Registration Dossier

Administrative data

Description of key information

Not sensitising to skin (OECD 406)

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:
2009-06-09 to 2009-07-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig maximisation studies that were carried out before 11 October 2016, and that meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4) shall be considered appropriate to address this standard information requirement.
Specific details on test material used for the study:
Batch No.: 20090226
Purity: 99.38%
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc., 3371-8 Kotoh-cho, Hamamastu, Shizuoka, 431-1103, Japan
- Age at study initiation: Approx. 6 wks
- Weight at study initiation: 227.9~265.7 g
- Housing: Stainless steel cages, ≤ 5 animals/cage
- Diet: Ad libitum, Harlan Teklad guinea pig pellets
- Water: Ad libitum, tap water
- Acclimation period: 12 d

ENVIRONMENTAL CONDITIONS
- Temperature:22 °C ± 3 °C
- Humidity: 55 % ± 10 %
- Air changes: 10~20 per hr
- Photoperiod: 12 hrs dark / 12 hrs light
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Route:
other: epicutaneous (unclear whether occlusive or semiocclusive; probably latter)
Vehicle:
corn oil
No. of animals per dose:
20 (T1 group, test item), 10 (V1 group, vehicle control), 20 (T2 group, positive control), 10 (V2 group, challenge control)
Details on study design:
RANGE FINDING TESTS:
- 2 guinea pigs range-finding test.
- The following concentrations were applied, intradermally to sites on their backs at 0.1 mL/site: 100 %, 50 %, 25 %, 12.5 %, 6.3 %, 3.1 %, 1.6 %, 0.8 %, 0.4 %, 0.2 %
- The highest concentration for without raising necrosis of the administration site was chosen for the dose level for intradermal administration. This was 100 %.
- For the patch administrations (i.e. 2nd induction and challenge), 100 %, 50 %, 25 % and 12.5 % of the test item were applied to their flanks. No skin reaction was observed at any concentration tested; therefore 100 % solution was chosen as the dose level for second induction and challenge.

MAIN STUDY
INDUCTION EXPOSURE
- Site: Back (lower shoulder), 2 cm × 4 cm. Site clipped before application. Into this shaved area three pairs of symmetrical intradermal injections were given. The intervals between 1 and 2 were closer than between 2 and 3.
- No. of injections: 2 sets of 3 injections as follows:
-- Group T1
--- (1) 1:1 (v/v) mixture of Freund's Complete Adjuvant (FCA)/distilled water (DW)
--- (2) Test material
--- (3) 1:1 (v/v) mixture of FCA/Test item
-- Group V1
--- (1) 1:1 (v/v) mixture of FCA/DW
--- (2) Corn oil
--- (3) 1:1 (v/v) mixture of FCA/Corn oil
-- Group T2
--- (1) 1:1 (v/v) mixture of FCA/DW
--- (2) 0.1 % DNCB
--- (3) 1:1 (v/v) mixture of FCA/0.1 % DNCB
-- Group V2
--- (1) 1:1 (v/v) mixture of FCA/DW
--- (2) 40 % ethanol
--- (3) 1:1 (v/v) mixture of FCA/40 % ethanol

SDS PRE-TREATMENT
- Time: Day 7
- The site of the 1st induction was shaved again and 10 % sodium dodecyl sulfate (SDS) in vaseline (about 0.5 g) applied again to induce mild skin irritation.

SECOND INDUCTION
- Time: Day 8
- The same site was clipped again and covered with a patch of filter paper (2 cm × 2 cm) saturated with test material for groups T1 and V1, and 0.1 % DNCB for T2 and V2. The filter paper was held in place with adhesive tape.
- Exposure period: 24 hrs

CHALLENGE EXPOSURE
- Time: Day 22
- Hairs were removed from a 5 cm × 5 cm area on the flank by close clipping and the area was covered with a patch of filter paper (2 cm × 2 cm) saturated with test material for groups T1 and V1, and 0.1 % DNCB for T2 and V2. The filter paper was held in place with adhesive tape.
- Exposure period: 24 hrs

SCORING
- 21 hrs after removal of challenge patch, the sites were cleared of hair.
- Approx 3 hrs and 27 hrs later (48 hrs and 72 hrs after application of challenge exposure, respectively), responses were scored according to the following scheme:
-- 0: No visible change
-- 1: Dispersed or blotchy erythema
-- 2: Moderate diffused erythema
-- 3: Marked erythema and oedema
- Classification of skin sensitisation level was assessed according to the following scale:
-- Grade I (weak): 0-8 % sensitisation rate
-- Grade II (mild): 9-28 % sensitisation rate
-- Grade III (moderate): 29-64 % sensitisation rate
-- Grade IV (strong): 65-80 % sensitisation rate
-- Grade V (extreme): 81-100 % sensitisation rate

STATISTICS
- Bodyweights collected during study were analysed with Fisher's F-test examine variance homogeniety.
- Student's t-test was used to determine whether the test item-sensitising group was different to the induction control-sensitisation group. The level of significance was taken as P<0.05 or 0.01. Statistical analysis was performed by using Path/Tox System 4.2.2 (Xybion Medical Systems Corporation, USA).
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene (DNCB)
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No adverse signs.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No adverse signs.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: positive control. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: No adverse signs.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: positive control. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: No adverse signs.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
other: Induction control (induced with corn oil, challenged with test material)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group:
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
other: Induction control (induced with corn oil, challenged with test material)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group:
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
other: Challenge control (induced with ethanol and challenged with DNCB)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group:
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
other: Challenge control (induced with ethanol and challenged with DNCB)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No adverse signs
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group:

Clinical signs and mortality

No treatment-related clinical signs or death were found during the observation period.

Body weights

A slight but statistically significant decrease in weight gains was observed in group T2 compared to those of the group V2. This was considered to be due to chance as there were no associated clinical signs.

Observation of application sites

Approximately 48 and 72 h after the start of the challenge application (24 and 48 h from the patch removal) the skin reaction was observed. The average skin reaction scores in the group T1, V1 , T2 and V2 at 48 h were 0.0, 0.0, 2.85 and 0.0 respectively. The sensitisation rates at the same time point were 0, 0, 100 and 0 & respectively. Thus, the skin sensitivity of the test substance was considered weak (Grade 1) while that of the positive control item was extreme (Grade V). The average skin reaction scores in the group T1, V1, T2 and V2 at 72 hours were 0.0, 0.0, 2.55 and 0.0 respectively. The sensitisation rates at the same time point were 0, 0, 100, 0 %, respectively.

Control results and validity

  • The positive control group T2, induced with DNCB and challenged with DNCB, was positive (grade V reaction), and therefore considered valid.
  • The induction control group V1, induced with corn oil and challenged with test material, was negative (grade I reaction), and therefore considered valid.
  • The challenge control group V2, induced with ethanol and challenged with DNCB, was negative (grade I reaction), and was therefore considered valid.
Interpretation of results:
not sensitising
Conclusions:
A guinea pig maximisation test was performed according to OECD Guideline 406 to determine the skin sensitization potential of the test substance. The concentration of the test item used in the intradermal administration (1st induction) was 100 % of the original solution, the highest concentration without raising necrosis of the administration site. The highest concentration of the test item (100 %) induced no skin irritation was used in the patch exposure for second induction and challenge. The animals were divided into four groups: Group T1, the test item-sensitisation group (20 animals), Group V1, the vehicle-sensitisation group (10 animals), Group T2, the positive control item-sensitisation group (20 animals) and Group V1, the ethyl alcohol-sensitisation group (10 animals). There were no dead animals and no clinical signs observed related with the test item sensitisation. Skin sensitising potential was only observed in the Group T2 with 2.85 of the average skin reaction score and 100 % of the sensitisation rate at 48 h after the challenge, and with 2.55 and 100 % at 72 h after the challenge. All other group showed no skin sensitizing activity.
The results indicate that the test substance is not a skin sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a guinea pig maximisation test to OECD guideline 406, the substance was found not to be sensitising to skin.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

The substance should not be classified as a skin sensitiser because the available test data do not meet the criteria set out in Regulation (EC) 1272/2008.

Respiratory sensitisation

No data are available for respiratory sensitisation, although this is not a concern given the very low vapour pressure and the lack of sensitisation shown in a dermal sensitisation study.