3,4-dihydro-2H-1,4-benzoxazin-6-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 19.5.94 to 25.8.94

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17th July 1992

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The LLNA method was not available yet by the time the study was conducted

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

EC Number:

247-415-5

EC Name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

Cas Number:

26021-57-8

Molecular formula:

C8H9NO2

IUPAC Name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: hydroxybenzomorpholine (A025) 2484. batch number: 0508918
- Purity, including information on contaminants, isomers, etc.:98.3%
- expiry date : september 2005

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at + 4°C, protected from light and under nitrogen gas
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: no data
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: the test item was prepared at the concentration of 2% (w/w) in the vehicle. Before preparation, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas, and kept under nitrogen atmosphere and were stored protected from light (using a glass beaker covered with aluminium foil) and under nitrogen atmosphere before use. they were used within the 6 hours following the preparation according to the known stability results.
The ph of the dosage form, measured at CIT, was approximately 7.5.2

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

species recommended by the international regulations for sensitization studies. The strain used has been shown to produce a satisfactory sensitization response using known positive sensitizers

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder - centre d'élevage lebeau, 78950 Gambais, France
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: no
- Age at study initiation: not specified
- Weight at study initiation: mean body weight 352 +/- 21 g
- Housing: on day 1, the animals were weighed and randomly allocated to 2 groups : a control group 1 consisting of 5 females and a treated group 2 consisting of 10 females. During acclimatizaion period, throughout the study, the animals were housed individually in polycarbonate cages (48x27x20) equipped with a polypropylene bottle. Sifted and dusted sawdust was provided as litter (SICSA, 92142 Alfortville, France). An analysis of potential residues and major contaminants is performed periodically (Laboratoire Wolff, 92110 Clichy, France)
- Diet (e.g. ad libitum): during the study the animals had free access to "guinea pig sustenance reference 106 diet" (U.A.R., 91360 VILLEMOISSON-sur-Orge, France). Food was periodically analysed (composition and contaminants) by the supplier.
- Water (e.g. ad libitum): drinking water filtered by a F.G. millipore membrane (0.22 micron) was contained in bottles.
- Acclimation period: at least 5 days before the beginning of the study
- Indication of any skin lesions: the animals were identified individually by an ear-tattoo

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 ° C
- Humidity (%): 30 to 70%
- Air changes (per hr): non-recycled and filtered
- Photoperiod (hrs dark / hrs light): 12h/12h
- IN-LIFE DATES: From: 19.05.1994 To: 20.06.1994

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: NaCl 0.9%

Concentration / amount:

6 intradermal injections were made into a clipper area (4cm x 2cm) in the scapular region, using a needle (diameter: 0.50 x16 mm, Terumo: C.M.L., 77140 Nemours, France) mounted on a 1 mL glass syringe (0.01 mL graduations, record: Carrieri, 7505 Paris, France) . Three injections of 0.1 mL were injected into each side of the animal, as follow:
control group:
- FCA diluted 50% (v/v) with an injectable isotonic solution (NaCl 0.9%),
- vehicle,
- a mixture of 50/50 (w/v) FCA diluted to 50% (v/v) with a sterile isotonic aqueous NaCl solution and the vehicle.
treated group:
- FCA diluted to 50% (v/v) with a sterile isotonic aqueous NaCl solution,
- test substance at a concentration of 1% (w/w) in the vehicle,
- a mixture 50/50 (w/v) of FCA diluted to 50% (v/v) with a sterile isotonic aqueous NaCl solution, and, the test substance at a concentration of 1% (w/w) in the vehicle.

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:

other: NaCl 0.9%

Concentration / amount:

On day 7, the scapular area was clipped. As the test substance is shown to be non-irritant after occlusive cutaneous treatment during preliminary test, the animals were treated with 0.5 mL of sodium laurylsulphate (10%) in vaseline to provoke irritation. On day 8, a cutaneous application on the 6 injection areas (4cm x 2 cm) of the scapular region was performed.
control group:
application of 0.5 mL of the vehicle
treated group:
application of 0.5 mL of a non-irritant concentration of the test substance i.e. at 25% (w/w) in the vehicle.
the test substance and the vehicle were prepared on a dry compress (Semes France, 54183 Heillecourt, France), which was then applied to the scapular region and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de pansements et d'hygiène, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Médicales, 92240 Malakoff, France). On removal of the dressing, any residual test substance was removed by means of a compress moistened using water for injections.

Day(s)/duration:

Day 7-8

Adequacy of induction:

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

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: NaCl

Concentration / amount:

Maximum Non Irritant Concentration (MNIC) 25%in the vehicle.
The animals form both groups received an application of 0.5 mL of the MNIC of test substance on the posteriori right flank, and 0.5 mL of the vehicle on the posterior left flank. This application was performed using a 1mL plastic syringe (0.01 mL graduations, Terumo: CML, 77140 Nemours, France) . The test substance and vehicle were prepared on a dry compress , the applied to a 4 cm² clipped area of the skin. The compress was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster. On removal of the dressing, any residual test substance was removed by means of a compress moistened using water for injections.

Day(s)/duration:

day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Preliminary study: 2 animals
Main study: 5 animals in control group and 10 animals in treated group

Details on study design:

RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: on day 1, 6 intradermal injections were made into a clipped area (4 cm x 2 cm) in the scapular region, using a needle (diameter: 0.50 x 16 mm, Terumo: C.M.L., 77140 Nemours, France) mounted on a 1 mL glass syringe
(0.01 mL graduations, record : carrieri, 75005 Paris, France).
Three injections of 0.1 mL were injected into each side of the animal, as follows:

control group :
- Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous Na Cl solution and the vehicle
- vehicle
- a mixture of 50/50 (w/v) Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCl solution and the vehicle.

treated group
- freund's complete adjuvant diluted to 50% (v/v) with a sterile aqueous Na Cl solution
- test substance at a concentration of 1% (w/w) in the vehicle
- a mixture 50/50 (w/v) Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCl solution, and, the test substance at a concentration of 1% (w/w) in the vehicle.

On day 7, the scapular area was clipped. As the test substance is shown to be non-irritant after occlusive cutaneous treatment during preliminary test, the animals were treated with 0.5 mL of sodium laurylsulphate (10%) in vaseline to provoke irritation. On day 8, a cutaneous application on the 6 injection areas (4cm x 2 cm) of the scapular region was performed.
control group:
application of 0.5 mL of the vehicle
treated group:
application of 0.5 mL of a non-irritant concentration of the test substance i.e. at 25% (w/w) in the vehicle.
the test substance and the vehicle were prepared on a dry compress (Semes France, 54183 Heillecourt, France), which was then applied to the scapular region and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de pansements et d'hygiène, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Médicales, 92240 Malakoff, France). On removal of the dressing, any residual test substance was removed by means of a compress moistened using water for injections.
one hour after removal of the occlusive dressing, cutaneous reactions were recorded.

- Site: for all animals and before each treatment, the application sites were clipped on days -1 and 7 (scapular area 4 cm x 2 cm), clipped again and shaved on da 21 (each flank 2 cm x 2 cm).


B. CHALLENGE EXPOSURE
At the end of the rest period on day 22, the test substance was applied at the Maximum Non Irritant Concentration (M.N.I.C.) at a concentration of 25% (w/w) in the vehicle.
The animals from both groups received an application of 0.5 mL of the MNIC of test substance on the posteriori right flank, and 0.5 mL of the vehicle on the posterior left flank. This application was performed using a 1mL plastic syringe (0.01 mL graduations, Terumo: CML, 77140 Nemours, France) . The test substance and vehicle were prepared on a dry compress , the applied to a 4 cm² clipped area of the skin. The compress was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster. On removal of the dressing, any residual test substance was removed by means of a compress moistened using water for injections.

Challenge controls:

control group received the test substance on day 22 at the concentration of 25%

Positive control substance(s):

yes

Remarks:

the sensitivity of Dunkin-Hartley guinea-pigs to a positive control dinitro 2,4 chlorobenzene was checked in January 1994 on 5 female at a concentration of 0.5%. it induced a positive skin sensitization reactions in 100% of the guinea pigs.

Results and discussion

Positive control results:

the sensitivity of Dunkin-Hartley guinea-pigs to a positive control dinitro 2,4 chlorobenzene was checked in January 1994 on 5 female at a concentration of 0.5%. it induced a positive skin sensitization reactions in 100% of the guinea pigs.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.05%

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

erythema, dryness of the skin

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

positive control

Dose level:

0.05%

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

erythema, dryness of the skin, crust

Results 3

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

beige coloration of the skin in 10 animals

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

beige coloration of the skin in 4 animals

Results 5

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

beige coloration of the skin in 3 animals

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

beige coloration of the skin in 3 animals

Any other information on results incl. tables

Individual body weight values (g)

			days
groups	sex	animals	-1	1	(1)	8	(1)	15	(1)	25
1	female	116	342	357	85	442	40	482	89	571
		117	356	355	4	359	63	422	56	478
		118	360	367	52	419	62	479	76	555
		119	372	372	63	435	38	473	53	526
		120	366	372	65	437	59	496	46	542
		M	359	365	54	418	52	470	64	534
		SD	11	8	30	34	12	28	18	36
2	female	121	313	323	62	385	46	431	61	492
		122	344	343	72	415	49	464	-9	455
		123	319	320	73	393	72	465	49	514
		124	320	309	31	340	52	392	48	440
		125	355	365	72	437	64	501	54	555
		126	348	343	57	400	60	460	54	514
		127	326	321	67	388	44	432	64	496
		128	334	341	52	393	63	456	80	536
		129	363	354	71	425	32	457	93	550
		130	358	350	64	414	45	459	53	512
		M	338	337	62	399	53	452	55	506
		SD	18	18	13	27	12	28	27	37

(1)  = body weight gain

M = mean

 SD = Standard Deviation

 

Macroscopic examination of cutaneous reactions – challenge application

 

			Day 24 scoring period (after 24 hours)				Day 25 scoring period (after 48 hours)
			Erythema		Oedema		Erythema		Oedema
Group	Sex	Animals	LF	RF	LF	RF	LF	RF	LF	RF
Control 1	Female	116	0	0/C	0	0	0	0/C	0	0
		117	0	0	0	0	0		0	0
		118	0	0	0	0	0		0	0
		119	0	0/C	0	0	0	0/C	0	0
		120	0	0/C	0	0	0	0/C	0	0
Treated 2	Female	121	0	0	0	0	0	0	0	0
		122	0	0	0	0	0	0	0	0
		123	0	0/C	0	0	0	0	0	0
		124	0	0	0	0	0	0	0	0
		125	0	0/C	0	0	0	0/C	0	0
		126	0	0	0	0	0	0	0	0
		127	0	0/C	0	0	0	0/C	0	0
		128	0	0/C	0	0	0	0	0	0
		129	0	0/C	0	0	0	0/C	0	0
		130	0	0/C	0	0	0	0/C	0	0

 

LF : left flank (control)

RF : right flank (treated)

C : beige colouration of the skin

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, hydroxybenzomorpholine was non-sensitising.

Executive summary:

The dilutions of IMEXINE OV used were selected on the basis of the results from preliminary
intra-dermal and topical experiments in which:
• 1% (w/w) hydroxybenzomorpholine in paraffin oil was the maximum practicable
concentration for intra-dermal injection; only slight irritation was observed at this
concentration;
• 25% (w/w) hydroxybenzomorpholine in paraffin oil was the maximum practicable
concentration for topical induction and challenge and it did not provoke any signs of irritation.

The application sites were clipped free of fur before each induction or challenge treatment. The induction procedure consisted of intra-dermal injections and one topical application, 7-day apart.
On a day designated as day 1, the animals received 3 pairs of intra-dermal injections in the scapular area, consisting of:
• 50% (v/v) Freund’s Complete Adjuvant in physiological saline (FCA, control and treated animals)
• hydroxybenzomorpholine at 0% or 1% (w/w) in paraffin oil (control and treated animals, respectively)
• a 50/50 (w/v) mixture of FCA and 0% or 1% hydroxybenzomorpholine (w/w) in paraffin oil (control and treated animals, respectively)
As hydroxybenzomorpholine at 25% (w/w) in paraffin oil (maximum practicable concentration) was non-irritating, the animals were treated on day 7 with 0.5 ml sodium lauryl sulphate in petrolatum (10%) to elicit local irritation. On day 8, 0.5 ml of hydroxybenzomorpholine at 0 or 25% (w/w) in paraffin oil was prepared on a dry compress and applied over the intra-dermal injection sites in control and treated animals, respectively. The compress was held under occlusion for 48 hours by means of surgical tape and elastic adhesive bandage (topical induction). Two weeks after the topical induction (day 22), 0.5 ml of hydroxybenzomorpholine
at 0 or 25% (w/w) in paraffin oil was prepared on a dry compress and applied to a 4 cm2 area to all control and treated animals on their left and right flanks, respectively (challenge topical application). These patches were kept for 24 hours under occlusion. The animals were examined and cutaneous reactions were assessed 24 and 48 hours after dressing removal.
Results
No skin reactions were observed at the vehicle (paraffin oil) or hydroxybenzomorpholine challenge application sites in control or treated animals, at any examination time-point.

Conclusion
Under the conditions of the present study, hydroxybenzomorpholine was non-sensitising.