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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
maximization test in guinea pig
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-12-06 to 2000-03-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted July 17, 1992
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An appropriate guinea pig maximisation test is available which would not justify conducting an additional LLNA due to animal welfare.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
443-010-4
EC Name:
-
Cas Number:
53641-10-4
Molecular formula:
C14H15ClN2O4
IUPAC Name:
N-[3-chloro-4-(3-oxobutanamido)phenyl]-3-oxobutanamide
Test material form:
solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: gpg, Pirbright-White HsdPoc:DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN WINKELMANN, Gartenstraße 27, 33178 Borchen, SPF breeding colony
- Weight at study initiation: males: 350 g - 410 g
- Housing: Groups of five animals in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate
- Diet (e.g. ad libitum): ssniff Ms-H (V 2233), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week
- Animal identification: fur marking with KMnO4 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3°C
- Humidity (%): 50 +/- 20%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Remarks:
deionized
Concentration / amount:
intradermal: 5 % in deionized water


Day(s)/duration:
day 1: induction
days 2-7: The administration area was examined for local tolerance. Systemic toxic
effects were recorded, when apparent.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
deionized
Concentration / amount:
25 % in deionized water


Day(s)/duration:
day 8: induction
The administration area was then kept under an occlusive
bandage covered with an impermeable film and an elastic bandage for
48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
deionized
Concentration / amount:
dermal: 25 % in deionized water
Day(s)/duration:
The administration area was then kept for
24 hours under an occlusive bandage covered with an impermeable film
and an elastic bandage.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Number of animals in test item group: 10
Number of animals in negative control group: 5
Details on study design:
Test procedure
The following preparations were used for the intradermal injections:
Control group:
Injection 1: a 1:1 mixture (v/v) FCA (Freund’s Complete Adjuvant)/ deionized water
Injection 2: deionized water
Injection 3: a 1:1 mixture (v/v) FCA (Freund’s Complete Adjuvant)/ deionized water (=vehicle)

Treatment group:
Injection 1: a 1:1 mixture (v/v) FCA/ deionized water
Injection 2: 5% of test chemical in deionized water
Injection 3: 5% of the test chemical in a 50% FCA solution

Determination of the primary non-irritant concentration:
Prior to the determination of the primary non-irritation concentration in a dermal occlusive test the animals received 4 intradermal injections of 50% FCA emulsion (4 x 0.1ml) into the dorsal area, since FCA may lower the threshold of primary irritation. Thereafter, each of the following test concentrations were administered to the flanks of two guinea pigs:
25% test item concentration in deionized water
5.0% test item concentration in deionized water
1.0% test item concentration in deionized water
The hair of the flanks of the animals was removed mechanically. 0.5 ml of the test substance preparation was administered to a 2x2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema.

Determination of the tolerance of the intradermal injections
To determine the tolerance of intradermal injections, each of the following preparations were administered twice by intradermal injection to 2 guinea pigs:
2 x 0.1 ml 5.0 % deionized water
2 x 0.1 ml 1.0 % deionized water
2 x 0.1 ml 0.2 % deionized water

The injection sites were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders. 24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.

Main test for the sensitizing properties
Day 0: The body weight of the animals was determined. The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.
Day 1: Intradermal induction treatment:
Two intradermal injections per animal of the following preparations were applied. The injection sites were all within the dorsal area and left uncovered.
Treatment group:
Site 1: 2 x 0.1 ml of a 1:1 mixture (v/v) FCA/ deionized water
Site 2: 2 x 0.1 ml of 5% of test chemical in deionized water
Site 3: 2 x 0.1 ml of 5% of the test chemical in a 50% FCA solution
Control group:
Site 1: 2 x 0.1 ml of a 1:1 mixture (v/v) FCA/ deionized water
Site 2: 2 x 0.1 ml of deionized water
Site 3: 2 x 0.1 ml of a 1:1 mixture (v/v) FCA / deionized water (=vehicle)

Day 2-7: The administered area was examined for local tolerance. Systemic toxic effects were recorded, when apparent.
Day 8: Dermal induction treatment:
An amount of 0.5 ml of the test substance (25% test substance in deionized water = treatment group) or vehicle (deionized water) for the control group was administered to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept under an occlusive bandage covered with an impermeable film and an elastic bandage for 48 hours.
Day 10: Occlusive bandage was removed, irritant effects were recorded, when apparent.
Day 11-21: Test animals were kept under observation.
Day 22: Dermal challenge treatment:
One area of 5 x 5cm on the left flank was shaved mechanically. An amount of 0.5 ml of the test substance 25% test substance in deionized water (treatment group and control group) was administered to a 2 x 2 cm cellulose patch. The administration area was then kept under an occlusive bandage covered with an impermeable film and an elastic bandage for 48 hours.
Day 23: Occlusive bandage was removed. Any remnants of the test substance were washed off with warm water.
Day 24: Examination of the skin approx. 24 hours after the removal of the patches.
Day 25: Examination of the skin approx.. 48 hours after the removal of the patches. Body weight of the test animals was determined.
Positive control substance(s):
yes
Remarks:
positive control assay with alpha-hexyl cinnamic aldehyde: Testing for the sensitizing properties of alpha-hexyl cinnamic aldehyde was performed in female guinea pigs according to the method of BUEHLER.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25% alpha-hexyl cinnamic aldehyde
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
other: Testing for the sensitizing properties of a/pha-hexyl cinnamic aldehyde was performed in female guinea pigs according to the method of BUEHLER.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25% alpha-hexyl cinnamic aldehyde
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
other: Testing for the sensitizing properties of a/pha-hexyl cinnamic aldehyde was performed in female guinea pigs according to the method of BUEHLER.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
polyethylene glycol 400
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Testing for the sensitizing properties of a/pha-hexyl cinnamic aldehyde was performed in female guinea pigs according to the method of BUEHLER.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
polyethylene glycol 400
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Testing for the sensitizing properties of a/pha-hexyl cinnamic aldehyde was performed in female guinea pigs according to the method of BUEHLER.

Any other information on results incl. tables

Main test for the sensitizing properties:


Body weight gains and clinical signs
The body weight gains of the animals were not impaired.
The treated animals showed no clinical signs of intoxication throughout the study.


 


Maximum concentration not causing irritating effects in test: 25 %

Signs of irritation during induction:

The intradermal injections (with and without test substance)
caused severe erythema and edema as well as indurations and
encrustations. The intradermal injection with test substance
in vehicel caused slight erythema and edema. The intradermal
injection of the vehicel alone exhibited no signs of
irritation. The dermal induced application with FCA caused
severe erythema and edema as well as indurations,
encrustations and necrosis. The test substance in vehicel
and the vehicel alone exhibited no signs of irritation.

Evidence of sensitisation of each challenge concentration:
treatment group: 0/10
control group: 0/5


Dermal induction treatment:
After the removal of the patches at day 10, severe erythema and edema, indurated,
and encrusted skin as well as necrosis were observed at the sites previously treated
with Freund's Adjuvant. The administration sites treated with the test substance or the
vehicle alone showed no signs of irritation.


Dermal challenge treatment:
No skin reactions were observed in the control and the treatment group 24 and 48
hours after removal of the occlusive bandage.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Based on the results of this study the test item showed no evidence for sensitizing properties.
Conclusions:
Under the conditions of the present study, none of the animals of the treatment group showed a positive skin response after the challenge procedure. Therefore, based on the results of this study the test item showed no evidence for sensitizing properties.
Executive summary:

Maximum concentration not causing irritating effects in test: 25 %

Signs of irritation during induction:
The intradermal injections (with and without test substance)
caused severe erythema and edema as well as indurations and
encrustations. The intradermal injection with test substance
in vehicel caused slight erythema and edema. The intradermal
injection of the vehicel alone exhibited no signs of
irritation. The dermal induced application with FCA caused
severe erythema and edema as well as indurations,
encrustations and necrosis. The test substance in vehicel
and the vehicel alone exhibited no signs of irritation.

Evidence of sensitisation of each challenge concentration:
treatment group: 0/10
control group: 0/5