3-acetoxymethylen-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 18, 1994 to November 08, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study (OECD 406) with acceptable restrictions.

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:

No guideline for a LLNA was available at the time of performing the presented test.

Test material

Test material information

Constituent 1

Reference substance name:

3-acetoxymethylen-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

213-485-0

EC Name:

3-acetoxymethylen-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

957-68-6

Molecular formula:

C10H12N2O5S

IUPAC Name:

3-acetoxymethylen-7-amino-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 of test material (as cited in study report): 7-ACA
- Physical state: white powder
- Analytical purity: 95%-102% (HPLC)
- Batch No.: 3701586
- Expiration date of the batch: stable under conditions of storage
- Storage condition of test material: in a refrigerator in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

Animals:
Hartley guinea pigs, Crl:(HA)BR.
Supplier: Charles River Wiga GmbH, D-32791 Sulzfeld.
Female healthy young adult and non pregnant animals.
Number in the preliminary study: 3
Number in the main study: 30
Body weight at receipt: ca. 300 g
Age at first application: ca. 6 weeks
Environmental conditions:
Hygiene: improved hygienic conditions.
Room number: EH1-21
Room temperature: average of ca. 22.5 °C.
Relative humidity: average of 45 %.
Control of room temp, and rel. humidity: continuous control and recording
Air exchange: ca. 12/h
Light: only artificial light from 6.00 a.m. to 6.00 p.m
Cages: Makrolon cages type m (23 cm x 39 cm x 15 cm) with wire mesh lids, single caging
Feed: Altromin Standard Diet No. 3022, ad libitum, offered in stainless steel containers. Analysis of the feed for ingredients and contaminants are performed randomly by Altromin GmbH, D-32791 Lage.
Bedding material: wood chips (aspen) from FINN TAPVEIKY, SF-73600 Kaavi. Reduction of microorganisms by autoclaving.
Water: tap water, acidified with HC1 to pH = 3, offered in Makrolon bottles with stainless steel canules, ad libitum.
Identification of the animals: numbers tattooed in the pinna of the right ear.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Remarks:

and white petrolatum

Induction 2

Concentration / amount:

The concentrations of the test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. The concentration used for the challenge exposure should be the highest non irritant one.

To obtain the appropriate concentrations of the test substance for the main study, a preliminary test was carried out with 3 female guinea pigs. 4 different concentrations of the test substance were administered epicutaneously in the lumbar region and 4 different concentrations were applied intradermally in the interscapular region. Mode of application was the same as in the main study. Duration of the epicutaneous exposure was 24 hours.

Intradermal injection:
Test substance concentrations were 5 %, 1 %, 0.1 % and 0.01 % in physiological saline. At the concentration of 5 % very slight to severe erythema and/or oedema to the skin were noted in all animals 24 and/or 48 hours after the application. No skin reaction occurred at the other concentrations. Therefore a concentration of 5 % in physiological saline was used for the intradermal induction in the main study.
Epicutaneous exposures:
Test substance concentrations were 40 %, 10 %, 2.5 % and 0.5 % in white petrolatum. No adverse skin reactions were observed at any of these concentrations. Therefore it was decided to use 40 % for the epicutaneous induction and for the challenge exposure. 40 % is also the highest technically feasible concentration for an applicable formulation in white petrolatum. According to B. Magnusson and A.M. Kligman, it was also decided to pretreat the animals with n-dodecylsulfate, sodium salt, before the epicutaneous induction exposure.

Challenge

Challenge 1

Route:

epicutaneous, open

Vehicle:

physiological saline

Remarks:

and white petrolatum

Challenge 2

Concentration / amount:

The concentrations of the test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. The concentration used for the challenge exposure should be the highest non irritant one.

To obtain the appropriate concentrations of the test substance for the main study, a preliminary test was carried out with 3 female guinea pigs. 4 different concentrations of the test substance were administered epicutaneously in the lumbar region and 4 different concentrations were applied intradermally in the interscapular region. Mode of application was the same as in the main study. Duration of the epicutaneous exposure was 24 hours.

Intradermal injection:
Test substance concentrations were 5 %, 1 %, 0.1 % and 0.01 % in physiological saline. At the concentration of 5 % very slight to severe erythema and/or oedema to the skin were noted in all animals 24 and/or 48 hours after the application. No skin reaction occurred at the other concentrations. Therefore a concentration of 5 % in physiological saline was used for the intradermal induction in the main study.
Epicutaneous exposures:
Test substance concentrations were 40 %, 10 %, 2.5 % and 0.5 % in white petrolatum. No adverse skin reactions were observed at any of these concentrations. Therefore it was decided to use 40 % for the epicutaneous induction and for the challenge exposure. 40 % is also the highest technically feasible concentration for an applicable formulation in white petrolatum. According to B. Magnusson and A.M. Kligman, it was also decided to pretreat the animals with n-dodecylsulfate, sodium salt, before the epicutaneous induction exposure.

No. of animals per dose:

group: K7 negative control, animal Nos.: 231 - 240, sex: f, exposure: induction exposure (intradermal with physiological saline, epicutaneous with white petrolatum), challenge exposure (left flank with 40 % test substance in white petrolatum, right flank with white petrolatum)
group: M test substance, animal Nos.: 241 - 260, sex: f, exposure: induction exposure (intradermal with 5% test substance in physiological saline, epicutaneous with 40% test substance in white petrolatum), challenge exposure (left flank with 40 % test substance in white petrolatum, right flank with white petrolatum)

Details on study design:

First induction exposure: 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: epicutaneous application of the test substance to the sites of the intradermal injections.

Positive skin reactions of the test substance treated sites after the challenge exposure indicate a sensitising effect of the test substance, if the scores are higher than those of the vehicle treated sites and if the rate of those - positively reacting - animals is higher than the corresponding percentage of animals in the negative control group.

Time Schedule:
Day 0 removal of hair, recording of body weight, intradermal induction exposure.
Day 1 examination of injection sites.
Day 6 removal of hair, treatment with n-dodecylsulfate, sodium salt.
Day 7 epicutaneous induction exposure.
Day 9 end of epicutaneous induction exposure.
Day 10 skin examination.
Day 21 removal of hair, epicutaneous challenge exposure.
Day 22 end of epicutaneous challenge period.
Day 23 approximately 21 hours after removing the patch cleaning of the challenge area, approximately 3 hours later skin examination.
Day 24 skin examination, recording of body weight, sacrifice of animals, fixation of the test substance treated- and vehicle sites, end of test

Clipping of hair:
On the respective days (see also 2.9.) the hair of the animals was clipped closely on the appropriate application sites with an Aesculap GH 204 electric hair clipper with a 1 mm and then with a 0.1 mm cutterhead.

Preparation and administration of the test substance:
All formulations of the test substance were prepared immediately before administration.

Intradermal induction exposure:
Stock solution of the test substance: 1000 mg of the test substance were suspended in 6.0 ml physiological saline. 4.0 ml of 1 m NaOH solution were added drop by drop under stirring to obtain a solution. The final concentration of the test substance was 10 %. This solution was further diluted (see below).
Cranial row: both groups: Freund's complete adjuvant (FCA), 1+1 (v/v) blended with physiological saline.
Middle row: control group: physiological saline
test substance group: test substance, final concentration: 5 % (w/v) in physiological saline.
Caudal row: control group: FCA, 1+1 (v/v) blended with physiological saline
test substance group: test substance, 5 % (w/v) final concentration in: physiological saline, 1+1 (v/v) blended with FCA.
A volume of 0.1 ml was applied for each injection site.

Epicutaneous induction exposure:
The highest technically feasible test substance concentration of 40 % in white petrolatum did not cause markable skin irritations in the preliminary test in the animals. According to B. Magnusson and A.M. Kligman, all animals of both groups were therefore pretreated with a formulation of n-dodecylsulfate, sodium salt (E. Merck, D-64274 Darmstadt, Art. No. 13760), 10 % (w/w) in white petrolatum, one day before the epicutaneous induction exposure. About 0.5 g per animal was gently applied with a plastic spatula onto the appropriate area to give a slight local hyperaemia. The area was left uncovered.
For the induction exposure, filter papers, about 2 cm x 4 cm, wer covered with the test substance, 40 % in white petrolatum (test substance group) and with white petrolatum (control group), resp. They were applied to the area of the intradermal injections and were fixed occlusively with a strip of non-irritating tape ("Blenderm®" surgical tape, hypoallergenic, 3M, made in USA, Medical Products Division, St. Paul, MN 551444). The area of administration was then covered occlusively with aluminium foil and finally fixed with "Fixomull® stretch" (self adhesive non woven fabric, hypoallergenic, made by Beiersdorf AG, D-20245 Hamburg). Exposure time was 48 hours. About 0.7 g of test substance formulation and of white petrolatum, resp., were applied to each animal.

Challenge controls:

Challenge exposure: epicutaneous application of the test substance to the left flanks and application of the vehicle to the right flanks of all animals.

2.11.3. Challenge exposure

Filter papers, about 2 cm x 2 cm, covered with the test substance, 25 % in white petrolatum, were applied to the left flanks of all animals of both groups. Filter papers of the same size, covered with white petrolatum, were applied to the right flanks of all animals. Mode of fixation was the same as for the epicutaneous induction exposure. About 0.7 g of test substance formulation and about 0.6 g of white petrolatum were applied to each animal.

Positive control substance(s):

yes

Remarks:

1,4-phenylenediamine

Results and discussion

Positive control results:

Results of the last check, performed in September/October 1994 (guinea pig maximisation test,
same methods as in the actual study):
Positive control substance: 1,4-phenylenediamine.
Concentrations:
0.5 % in physiological saline for intradermal induction exposure 10 % in white petrolatum for epicutaneous induction exposure 5 % in white petrolatum for epicutaneous challenge exposure.

Number of animals: 5 female guinea pigs for 1,4-phenylenediamine and 5 female guinea pigs for a control group (same strain of animals, supplier, animal maintenance, feed etc. as in the actual study)
Results after 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)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No animal had a "positive skin reaction".

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No animal had a "positive skin reaction"..

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No animal had a "positive skin reaction".

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No animal had a "positive skin reaction"..

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

20/20 animals were regarded as sensitised

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: 20/20 animals were regarded as sensitised.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

20/20 animals were regarded as sensitised

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: 20/20 animals were regarded as sensitised.

Results 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:

10

Results 6

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:

10

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5 %

No. with + reactions:

5

Total no. in group:

5

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

5 %

No. with + reactions:

5

Total no. in group:

5

Any other information on results incl. tables

Mortality: All animals survived till the end of the study.

Body weight: There were no significant differences in mean body weights between the test substance group and the control group on Days 0 and 24.

General observations: 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 of the observations described above no abnormal behaviour or clinical signs were detected during the experiment in the animals.

Skin reactions:

Skin reactions after 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. Lesions did not heal until the end of the study. This local alterations are known effects of Freund's adjuvant.

Middle injection sites: No irritative reactions were observed 24 hours after induction exposure in the negative control group. Very slight to severe erythema and/or oedema were noted in 15/20 animals of the test substance group.

Skin reactions after epicutaneous induction exposure: The reactions caused by the adjuvant obscured the reading of reactions following the epicutaneous induction exposure in this area. All animals had severe erythema and oedema in the interscapular region (score "3"), which were attributed to the effects of the adjuvant.

Skin reactions after 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: no positive skin reaction in any animal at any reading time.

Test substance site: very slight to severe erythema and/or oedema in all animals 24 and/or 48 hours after the end of the challenge exposure, eschars in one animal.

20/20 animals were regarded as sensitised.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test substance "7-ACA" is considered to be a skin sensitizer.

Executive summary:

The "maximisation test" of Magnusson and Kligman was performed to reveal a possible sensitising potential of "7-ACA". 20 female guinea pigs were used as a test substance group and another 10 females were used as a negative control group. There were two induction exposures (intradermally and epicutaneously) and one epicutaneous challenge exposure.

Results: In the negative control group, the control sites and also the test substance treated sites of all animals were normal at any observation time. In the test substance group, the control sites of all animals were also normal. At the test substance treated sites, positive skin reactions were noted in 20/20 animals. These animals, i.e. 100 % of the test substance group animals, were regarded as sensitised. The test substance "7-ACA" is considered to be a skin sensitizer.