(4aR*, 8aR*)-1-bromo-4a,5,9,10,11,12-hexahydro-3-methoxy-6H-benzofuro[3a,3,2-ef] [2]benzazepin-6-one hydrochloride (1:1)

Endpoint summary

Description of key information

In a guinea pig maximisation test according to OECD Guideline 406 and EC method B.6, the substance is observed to be sensitising to the skin (Arcelin G., 2001). Another guinea pig maximisation test was performed according to the same guidelines (Arcelin G., 1998), concluding on the absence of skin sensitization potential of the substance. However, due to the lack of solubility of T002102 in water, the substance was finally considered moderate skin sensitizer based on the results of the first challenge.

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:

2001-06-27 to 2001-08-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from 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.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT002102G4A311
- Expiration date of the lot/batch: 25 March 2002 (retest date)
- Purity: 99.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container, at room temperature (range 20 ± 3°C), away from direct sunlight
- Solubility and stability of the test substance in the solvent/vehicle: Stable at 20% (w/v) in PEG 300 for at least 7 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item and vehicle or auxillary compound were placed into a glass beaker on a tared Mettler PM 460 balance and a weight dilution was prepared. Homogeneity of the test item preparation was ensured and maintained during treatment using a magnetic stirrer and/or Ultra-Turrax. The preparations were made immediately prior to dosing.

Species:

guinea pig

Strain:

other: Ibm: GOHI guinea pigs (synonym: Himalayan spotted)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: SPF-Quality, RCC Ltd., Biotechnology & Animal Breeding Division, Switzerland
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 4-6 weeks at pretest start/beginning of acclimatization period, 5-7 weeks at main study start
- Weight at study initiation: 346-362 g (pretest group) at pretest start, 308-380 g (control and test groups) at beginning of acclimatization period.
- Housing: individually in Makrolon type-4 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418, batch nos. 90/01 and 91/01, guinea pig breeding/maintenance diet, containing Vitamin C, ad libitum
- Water (e.g. ad libitum): Community tap water from Fűllinsdorf, 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 for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): approx 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light ):12 hours light and 12 hours dark. Music was played during the light period

Route:

intradermal

Vehicle:

polyethylene glycol

Concentration / amount:

volume: 0.1 mL/site
test group: 5% test item in PEG 300 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 concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Concentration / amount:

concentration: 75% 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

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

volume: 0.2 g
test item: 75%: applied to the left flank
vehicle only (bi-distilled) 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:
- Intradermal injections: 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 A=10%, B=5%, and C=3% of the test substance in PEG 300. The three concentrations were determined during non-GLP formulation trials performed prior to the pretest. Dermal reactions were assessed 24 hours later. Based on the results, the test substance concentration of 10% was selected for intradermal induction in the main study.
-Epidermal application: 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 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 (3 x 3 cm) were saturated with the test substance at D=50%, E=25%, F=15%, and G=10% in PEG 300 and applied to the clipped and shaved flanks. The volume of the test substance preparation applied was approximately 0.2 mL. 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. The dressings were removed after an exposure period of 24 hours. Twenty-four hours after removal of the dressing, the application site was depilated with an approved depilatory cream for 3-5 minutes in order to visualize any resulting erythema. The cream was then thoroughly washed off with water. The reaction sites were assessed 24 and 48 hours after removal of the bandage according to the method of Magnusson and Kligman. Based on the results obtained, the concentration selected for induction and challenge in the main study was 50% and 15%, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Intradermal injections were performed on test Day 1; Epidermal applications were performed on test Day 8.
- Test groups:
intradermal injections (day1):
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline,
2) the test substance at 10% in PEG 300, and
3) The test substance at 10% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
epidermal applications (day 8)
One week after the injection, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to application. A 2 x 4 cm patch of filter paper was saturated with approximately 0.3 mL of the test substance (50% in PEG 300) and placed over the injection sites of the test animals. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of animal and secured with impervious adhesive tap. The occlusive dressing was left in place for 48 hours. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.
- Control group: Three pairs of intradermal injections (0.1 mL/site) as follows:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline,
2) PEG 300, and
3) 1:1 (v/v) mixture of PEG 300 in a 1:1 (v/v) mixture Freund's Complete Adjuvant and physiological saline. For the epidermal exposure, approximately 0.3 mL of PEG 300 was applied.
- Site: Intradermal injection: an area of dorsal skin from the scapular region; epidermal induction: scapular area.
- Frequency of applications: one time intradermal, one time epidermal
- Duration: one time intradermal injection, and 48 hours for epidermal exposure
- Concentrations: 10% for intradermal injection; 50% for epidermal application

B. CHALLENGE EXPOSURE
- No. of exposures: one exposure
- Day(s) of challenge: Test Day 22
- Exposure period: 24 hours
- Test groups: Test substance (15% in PEG 300; 0.3 mL) was applied to the shaved left flank. 21 hours after removal of the dressing the test sites treated with the test substance were depilated as described in the epidermal pretest.
- Control group: PEG 300 (0.3 mL) was applied to the right flank.
- Site: left (test item) and right (control group) flank
- Concentrations: 15%
- Evaluation (hr after challenge): The reaction sites were assessed 24- and 48-hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.
The scoring system was performed by visual scoring of erythema, oedema and other clinical changes of skin conditions. They were assessed using the following Magnusson and Kligman grading scale:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
Grading of all animals was done by positioning the animal under true-light.

OTHER:
Rating of allergenicity according to Magnusson and Kligman
Based on the percentage of animals sensitized (24- and 48-hour readings), the test substance was assigned to one of five grades of allergenic potency according to Magnusson and Kligman.

Challenge controls:

A patch of filter paper saturated with the vehicle only (PEG 300) was applied to the right flank.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

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.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

15% in PEG 300 (left flank)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

no signs of systemic toxicity were observed in the animals

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15% in PEG 300 (left flank)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

no signs of systemic toxicity were observed in the animals

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

PEG 300 (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no signs of systemic toxicity were observed in the animals

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

PEG 300 (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no signs of systemic toxicity were observed in the animals

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.5 % in mineral oil (left flank)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

no toxic symptoms were evident in the guinea pigs

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

28

Group:

positive control

Dose level:

0.5 % in mineral oil (left flank)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

no toxic symptoms were evident in the guinea pigs

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

Mineral oil only (right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no toxic symptoms were evident in the guinea pigs

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

Mineral oil only (right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No toxic symptoms were evident in the guinea pigs

Remarks on result:

no indication of skin sensitisation

Skin effects after intradermal induction - performed on test day 1:

The expected and common findings were observed in the control and test groups after the different applications using FCA intradermally and consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

Skin effects after epidermal induction - performed on test day 8:

No erythematous or oedematous reactions were observed in the animals treated with PEG 300 only in the control group. Discrete/patchy erythema was observed in all animals at the 24 -hour reading, and in 9 out of 10 animals at the 48 -hour reading after treatment with the test substance at 50% in PEG 300 in the test group.

Skin effects after the challenge - performed on test day 22:

No skin reactions were observed in the animals when treated with either PEG 300 only, or when treated with the test substance at 15% in PEG 300. In the test group, discrete/patchy erythema was observed in all animals at the 24- and 48 -hour readings after treatment with the test substance at 15% in PEG 300, and no skin reactions were observed in the animals when treated with PEG 300 only.

There were no deaths during the course of the study. No signs of systemic toxicity were observed in any animals. The body weights of the animals were within the normal range for animals of this strain and age.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the above mentioned findings in an adjuvant sensitization test in guinea pigs and in accordance to Commission Directive 96/54/EEC, the test substance does have to be classified and labelled as an extreme skin sensitizer. With a response of > 30% at >1% intradermal induction dose, the substance is classified as a skin sensitiser category 1B according to the criteria of the CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In order to assess the cutaneous allergenic potential of the substance, two Guinea Pig Maximization Tests were performed in 15 (10 test and 5 control) female albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6 (Arcelin G., 2001 and Arcelin G., 1998).

In a key K1 GLP compliant study (RCC), the intradermal induction of sensitization was conducted as follows in the test group: three pairs of intradermal injections (0.1 mL/site) as follows: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline, 2) the test substance at 10% in PEG 300, and 3) the test substance at 10% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.The intradermal induction of sensitization was conducted as follows in the control group:

three pairs of intradermal injections (0.1 mL/site) as follows: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline, 2) PEG 300, and 3) 1:1 (v/v) mixture of PEG 300 in a 1:1 (v/v) mixture Freund's Complete Adjuvant and physiological saline. For the epidermal exposure, approximately 0.3 mL of PEG 300 was applied.The epidermal induction of sensitization was conducted for 48 hours under occlusion with 0.3 mL of the substance at 50 % in PEG 300 one week after the intradermal induction.Two weeks after epidermal induction the test and control animals were challenged by epidermal application of the test item at 15 % in PEG 300 and PEG 300 alone under occlusive dressing. After the intradermal induction, the expected and common findings were observed in the control and test groups after the different applications using FCA intradermally and consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation. After the epidermal induction, no erythematous or oedematous reactions were observed in the animals treated with PEG 300 only in the control group. Discrete/patchy erythema was observed in all animals at the 24 -hour reading, and in 9 out of 10 animals at the 48 -hour reading after treatment with the test substance at 50% in PEG 200 in the test group. After the challenge, in the control group, no skin reactions were observed in the animals when treated with either PEG300 only, or when treated with the test substance at 15% in PEG 300. In the test group, discrete/patchy erythema was observed in all animals at the 24- and 48 -hour readings after treatment with the test substance at 15% in PEG300, and no skin reactions were observed in the animals when treated with PEG 300 only.

There were no deaths during the course of the study. No signs of systemic toxicity were observed in any animals. The body weights of the animals were within the normal range for animals of this strain and age.

In the second guinea pig maximisation test study performed (Arcelin, 1998), the intradermal induction of sensitization was performed with a 5% dilution of the test article in bi-distilled water and in an emulsion of Freund's Complete Adjuvent (FCA)/physiological saline. The epidermal induction of sensitization was conducted under occlusion with the test article at 50% in bi-distilled water. Two weeks after the epidermal induction application, the challenge was completed by epidermal application of the test article at 50% in bi-distilled water under occlusive dressing. The animals of the control group were induced with bi-distilled water and FCA/physiological saline and challenged similarly to those of the test group. A second challenge was also performed 9 days after the first challenge using the same test animals and the same concentration of 50% in bi-distilled water. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal of the dressing.

After the intradermal induction, the expected local symptoms were observed in the animals of the control and test group after intradermal injections.

After the epidermal induction, very slight erythematous reaction was observed at the 24- and 48- hour reading in 3 out of 5 animals treated with bi-distilled water in the control group. In the test group, very sligth erythematous reaction was observed at the 24- and 48- hour reading in 6 out of 10 animals treated with the test article at 50 % in bi-distilled water.

After the challenge, no positive reactions were observed in the positive control animals either when treated with bi-distilled water only or when treated with the test article at 50% in bi-distilled water. Regarding the test groups, skin reactions were observed in 3 out of 10 animals when treated with the test article at 50% in bi-distilled water, at the 48 -hour reading. Following the 2nd challenge, no positive reactions were observed in the animals when treated with the test article at 50% in bi-distilled water.

Additionnal expert statement based on deviant conclusions on the two reports

T002102 was evaluated for its sensitization potential using the maximization test described by Magnusson et al. Intradermal and epicutaneous induction followed by epicutaneous challenge resulted in 30% positives when T002102 was tested as a watery formulation and in 100% positives when a PEG 300 formulation was employed. Although a second challenge with the watery formulation demonstrated no further positive reactions, the data of the study with PEG 300 as a vehicle confirmed the potential of T002102 to induce a dermal reaction after epidermal challenge with a non-irritating dilution.

Based upon these results it is therefore concluded that T002102 has a sensitization potential. The sensitization potential of the T002102 formulations according to the number of positive animals (Kligman classification) revealed a classification of moderate sensitizer (with the watery formulation; first challenge) to extreme sensitizer (with the PEG 300 formulation).

None of the vehicles showed dermal irritation or signs of sensitization potential as evidenced by the negative results of the vehicle-induced groups at challenge.

Justification for selection of skin sensitisation endpoint:
Only one reliable key study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the guinea pig maximisation test and the criteria of the CLP Regulation as skin sensitizer category 1B (H317). The substance is classified as it was determined in the guinea pig maximisation test that 100 % of the test animals responded at > 1 % intradermal induction dose.