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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:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A study according to the OECD method, including GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The LLNA-method was not available at the time of initiation the study.

Test material

Constituent 1
Chemical structure
Reference substance name:
(6R-trans)-7-amino-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
EC Number:
261-491-7
EC Name:
(6R-trans)-7-amino-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
Cas Number:
58909-56-1
Molecular formula:
C12H13N5O5S2
IUPAC Name:
7-amino-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)sulfanyl]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: 7-ACT
Chemical name: (6R, 7R)-7-Amino-3-(2,5-dihydro-2-methyl-5-oxo-6-hydroxy-1,2,4-triazin-3-yl) thiomethyl-ceph-3-em-4-carbonic acid
CAS No.: 58909-56-1
Supplier: Biochemie Ges.m.b.H.
Batch No.: 3201530
Purity: 99.5 % (content related to waterfree substance)
Water content: 1.5 %
Impurities: Heavy metals: < 20 ppm, by-products: < 0.2 % each
Appearance: white powder
pH: 3.97 (1 % aqueous suspension, measured by pH-meter)
Stability of the test substance: stable under conditions of storage
Disintegration: from 180 *C on
Storage: in a refrigerator in the dark
Date of handing over: July 26, 1994



In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga. D-97633 Sulzfeld. Germany.
- Age at study initiation: 6 weeks.
- Weight at receipt: ca. 300 g.
- Housing: single caged.
- Diet: Altromin No. 3022, ad libitum
- Water: tap water, acidified with HCl to pH 3, ad libitum
- Acclimation period: ca. 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): average of ca. 22
- Humidity (%): average of ca. 45
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
petrolatum
Concentration / amount:
Negative control, induction, intradermal: 0.1 mL each of physiological saline; FCA/saline 1+1 v/v; and FCA/saline 1+1 v/v .
Negative control, induction, epicutaneous: 0.5 g white petrolatum.
Negative control, challenge, epicutaneous: 0.6 g of a 20 % test substance formulation in white petrolatum on the left side + 0.5 g white petrolatum on the right side.

Test substance group, induction, intradermal: 0.1 mL each of 5 % test substance in physiological saline, w/v; FCA/saline 1+1 v/v; and 5 % final test substance in a mixture of FCA/physiological saline 1 + 1, v/v). 500 mg of the test substance were suspended in 7.2 ml physiological saline. 2.8 ml of 1 m NaOH solution were then added drop by drop under stirring to obtain a solution.
Test substance group, induction, epicutaneous: 0.6 g of a 40 % test substance formulation in white petrolatum
Test substance group, challenge, epicutaneous: 0.6 g of a 20 % test substance formulation in white petrolatum on the left side + 0.5 g white petrolatum on the right side.

The test substance concentrations were based on the result of a preliminary experiment. At the concentration of 40 % in epicutaneous exposure 2/3 animals had very slight erythema 48 hours after the start of the exposure.
FCA: Freund's complete adjuvant (Difco Laboratories, Article No 0638-60-7).
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
Negative control, induction, intradermal: 0.1 mL each of physiological saline; FCA/saline 1+1 v/v; and FCA/saline 1+1 v/v .
Negative control, induction, epicutaneous: 0.5 g white petrolatum.
Negative control, challenge, epicutaneous: 0.6 g of a 20 % test substance formulation in white petrolatum on the left side + 0.5 g white petrolatum on the right side.

Test substance group, induction, intradermal: 0.1 mL each of 5 % test substance in physiological saline, w/v; FCA/saline 1+1 v/v; and 5 % final test substance in a mixture of FCA/physiological saline 1 + 1, v/v). 500 mg of the test substance were suspended in 7.2 ml physiological saline. 2.8 ml of 1 m NaOH solution were then added drop by drop under stirring to obtain a solution.
Test substance group, induction, epicutaneous: 0.6 g of a 40 % test substance formulation in white petrolatum
Test substance group, challenge, epicutaneous: 0.6 g of a 20 % test substance formulation in white petrolatum on the left side + 0.5 g white petrolatum on the right side.

The test substance concentrations were based on the result of a preliminary experiment. At the concentration of 40 % in epicutaneous exposure 2/3 animals had very slight erythema 48 hours after the start of the exposure.
FCA: Freund's complete adjuvant (Difco Laboratories, Article No 0638-60-7).
No. of animals per dose:
10 in the control group.
20 in the test substance group.
Details on study design:
Hair was clipped on Days 0 and 21 on the treatment areas.

First induction exposure on Day 0: intradermal injections of the test substance, of FCA (to enhance a possible sensitisation) and of the test substance diluted with FCA. Application site was an area of approx. 2 cm x 4 cm in the interscapular region.
Second induction exposure on Day 7: epicutaneous application of the test substance to the sites of the intradermal injections for 48 hours.

Challenge exposure on Day 21 for 24 hours: epicutaneous application of the test substance to the left flanks and application of the vehicle to the right flanks of all animals.
Readings of skin reactions at 24 and 48 h after challenge, i.e. on Days 23 and 24.

Challenge controls:
The method was checked periodically with 1,4-phenylenediamine
Positive control substance(s):
yes

Results and discussion

Positive control results:
Last positive control test:
Results after the challenge exposure:
The control areas of all animals were normal. There were no positive skin reactions at the 1,4-phenylenediamine treated sites of the negative control group. In the positive control group 5/5 animals, i.e. 100 %, had positive reactions on the 1,4 phenylenediamine treated sites 24 and 48 hours after the end of the challenge exposure and were regarded as sensitised.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 % test substance
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: 20 % test substance. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 % test substance
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 20 % test substance. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20 % test substance
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
Score 2 and 3 were observed. Eschar was recorded in 7 cases.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20 % test substance. No with. + reactions: 19.0. Total no. in groups: 20.0. Clinical observations: Score 2 and 3 were observed. Eschar was recorded in 7 cases..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 % test substance
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
Score 1 and 3 were observed. Eschar was recorded in 14 cases.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20 % test substance. No with. + reactions: 19.0. Total no. in groups: 20.0. Clinical observations: Score 1 and 3 were observed. Eschar was recorded in 14 cases..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5 %
No. with + reactions:
5
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
5 %
No. with + reactions:
5
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
other: negative control to the positive control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
other: negative control to the positive control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
5

Any other information on results incl. tables

One animal of the negative control group died spontaneously on day 7. A post mortem examination did not reveal the cause of death. As since the start of this study one spare animal had been kept and administered under the same conditions as the other control animals, the deceased animal was replaced by the spare animal. The outcome of this study was not adversely affected by this event.

All other animals survived till the end of the study.

There were no significant differences in the mean body weights between the test substance group and the control group on Days 0 and 24. Immediately after the beginning of all epicutaneous exposures (induction, challenge) the motor activities of all animals were increased. This is due to the dressings which restrict the freedom of movement. Soon afterwards the behaviour was regular again. Except for this observations no abnormal behaviour or clinical signs were detected during the experiment.

 

Skin reactions:

Skin reactions after the intradermal induction exposure:

Cranial and caudal injection sites: Local irritations were observed in all animals beginning on the day after the injections. The irritations started with local erythema, which became more severe and led to ulcerations. This local alterations are known effects of Freund's adjuvant.

Middle injection sites: No irritant reactions were observed 24 hours after induction exposure in the negative control group, whereas very slight to well defined erythema were noted in 8/20 animals of the test substance group.

Skin reactions after the epicutaneous induction exposure:

All animals had severe erythema and oedema in the interscapular region, which were attributed to the effects of the adjuvant.

 

Skin reactions after the challenge exposure

These are the reactions of major interest for the grading of an allergenic potency of the test substance.

Negative control group:

Vehicle site: no positive skin reaction in any animal at any reading time.

Test substance site: no positive skin reaction in any animal at any reading time.

No animal had a "positive skin reaction".

Test substance group:

Vehicle site: A post mortem examination did not reveal the cause of death..

Test substance site: Very slight to severe erythema and/or oedema in 19/20 animals 24 and/or 48 hours after the end of the challenge exposure and, additionally, eschars in 14 of these animals at the first and/or second reading time.

19/20 animals had a "positive skin reaction", they are regarded as sensitised.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
19/20 animals of the test substance group, i.e. 95 % of the animals, were regarded as sensitised.
Executive summary:

The maximisation test of B. Magnusson and A. M. Kligman was performed according to the OECD-method 406 to detect a possible sensitising potential of 7 -ACT. 20 female guinea pigs were used as a test substance group and another 10 females as a negative control group. There were two induction exposures (intradermally and epicutaneously) and one epicutaneous challenge exposure. Test substance concentrations were:

5 % in physiological saline for the intradermal induction

40 % in white petrolatum for the epicutaneous induction and

20 % in white petrolatum for the challenge exposure.

Application of Freund's complete adjuvant was included in the intradermal exposure of both groups to enhance a possible sensitisation.

Result: 19/20 animals of the test substance group, i.e. 95 % of the animals, were regarded as sensitised. None in the negative control group.