9-Anthracenecarboxylic acid, (triethoxysilyl)methyl ester

Endpoint summary

Description of key information

Skin sensitisation: not sensitising (OECD 406; GLP)

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:

2002-12-09 to 2003-04-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Deviations from the OECD guideline 406 (1992): positive control (i.e., alpha-hexylcinnamicaldehyde) was evaluated after 24 hours as being sensitising (5/10 animals; 20 %), after 48 hours 2/10 animals showed a skin reaction; semi-occlusive dressing was used instead of occlusive dressing; during the induction phase; concentrations of the intradermal injections 2 and 3 shall be the same, but in this study injection 2 was 5 % of the test item and injection 3 was 10 % of the test item; a modified Magnusson and Kligman grading scale was used.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992-07-17

Deviations:

yes

Remarks:

please refer to the field "Rationale for reliablity incl. deficiencies" above

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

According to the REACH regluation (EC) No. 1907/2006, the LLNA test is the first-choice method for in vivo testing and in exceptional circumstances another test can be used. Since the study was carried out before the regulation entered into force and the guinea pig maximisation test according to OECD 406 is an acceptable method for testing skin sensitisation, it is not justified to conducted an LLNA test due to animal welfare in addition.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the freezer protected from light, under nitrogen

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 4 weeks old
- Weight at study initiation: test animals: 311 to 367 g; control animals: 326 - 358 g
- Housing: group hosuing of maximally 5 animals/cage (74 cm x 54 cm x 25 cm height); bedding material: purified sawdust (SAWI, Jelu Werk, Rosenberg, Germany)
- Diet (ad libitum): standard guinea pig diet, including ascorbic acid (1000 mg/kg)(Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Lage; Germany); Pressed hay (B.M.I., Helmond, The Netherlands) was provided twice a week.
- Water (ad libitum): tap water (200 mg/L vitamin C was added to the water )
- Acclimation period: at least 5 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Relative humidity: 30 - 70 %
- Air changes: approx. 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Concentration / amount:

1:1 w/w mixture of Freunds' complete adjuvant with water for injection

Day(s)/duration:

Day 0

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

5 % test item

Day(s)/duration:

Day 0

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

1:1 w/w mixture of 10 % of the test item and Freunds' Complete Adjuvant

Day(s)/duration:

Day 0

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

epicutaneous, semiocclusive

Vehicle:

corn oil

Concentration / amount:

20 % test item

Day(s)/duration:

Day 7

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

corn oil

Concentration / amount:

20 % test item

Day(s)/duration:

Day 21

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 (test substance) + 5 (controls)

Details on study design:

RANGE FINDING TESTS:
a) Interdermal injections:
A series of four test substance concentrations (2, 5, 10 and 20* %) was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two differen concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.

b) Epidermal application:
A series of four test substance concentrations (2, 5, 10, 20* %) was used, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2 x 3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage. The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations.
After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water.
The treated skin areas were assessed for irritation 24 and 48 hours after exposure.

Results:
Based on the results, the test substance concentrations selected tor the main study were a 5% concentration for the intradermal induction and a 20% concentration for the epidermal inductionexposure.
No signs of irritation were observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction.
A 20% test substance concentration was selected for the challenge phase.

*Trail formulations showed that a 20 % concentration was the highest concentration that could be prepared homogeneously to visually accpetable levels.
Injection of a 20 % concentration was difficult and the reliability of the injected volume could not be esablished. A 10 % test substance concentration was considered the highest concentration that could reproducibly be injected.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal injection and dermal application)
- Site: the scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area on day 1. On day 3, the dermal reactions caused by the intradermal injections were assessed for irritation. Then, the scapular area between the injection sites was clipped and subsequently rubbed with 10 % sodium-dodecyl-sulfate (SDS) in vaseline using a spatula on day 7. On day 8, the 10 % SDS treated area between the injection sites was treated with 0.5 mL of a 20 % test substance concentration using a Metalline patch (2 x 3cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal rections caused by the epidermal exposure were assessed for irritation.
- Concentrations:
Test animals:
Intradermal:
i) 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection
ii) the test substance in corn oil at a 5 % concentration
iii) a 1:1 w/w mixture of the test substance, at twice the concentration used in (ii) and Freunds' Complete adjuvant.

Control animals:
Intradermal injection:
i) 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection
ii) 0.1 mL corn oil
iii) a 1:1 w/w mixture of corn oil and Freunds' Complete adjuvant.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (two weeks after the topical application (induction))
- Exposure period: 24 hours
- Site (test animals and controls): one flank of all animals was clipped and treated by epidermal application of a 20% test substance concentration and the vehicle (0.1 mL each)
- Evaluation (hr after challenge): 24 and 48 hours

OTHER OBSERVATIONS:
- Mortality/Viability: twice daily
- Toxicity: at least once daily
- body weights: prior to start and at termination of the study
- skin reactions: skin reactions were graded according to the Draize scale, Furthermore, a description of all other (local) effects was recorded. Whenever necessary, the treated skin-areas were clipped at least 3 hours before the next skin reading to facilitate scoring.

Challenge controls:

5 guinea pigs were used as control animals.
Challenge dose: one flank of all animals was clipped and treated by epidermal application of a 20% test substance concentration and the vehicle (0.1 mL each)

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamicaldehyde, tech. 85 % (study conducted not more than 6 months prior to current study; 10 test & 5 control animals; intradermal (induction): 20 % sol. in H2O (w/w); topical (induction): undiluted; challenge: 20 % sol. in H2O (w/w))

Positive control results:

The skin reactions (grade 1 to 2; 24 hour reading) observed in five experimental animals in response to the 20 % test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 50 % to the 20 % concentration.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

20 % test item

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were evident after the challenge exposure. No mortality occurred & no clinical signs were observed in the animals of the main study. Body weights & body weight gain of experimental animals remained in the same range as controls.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

20 % test item

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were evident after the challenge exposure. No mortality occurred & no clinical signs were observed in the animals of the main study. Body weights & body weight gain of experimental animals remained in the same range as controls.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

20 % test item

No. with + reactions:

0

Total no. in group:

5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

20 % test item

No. with + reactions:

0

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20 % positive control

No. with + reactions:

5

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

20 % positive control

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

Some animals showed scaliness.

MAIN STUDY -INDUCTION

The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the Regulation (EC) 1272/2008 and subsequent adaptations, the substance is not sensitising to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation

The substance does not possess a skin sensitisation potential and does not require classification as skin sensitiser according to Regulation (EC) No 1272/2008 and subsequent adaptations.