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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-07-10 to 1990-08-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
June 1989
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig test was performed in 1989. At that time, a LLNA guideline was not available.

Test material

Constituent 1
Chemical structure
Reference substance name:
4H-3,1-benzoxazine-2,4(1H)-dione
EC Number:
204-255-0
EC Name:
4H-3,1-benzoxazine-2,4(1H)-dione
Cas Number:
118-48-9
Molecular formula:
C8H5NO3
IUPAC Name:
4H-3,1-benzoxazine-2,4(1H)-dione
Details on test material:
- Name of test material (as cited in study report): Isatosäureanhydrid (Isatoic anhydride)
- Physical state: solid (powder, beige-brown)
- Analytical purity: 99.81% by HPLC (non-GLP)
- Impurities (identity and concentrations): no data
- Lot/batch No.: 05-0617
- Expiration date of the lot/batch: no data
- Stability under test conditions: yes
- Storage condition of test material: room temperature
No further data

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, Hagemann GmbH & Co . KG, D-4923 Extertal 1, FRG
- Age at study initiation: no data
- Weight at study initiation: 270 - 326 g
- Housing: 5 per cage (Makrolon, type I V)
- Diet (ad libitum): Kliba 341.4 mm (Kaninchen-Meerschweinchen-Haltungsdiät), Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland
- Water (ad libitum): tap water, about 2 g of ascorbic acid per 10 L water was added to the drinking water twice a week
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
5% intradermal induction
25 % epicutanoeus induction (occlusive)
10% challenge (occlusive)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
5% intradermal induction
25 % epicutanoeus induction (occlusive)
10% challenge (occlusive)
No. of animals per dose:
10 (test group)
5 (control groups 1 and 2)
Details on study design:
RANGE FINDING TESTS:
- Amount applied: 2 x 2 cm filter paper strips containing the test material were applied to the skin of the flanks under an occlusive dressing. The animals were exposed to about 0.15 g of the test substance formulation.
- Exposure period: The test substance was applied 2 times for 24 hours within a period of 96 hours in order to detect non-specific phenomena that are not caused by a sensitization reaction but could possibly be attributed to a shift in the irritation threshold.
- Site of application: flank, respective on the same area
- Number of test animals: 4 per test concentration
- Readings: about 24 and 48 h after the beginning of application

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal induction :
- 6 intradermal injections in groups of two per animal
- Injections for the test group:
A) front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCl-solution in a ratio of 1:1
B) middle row: 2 injections each of 0.1 ml of the test substance formulation
C) back row: 2 injections each of 0.1 ml Freund's adjuvant / 0.9 % aqueous NaCl-solution (1: 1) with test substance
- Injections for control groups 1 and 2: The animals were given the same injections (A, B, C) but without test substance, only with the formulating agent.
- Site of application: shoulder
- Readings: 24 h after the beginning of application
- Assessment of the skin findings: Draize scheme
- Application of the n-Dodecylsulphate sodium salt: Because of slight skin changes at the original injection sites these areas were treated with a 10% formulation of n-Dodecylsulphate sodium salt in white vaseline one day before the percutaneous induction.

Percutaneous induction:
- Percutaneous induction was carried out one week after intradermal induction
- Amount applied: 2 x 4 cm filter paper strips were applied to the skin of the shoulder under an occlusive dressing. The animals were exposed to about 0.3 g of the test substance formulation.
- The control groups were treated analogously to the test group but only with the solvent without the test substance.
- Duration of exposure: 48 hours
- Site of application: shoulder, same area as in the case of the previous intradermal application
- Readings: 48 h after the beginning of application
- Assessment of the skin findings: Draize scheme

B. CHALLENGE EXPOSURE
- Test concentration: non-irritant concentration
- Challenge 21 days after intradermal induction.
- Amount applied: 2 x 2 cm filter paper strips were applied to the skin of the flank under an occlusive dressing. The animals were exposed to about 0.15 g of the test substance formulation.
- Challenge: treatment of the test group and of control group 1 with the test substance formulation. Additionally olive oil DAB 9 was applied as a vehicle. Control group 2 only received olive oil DAB 9.
- Duration of exposure: 24 hours
- Site of application: intact clipped flank
- Readings: 24 and 48 h after the removal of the patch
- Assessment of the skin findings: Draize scheme
Challenge controls:
no data

Study design: in vivo (LLNA)

Statistics:
no data

Results and discussion

Positive control results:
no data

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
scaling, superficial scabbing
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: scaling, superficial scabbing.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle control
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: vehicle control. No with. + reactions: 6.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle control
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: vehicle control. No with. + reactions: 4.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle control. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Induction findings:

After intradermal induction distinct erythema and edema were observed at the injection sites of the control and test animals, at which only Freund's adjuvant / saline (1:1) was applied. Injection of the test substance preparation in olive oil DAB 9 resp. in Freund's adjuvant / saline (1:1) caused distinct erythema and edema in the test animals. The control animals injected with olive oil DAB 9 (vehicle) showed distinct erythema .

After percutaneous induction incrustation, partially open (caused by the intradermal induction) in addition to distinct erythema and edema could be observed in the test animals. The animals of control group 1 and 2 which were injected with olive oil DAB 9 (vehicle) exhibited incrustation, partially open (caused by the intradermal induction) in addition to distinct erythema and edema.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Classification: sensitizing
Executive summary:

Report Summary

The substance Isatosäureanhydrid was tested for its sensitizing effect on the skin of the guinea pig in the maximization test based on the method of Magnusson and Kligman.

After intradermal induction distinct erythema and edema were observed at the injection sites of the control and test animals, at which only Freund's adjuvant / 0.9% aqueous NaCl-solution (1:1) was applied. Injection of the test substance preparation in olive oil DAB 9 resp. in Freund's adjuvant / 0.9 % aqueous NaCl-solution (1:1) caused distinct erythema and edema in the test animals. The control animals injected with olive oil DAB 9 (vehicle) showed distinct erythema.

After percutaneous induction incrustation, partially open (caused by the intradermal induction) in addition to distinct erythema and edema could be observed in the test animals. The animals of control group 1 and 2 which were injected with olive oil DAB 9 (vehicle) exhibited incrustation, partially open (caused by the intradermal induction) in addition to distinct erythema and edema.

The number of animals with skin findings after the challenge (21 days after intradermal induction) is summarized in the following table:

      Challenge
   10% in olive oil DAB 9  olive oil DAB 9
 Control group 1  0/5  0/5
 Control group 2 *  no application of test substance  0/5
 Test group  10/10  6/10

x/y: number of positive reactions/number of animals tested; readings 24 h after the removal of the patch

Control group 2 that had been intended for a potential 2nd challenge was not needed, since a 2nd challenge was not necessary on the basis of the unambiguous results of the 1st challenge.

After the challenge with the 10% test substance preparation in olive oil DAB 9 in 4 out of 10 test animals severe erythema in addition to slight edema could be observed. 6 out of 10 test animals showed distinct erythema. The animals of control group 1 did not show any skin reactions.

Olive oil DAB 9, which was applied as a vehicle caused slight erythema in 6 out of 10 test animals. Control group 1 and 2 did not show any skin reactions.

Under these test conditions and following the results described above Isatosäureanhydrid (i.e. isatoic anhydride) has a sensitizing effect on the skin of the guinea pig.