Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
End of the study period: 10 February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 20.6 +/- 1.3 g
- Housing: the animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-2°C
- Humidity: 30 to 70%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hrs light / 12 hrs dark

IN-LIFE DATES: From: 16 July 2002 To: 19 August 2002
Vehicle:
other: ethanol/water (50/50)
Concentration:
0, 1, 2.5 and 5%
positive control: 25%
No. of animals per dose:
4 females
Details on study design:
PRELIMINARY TEST:
To assess the irritant potential of the test substance (through ear thickness measurement), a preliminary test was performed on 4 animals, as follows:
- the test substance was prepared at the concentrations of 50, 25, 10 and 5%,
- for three consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear),
- measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Kimber and Dearman
- Criteria used to consider a positive response: The test substance was considered as a skin sensitizer when the Stimulation Indices (SI) for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
In the main test, twenty female CBA/J mice were allocated to five groups:
- three treated groups of four animals receiving the test substance 4-tert-butylpyrocatechol 85% in water at the concentrations 1, 2.5 and 5%
- one negative control group of four animals receiving the vehicle
- one positive control group of four animals receiving the reference item, alpha-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%

On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test substance, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of tritiated methyl-thymidine (3H-TdR) via the tail vein. Lymph node cell proliferative responses were measured on day 6. The lymph nodes were pooled for each experimental group.
The obtained values were used to calculate stimulation indices (SI).
The irritant potential of the test substance was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold positive value of 3 (SI = 27.37) were noted. The study was therefore considered valid.
Parameter:
SI
Remarks on result:
other: In the treated groups, a dose-related increase in the SI was noted and the threshold positive value of 3 for the SI was exceeded at all tested concentrations, leading to a maximum SI of 63.26 at the concentration of 5%.

Study results:

Groups

1

2

3

4

5

Concentrations (%)

0

(ethanol/water)

1

2,5

5

HCA at 25%

Cell count viable

97

330

394

577

318

Cell count dead

5

14

12

24

11

Viability (%)

95,1

95,93

97,04

96,01

96,66

Amount of cells (x106cells)

4,85

16,5

39,4

57,7

31,81

Cellularity index

 

3,4

8,12

11,9

6,56

Number of nodes per group

8

8

8

8

8

Disintegrations per min per group (dpm)

475,54

7299,34

22376,37

30084,64

13014,36

Disintegrations per min per node (dpm)

59,44

912,42

2797,05

3760,58

1626,8

stimulation index (SI)

 

15,35

47,05

63,26

27,37

Increase in ear thickness (% between day 1 and day 6)

-7,08

0

28,57

149,53

 

Preliminary assay:

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/oil (4/1, v/v)), a mixture ethanol/water (50/50, v/v) was chosen among the other proposed vehicles. A homogeneous dosage form preparation was obtained whatever the proportion.

During the preliminary assay, a marked increase in the ear thickness was noted in the animals given the test item at the concentration of 10% (+ 60%).

At the two highest tested concentrations, the increase in ear thickness was so important that the animals were killed for ethical reasons after the second cutaneous application.

Since the test item was severely irritant at the concentrations ≥ 10%, the highest tested concentration retained for the main test was 5%.

Main test:

No systemic clinical signs and no mortality were observed during the study.

The body weight gain of the treated animals was not affected by the treatment with the test item.

Dryness of the skin was noted on day 6 on the ears of the animals given the test item at the concentration of 5%. In addition, a moderate or severe increase in ear thickness (+ 28.57% and + 149.53%) was recorded in the animals given the test item at the concentrations of 2.5 or 5% respectively.

The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. The cell viability was higher than 80% in each group.

In the absence of local irritation, the positive lymphoproliferative response observed at the concentration of 1% was attributed to delayed contact hypersensitivity.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under our experimental conditions, the test substance 4-tert-butylpyrocatechol 85% in water should be considered as a skin sensitizer (EU GHS Classification: Skin sensitisation Category 1 (H317)).
However, as this conclusion is based on the results obtained at one concentration only (due to the irritants effects at higher concentrations), a challenge study would be necessary to conclude on the potential of the test item to induce delayed contact hypersensitivity or not.
Executive summary:

In a dermal sensitization study using the method of Local Lymph Node assay (LLNA), (CIT, 2003) with 4 -tert-butylpyrocatechol 85% in water, in a mixture ethanol/water, 9-week CBA/J mice (4 females per group) were tested according to the guideline OECD 429, at the concentrations of 1%, 2.5%, 5% and 25%. The positive control group received alpha-hexylcinnamaldehyde.

No mortality and no systemic clinical signs were observed during the study. Dryness of the skin was noted on day 6 on the ears of the animals given the concentration of 5%. In addition, a moderate or severe increase in ear thickness was recorded in the animals given the test substance at the concentrations of 2.5 and 5%, respectively.

A dose-related increase in the stimulation indices (SI) (SI = 15.35 / 47.05 / 63.26 at 1% / 2.5% / 5% respectively) was noted and the thresold positive value of 3 was exceeded at all tested concentrations. In the absence of local irritation, the positive lymphoproliferative response observed from the concentration of 1% could be attributed to delayed contact hypersensitivity. However irritation effects were observed at concentrations above 1% and the evaluation of skin sensitization was made at the only concentration of 1% for which no increase in ear thickness was observed.

In this study, 4 -tert-butylpyrocatechol 85% in water was a dermal sensitizer but these results should be considered with caution.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Academic study, well documented, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study has been performed before the LLNA study and since it is a publication it is publicly available.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS:
- Strain: Dunkin-Hartley
- Sex: female
- Source: J.A. Sahlin, Malmö, Sweden
- Age at study initiation: no data
- Weight at study initiation: 300-400 g
- Acclimation period: no data
- Housing: no data
- Diet: no data
- Water: no data

ENVIRONMENTAL CONDITIONS
- Temperature (degrees C): no data
- Humidity (%): no data
- Air changes: no data
- Photoperiod: no data

In life dates: no data
Route:
other: intradermal and topical
Vehicle:
other: propylene glycol/acetone 90/10% v/v (intradermal)
Route:
epicutaneous, occlusive
Vehicle:
other: propylene glycol/acetone 90/10% v/v (intradermal)
No. of animals per dose:
24: test group
12: control group
Details on study design:
TOPICAL IRRITANCY:
It was studied by means of a 48-h closed patch test in 4-8 animals on the flank. Testing was performed 1 week after pretreatment of the animals with Freund's complete adjuvant (FCA).

INDUCTION / CHALLENGE SCHEDULE:
* Induction procedure:
For intradermal sensitisation, 3 injections were given in a row on each side of the shoulder: (I) 0.1 mL of FCA in water 50% v/v; (II)0.1 mL of TBC in propylene glycol/acetone 90/10% v/v; (III) 0.1 mL of a preparation consisting of a mixture of TBC and FCA in propylene glycol/acetone 90/10% v/v in which the concentration for FCA was the same as that in (I) and the concentration of TBC was the same as that in (II).
24h before the topical sensitisation, all animals were treated on 2x4 cm area on the shoulder with 200 µL of a preparation consisting of sodium lauryl sulphate 10% w/v in dimethylacetamide/acetone/ethanol 4/3/3 v/v/v.
Topical sensitisation on the same skin area was then performed with 20 µL of a solution of 16.7% TBC on a 2x4 cm patch of Munktell 1002 filter paper.
The patch was covered with overlapping, impermeable plastic adhesive tape. This in turn was firmly secured by adhesive bandage. The dressing was left in place for 48h.

* Challenge procedure:
Challenge I was performed 2 weeks after the end of the induction stage, and a 24-h occluded patch test was performed on the right flank with 25 µL of TBC 7.5% (w/v) on each of the 2 patches near the back.
The Al-test on Durapore was used for patch-testing and was firmly secured by Acrylastic and an outer layer of Durapore.
12 test animals received TBC on both patches; 6 test animals received TBC only on the cranially located patch, while the vehicle alone was applied to the other patch. 6 other test animals received the vehicle alone on the cranially located patch, while TBC was applied to the other patch.
Challenge II was performed at the same time as challenge I but on the left side of the flank. 6 patches were tested on 24 test and 12 control animals: 2 patches near the back, 2 near the abdomen, and 2 between the back and the abdomen. The animals were challenged with the sensitiser (TBC: 10%).
A challenge was performed with nonirritant solutions.

GRADING SYSTEM:
The reactions were evaluated in a blind manner 24h after the removal of the patches.
The minimal criterion of an allergic (positive) reaction was a confluent erythema. The number of positive animals in each test group was statistically compared with the number of positive animals in the corresponding control group and also with the number of positive test animals tested with the vehicle alone.
When both comparisons yielded significant values, the compound was considered to be a contact sensitiser.
For challenge II, a comparison was only made between the number of animals positive for each substance in the test and control groups.
Challenge controls:
Challenge I & II: 12 females
no more data available
Positive control substance(s):
yes
Remarks:
2-methylol phenol
Positive control results:
Challenge I: 3/6 positive animals
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
7.5 %
No. with + reactions:
20
Total no. in group:
24
Clinical observations:
No data
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
No data
No. with + reactions:
2
Total no. in group:
12
Clinical observations:
No data
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
No data
No. with + reactions:
3
Total no. in group:
6
Clinical observations:
No data
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
18
Total no. in group:
24
Clinical observations:
No data
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
No data
No. with + reactions:
1
Total no. in group:
12
Clinical observations:
No data

CHALLENGE I:

Group                        Number of positive animals
Negative control/vehicle        2/12
TBC (7.5%)               20/24

CHALLENGE II:

                        Nb positive animals after challenge
Challenge substance        TBC group (10%)        Control group
TBC                18/24                1/12


Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study, TBC was shown to be a potent sensitizer in guinea pigs.

EU GHS classification: skin sensitisation Category 1 (H317)



Executive summary:

In a dermal sensitization study using the method of Maximization test (Zimerson et al., 1999) with 4 -tert-butylpyrocatechol (TBC) in propylene glycol / acetone (90/10%), Dunkin-Hartley guinea pigs (24 females) were tested under test conditions similar to the guideline OECD 406. The positive control group received 2 -methylol phenol.

On day 1, three intradermal injections were performed in a row on each side of the shoulder. 24 hours before the topical sensitization (day 7), all the animals were treated on the shoulder with a preparation consisting of sodium lauryl sulphate (SLS) 10% in dimethyl acetamide/acetone/ethanol. On day 8, topical sensitization on the same skin area was then performed with a solution of the suspected sensitizer. The patch was covered by an occlusive dressing for 48 hours. On day 22 (= Challenge I), a 24 -hour occluded patch was performed on the right flank with 25µl of TBC, 2 patches near the back. Challenge II was performed at the same time as challenge I but on the left side of the flank.

The concentrations used for induction phase were 3.40% (intradermal) and 16.7% (topical) and those used for challenge phase were 7.50% (challenge I) and 10% (challenge II).

In challenge I, 20/24 treated animals showed positive reactions. In challenge II, 18/24 treated animals showed positive reactions.

In this study, 4 -tert-butylpyrocatechol was a dermal sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Three studies relative to skin sensitization potential are available.

In an in vivo assay (Guinea Pig Maximization Test or GPMT) of reliability 2 according to the Klimisch criteria, selected as the key study, 4 -tert-butylpyrocatechol was a potent skin sensitizer. Following an induction phase (at 3.4% intradermal + 16.7% topical), challenge I using a 7.5% test concentration resulted in 83% (20/24) of animals showing positive skin reactions, and challenge II using a 10% test concentration resulted in 75% (18/24) of animals showing positive skin reactions. In an ex vivo method (LLNA) used as supportive study, of validity 1 according to Klimisch cotation criteria, positivity threshold of 3 was exceeded at all test concentrations (from 1% to 5 %) but irritation was observed at concentrations above 1%. The evaluation of sensitization potential could therefore be made only at the lowest concentration tested, 1%, for which a stimulation index of 15.35 and no signs of ear thickness increase was noted. The corrosive properties of the test substance have to be taken into consideration when evaluating the need for classification for skin sensitisation.

However, several well controlled studies in workers (see section 7.10.4.) have shown that 4 -tert-butylpyrocatechol exerts skin sensitization activity with a variable incidence. Some studies showing a low incidence of positive skin reactions among workers are inconclusive but other studies demonstrate that all individuals tested using a patch-test technique develop positive reactions.

4 -tert-butylpyrocatechol is therefore classified as a skin sensitizer according to the criteria of Annex VI Directive 67/548/EEC or UN/EU GHS, based on the results of the GPMT and the observations in humans.


Migrated from Short description of key information:
Positive in GPMT, equivocal in LLNA, but sensitizing in some human cases (workers) and therefore classified as skin sensitizer.

Justification for selection of skin sensitisation endpoint:
The study was a GLP-compliant OECD-guideline study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Respiratory sensitisation has not been formally assessed.

Justification for classification or non-classification

Based on the positive results in validated animal tests (GPMT and LLNA), 4 -tert-butylpyrocatechol is classified as a skin sensitizer.