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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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
reliability scoring based on 1992 guideline
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The maximization test was a validated test method before REACH came into force.

Test material

Constituent 1
Chemical structure
Reference substance name:
4-tert-butylphenol
EC Number:
202-679-0
EC Name:
4-tert-butylphenol
Cas Number:
98-54-4
Molecular formula:
C10H14O
IUPAC Name:
4-tert-butylphenol
Details on test material:
- Name of test material (as cited in study report): para-tertiary butylphenol, UCAR Butylphenol 4T-Flake (para tert-Butyl Phenol), PTBP.
- Physical state: White flakes.
- Analytical purity: Not reported.
- Lot/batch No.: Charge No. 496500; BRRC Sample No. 48-186.
- Expiration date of the lot/batch: Not reported.
- Stability under test conditions: Not reported.
- Storage condition of test material: Not reported.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Dunkin Hartley, Pirbright White
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Versuchstierzucht, Gartenstraße 27, 33176 Borchen
- Age at study initiation: young adult
- Weight at study initiation: < 500 g
- Housing: Conventional, maximum 5 animals/Type IV Makrolon cage
- Diet (e.g. ad libitum): Ssniff G 4 diet in pellet form (laboratory standard guinea pig diet), produced by Sniff, Spezialfutter GmbH, 59494 Soest. Food was administered ad libitum.
- Water (e.g. ad libitum): Animals received tap water ad libitum
- Acclimation period: minimum of 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): Artificial light, 12-hour light/dark rhythm


Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: see concentration
Concentration / amount:
0.5% in corn oil (intradermal induction)
10% in vaseline (epidermal induction)
1% in vaseline (epidermal challenge)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: see concentration
Concentration / amount:
0.5% in corn oil (intradermal induction)
10% in vaseline (epidermal induction)
1% in vaseline (epidermal challenge)
No. of animals per dose:
Test group: 10 males
Control group: 5 males

Control group: one side of flank induced with vehicle and challenged with vehicle; other side of flank induced with vehicle and challenged with test substance.
Test group: one side of flank induced with test substance and challenged with vehicle; other side of flank induced with test substance and challenged with test substance.
Details on study design:
RANGE FINDING TESTS:
Preliminary test:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.

Solubility test:
In the solubility test a 10 and 50% concentration of the test compound in deionized water, a 10, 50, and 75% concentration in corn oil and a 50 and 60% concentration in vaseline were formulated.

lntracutaneous injection:
To determine a well-tolerated concentration for intracutaneous induction the following concentrations of the test substance in corn oil (percentage w/w) were injected intracutaneously into the left flank of two animals:

Animals 1 and 2:
i.c. injection; dose. - volume. (cubic cm); conc. (%); vehicle
1.; 1 x 0.1; -; corn oil
2.; 1 x 0.1; 0.01; corn oil
3.; 1 x 0.1; 0.05; corn oil
4.; 1 x 0.1; 0.10; corn oil
5.; 1 x 0.1; 0.50; corn oil
6.; 1 x 0.1; 1.00; corn oil
7.; 1 x 0.1; 5.00; corn oil

The intracutaneous injections of the pilot study were assessed 24 hours post applicationem using the grading scale by Magnusson and Kligman

Dermal administration:
2-3 hours before the administration, the fur was mechanically removed from the left and right flanks of 3 animals. The animals received about 0.2 g of each of the test substance concentrations (5; 10, 25, and 50% in vaseline) applied to patches of filter paper (2 x 2 cm). These were placed onto the clipped flanks of each animal. Each animal received two patches on each flank. The patches were covered by a strip of an occlusive plaster and firmly secured by a bandage for 24 hours.

Animals 3, 4, 5:
site of administration; concentration (wlw); vehicle
left flank, front; 5; vaseline
left flank, rear; 10; vaseline
right flank, front; 25; vaseline
right flank, rear; 50; vaseline

After removal of the patch, the residual test substance was washed off with corn oil. The dermal reactions were assessed 48 and 72 hours after start of treatment, using the grading scale by Magnusson and Kligman.

Determination of the challenge concentration:
To determine the highest non-irritant concentration of the test substance for the challenge exposure, the adjuvant treated animals of the accompanying group were used in the 3rd week of the test. This additional testing of the concentration was carried out because it was suspected that the sensitivity of the skin changed after the treatment with Freund's Complete Adjuvant (FCA). In this test, the concentrations administered were 0.5, 1.0, 5.0, and 10.0% in Vaseline. The experimental conditions corresponded to those of the preliminary test.


MAIN STUDY: The body weights of all animals were recorded at the start of administration and then each week and at the end of the investigation. In addition the occurrence of systemic effects was monitored.

A. INDUCTION EXPOSURE
lntracutaneous induction (day 0)
2-3 hours before the administration, fur was mechanically removed from the shoulder region of the animals. Three pairs of intracutaneous injections of 0.1 cubic cm volume were given in the shoulder region, one of each pair on either side of the midline. The skin reactions were observed and recorded 1 hour and 24 hours after administration, according to the grading scale by Magnusson and Kligman.

SDS-treatment (day 6)
As the test substance concentration used for the topical induction caused skin irritations, pretreatment with sodium dodecylsulfate (SDS) was not necessary.

Topical induction procedure (day 7)
One week after the injections the same area was clipped 2-3 hours before the administration. A filter paper (2 x 4 cm) was covered with the 10% concentration of the test substance or with the vehicle (about 0.4 g/patch) and placed onto the shoulder region of the animals on top of the injections and held in place by an occlusive patch and fixed with a bandage for 48 hours.

After removing the dressing, the administration areas (residual substance) were washed with corn oil. The skin reactions of the injection area were observed and recorded 49 and 72 hours after administration, according to the grading scale by Magnusson and Kligman.
B. CHALLENGE EXPOSURE
Challenge treatment (day 21)
For the challenge treatment, the flanks of test and control animals were clipped free of hair 2 to 3 hours before administration. Surgical gauze (2 x 2 cm) was covered with 0.2 g of the 1% test substance in Vaseline and placed onto the clipped right flank of the animals. An occlusive patch with the vehicle alone was applied to the left flank. The patches were held in contact with the skin by an occlusive patch and fixed with a bandage for 24 hours.

After removing the dressing, the administration areas were washed with corn oil. Assessment took place 48 and 72 hours after administration according to the grading scale by Magnusson and Kligman.

Rechallenge treatment (day 28)
A rechallenge was not performed. It was not necessary to clarify the results obtained in the first challenge.

Magnusson and Kligman grading scale for evaluation of challenge patch test reaction
0=no visible change
1=discrete or patchy erythema
2=moderate and confluent erythema
3 =intense erythema and swelling
Challenge controls:
To determine the highest non-irritant concentration of the test substance for the challenge exposure, the adjuvant treated animals of the accompanying group were used in the 3rd week of the test.

This additional testing of the concentration was carried out because it was suspected that the sensitivity of the skin changed after the treatment with FCA. In this test, the concentrations administered were 0.5, 1.0, 5.0, and 10.0% in Vaseline. The experimental conditions corresponded to those of the preliminary test.
Positive control substance(s):
yes
Remarks:
¿- hexycinnamaldehyde or 2-mercaptobenzothiazole

Study design: in vivo (LLNA)

Vehicle:
other: not applicable
Details on study design:
Not applicable
Statistics:
Not applicable

Results and discussion

Positive control results:
In order to show any reaction to or any effect of known sensitizing agents (¿- hexycinnamaldehyde or 2-mercaptobenzothiazole), the guinea pig strain employed for the sensitization test must be sensitive to these agents. This sensitivity is checked at regular intervals. The results of the latest reliability check was not provided in the report.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1.0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1.0% . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1.0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 1.0%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
other: control
Dose level:
1.0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: control. Dose level: 1.0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
other: control
Dose level:
1.0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: other: control. Dose level: 1.0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.

Any other information on results incl. tables

Individual animal data was not provided in the report.

Results from the Main test:

Body weight gain and systemic effects:

The test and control animals showed a normal body weight gain throughout the investigation. Occasional fluctuations in body weight are physiological for guinea pigs and were not caused by the test substance. No systemic effects were detected in treated animals during the observation period.

lntracutaneous induction:

1 hour and 24 hours after injection all induction sites of test and control animals treated with FCA showed intense erythema and swelling, after 1 hour combined with well defined pustulae and after 24 hours with necrosis in all animals.

 

1hour and 24 hours after injection the injection sites treated with 0.5% test

substance (test group) or with vehicle (control group and accompanying group) showed moderate and confluent erythema, combined with well defined pustulae after one hour and discrete erythema after 24 hours.

Topical induction:

49 and 72 hours after application all induction sites of test and control animals and of the animals of the accompanying group, treated with FCA a week before intracutaneously, showed intense erythema and swelling and eschar formations partially combined with bloody scratch wounds.

 

49 and 72 hours post applicationem the injections, a week before treated with 0.5% test substance or vehicle, showed no reactions in all test and control animals (including accompanying group).

 

Challenge treatment:

The challenge treatment was carried out with the 1.0% test compound. 48 and 72 hours post application the treatment did not cause any skin reactions within the meaning of contact hypersensitivity in all animals on the right flank of the test group. No control animals did show any skin reactions 48 and 72 hours post application. Neither did the vehicle patch lead to skin reactions in any animal of the test and control group on the left flank.

Rechallenge treatment:

A rechallenge was not performed.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
From the results obtained after the challenge treatment, P-TERT-BUTYLPHENOL showed no evidence of contact skin sensitization.