N,N'-ethylenebis[N-acetylacetamide]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to approriate test guideline and GLP. However, as the report does not indicate whether a positivie control was performed, reliability 2 is assigned.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethylenebis[N-acetylacetamide]

EC Number:

234-123-8

EC Name:

N,N'-ethylenebis[N-acetylacetamide]

Cas Number:

10543-57-4

Molecular formula:

C10H16N2O4

IUPAC Name:

N,N'-ethylenebis[N-acetylacetamide]

Details on test material:

- Name of test material (as cited in study report): T.A.E.D
- Substance type: powder
- Physical state: white powder
- Lot/batch No.: reference 083

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Bantin &Kingmn LtD, Grimston, Hull, Yorks
- Weight at study initiation: 250-450 g
- Housing: in groups of up to four animals in solid floor polypropylene cages furnished with softwood sawdust
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3°C
- Humidity (%): no data
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 12.03.1982 To: 15.04.1982

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: arachis oil for intradermal induction, petroleum jelly B.P. for topical applications

Induction 2

Concentration / amount:

intradermal induction: 5%
topical induction: 25%
topical challenge: 25%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: arachis oil for intradermal induction, petroleum jelly B.P. for topical applications

Challenge 2

Concentration / amount:

intradermal induction: 5%
topical induction: 25%
topical challenge: 25%

No. of animals per dose:

10 animals per group

Details on study design:

RANGE FINDING TESTS

Intradermal injection
An area measuring 4 x 6cm in the shoulder region of 2 guinea pigs was clipped free of hair.Into these areas were injected simultaneously four 0.1 ml intradermal injections of the test material in an appropriate vehicle as follows:
Guinea pig / Concentration of TAED injected / No. of 0.1 ml intradermal injections
A / 1% / 4
B / 5% / 4
Animals were observed 24, 48 and 72 hours and 7 days following treatment and any evidence of localized necrosis or systemic toxicity was recorded.
Using the information obtained from this preliminary test, a concentration of the test material which was well tolerated both topically and systemically was selected for the Main Study.

Topical application
The test material at two concentrations was topically applied under an occlusive patch to two sites on the shaved flanks of each of a group of two guinea pigs.
These animals had previously been intradermally injected with Freunds Complete Adjuvant. Following a 24 hour exposure period the patches were removed and the degrees of irritancy at the test sites were evaluated for erythema immediately after removal of the patches and then at 24 and 48 hours.
The concentrations of the test material to be used on two further animals were chosen, applied and then evaluated after similar time intervals.
Using the information obtained from this preliminary test, the concentrations .of the test material to be used for Topical Induction and Topical Challenge in the Main Study were selected.



MAIN STUDY

The method involved a two stage induction procedure followed by a topical challenge two week's later .

Induction Procedure - Day 0
An area measuring 4 x 6 cm in the shoulder region of each of 10 guinea pigs was clipped short of hair using Oster A5 electric animal clippers. Into this area three pairs of intradermal injections were given simultaneously as follows:
(1) (1)
(2) (2)
(3) (3)

(1) 0.1ml of Freunds complete adjuvant alone.
(2) 0.1ml of the test material alone
(3) 0.1ml of the test material emulsified in the adjuvant.

Induction Procedure - Day 7
The second stage of the induction procedure involved the topical application of the test material to the injection site one week after the injections. The area was pretreated with 10% sodium lauryl sulphate (SLS) in petrolatum 24 hours prior to topical application. This concentration of SLS enhances sensitization by provoking a mild inflammatory reaction.
The test material was applied to a 2 x 4cm patch of Whatman No.4 filter paper. This patch was covered with an overlapping 7.5cm x 4cm length of Sleek Waterproof adhesive strapping and applied to the area injected one week previously. This patch was further secured by a 7.5cm x 25cm length of Elastoplast elastic adhesive bandage, which was wound in double layer around the torso of the animal. The dressing and patch was removed after-a 48 hour exposure period.

Challenge Procedure – Day 21
The test animals were challenged by topical application two weeks following the Topical Induction stage. Hair was removed from an area measuring 5cm x 5cm, on both flanks, by clipping and closely shaving. The test material, at the highest non-irritant concentration, was applied to the right flank under an occlusive patch which consisted of a 2cm x 2cm patch of Whatman No .4 filter paper, backed by a 4cm strip of 2.5cm wide Sleek waterproof adhesive strapping. A similar patch was used to apply the vehicle alone to the shaved area on the left flank. Both patches were held firmly in position for 24 hours by means of a 7.5cm x 25cm length of Elastoplast elastic adhesive bandage, wound in a double layer around the trunk of the animal. All dressings and patches were removed following the 24 hour exposure period.

Challenge Procedure – Day 23 & 24
The skin reactions observed at both test material and vehicle alone challenge sites (i.e. right and left flanks respectively) were recorded 24 hours and 48 hours following removal of the patches.
Reactions were scored according to the following scale:
no reaction - 0; scattered mild redness - 1; moderate and diffuse redness - 2; inense redness and swelling - 3

Procedure for Main Study – Control Group
An additional group of 10 guinea pigs received similar treatment to the test group except that they were exposed to the vehicle, without the test material, at the Intradermal Induction and Topical Induction stages.
Each of the control group animals, therefore, received the following treatment schedule
Day 0 - Intradermal-Induction:
2 x 0.1 ml injection of Freunds complete adjuvant.
2 x 0.1 ml injection of the vehicle alone
2 x 0.1 ml injection of the vehicle emulsified in the adjuvant.

Day 7 -Topical Induction
The vehicle alone applied for 48 hours under an occlusive patch.

Day 21 - Topical Challenge
Right flank - the test material at the same concentration used in the test group.
Left flank - the vehicle alone.

Day 23 & 24 – The skin reactions at both challenge sites of each animal were recorded.


INTERPRETATION OF RESULTS

The skin reactions at both challenge sites of each animal were recorded. The number of animals in the test group showing a more severe reaction at the test material challenge site (i.e. right flank) than the most severe reaction seen at the test material challenge site in the control animals were noted.
These effects seen in the test group were attributed to contact sensitization and the test material was assigned a Maximization Grading according to Magnusson & Kligman 1969.
The reactions observed at the vehicle alone challenge sites (i.e. left flank) in both the test and control groups were recorded. This information enables elimination of false positive results due to either primary irritation or contact sensitization caused by the vehicle.

Challenge controls:

see "details on study design"

Positive control substance(s):

not specified

Results and discussion

Positive control results:

no data

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% TAED at challenge

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% TAED at challenge. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% TAED at challenge

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

the 10th animal had died on day 8

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% TAED at challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: the 10th animal had died on day 8.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25% TAED at challenge

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% TAED at challenge. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% TAED at challenge

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

the 10th animal had died on day 8

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% TAED at challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: the 10th animal had died on day 8.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

TAED is considered to be not sensitising in guinea pigs under the conditions of the test.