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Description of key information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Kisslegg, Germany
- Age at study initiation: 5 to 6 weeks; 7 to 8 weeks control group II
- Weight at study initiation: 349 to 380 g control group I and test group; 417 to 446 g control group II
- Housing: Individually in macrolon cages (type 4) with standard softwood bedding
- Diet: Pelleted standard Provimi Kliba 3418 guinea pig breeding/ maintenance diet (batch no. 74/05; provided by Provimi Kliba AG, CH-4303 Kaiseraugst); ad libitum
- Water: Community tap water from Füllinsdorf, ad libitum
- Acclimation period: Thirteen days under laboratory conditions after health examination for the control group I and test group. No acclimatization for the animals of the pretests and control group II

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
According to the findings of the intra- and epidermal pretests, the concentrations for the main study were selected.

3 % for the intradermal induction
50 % for the epidermal induction
3 % in PEG 300 for the first challenge
1 % and 0.3 % in PEG 300 for the second challenge
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
According to the findings of the intra- and epidermal pretests, the concentrations for the main study were selected.

3 % for the intradermal induction
50 % for the epidermal induction
3 % in PEG 300 for the first challenge
1 % and 0.3 % in PEG 300 for the second challenge
No. of animals per dose:
Intradermal Pretest: 1
Epidermal Pretest: 2
Control Group I: 5
Test Group: 10
Epidermal Pretest II: 2
Control Group II: 5
Details on study design:
A. INDUCTION EXPOSURE
Intradermal induction, performed on test day 1:

An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair.

- Exposure period: 48 hours for the epidermal induction (occlusive dressing)

- Test group:
Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item at 3 % in PEG 300.
3) The test item at 3 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Control group:
Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) PEG 300
3) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal application, performed on test day 8:

An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair.

- a 2 x 4 cm patch of filter paper was saturated with the test item 50 % in PEG 300 and placed over the injection sites of the test animals. ( The amount of test item preparation applied for the second administration was approximately 0.3 g). The patches were covered by occlusive dressings and left for 48 h.
- the guinea pigs of the control group I were treated as described above with PEG 300 only, applied at a volume of approximately 0.3 mL.
- Evaluation: The reaction sites were assessed approximately 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman:

0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling)

B. CHALLENGE EXPOSURE
- No. of exposures: 2

- Day of first challenge: 22
- Exposure period: 24 hours (occlusive dressing)
- Test groups: test item in PEG 300
- Control group I: test item in PEG 300
- Concentration: 3 % test item in PEG 300;
- Site: left side (at the right site a saturated filter paper with PEG only was applied)

- Day of second challenge: 36
- Exposure period: 24 hours (occlusive dressing)
- Test groups: test item in PEG 300
- Control group II: test item in PEG 300
- Concentrations: 1 % and 0.3 % test item in PEG 300
- Site: on the right cranial and caudal flank respectively

- Evaluation (hr after challenge): 24, 48 (approximately 48 and 72 hours from the start of the challenge application respectively)
Challenge controls:
During the second challenge, which was performed in the same way as the previous challenge, five naïve control animals (control group II) were included into the test.
Positive control substance(s):
yes
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE
Positive control results:

Summary of results:
- first challenge with ALPHA-HEXYLCINNAMALDEHYDE at 0.1 % (w/w) in PEG 300: 1/10 test animals showed discrete/patchy erythema at the 24- and 48-hour reading after the treatment. No skin effect was observed in the control group.
- second challenge treatment with ALPHA¬HEXYLCINNAMALDEHYDE at 3 % (w/w) in PEG 300.: 10/10 animals showed discrete/patchy to moderate/confluent erythema with/without scales or oedema at the 24- and 48-hour reading. Discrete/patchy erythema was observed in 1/5 control animals at the 24-hour reading when treated under the same conditions.
- second challenge treatment with ALPHA¬HEXYLCINNAMALDEHYDE at 1 % (w/w) in PEG 300: 7/10 test animals showed discrete/patchy to moderate/confluent erythema at the 24-hour reading and in 6/10 test animals discrete/patchy erythema with/without scales at the 48-hour reading were noted. No skin effect was observed in the control group.
- No toxic signs were evident in the guinea pigs of the control or test group.
- No deaths occurred.

Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea pigs ALPHA-HEXYLCINNAMAL-DEHYDE was considered to be a skin sensitizer.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
3 % in PEG 300
No. with + reactions:
7
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 3 % in PEG 300. No with. + reactions: 7.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
PEG 300 only
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: PEG 300 only. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: control group I
Dose level:
3 % in PEG 300
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group:
Reading:
1st reading
Hours after challenge:
24
Group:
other: control group I
Dose level:
PEG 300 only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group:
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
3 % in PEG 300
No. with + reactions:
3
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 3 % in PEG 300. No with. + reactions: 3.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
PEG 300 only
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: PEG 300 only. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control group I
Dose level:
3 % in PEG 300
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group:
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control group I
Dose level:
PEG 300 only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group:
Reading:
rechallenge
Hours after challenge:
24
Group:
other: control group II
Dose level:
1 % in PEG 300
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group:
Reading:
rechallenge
Hours after challenge:
24
Group:
other: control group II
Dose level:
0.3 % in PEG 300
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group:
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
1 % in PEG 300
No. with + reactions:
2
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 1 % in PEG 300. No with. + reactions: 2.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
0.3 % in PEG 300
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 0.3 % in PEG 300. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.
Reading:
rechallenge
Hours after challenge:
48
Group:
other: control group II
Dose level:
1 % in PEG 300
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group:
Reading:
rechallenge
Hours after challenge:
48
Group:
other: control group II
Dose level:
0.3 % in PEG 300
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of clinical toxicity
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group:
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
1 % in PEG 300
No. with + reactions:
2
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 1 % in PEG 300. No with. + reactions: 2.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0.3 % in PEG 300
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 0.3 % in PEG 300. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: spontaneous death of 1 animal, no signs of clinical toxicity in surviving animals.

Table 1: pretest intradermal injection

Animal No

Concentration [%]

Reaction Reading after 24 h

737

5

2

3

1

1

1

Table 2: epidermal pretest I

Animal No

Concentration [%]

Reaction Readings after removal of bandage

 

 

24 h

48 h

738

50

1

1

25

1

1

15

1

1

10

1

1

739

50

1

1

25

1

1

15

1

1

10

1

1

Table 3: epidermal pretest II

Animal No

Concentration [%]

Reaction Readings after removal of bandage

 

 

24 h

48 h

755

5

0

0

3

0

0

1

0

0

0.3

0

0

756

5

1

0

3

0

0

1

0

0

0.3

0

0

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the above mentioned findings in an adjuvant sensitization test in guinea pigs the test item is not considered to be sensitising to skin.
Executive summary:

Testing for sensitising properties of the test item was performed in female guinea pigs according to the adjuvant sensitisation test by Magnusson and Kligman (Guinea pig Maximisation Test). Intradermal induction was performed using 3 % test item in PEG 300 or in a mixture of Freund's Adjuvant and physiological saline (1:1 (v/v)). Dermal induction was carried out with 50 % test item in PEG 300. Using 3 % test item in PEG 300 for the first challenge treatment resulted in a high incidence of animals with skin reactions (with either erythema scores of 1 or 2) in both the test group as well as the control group.

Therefore a second challenge was conducted using either 1 % or 0.3 % test item in PEG 300. This second challenge included also a second control group consisting of 5 naive animals. At the 24 hour reading (1 % test item in PEG 300) ) 2 out of 5 control group II animals showed skin reactions, while 2 out of 9 test group animals showed positive results. Skin reactions were less severe than in the first challenge (only erythema score 1). At the 48 hour reading all effects in the control groups were vanished and persisted only in 2 out of 9 test animals. No skin reactions were observed after application of 0.3 % test item in PEG 300.

The incidence of animals with skin reactions for the test group and control groups were:

78 % and 100 % at a challenge concentration of 3 %,

22 % and 40 % at a challenge concentration of 1 % and

0 % and 0 % at a challenge concentration of 0.3 %.

The positive control from the test lab showed distinctive positive results under the conditions applied in the lab.

Taken the results together (comparable effects in control as well as test animals; valid positive control) the overall conclusion is that the test item is not sensitising.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
Pigment Red 254 was found to be not a skin sensitiser in the GPMT (OECD 406)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Migrated from Short description of key information:
Based on available data the substance is considered not to exert any sensitizing effects on the respiratory tract; when aerosolized in respirable form, the substance is considered likely to behave like an inert dust. Therefore, it is concluded that classification is not warrantable.

Justification for classification or non-classification

Pigment Red 254 did not show skin sensitising properties in a Guinea Pigs, Maximization-Test according to OECD TG 406. Likewise, allergic skin reactions or case reports of acute contact dermatitis caused by Pigment Red 254 are not published. Therefore, Pigment Red 254 does not have to be classified as a skin sensitizer according to the criteria laid down in Directive 67/548/EEC and Regulation (EC) No 1272/2008.

 

It can reasonably be deduced that Pigment Red 254 does not cause respiratory tract sensitization and thus does not have to be classified according to the criteria laid down in Directive 67/548/EEC and Regulation (EC) No 1272/2008, because

- Pigment Red 254 did not cause skin sensitization, and

- it is unlikely that Pigment Red 254 can interact with the immune system due to its extremely low solubility in water and n-octanol.