2-bromo-5-hydroxy-4-methoxybenzaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-12-23 to 1998-04-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

: Certificate issued by the Swiss GLP monitoring authorities.

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Murine Local Lymph Node Assay (LLNA) is the first-choice method for in vivo testing according to the REACH Regulation. However, this reliable GPMT test was performed before entry into force of the REACH Regulation.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

426-540-0

EC Name:

-

Cas Number:

2973-59-3

Molecular formula:

C8 H7 Br O3

IUPAC Name:

2-bromo-5-hydroxy-4-methoxybenzaldehyde

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00265786
- Expiration date of the lot/batch: 01 -JULY- 1998 (Retest date)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container at room temperature (approx. 20 °C), away from direct sunlight.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test article and vehicle were placed into a glass beaker on a tared Mettler PM 460 balance and a weight by weight dilution was prepared. Homogeneity of the test article in a suitable vehicle was maintained during treatment using a magnetic stirrer. The preparations were
made immediately prior to each dosing.

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Ibm: GOHI; SPF-quailty guinea ppigs (Himalayan spotted)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. (Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland)
- Age at study initiation: 4-6 weeks (at pretest/beginning of acclimation period)
- Weight at study initiation: 331-469 g (at beginning of acclimation period)
- Housing: individually in Makrolon type-4 cages with standard softwood bedding
- Diet: pelleted standard Nafag Ecosan 845 25W4, Batch nos. 102/97 and 112/97 guinea pig breeding / maintenance diet, ad libitum
- Water: community tap water from Füllinsdorf, ad libitum
- Acclimation period: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12, music was played during the light cycle

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

polyethylene glycol

Concentration / amount:

volume: 0.1 mL/site
test group: 5% test item in PEG 400 or 5% test item in a 1/1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline

Day(s)/duration:

Day 1

Adequacy of induction:

highest technically applicable concentration used

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG400

Concentration / amount:

concentration: 50% test item in bi-distilled water
volume applied: 0.3 mL

Day(s)/duration:

Day 8- 48 Hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

volume: 0.2 g
test item: 15%: applied to the left flank
vehicle only (PEG 400) applied to the right flank

Day(s)/duration:

Day 22 - 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

1 for intradermal pretest, 2 for epidermal pretest, 5 for control group, 10 for the test group.

Details on study design:

RANGE FINDING TESTS:
In a preliminary test, the maximum tolerated concentration of the test substance suitable for the induction phase of the main study was determined on 2 guinea pigs. In addition, a suitable non-irritant concentration of the test substance, by the topical route of administration, was identified for the challenge application. To accomplish these goals, both intradermal injections and epicutaneous applications were made.

Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund’s Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later, intradermal injections (0.1 mL/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1 % in PEG 400. The resulting dermal reactions were assessed 24 hours later. For intradermal induction application in the main study, a 5 % test substance concentration was selected.

Four epidermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund’s Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later, both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter, 4 patches of filter paper (2 x 2 cm) were saturated with the test substance at Site A = 50 % (this concentration was found to be the most qualified to assure an optimum technical application procedure), Site B = 25 %, Site C = 15 % and Site D = 10 % in PEG 400 and applied to the clipped and shaved flanks. The volume of test substance was approximately 0.2 g for the concentrations of 50 % and 25%, and 0.2 mL for the remaining concentrations. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test substance. The dressings were removed after an exposure period of 24 hours.

Approximately 21 hours after removal of the dressing, the application site was depilated with an approved depilatory cream to clean the application site, so that possible erythema reactions were clearly visible at that time. The depilatory cream was placed on the patch sites and surrounding areas and left on for 3 -5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.

The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and edema on a numerical basis according to the Draize scale. The allocation of the different test dilutions to the sites (A, B, C, D) on the animals was alternated in order to minimize site-to-site variation in responsiveness. The concentrations selected for the induction period and challenge procedure were 50 % and 15 %, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: There were two exposures. On Day 1, intradermal injections were given, and on Day 8 epicutaneous applications were applied.
- Exposure period: not applicable
- Test groups: On Day 1, test groups received three intradermal injections (0.1 mL/site) made at the border of a 4 x 6 cm area in the clipped region:
1) 1:1 (v/v) mixture of Freund’s Complete Adjuvant and physiological saline;
2) the test substance, diluted to 5% with PEG 400;
3) the test substance diluted to 5% by emulsion in a 1:1 (v/v) mixture of Freund’s Complete Adjuvant and physiological saline

On test day 8, a 2 x 4 cm patch of filter paper was saturated with the test substance (50 % in PEG 400) and placed between the injection sites of the test animals. The volume of test substance applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test substance. Reaction sites were assessed for erythema and edema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

- Control group: The control animals received three intradermal injections (0.1 mL/site) made at the border of a 4 x 6 cm area in the clipped region:
1) 1:1 (v/v) mixture of Freund’s Complete Adjuvant and physiological saline;
2) PEG 400;
3) 1:1 (w/w) mixture of PEG 400 in a 1:1 (v/v) mixture of Freund’s Complete Adjuvant and physiological saline.

The guinea pigs of the control group were treated as the test animals, except the test substance was replaced by PEG 400 only. The volume applied was approximately 0.3 mL. Reaction sites were assessed for erythema and edema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

- Site: an area of dorsal skin from the scapular region (approx. 6 x 8 cm)
- Frequency of applications: 2 induction applications, made 7 days apart
- Duration: not applicable (intradermal); 48 hours (epicutaneous)
- Concentrations: 5% (intradermal); 50% (epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22 (2 weeks after the epicutaneous induction application)
- Exposure period: 24 hours
- Test groups: Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 15% (left flank) and the vehicle only (PEG 400 applied to the right flank) using the same method as for the epidermal application. The volume of test substance applied was approximately 0.2 mL. The dressings were left in place for 24 hours. Approximately 21 hours after removal of the dressing, the test sites treated with the test substance were depilated. The depilatory cream was placed on the patch sites and surrounding areas and left on for 3 -5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.
- Control group: Treated in the same manner as the test animals.
- Site: the left and right flanks
- Concentrations: 15% (left flank); vehicle only (right flank)
- Evaluation (hr after challenge): Approximately 24- and 48-hours after the removal of the dressing, the application sites were assessed for erythema and edema under artificial fluorescent light (daylight spectrum). The numerical scoring system according to Draize was used. Based upon the percentage of animals sensitized (24- and 48-hour reading), the test substance was assigned an allergenic potency classification according to the Magnusson and Kligman classification scheme. A response of at least 30% positive animals is considered positive “R43”: may cause sensitization by skin contact, per EEC Commission Directive 96/54/EEC, July 30, 1996.

Challenge controls:

Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 15 % (left flank) and the vehicle only (PEG 400 applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 ml.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

Results and discussion

Positive control results:

The study was performed with 15 (10 test and 5 control) male albino guinea pigs. The intradermal induction of sensitization in the test group was performed in the nuchal region with a 5% dilution of the test item in mineral oil and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of skin sensitization was conducted for 48 hours under occlusion with the test item at 50% in mineral oil one week after the intradermal induction. The animals of the control group were intradermally induced with mineral oil and FCA/physiological saline and epidermally induced with mineral oil under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 0.5% in mineral oil and mineral oil alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing. No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.
All 10 test animals showed discrete/patchy to intense erythema and swelling at the 24-and 48-hour reading after the challenge treatment with 2-mercaptobenzothiazole at 0.5% (w/w) in mineral oil.
No skin effect was observed in the control group.
Based on these findings, 2-mercaptobenzothiazole has to be classified and labelled as a skin sensitizer.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0% in PEG 400

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0% in PEG 400

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

15% in PEG 400

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Remarks on result:

positive indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15% in PEG 400

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Remarks on result:

positive indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

2-mercaptobenzothiazole at 0.5% (w/w) in mineral oil

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

No toxic symptoms were evident. No deaths occurred.

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

2-mercaptobenzothiazole at 0.5% (w/w) in mineral oil

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

No toxic symptoms were evident. No deaths occurred

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Following the Day 1 intradermal injections, the expected local symptoms were observed in the animals of the control and test group after the intradermal injections.

The Day 8 epicutaneous application of the test substance stained the skin brown; therefore, it was not possible to determine whether erythema was present or not. However, no edema was observed when treated with the test substance at 50% in PEG 400. There were no erythematous or edematous reactions in the control animals following the Day 8 applications.

Folliwng the Challenge on Day 22, slight to moderate erythematous reactions were observed in 8/10 and 7/10 animals at the 24- and 48-hour readings, respectively, when treated with the test substance at 15% in PEG 400. In the control group, no positive reactions were observed in the animals either when treated with PEG 400 only, or when treated with the test substance at 15% in PEG 400.

As there were no deaths during the course of the treatment period, no necropsies were performed. No symptoms of systemic toxicity were observed in the animals. One animal of the epidermal pretest lost weight during its treatment period. The body weight of the other animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study 80 % of the animals of the test group were observed with slight to well defined erythematous reactions after treatment with a non-irritant test article concentration of 15 % in PEG 400. No skin reactions were observed in the control group.

A response of at least 30% positive animals is considered positive. Based on these results and according to the criteria of the CLP Regulation, T002019 applied at a concentration of 15% in PEG 400 is considered to be a sensitizer category 1 when used under the described test conditions.