Reaction mass of 3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-4-yl)propanal

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January to July 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

Taking effects as from 22nd September 1999, until 21st June 2000

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

At the time of study completion (2000), the LLNA OECD test method was not adopted.

Test material

Test material information

Constituent 1

Reference substance name:

3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Cas Number:

173445-65-3

Molecular formula:

C14H18O

IUPAC Name:

3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Constituent 2

Reference substance name:

3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Cas Number:

173445-44-8

Molecular formula:

C14H18O

IUPAC Name:

3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): ST 22 C 99
- Physical state: colourless liquid
- Storage condition of test material: at room temperature, protected from light and under nitrogen gas

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS: Hartley Crl: (HA) BR, Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS - VAF®)
- Source: Charles River France, 76410 Saint-Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 3 months old
- Weight at study initiation: M: 369 ± 20 g; F: 369 ± 12 g
- Housing: individually in polycarbonate cages equipped with polypropylene bottle.
- Diet (e.g. ad libitum): free access to "106 pelleted diet" (UAR, 91360 Villemoisson-sur-Orge, France). Contaminants-free.
- Water (e.g. ad libitum):drinking water filtered by a FG Millipore membrane (0.22 µm) ad libitum. Contaminants-free.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): approximately 12 cyles/hour of filtered non-recycled air.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2000-03-02 To: 2000-04-03

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

1 % w/w

Day(s)/duration:

Day 1

Adequacy of induction:

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

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% w/w

Day(s)/duration:

D8 / 48 hrs

Adequacy of induction:

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

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100 % w/w

Day(s)/duration:

D22 / 24 hrs

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

2 preliminary/sex, 10 treated/sex, 5 control/sex

Details on study design:

RANGE FINDING TESTS:
- Intradermal injections: 75, 50, 25, 10, 5 and 1 % (w/w); 2 animals/sex. The concentration chosen for the main study was 1% w/w (concentration well-tolerated systemically and locally, intradermal injections cause moderate irritant effects but no necrosis or ulceration of the skin).
- Topical application: 100 and 50 % (w/w); 1 animal / sex. The concentration chosen for the topical application of the induction phase and for the challenge application was 100% (concentration well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or to be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal injections + topical application
- Exposure period: 48 hours for topical application
- Test groups:
INTRADERMAL: 3 pairs of intradermal injection (0.1 mL) on Day 1 as follows:
- 1/ FCA 50% (v/v) in 0.9% NaCl
- 2/ test substance 1% in corn oil
- 3/ test substance 1% in 50:50 FCA/0.9% NaCl
TOPICAL: On Day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10% w/w), in order to induce local irritation. On Day 8, a pad of filter paper (approximately 8 cm²) was fully-loaded with the undiluted test substance and was then applied to the intrascapular region of the animals of the treated group. The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive analergenic waterproof plaster.
- Control group: similarly treated with the exception that corn oil was topically applied instead of the test substance.
- Site: intrascapular area
- Other: the presence of cutaneous irritation was checked on Day 10, after removal of the dressings of the cutaneous application.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 hours
- Test groups: The animals of treated group received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chamber®) was fully-loaded with the undiluted test substance and was then applied to a clipped area of the skin of the posterior right flank of all animals). The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. the chambers were held in contact with the skin for 24 hours by means of an adhesive analergenic waterproof plaster.
- Control group: similarly treated
- Site: right and left flank
- Concentrations: 100 %
- Evaluation (hr after challenge): approximately 48 and 72 hours (24 and 48 hours after removal of the dressing)

Challenge controls:

None

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole (recent reliability check)

Study design: in vivo (LLNA)

Positive control substance(s):

mercaptobenzothiazole (CAS No 149-30-4)

Results and discussion

Positive control results:

In a recent study (December 1999) performed under CIT experimental conditions, Mercaptobenzothiazole at the concentration of 20 % (w/w) induced positive skin sensitization in 80% guinea pigs, showing the sensitivity and reliability of the experimental technique.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

discrete erythema (score 1) in 8/20 animals, moderate erythema (score 2) in 5/20 animals

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

discrete erythema in 3/20 animals, moderate erythema in 7/20 animals, dryness of the skin in 14/20 animals and crusts in 1/20 animals

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

1 male with discrete erythema and dryness of the skin

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 1 male with discrete erythema and dryness of the skin.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20%

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

Yellow coloration. 2 animals with score 1, 6 with score 2, 2 with score 3

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 20%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: Yellow coloration. 2 animals with score 1, 6 with score 2, 2 with score 3 .

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

20%

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

Yellow coloration of the skin. 2 animals with score 0, 3 with score 2, other scorings masked by dryness of the skin

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 20%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: Yellow coloration of the skin. 2 animals with score 0, 3 with score 2, other scorings masked by dryness of the skin.

Any other information on results incl. tables

No clinical signs and no deaths were observed during the study. The body weight gain of the treated animals was similar to that of the control animals.

On Day 2, 24 hours after the intradermal injections and on Day 10, after removal of the dressings of the cutaneous application, signs of irritation were observed at the treatment site in the animals of the control and treated groups.

On removal of the challenge dressing, no residual test substance was observed.

In the control group, only a discrete (grade 1) erythema, associated with dryness of the skin, was observed at the 48 -hour reading in 1/10 animals.

In the treated group, a discrete erythema was noted in 8/20 animals at the 24 -hour reading and in 3/20 animals at the 48 -hour reading; a moderate erythema (score 2) was obseved in 5/20 animals at the 24 -hour reading and in 7/20 animals at the 48 -hour reading.

At the 48 -hour reading, dryness of the skin was observed in 14/20 animals and crusts were noted in 1/20 animals.

The moderate cutaneous reactions observed in 7/20 (35%) of animals of the treated group, which were of higher incidence and severity than those recorded in the animals of the control group, were attributed to delayed contact hypersensitivity.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on the overall sensitisation rate (35%), the test material is classified as skin sensitiser Category 1B according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a dermal sensitisation study performed according to the OECD test method No. 406 and in compliance with GLP, the test material was tested in male and female Hartley guinea-pigs using the Guinea-Pig Maximisation Test method (20 treated animals + 10 controls).

The preliminary study determined the concentration to be used for the induction and challenge phases of the main study.

The test material diluted in corn oil at 1 %( w/w) was administered by injection for intradermal induction. On Day 7, the animals received a topical application of sodium lauryl sulfate in vaseline (10% w/w), in order to induce local irritation. On Day 8, topical induction was performed with the test material as supplied. For the challenge, on Day 22, the test material was tested undiluted.

During a recent reliability check performed at CIT, 80% of the animals gave positive response to both challenge concentrations of the positive control, Mercaptobenzothiazole, at both the 48- and 72-hour examinations. The experimental technique used was therefore considered to be valid.

There were no responses apparent in the control group

No clinical signs and no deaths were noted during the study. The body weight gain of the treated animals was similar to that of the control animals.

After the challenge application, only a discrete (grade 1) erythema, associated with dryness of the skin, was observed at the 48 -hour reading in 1/10 animals in the control group.

In the treated group, a discrete erythema was noted in 8/20 animals at the 24 -hour reading and in 3/20 animals at the 48 -hour reading; a moderate erythema (score 2) was obseved in 5/20 animals at the 24 -hour reading and in 7/20 animals at the 48 -hour reading.

At the 48 -hour reading, dryness of the skin was observed in 14/20 animals and crusts were noted in 1/20 animals.

The moderate cutaneous reactions observed in 7/20 (35%) of animals of the treated group, which were of higher incidence and severity than those recorded in the animals of the control group, were attributed to delayed contact hypersensitivity.

The overall sensitisation rate was 35 %.

Based on the overall sensitisation rate, the test material is classified as skin sensitizer

- H317: May cause an allergic skin reaction (Sub-Category 1B) according of the Regulation EC No. 1272/2008 (CLP).

- Sub-Category 1B according to the GHS.

This study is considered as acceptable and satisfies the requirement for the skin sensitisation endpoint.