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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Buehler Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 December 2015 to 17 March 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The results were confounded by the observation of irritation in the control animals. The absence of re-challenge data, due to failings of the CRO in study conduct, for either of the tests made interpretation of the data difficult because of the variability that was seen in the irritation responses of the control animals and the animals used in the two determinations of the minimal irritant concentration. There were a number of issues with the conduct of this study which resulted in a degree of doubt about the level of sensitisation in the animals, however on balance having reviewed all of the information from all tests, it was considered that the most probable conclusion for this substance was a positive result, Category 1. The study was quality assessed to be Klimisch 2, i.e. result reliable, however with restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
The LLNA is the study of choice for skin sensitisation. As detailed in the OECD 429 guideline, despite the advantages of the LLNA, it should be recognised that there are certain limitations that may necessitate the use of TG 406. Chemical groups such as metal salts, organometal, unsaturated compounds and surfactants have been known to be linked to false positive. The test material is considered to be amphiphilic in nature and therefore would likely have surfactant characteristics, which are known to be falsely positive in the LLNA. Therefore a Guinea Pig Study, OECD 406, was selected to decrease the uncertainty of possible falsely positive effects.

Test material

1
Chemical structure
Reference substance name:
1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]
EC Number:
264-637-8
EC Name:
1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]
Cas Number:
64051-50-9
Molecular formula:
C52H95N5O4
IUPAC Name:
3-octadecyl-1-[2-({2-[(2-{[2-(3-octadecyl-2,5-dioxopyrrolidin-1-yl)ethyl]amino}ethyl)amino]ethyl}amino)ethyl]pyrrolidine-2,5-dione
Test material form:
liquid
Details on test material:
Appearance: Brown liquid
Storage: room temperature, darkness

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 3, 4 or 5 weeks old at the beginning of the main test.
- Weight at study initiation: Between 238 g and 287 g
- Housing: The animals were housed in groups of 2 or 3 in polycarbonate containers. The flooring of the cages was covered with dust-free wood shavings and the top fitted a stainless steel lid containing with a feeding device and drinking device of 500 mL.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Minimum acclimatization period of 5 days, under housing and diet conditions identical to those of the test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3 °C
- Humidity (%): from 30 % to 70 %
- Air changes (per hr): At least 10 cycles per hour.
- Photoperiod (hrs dark / hrs light): Circadian cycle (12 hrs day/ 12 hrs darkness)

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
liquid paraffin
Concentration / amount:
100%
Day(s)/duration:
The test material was left on for 6 hours; 3 local applications were performed on D0, D6 and D13, then the area was washed with liquid paraffin after removal of the dressing.
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
other: an application on either side of the spine under occlusive dressing.
Vehicle:
paraffin oil
Remarks:
liquid paraffin
Concentration / amount:
25% in liquid parrafin (w/w)
Day(s)/duration:
The test material was left on for 6 hours then the area was washed with liquid paraffin after removal of the dressing.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 10 animals
Test material group: 20 animals
Details on study design:
RANGE FINDING TESTS:
Three guinea pigs were treated with the test material placed onto the selected treatment sites and covered with an occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for a period of 6 hours at 4 different concentrations: 100 %, and diluted at 75 %, 50 % and 25 % in liquid paraffin.
All animals received 0.5 mL of the corresponding preparation. Washing of the skin after removal of the dressing was done with liquid paraffin.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. The skin reaction was observed and recorded according to the grades described hereafter.
As irritation was only observed in treated area of 100% which was not in accordance with the pre-test of the 1st test (irritation noted on the treated area of 100 %, 75 % and 50 %), the same three guinea pigs were treated with the same test material concentrations under the same experimental conditions, 10 days after the 1st application and before the challenge of the main test, in order to assure the appropriate Maximum Non Irritant Concentration (MNIC) for use for the challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Three
- Exposure period: 6 hours
- Site: The scapular zone.
- Test groups: Received 0.5 mL of the test item as supplied.
- Control group: Received 0.5 mL liquid paraffin.
- Frequency of applications: Applications were performed on D0, D6 and D13.
- Concentrations: 100 %
- Dressing: Occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M).
- Washing: Washing of skin after removal of the dressing was done with liquid paraffin.
- Rest phase: The animals of both groups were left untreated for 13 days.

B. CHALLENGE EXPOSURE
- No. of exposures: One
- Exposure period: 6 hours
- Test groups: 1 area containing 0.5 mL of the test item diluted at 25 % (MNIC) on the left flank and one area containing 0.5 mL liquid paraffin on the right flank.
- Control group: 1 area containing 0.5 mL of the test item diluted at 25 % (MNIC) on the left flank and one area containing 0.5 mL liquid paraffin on the right flank.
- Site: On the previously shorn dorso-lumbar zone, an application was made on either side of the spine.
- Dressing: Occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M).
- Washing: Washing of skin after removal of the dressing was done with liquid paraffin.
- Evaluation (hr after challenge): Approximately 24 h after removal of the occlusive dressing, the cutaneous reactions were observed and graded. Approximately 24 h later (i.e. 48 h after removal of the occlusive dressing) a second observation was made.

OTHER:
The percentage of animals that showed a sensitivity contact potential is calculated 24 and 48 hours after the removal of the occlusive dressings.
A comparison of the intensity and persistence of reactions at the test material challenge sites in the test and control animals permits identification of sensitisation reactions.
- If the test material at the maximum non-irritant concentration produces reactions in test group animals at the 24 or 48-hour readings, these reactions are attributed to skin sensitisation. This pre-supposes that no similar reactions are observed in the test material challenge sites of any of the control group animals. If irritation is observed in the control group animals, only reactions in the test group animals that exceed the most severe reaction seen in the control group animals are attributed to skin sensitisation. The results are expressed in terms of incidence and severity of responses, i.e.:

Incidence Score: The number of test group animals showing skin reactions greater than the most severe reaction observed in the control group animals, expressed as a fraction of the total number of test group animals.

Severity Score: The sum of the values assigned to the skin responses at the test material challenge sites of the test and control group animals, at each evaluation, divided by the number of animals in that group.

- The animals were weighed at the beginning, before the second induction and at the end of the study.
Challenge controls:
liquid paraffin
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
12
Total no. in group:
20
Clinical observations:
Erythema was noted.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
Erythema was noted.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
25 %
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
Erythema was noted.
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle (liquid parrafin)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No erythema was noted
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle (liquid parrafin)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No erythema was noted
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
vehicle (liquid parrafin)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No erythema was noted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Grade 1 erythema was noted
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Grade 1 erythema was noted
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema was noted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle (liquid parrafin)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema was noted
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle (liquid paraffin)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema was noted
Key result
Reading:
other: 3rd challenge
Hours after challenge:
72
Group:
negative control
Dose level:
vehicle (liquid paraffin)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema was noted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50% alpha-hexylcinnamaldehyde
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
Erythema was noted
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% alpha-hexylcinnamaldehyde
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
Erythema was noted
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 1. Macroscopic Evaluation (Readings at 24, 48 and 72 Hours) of Cutaneous Reactions

Groups

Reading time

Concen-trations

Incidence

 

% of positive responses =1

% of animals

sensitized#

0

1

2

3

Control

Group 1

24

25 %

5

1

0

0

50 %

- 

48

25 %

8

5

0

0

20 %

 

72

25 %

10

2

0

0

0 %

 

24

0 %

10

0

0

0

0 %

 

48

0 %

10

0

0

0

0 %

 

72

0 %

10

0

0

0

0 %

 

Treated

Group 2

24

25 %

2

6

12

0

90 %

60 %

48

25 %

5

12

3

0

75 %

15 %

72

25 %

13

6

1

0

35 %

35 %

24

0 %

20

0

0

0

0 %

0 %

48

0 %

20

0

0

0

0 %

0 %

72

0 %

20

0

0

0

0 %

0 %

# A comparison of the intensiy and persistence of reactions at the test item challenge sites in the test and control animals permits identification of sensitisation reactions.

If the test item at the maximum non-irritant concentration produces reactions in test group animals at the 24 or 48-hour readings, these reactions are attributed to skin sensitisation. This pre-supposes that no similar reactions are observed in the test item challenge sites of any of the control group animals. If irritation is observed in the control group animals, only reactions in the test group animals that exceed the most severe reaction seen in the control group animals are attributed to skin sensitisation. Results are reported as the number of test group animals showing skin reactions greater than the most severe reaction observed in the control group animals, expressed as a percentage of test group animals.

MNIC Determination

In the first study, no cutaneous reactions were noted following application of the test item at concentrations of 50 % and 25 % whereas a discrete erythema was noted only 24 hours after removal of the patches at the tested concentrations of 100 % and 75 %.

In the first preliminary study for the repeated main study, a discrete erythema was recorded in all animals both 24 and 48 hours after removal of the patches only at the tested concentration of 100 %.

Given that the concentration of 50 % caused cutaneous responses in the negative control group after the challenge in the first main study, the same animals were treated with the same test item concentrations under the same experimental conditions, 10 days after the 1st application and before the challenge of the main groups, in order to assure the appropriate MNIC for use for the challenge exposure.

24 hours after removal of the patches, at the tested concentrations of 100 %, 75 % and 50 %, a moderate erythema was noted in all animals (3/3) and no cutaneous reaction was noted at the tested concentration of 25 %.

48 hours after removal of the patches, at the tested concentration of 100 %, a moderate erythema was noted in two animals (2/3) and a discrete erythema in one animal (1/3).

48 hours after removal of the patches, at the tested concentration of 75 %, a moderate erythema was noted in one animal (1/3) and a discrete erythema in two animals (2/3).

48 hours after removal of the patches, at the tested concentration of 50 %, a moderate erythema was noted in one animal (1/3), a discrete erythema in one animal (1/3) and no cutaneous reaction was noted in the last animal.

48 hours after removal of the patches, at the tested concentration of 25 %, no cutaneous reaction was noted in all animals (3/3).

In view of these results, the concentration selected was 100 % for the 3 inductions of the main study and the concentration selected was 25 % (MNIC) for the challenge phase.

 

Main Study

- Induction phase

In the treated group, a moderate erythema was noted in three animals (3/20) and a discrete erythema was noted in seventeen animals (17/20) after the first induction.

 A moderate erythema was noted in ten animals (10/20) and a discrete erythema was noted in ten animals (10/20) after the second induction.

 A moderate erythema was noted in fourteen animals (14/20) and a discrete erythema was noted in six animals (6/20) after the third induction. 

No cutaneous reaction was recorded during the induction phase in the control group.

 

- Challenge phase:

In the treated group (treatment dose of 25 %), a discrete to intense erythema was recorded on the treated area in 90 % (18/20), 75 % (15/20) and 35 % (7/20) of the animals, respectively 24, 48 and 72 hours after the challenge phase.

No cutaneous reaction was recorded in animals from the treated group after the challenge phase, on the treated area with liquid paraffin (control item).

In the control group (associated with the treatment dose of 25 %), a discrete erythema was recorded on the treated area in 50 % (5/10) and 20 % (2/10) of the animals, respectively 24 and 48 hours after the challenge phase.

No cutaneous reaction was recorded in animals from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted on the area challenged with the test item at 25 %, in 60 %, 15 % and 35 % of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, respectively.

The severity of the reactions was 1.5, 0.9 and 0.4 in the treated group versus 0.5, 0.2 and 0 in the control group 24, 48 and 72 hours after the challenge phase, respectively.

 

Weight Evolution

No abnormalities and no differences in the body weight between the control and the treated group were observed.

 

Mortality

No mortality was registered during the main test.

 

Clinical Signs

No abnormal clinical signs related to the administration of the test item were observed.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the conditions of the study, the test material was considered a skin sensitizer.
Executive summary:

A study was conducted on Dunkin-Hartley guinea pigs to asses the skin sensitization potential of the test material. The study was conducted according to OECD 406, EU method B.6 and EPA OPPTS 870.2600 under GLP conditions.

Based on the results of the preliminary study, the concentration selected was 100 % for the 3 inductions of the main study and the concentration selected was 25 % (MNIC) for the challenge phase. 10 female guinea pigs were used for treatment with the negative control (liquid paraffin) and 20 female guinea pigs for the test material. For the induction phase, after shearing of the scapular zone, three local applications were performed on Day 0, Day 6 and Day 13 for 6 hours under occlusive dressing. The animals of the control group received 0.5 mL of liquid paraffin and the animals of the treated group received 0.5 mL of the test material as supplied (i.e. 100 %). Washing of the skin after removal of the dressing was done with liquid paraffin. The animals of both groups were left untreated for 13 days. On the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing, was performed for 6 hours for the challenge phase. The animals were treated with 0.5 mL of the test material diluted at 25 % (MNIC = maximal non-irritant concentration) on the left flank and 0.5 mL of liquid paraffin on the right flank. Washing of the skin after removal of the dressing was done with liquid paraffin. Readings were performed 24, 48 and 72 hours after removal of the patches.

In the treated group (treatment dose of 25 %), a discrete to intense erythema on the treated area was recorded in 90 % (18/20), 75 % (15/20) and 35 % (7/20) of the animals, respectively 24, 48 and 72 hours after the challenge phase.

No cutaneous reaction was recorded in animals from the treated group after the challenge phase, on the area treated with liquid paraffin (vehicle control).

In the control group (associated with the treatment dose of 25 %), a discrete erythema on the treated area was recorded in 50 % (5/10) and 20 % (2/10) of the animals, respectively 24 and 48 hours after the challenge phase. No cutaneous reaction was recorded in animals from the control group after the challenge phase, on the treated area with liquid paraffin (control item). As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted on the area challenged with the test item at 25 %, in 60 %, 15 % and 35 % of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, respectively. The severity of the reactions were 1.5, 0.9 and 0.4 in the treated group versus 0.5, 0.2 and 0 in the control group 24, 48 and 72 hours after the challenge phase, respectively.

Under the conditions of the study, the test material was considered a skin sensitizer.