Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin sensitisation (OECD 406): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Jan - 21 Feb 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1992
Deviations:
yes
Remarks:
The challenge sites were evaluated 72 h after respective application in addition to the 24 and 48 h reading time points.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
adopted in 1998
Qualifier:
according to guideline
Guideline:
other: JMAFF, 59 NohSan No. 4200
Version / remarks:
adopted in 1985
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The study was conducted prior to the current requirement in Regulation (EC) 1907/2006 to perform a LLNA study (OECD 429) as the preferred in vivo skin sensitisation study.
Specific details on test material used for the study:
Appearance: white powder
Species:
guinea pig
Strain:
other: Crl:(HA)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 5 - 7 weeks (males and females)
- Weight at study initiation: 362 - 466 g (males and females)
- Housing: individually in suspended, stainless-steel cages with carrot stick as enrichment
- Diet: certified guinea pig diet (#5026, Purina Mills, Inc.), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
10% (w/v) (0.4 mL/site)
Day(s)/duration:
6 h; one application per week for 3 weeks
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
5% (w/v) (0.4 mL/site)
Day(s)/duration:
6 h
Adequacy of challenge:
other: in an irritation screening study a very faint erythema reaction (score of 0.5) was seen at one of the four sites treated with 5% test substance in propylene glycol
No. of animals per dose:
Irritation screening study: 2 per sex
Mean study: 5 per sex (control), 10 per sex (test groups)
Details on study design:
RANGE FINDING TESTS: For the determination of the irritation threshold the test substance was administered epicutaneously to four animals at concentrations of 1, 5, 10 and 25% (w/v) in propylene glycol with each animal receiving all doses. After 6 h the patches and any residual test substance were removed. The observation of dermal reaction at 24, 48 and 72 h after test substance application showed no dermal irritation at 1%, a very faint erythema (score of 0.5) at one of the four sites at 5% and faint (score of 1.0 at two sites) to moderate (score of 2.0 with blanching at one site) at 10%. Moderate (score of 2.0 with blanching at one site) to strong (score of 3.0 with subcutaneous hemorrhaging and necrotic appearing areas at three sites) erythema reactions were observed at the highest concentration (25%). Based on the results of the screening test a concentration of 10% (w/v) was applied in the induction phase and 5% (w/v) was applied in the challenge phase of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: one week
- Test groups: 10% (w/v) test substance in dermal vehicle (propylene glycol)
- Control group: no treatment
- Site: along the dorsal anterior left quadrant
- Frequency of applications: one application per week


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: not specified
- Exposure period: 24 h
- Test groups: 5% (w/v) test substance in dermal vehicle (propylene glycol)
- Control group: 5% (w/v) test substance in dermal vehicle (propylene glycol)
- Site: along the dorsal anteriar right quadrant
- Evaluation (hr after challenge): 24 h

OTHER:
- Clinical signs: The animals were observed for clinical signs daily throughout the study.
- Body weights: The body weights of the animals were recorded before the initial treatment and at termination. Animals of the irritation screening study were weighed only on the day of treatment.
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde (epicutaneous induction: 2.5% (w/v) in ethanol, challenge: 1.0, 2.5 and 5.0% (w/v) in acetone)
Positive control results:
Challenge with alpha-hexylcinnamaldehyde resulted in a positive response in test animals previously sensitised. Very faint to faint erythema reactions (scores of 0.5 - 1.0) were observed in 5/10 test animals at 1.0 and 2.5% (w/v) test substance in acetone. Very faint (score of 0.5 in 2/10 animals), faint (score of 1.0 in 3/10 animals) and moderate (score of 2.0 in 2/10 animals) erythema reactions were observed at 5.0% (w/v). In the control animals no erythema reactions were found following challenge with the test substance in acetone at 1.0 and 2.5%. Very faint erythema reaction was seen in 1/5 control animal at 5% at challenge.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 10%, challenge: 5% (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No clinical signs were observed.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction 10%, challenge 5% (w/v)
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
No clinical signs were observed.
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%, challenge: 5% (w/v)
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
No clinical signs were observed.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction 10% (w/v), challenge: 5% (w/v)
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
No clinical signs were observed.
Remarks on result:
positive indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
Induction: 0%, challenge: 5% (w/v)
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
No clinical signs were observed.
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
Induction: 10% (w/v), challenge: 5% (w/v)
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
No clinical signs were observed.
Remarks on result:
positive indication of skin sensitisation

Table 1: Individual Dermal Reactions – Challenge Phase

Animal number

Test substance (5% (w/v) mixture)

Vehicle

24 h

48 h

72 h

24 h

48 h

72 h

Test group

1

0.5

1.0

1.0

0.0

0.0

0.0

2

0.5

1.0

1.0

0.0

0.0

0.0

3

1.0

1.0

1.0

0.0

0.0

0.0

4

0.0

0.5

0.5

0.0

0.0

0.0

5

1.0

1.0

1.0

0.0

0.0

0.0

6

0.5

1.0

1.0

0.0

0.0

0.0

7

0.0

0.0

0.5

0.0

0.0

0.0

8

0.5

1.0

1.0

0.0

0.0

0.0

9

2.0a

1.0

1.0

0.0

0.0

0.0

10

1.0

1.0

1.0

0.0

0.0

0.0

11

0.0

0.5

0.5

0.0

0.0

0.0

12

1.0

0.5

0.5

0.0

0.0

0.0

13

0.0

0.5

1.0

0.0

0.0

0.0

14

1.0

1.0

0.5

0.0

0.0

0.0

15

0.0

0.0

0.0

0.0

0.0

0.0

16

1.0

1.0

1.0

0.0

0.0

0.0

17

1.0

1.0

1.0d

0.0

0.0

0.0

18

1.0

1.0

1.0

0.0

0.0

0.0

19

1.0

2.0

2.0f

0.0

0.0

0.0

20

0.5

1.0

1.0

0.0

0.0

0.0

 

Control group

1

0.0

0.0

0.0

0.0

0.0

0.0

2

0.0

0.0

0.0

0.0

0.0

0.0

3

0.0

0.0

0.0

0.0

0.0

0.0

4

0.0

0.5

1.0

0.0

0.0

0.0

5

0.0

0.5

0.5

0.0

0.0

0.0

6

0.0

0.0

0.0

0.0

0.0

0.0

7

0.0

0.0

0.0

0.0

0.0

0.0

8

0.0

0.0

0.0

0.0

0.0

0.0

9

0.0

0.0

0.0

0.0

0.0

0.0

10

0.0

0.0

0.0

0.0

0.0

0.0

Score 0.0 = No reaction

Score 0.5 = Very faint erythema, usually nonconfluent

Score 1.0 = Faint erythema, usually confluent

Score 2.0 = Moderate erythema

a = Subcutaneous hemorrhaging

d = desquamation

f = fissuring

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
CLP: Skin sens. 1, H317
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Reliable data regarding skin sensitisation are available for the test substance. Therefrom, the test conducted and reported by Glaza 2001 is considered the most suitable as key study. A study for respiratory sensitisation potential is not available.

 

With respect to the key study, the test was done according to the OECD Guideline 406 (Buehler test; Glaza, 2001). Skin sensitisation was assessed in guinea pigs using 10% solutions of the test substance for the 3 induction exposures and a 5% solution for the challenge exposure. Groups of 20 (test group) and 10 (naïve controls) animals were used in the main study. The induction exposures were conducted in weekly intervals and the challenge treatment was applied 2 weeks after the last induction treatment. Skin reactions to the challenge treatment were scored at 24, 48, and 72 h after the end of the 6-h challenge. For positive control, the study report refers to a reliability check using alpha-hexylcinnamaldehyde. The reliability check of the applied method and strain with alpha-hexyl cinnamaldehyde clearly detected the skin sensitizing property of this positive control substance. With respect to the test substance, moderate to strong erythema reactions were observed at the sites treated with the 10% w/v mixture during the induction phase. Subcutaneous haemorrhaging, blanching, and/or necrotic appearing areas were also observed within the induction sites of all test animals. After the challenge with the 5% test substance mixture, 19 out of 20 of the test compound group showed very faint to moderate redness. Subcutaneous haemorrhaging, desquamation or fissuring was also observed within the challenge sites of 3 test animals. Very faint to faint erythema was observed in 2 of the 10 naïve control animals after challenge. The test substance showed a skin sensitizing potential in the Buehler test on guinea pigs. Thus, under the conditions of the study, a classification as Skin Sensitising Category 1 according to Regulation (EC) No 1272/2008 is triggered.

 

In a further study the potential of the test substance to induce skin sensitization was investigated using the Buehler test according to OECD Guideline 406 and in compliance with GLP (Vohr, 2002). A group of 20 female guinea pigs were exposed topically to 50% formulations of the test substance in polyethylene glycol during induction as well as challenge phase. Further 10 animals served as control group and were treated with the vehicle. Three induction exposures were performed in a weekly interval for 6 h and the challenge exposure was performed 4 weeks after the first dermal induction for 6 h. Skin reactions to the challenge treatment were scored 30 and 54 h after start of exposure. The test was checked for reliability using alpha-hexylcinnamaldehyde. The check clearly confirmed the sensitivity as well as the reliability of the experimental technique. With respect to the test substance, skin effects (grade 1) was observed in 7/20 animals of the treated group after the 3rdinduction. The challenge exposure caused skin effects (grade 1 – 2) in 9/20 animals (45%). No skin reactions were observed in the control group after induction or challenge exposure. Thus, under the conditions of the study, the test substance showed a skin sensitizing potential in the Buehler test on guinea pigs.

 

Another test for the skin sensitizing potential of the test substance according to the Buehler method was performed according to OECD Guideline 406 and in compliance with GLP (Vegarra, 2005). Deviating from the Guideline, the treatment and the control group consisted of only 10 male guinea pigs whereas the guideline recommends a minimum of 20 animals. In terms of induction, the animals received 0.50% of the test substance formulated in 80% ethanol/water solution topically for 6 h once a week for 3 consecutive weeks. The control animals were not treated during the induction phase. Two weeks after the 3rd induction administration a challenge dose of the test substance of 0.25% in acetone was topically applied to all animals. For reliability check the positive control alpha-hexylcinnamaldehyde was used. Challenge with alpha-hexylcinnamaldehyde resulted in a positive response in test animals previously sensitised. At the end of the induction phase very faint to faint erythema were observed at the application sites of animals treated with the test substance. After the challenge phase the test animals exhibited no (60%) or very faint (40%, grade 0.5) erythema at 24 and 48 h post-application. In control animals treated with the test substance during challenge phase dermal reactions (grade 0.5) were observed in 20% animals at 24 h and in 10% animals at 48 h after administration. Thus, under the conditions of this study, the test substance did not show a skin sensitizing potential.

 

In addition to the Buehler assays, the test substance was assessed for a potential sensitising effect on skin of female guinea pigs in an open epicutaneous test according to Klecak (Schmidt, 1986). Three treatment groups consisted of 8 animals each which were exposed to different test substance concentrations. Eight further animals served as control groups (one control group for the 1st and one control group for the 2nd challenge). During the induction phase of 4 weeks animals were dermally exposed to 1, 3 and 10% formulations of the test substance in 1,2-propanediol on 20 days. Exposure concentration of 10% was reduced to 3% after 5 treatments due to severe skin reactions. Control animals were not treated during induction phase. Formulations of 0.3, 1 and 3% test substance in 1,2-propanediol were applied epicutaneously at the end of the induction phase to the skin of all animals of the treatment group and one control group in terms of challenge exposure. Two weeks thereafter a second challenge phase (rechallenge) was conducted in the same way as the first induction on all animals of the treatment group and the second control group. All animals exposed to 3% during the induction phase showed skin reactions (grade 0.5 or 1) from the 4thtreatment up to the end of treatment. In animals exposed to 1% during the induction phase less pronounced skin reactions were observed at individual time points only. During the first challenge 2/8 animals treated with 3% of the test substance during induction showed erythemas of grade 0.5 and 1 to the 1 and 3% challenge formulations after 24 h. 1/8 animal treated with 1% during induction showed erythema of grade 0.5 to the 1% challenge formulation at the same time. During rechallenge, the 1% challenge formulation induced skin reactions in 1/8 animal initially treated with 10% during induction and in 1/8 animal treated with 3% during induction (both grade 0.5). The 3% challenge formulation induced skin reactions in 2/8 animals induced with 3% test substance and in 1/8 animal induced with 1% test substance formulation. Thus, under the conditions of this study, the test substance was not considered skin sensitizing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation meet the criteria for classification according to Regulation (EC) 1272/2008. The test substance is classified as Skin Sensitising Category 1, H317: May cause an allergic skin reaction.

In fact, this is further in line with the conclusion of the ECHA Committee for Risk Assessment (RAC) Opinion for harmonised EU classification and labelling of Chlorophene based on the same reliable data, which was adopted in 2015 ( RAC CLH-O-0000001412-86-58/F, 12 March 2015). As a result, and referring to the Adaptation to Technical Progress (ATP) to CLP Regulation, Chlorophene was inserted in ATP10 with following classification and labelling for skin sensitisation: Skin sens. 1, H317.