area was divided into three application sites, which were dosed on a rotating basis. The test material was applied beneath a 20 x 20 mm Webril pad on a 37 x 40 mm Readi-Bandage, and covered with dental dam held in place with a suitable bandage. Animals were exposed for 6 hours after which the dams and patches were removed. The treated sites were examined after each dosing day and scored at 24 and 48 hours. This procedure was performed three times weekly for three weeks (total 96-hour insults)

- Concentration: 60% in ethanol (control animals: ethanol 95% (negative) or 0.3% DNCB in 80% ethanol (positive))
- Amount: 0.4 mL
- Area: 4 cm2
- Exposure period: 6 hours (occlusive)
- Readings: 24 and 48 hours after patch removal

B. CHALLENGE EXPOSURE (control and test group)
Twenty-four hours after the challenge all animals were depilated with Neet Cream Hair Remover (Whitehalll Laboratories) for no more than 30 minutes after which the depilatory was thoroughly washed off.

- Day of challenge: approximately 17 days after last epidermal induction application
- Concentrations: 60%
- Exposure period: 6 hours (occlusive)
- Sites: site of sensitizing exposure and second naive site
- Amount: 0.4 mL
- Readings: 24 (a minimum of two hours after depilation) and 48 hours after patch removal
Challenge controls:
Control group was treated with 0.4 mLvehicle (Ethanol 95%) during induction phase and challenged with 0.4 mL 60% test substance.
Positive control substance(s):
yes
Remarks:
DNCB
Positive control results:
The results of the positive control animals show that the system is responsive (9/10 and 5/10 animals reacted positive at the induction site and naive site, respectively)
Reading:
1st reading
Hours after challenge:
24
Group:
other: negative control - induction and naive site
Dose level:
60%
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
other: negative control - induction and naive site
Dose level:
60%
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
other: test group - induction site
Dose level:
60%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Animals showed slight or confluent or moderate patchy erythema (mean score 0.9)
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: test group - naive site
Dose level:
60%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Animals showed slight or confluent or moderate patchy erythema (mean score 0.8)
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test group - induction site
Dose level:
60%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Animals showed slight or confluent or moderate patchy erythema (mean score 1.2)
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test group - naive site
Dose level:
60%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Animals showed slight or confluent or moderate patchy erythema (mean score 0.7)

Observations:

Slight edema was present and erythema was observed during induction at 48 hours post dose 3 with test material. In positive control animals erythema was observed during induction at 24 hours post dose 9 and 48 hours post dose 3, 4, and 8. No irritation was observed in the negative control animals during induction or challenge periods.

One control animal was found dead on day 29.

Interpretation of results:
other: Sensitising
Remarks:
According to Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
In a Buehler test performed similar to OECD 406 (1981) and according to GLP principles, the substance is considered a skin sensitiser.
Executive summary:

The skin sentisation potential of the substance was investigated by performing a Buehler test similar to OECD 406 (1981) and according to GLP principles. Ten animals were used in the treatment group and 5 in the negative control group (instead of 20 resp. 10 animals according the guideline). A concentration of 60% was used for the inductions (total of 9) and the challenge. After the inductions, patchy erythema was observed among the animals. After the challenge with the substance 8 of the 10 and 7 of 10 animals showed slight or confluent or moderate patchy erythema (score 1) at the naive site at 24 and 48 hours, respectively. At the induction site slightly more effects were observed. Reliable negative (no response during challenge) and positive controls were included. Based on the positive results, the substance is considered to be a sensitiser under the conditions of the test.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004, August 18 - 2004, August 24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, UK
- Age at study initiation: 6 - 8 weeks (beginning of acclimatization period)
- Weight at study initiation: 15.1 to 18.3 g (beginning of acclimatization period)
- Housing: In groups of four in appropriate cages with environmental enrichment (tents, bases and nestlets).
- Diet: Free access to pelleted RM1 diet (Special Diet Services Limited, Witham, Essex, UK)
- Water: Free access to community tap water
- Acclimation period: Under test conditions, at least 5 days.

ENVIRONMENTAL CONDITIONS (target ranges)
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: 1:3 EtOH:DEP
Concentration:
the test item at concentrations of 1, 2.5, 5, 10 or 25 % w/v in vehicle
No. of animals per dose:
Groups of four mice were treated
Details on study design:
TREATMENT PROCEDURES:
TOPICAL APPLICATION:
Each test group of mice was treated with different test item concentrations of 1, 2.5, 5, 10 or 25% in EtOH:DEP, 1:3 (w/v). The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using a variable volume micropipette.
A further group of four mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µl of the positive control item, α-Hexylcinnamaldehyde, at a concentration of 5, 10 or 25 % w/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration
Three days following the third topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were checked at least once daily for systemic toxicity.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to injection fo 3HTdR).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by inhalation of halothane followed by cervical dislocation. For each individual animal of each group the draining auricular lymph nodes were excised and processed together with the nodes from the other animals in te group.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells was prepared by mechanical disaggregation through a 200 mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase). 3HTdR incorporation was measured by beta scintillation counting using a Packard Tri-Carb Liquid Scintillation Counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistics were performed.

Positive control results:
The positive control item, Hexylcinnamaldehyde, gave a Stimulation Index of 3.3 and 10.9 when tested at a concentration of 10% and 25 % v/v, respectively, in acetone/olive oil 4:1.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentration 1, 2.5, 5, 10 and 25% were 1.0, 1.5, 2.0, 1.5 and 2.3, respectively.
Key result
Parameter:
EC3
Remarks:
%
Value:
> 25

The following results were obtained:

 Concentration of test substance (% w/v)

 Desintegrations per minute (DPM) Dpm per lymph node a)  Test:control ratio
 0 (vehicle only)  3686  461  N/A
 1  3832  479  1.0
 2.5  5171  646  1.4
 5  7383  923  2.0
10   5332  667  1.5
 25  8641  1080  2.3

a) Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured DPM by 8.

EC3: estimated to be greater than 25% w/v (>6250 µg/cm3 )

Body weights

Treatment group (%(w/v))

Animal number

Body weight (g)

Day 1

Day 6

0

1

17.5

19.0

2

18.3

20.9

3

16.1

17.6

4

16.7

18.1

1

5

15.1

16.9

6

16.3

17.3

7

16.9

17.9

8

16.9

17.6

2.5

9

16.0

16.7

10

18.3

18.9

11

17.8

18.1

12

17.2

18.0

5

13

16.1

17.9

14

17.6

18.3

15

16.8

17.8

16

17.3

18.1

10

17

15.9

17.2

18

16.2

17.8

19

16.7

17.9

20

18.1

19.4

25

21

17.8

19.3

22

16.4

18.7

23

16.9

19.0

24

17.0

18.6

Interpretation of results:
other: Not sensitising.
Remarks:
According to Regulation (EC) No. 1272/2008.
Conclusions:
The test item was considered not to be a sensitiser under the conditions of the test.
Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: "Local Lymph Node Assay" method. At 1, 2.5, 5, 10, and 25% the substance showed SI values of 1.0, 1.4, 2.0, 1.5 and 2.3, respectively. EC3 was estimated to be >25% indicating a NOAEL of 25%. Reliable negative and positive controls were included. The test substance was considered not te be a sensitiser under the conditions of the test.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Related information:
Composition 1
Reason / purpose:
data waiving: supporting information
Related information:
Composition 1
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Two skin sensitisation studies are performed one according to the Buehler protocol and one LLNA. The Buehler is considered the key study because the reliability is 1, the study is positive and tested at higher concentrations than the LLNA (60 and 25%, respectively). Both tests are summarised below.

Key information: Skin sensitisation in a Buehler test:

The skin sentisation potential of the substance was investigated by performing a Buehler test similar to OECD 406 (1981) and according to GLP principles. Ten animals were used in the treatment group and 5 in the negative control group (instead of 20 resp. 10 animals according the guideline). A concentration of 60% was used for the inductions (total of 9) and the challenge. After the inductions, patchy erythema was observed among the animals. After the challenge with the substance 8 of the 10 and 7 of 10 animals showed slight or confluent or moderate patchy erythema (score 1) at the naive site at 24 and 48 hours, respectively. At the induction site slightly more effects were observed. Reliable negative (no response during challenge) and positive controls were included. Based on the positive results, the substance is considered to be a sensitiser under the conditions of the test.

Supporting information: Skin sensitisation in an LLNA (OECD TG 429)

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: "Local Lymph Node Assay" method. At 1, 2.5, 5, 10, and 25% the substance showed SI values of 1.0, 1.4, 2.0, 1.5 and 2.3, respectively. EC3 was estimated to be >25%. The test substance was considered not to be a sensitiser under the conditions of the test.

Based on the Buehler study the substance is considered a sensitizer and classified catergory 1B (>15% responding at >20% topical induction dose). Classification in this sub-category is supported by the LLNA test which showed an EC of >25%.

 

Justification for classification or non-classification

The substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008 and GHS.