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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP standards

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study conducted according to OECD Guideline 406 for skin sensitisation that predates adoption of OECD 429 in 2010.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Aqueous solution of TBHP (tertiary butyl hydroperoxide solution (70%))
- TBHP content 70%
- Physical state: clear colourless liquid
- Lot/batch No.: SGS REF 534326

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall, England
- Age at study initiation: circa 3 weeks
- Weight at study initiation: 215 - 262g
- Housing: in a mobile battery, containing 6 cages; maximal 10 animals per cage.
- Diet (e.g. ad libitum): Standard laboratory diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 C
- Humidity (%): 30 - 70% (occasionally up to 100% for a short period, because of wet cleaning of the room or meteorological circumstances).
- Air changes (per hr): ca. 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12hours dark cycle.


IN-LIFE DATES: From: 26 June 2001 To: 20 July 2001

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
10 male guinea pigs; induction with intradermal injection on day 1 of Freunds Complete Adjuvant (FCA) 1:1 diluted with isotonic saline, a 1% test dilution of TBHP-70 in physiological saline and 1% test dilution of TBHP in FCA followed by topical induction on day 8 with 30% TBHP; challenge 14 days after with 3% TBHP (Day 22).
Challengeopen allclose all
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
10 male guinea pigs; induction with intradermal injection on day 1 of Freunds Complete Adjuvant (FCA) 1:1 diluted with isotonic saline, a 1% test dilution of TBHP-70 in physiological saline and 1% test dilution of TBHP in FCA followed by topical induction on day 8 with 30% TBHP; challenge 14 days after with 3% TBHP (Day 22).
No. of animals per dose:
15 male guinea pigs were randomly divided into 2 groups, one test group of 10 males and one control group of 5 males.
Details on study design:
RANGE FINDING TESTS:
The irritation response to intradermal injection of various concentrations of the test substance was examined in 2 guinea pigs. Amounts of 0.1 mL of the selected concentrations were applied by intradermal injection. 24 hours post injection, animals were examined for signs of irritation. A concentration causing slight to moderate irritation but otherwise well-tolerated by the animals (1% Aq. TBHP-70) was chosen for intradermal injection in the induction phase of the main study.

The irritation reponse to topical treatment of various concentrations of the test substance was examined in 2 other guinea pigs. Patches were loaded with the test material and placed on the clipped skin of each animal and covered with a piece of hypoallergenic paper bandage that was secured by the elastic adhesive bandage wound around the torso of the animal. The dressing was left in place for 24 hours. 24-48 after removal of the dressing, the animals were examined for signs of skin irritation. A concentration causing slight to moderate skin irritation is usually chosen for topical induction and a non-irritant concentration for topical challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 10
- Exposure period: Topical induction - 48 hour exposure
- Test groups: 1 (10 animals)
- Control group: 1 (5 animals)
- Site: Dorsal skin
- Duration: Topical induction for 48 hours then topical challenge on day 14.
- Concentrations: 1% TBHP-70


B. CHALLENGE EXPOSURE
- No. of exposures: 10
- Day(s) of challenge: 22
- Exposure period: Topical challenge - 24 hour exposure
- Test groups: 1 (10 animals)
- Control group: 1 (5 animals)
- Site: Dorsal skin
- Concentrations: 3% test concentration of TBHP-70



Challenge controls:
Physiological saline
Positive control substance(s):
yes
Remarks:
Formaldehyde

Results and discussion

Positive control results:
Challenge treatment with 10% and 3% test dilution of formaldehyde (37%) induced positive reactions in 100% and 60% of the test animals respectively.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
3% TBHP-70
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 3% TBHP-70. No with. + reactions: 6.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
3% TBHP-70
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 3% TBHP-70. No with. + reactions: 6.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
3% formaldehyde
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 3% formaldehyde. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
3% formaldehyde
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 3% formaldehyde. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% formaldehyde
No. with + reactions:
4
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 10% formaldehyde. No with. + reactions: 4.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10% formaldehyde
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 10% formaldehyde. No with. + reactions: 5.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Clinical observations:

Induction

Intradermal injections caused the following skin reactions:

1) In test animals:

- FCA/saline (1:1): moderate erythema

- selected test concentration: no skin reactions

- selected test concentration in FCA/diluent (1:1): moderate erythema

2) Control animals

- FCA/saline (1:1): moderate erythema

- vehicle: no skin reactions

- FCA/diluent (1:1): moderate erythema

Challenge

After the challenge treatment with the 3% test concentration of Aq. TBHP-70, slight moderate or severe erythema was observed in six test animals at 24 hours after challenge. At 48 hours after challenge, the 6 test animals showed moderate or severe erythema at the test site. The controls did not show any skin reactions.

Table 1 Skin scores in the test group (challenged with 3% TBHP-70).

Skin scores obtained 24 and 48 hours after removal of the dressing. 
Animal No. 24 hours 48 hours
Control group
22 0 0
24 0 0
26 0 0
28 Sacrified in extremis, unrelated to test article application
30 0 0
Test group
2 1 2
4 2 3
6 2 3
8 0 0
10 3 3
12 0 0
14 2 2
16 0 0
18 2 3
20 0 0

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Aq. TBHP-70 caused skin sensitisation reactions in the test Guinea pigs. The induction and challenge regime employed corresponded to to TBHP treatment at 0.7% and 2.1% (induction) and 21% (challenge). It is therefore concluded that TBHP caused delayed contact sensitisation in Guinea pigs and must be considered to be a skin sensitiser.
Executive summary:

TBHP caused delayed contact sensitisation in Guinea pigs and must be considered to be a skin sensitiser. Under EC Regulation 1272/2008, TBHP is classified as a skin sensitizer Category 1A.