Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 October 2012 to 23 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
other: FIFRA PR86-5 Compliant
Deviations:
not specified
Principles of method if other than guideline:
The temperature of the room in which the animals were maintained on occasion exceeded the recommended maximum value in the guideline.
GLP compliance:
yes
Type of study:
Buehler test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid (unspecified)
Details on test material:
- Appearance: brown liquid
- Storage conditions: ambient

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adult.
- Weight at study initiation: 337 to 470 g.
- Housing: Animals were individually housed in wire mesh suspension cages.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum tap water.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26 °C.
- Humidity (%): 30 to 70 %.
- Photoperiod (hrs dark / hrs light): 12 hour cycle in a light controlled room.

IN-LIFE DATES: From: 24 October 2012 To: 23 November 2012

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
15 % concentration for the induction phase.
7.5 % concentration for the challenge phase.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
15 % concentration for the induction phase.
7.5 % concentration for the challenge phase.
No. of animals per dose:
20 animals were subjected to the induction and challenge doses.
Details on study design:
RANGE FINDING TESTS: Irritation Screening (Pilot)
The irritation potential of the test material at levels of 60, 30, 15 and 7.5 % were evaluated in one group of two animals. Four levels of test material were evaluated per animal. Dilutions of the test material were formulated w/w in distilled water. The position of the different concentrations of the test material on the animals was varied to adjust for possible site-to-site variation in response. The exposure technique was the same as that followed for the induction and challenge phases of the study.

The day prior to test material exposure, the hair was removed from each animal's back using a small animal clipper. 0.4 mL of each test preparation was applied directly into a 25 mm Hilltop Chamber and the chambers applied to the clipped left shoulder. The chambers were secured with Micropore tape followed by Kendall adhesive tape. Approximately six hours later, the tape and chambers were removed. The day following the irritation exposure all animals were scored. The dose levels for the test were selected based upon these results.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 applications.
- Exposure period: 6 hours (occlusive).
- Control group: Yes. 10 animals remained untreated throughout the induction as a naïve control.
- Site: The left shoulder of each animal (clipped the day prior to treatment).
- Frequency of applications: Weekly.
- Duration: 3 applications. After the last induction, animals remained untreated for 2 weeks.
- Concentrations: The induction was with a dose of 15 % w/w in distilled water.

B. CHALLENGE EXPOSURE
- No. of exposures: A single challenge exposure.
- Day(s) of challenge: 14 days after the last induction exposure.
- Exposure period: 6 hours.
- Control group: Yes. Five naive control animals received the primary challenge dose at a 7.5 % concentration. The five remaining guinea pigs were designated for a re-challenge, if necessary.
- Site: The right shoulder of each animal, a site that had not been previously exposed (clipped the day prior to treatment).
- Concentrations: The primary challenge was with a dose of 7.5 % w/w in distilled water.
- Evaluation (hr after challenge): 24 hours following the primary challenge exposure. The evaluation was repeated at 48 hours.

OTHER
A. OBSERVATIONS
The test sites were graded using the following scale:
0 = no perceptible reaction
± = slight, diffuse or ill-defined erythema
1 = slight but confluent, or moderate patchy erythema
2 = moderate erythema
3 = severe erythema with or without oedema
A score of 1 or greater was considered positive.

B. BODYWEIGHTS
Initial bodyweights were measured just prior to the first exposure. Final bodyweights were taken at the termination of the study.

C. TERMINAL PROCEDURES
Animals were euthanised by carbon dioxide inhalation at the termination of the study.
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene

Results and discussion

Positive control results:
POSITIVE CONTROL ANIMALS
- Animal strain was tested every six months using 0.3% of 1-chloro-2,4-dinitrobenzene for induction and 0.1% of 1-chloro-2,4-dinitrobenzene for challenge. In the most recent test, conducted simultaneously with the test of the test substance, 20/20 guinea pigs tested positive with the positive control substance.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
7.5 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
3 animals showed slight, diffuse or ill-defined erythema.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 7.5 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 3 animals showed slight, diffuse or ill-defined erythema. .
Reading:
1st reading
Hours after challenge:
24
Group:
other: naive control
Dose level:
7.5 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
1 animal showed slight, diffuse or ill-defined erythema.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: naive control. Dose level: 7.5 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: 1 animal showed slight, diffuse or ill-defined erythema. .
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
7.5 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 7.5 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: naive control
Dose level:
7.5 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: naive control. Dose level: 7.5 %. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Table 1 Skin Grades Following Primary Challenge - Test Group

Animal Number

24 Hours

48 Hours

562

0

0

563

0

0

564

0

0

565

0

0

566

0.5

0

567

0

0

568

0

0

569

0

0

570

0

0

571

0

0

572

0

0

573

0

0

574

0.5

0

575

0

0

576

0.5

0

577

0

0

578

0

0

579

0

0

580

0

0

581

0

0

Mean

0.08

0

 

Table 2 Skin Grades Following Primary Challenge - Naïve Control Group

Animal Number

24 Hours

48 Hours

582

0

0

583

0

0

584

0

0

585

0.5

0

586

0

0

Mean

0.10

0

 

Table 3 Induction Scores of the Test Group

Animal Number

Induction 1

Induction 2

Induction 3

24 Hours

48 Hours

24 Hours

48 Hours

24 Hours

48 Hours

562

1

0

1

0

1

0

563

1

0

1

0

1

0.5

564

1

0

1

0

2

1

565

2

0.5

1

0.5

1

0

566

1

0

1

0

1

0

567

0.5

0

0.5

0

1

0

568

1

0.5

2

1

2

1

569

1

0

2

0.5

1

0.5

570

1

0

1

0

1

0.5

571

1

0

1

0

1

0

572

1

0.5

2

1

1

0

573

1

0

0.5

0

1

0

574

0.5

0

1

0

0.5

0

575

1

0

1

0

1

0.5

576

1

0

1

0

1

0

577

1

0

1

0

1

0.5

578

1

0

2

1

2

1

579

1

0.5

0.5

0

1

0

580

1

0

1

0

1

0

581

1

0

1

0

1

0

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, it was determined that the test material did not induce sensitisation in accordance with EU criteria.
Executive summary:

The test material was investigated for dermal sensitisation potential utilising a Buehler guinea pig test in accordance with the standardised guidelines OECD 406 and EPA OPPTS 870.2600. The test was also PR 86-5 compliant.

In the induction phase of the exposure, 0.4 mL of the test material was applied at a 15 % w/w concentration in distilled water to the clipped left shoulder of 20 Hartley guinea pigs. The application was done using a Hilltop Chamber secured in an occlusive fashion for 6 hours. Two additional induction doses were conducted following the same procedure, at weekly intervals. 

Two weeks after the final application, the animals received a topical primary challenge dose (6 hour occlusive contact) of the test material at 7.5 % concentration w/w in distilled water on a naive site located on the right shoulder. Animals were scored for irritation at 24 and 48 hours after initiation of the primary challenge application.

Ten guinea pigs served as a naive control group, and remained untreated through the induction phase. Five naive control animals received the primary challenge dose, at a 7.5 % concentration. 

The incidence and severity of the responses in the test animals were not significantly greater than those produced by the naive control group, indicating that sensitisation had not been induced.

Under the conditions of this study, it was determined that the test material did not induce sensitisation in accordance with EU criteria.