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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:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to valid methods. Therefore it is considered relevant, reliable and adequate for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
The procedure described by Magnusson and Kligman was followed, comprising intradermal induction with Freund's Complete Adjuvant (FCA) on day 0; topical induction on day 7 and a subsequent chaIlenge on day 20 and rechaIlenge on day 27 by applying the test article in Finn chambers to the flank of the animal. The sensitization was carried out using the dose response modification of the guinea pig maximization test method developed by Andersen and Vølund.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-chloroacetamide
EC Number:
201-174-2
EC Name:
2-chloroacetamide
Cas Number:
79-07-2
Molecular formula:
C2H4ClNO
IUPAC Name:
2-chloroacetamide
Details on test material:
- Name of test material (as cited in study report): Chloroacetamide
- Composition of test material, percentage of components: Analytical grade
- Other: Purchased from Merck, Darmstadt (Germany)

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mollegaard Breeding and Research Centre Ltd, Ejby, DK-4623 Lille Skensved
- Age at study initiation: Young adult
- Weight at study initiation: 250-450 g
- Fasting period before study: No data
- Housing: PPL (Type IV) cages, 2 or 3 per cage; bedding was softwood sawdust “Hahnflock H ¾” from Hahn & Co, D-24796 Bredenbek-Kronsburg or aspen woodchips from Beekey Bedding, B & K Universal AB, Sollentuna
- Diet: Pelleted diet, "3113 Altromin", from Chr. Petersen, DK-4100 Ringsted or Special Diets Services, ad libitum
- Water: Bottles with domestic quality drinking water enriched with vitamin E and acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth, ad libitum
- Acclimation period: At least 4 days (to reject animals in poor condition)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 3°C
- Humidity (%):55% ± 15%
- Air changes (per hr): 10 (filtered air)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: intradermal: water; epicutaneous: PEG/water 1:7
Concentration / amount:
Induction, test I + test II, intradermal: 0%; 0.003%; 0.01%; 0.03%; 0.1%; 0.3%
Induction: test I, epicutaneous: 0%; 0.5%; 50%; 0.5%; 50%; 0.5% / test II, epicutaneous: 0%; 0.3%; 30%; 0.3%; 30%; 0.3%
Challenge: test I, 30 % in PEG/water 1:7/test II, 5 % in PEG/water 1:7
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: intradermal: water; epicutaneous: PEG/water 1:7
Concentration / amount:
Induction, test I + test II, intradermal: 0%; 0.003%; 0.01%; 0.03%; 0.1%; 0.3%
Induction: test I, epicutaneous: 0%; 0.5%; 50%; 0.5%; 50%; 0.5% / test II, epicutaneous: 0%; 0.3%; 30%; 0.3%; 30%; 0.3%
Challenge: test I, 30 % in PEG/water 1:7/test II, 5 % in PEG/water 1:7
No. of animals per dose:
5
Details on study design:
INDUCTION
Day 0 - Intradermal induction
An area (4 x 6 cm) of dorsal skin in the shoulder region was clipped free of hair with an electric clipper the day prior to treatment.
Three pairs of intradermal injections (dose volume 0.1 mL) were given simultaneously into this area. The two first pair of injections (injection 1 and 2) were given close to each other and most cranially, while the third pair of injections (injection 3) were given towards the caudal part of the test area.
Control group
Injection1, a 1:1 mixture (w/w) FCA/water
Injection2, physiological saline (0.9% NaCl)
Injection3, a 50% w/w formulation of saline (0.9% NaCl) in a 1:1 mixture (w/w) FCA/water
Test groups
Injection1, a 1:1 mixture (w/w) FCA/water
Injection2, chloroacetamide in vehicle at the specified concentration.
Injection3, Test article at the double of the specified concentration formulated in a 1:1 mixture (w/w)FCA

Day 6 - Sodium lauryl sulphate exposure
The skin area used for intradermal injection was again clipped free of hair. Approximately 0.5 g sodium lauryl sulphate (10% in petrolatum) was massaged into the skin in order to induce a mild inflammatory reaction.

Day 7 - Topical induction
A paper patch (Whatman No. 3 tvfM, 2 x 4 cm) was saturated with about 0.25 mL of test article and placed on the skin and fixed with impermeable tape (Blenderm 3M, 5 x 7 cm). The trunk of the animal was wrapped with tape (Micropore, width 5.0 cm, or Elastoplast). Tapes and patch were removed after 48 hours. The guinea pigs in the control group were treated in a similar manner, with vehicle only.

CHALLENGE AND RECHALLENGE
Day 20 and 27 - Topical application at the flank region
An area (5 x 5 cm) of skin in each flank region was clipped free of hair and shaved with an electric razor. Finn Chambers ® (Epitest Ltd., Tuusula, Finland) on Scanpor® (Norgesplaster AIS, Askim, Norway) were used for challenge. Two patches were mounted side by side, one with the challenge preparation and one with vehicle control. Two pieces of filter paper were placed in the Finn Chambers ® and filled to saturation (15 30µI). The preparations were dosed using an EDP Digital pipette. The Finn chambers were applied to the skin of the left flank for 24 hours secured with Acrylastic® (Beiersdorf, Hamburg, Germany) and tape (Micropore 7.5 cm) wrapped around the trunk.
Day 21 and 28 - Preparation of treatment sites
At least three hours before the first observation, the skin of the flank was shaved with an electric razor, in order to facilitate the evaluation.

OBSERVATION OF SKIN REACTIONS
Each chaIlenge site was examined 24, 48 and 72 hours after challenge and rechaIlenge. Reactions were scored in artificial daylight.

CHALLENGE SKIN REACTIONS
The chaIlenge skin reactions were read blindly after 24, 48 and 72 hours using the routine grading scale (Magnusson and Kligman) :
Skin reaction Score
No visible change, 0
Slight or discrete erythema, 1
Moderate and confluent erythema, 2
Intense erythema and swelling, 3
A grade 1 reaction was not regarded as a positive challenge as patchy erythema may represent nonspecific irritancy caused by clipping, shaving and patching the animal The number of sensitized animals (score 2 and 3) in each group was used in the statistical analyses.
Challenge controls:
Methyl acrylic acid (negative control)

Study design: in vivo (LLNA)

Statistics:
The program for multi-dose response analysis developed by Andersen and Vølund was used. Monotonous and non-monotonous dose response relations can be analysed, as well as positive reactions among control animals. Briefly, the logistic regression analysis can be performed stepwise to evaluate:
1) The effect of both intradermal and topical doses on the sensitization rate.
2) The effect of the various intradermal doses alone on the frequency of sensitization.
3) The effect of the various topical induction doses alone on the frequency of sensitization.
The analysis presents the regression coefficients for the constant and for the logarithm to the induction treatment chosen for analysis.
Further, the goodness of fit and dose response relation for the fitted curve is shown as chi-square values.
The logistic regression analysis is accepted if the goodness of fit is not significant and the standardized residuals are below 2, indicating that the observed and calculated values are in sufficient agreement.
A significant chi-square for goodness of fit and large residuals indicate that the model cannot describe the responses statistically acceptably. The logistic regression analysis showing the lowest goodness of fit chi-square value and the lowest residuals is usually chosen.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: challenge - mean of readings 24, 48, 72 h
Group:
negative control
Dose level:
controls, challenge conc. 5 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: other: challenge - mean of readings 24, 48, 72 h. Group: negative control. Dose level: controls, challenge conc. 5 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.003 %, topical 0.3%, challenge conc.5%
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: other: challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.003 %, topical 0.3%, challenge conc.5%. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
other: challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.01 %, topical 30%, challenge conc.5%
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: other: challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.01 %, topical 30%, challenge conc.5%. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
other: challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.03 %, topical 0.3%, challenge conc.5%
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: other: challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.03 %, topical 0.3%, challenge conc.5%. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
other: challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.1 %, topical 30%, challenge conc.5%
No. with + reactions:
1
Total no. in group:
5
Remarks on result:
other: Reading: other: challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.1 %, topical 30%, challenge conc.5%. No with. + reactions: 1.0. Total no. in groups: 5.0.
Reading:
other: challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.3 %, topical 0.3%, challenge conc.5%
No. with + reactions:
4
Total no. in group:
5
Remarks on result:
other: Reading: other: challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.3 %, topical 0.3%, challenge conc.5%. No with. + reactions: 4.0. Total no. in groups: 5.0.
Reading:
other: re-challenge - mean of readings 24, 48, 72 h
Group:
negative control
Dose level:
controls, challenge conc. 5 %
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: other: re-challenge - mean of readings 24, 48, 72 h. Group: negative control. Dose level: controls, challenge conc. 5 %. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
other: re-challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.003 %, topical 0.3%, challenge conc.5%
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: other: re-challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.003 %, topical 0.3%, challenge conc.5%. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
other: re-challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.01 %, topical 30%, challenge conc.5%
No. with + reactions:
1
Total no. in group:
5
Remarks on result:
other: Reading: other: re-challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.01 %, topical 30%, challenge conc.5%. No with. + reactions: 1.0. Total no. in groups: 5.0.
Reading:
other: re-challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.03 %, topical 0.3%, challenge conc.5%
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: other: re-challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.03 %, topical 0.3%, challenge conc.5%. No with. + reactions: 3.0. Total no. in groups: 5.0.
Reading:
other: re-challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.1 %, topical 30%, challenge conc.5%
No. with + reactions:
2
Total no. in group:
5
Remarks on result:
other: Reading: other: re-challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.1 %, topical 30%, challenge conc.5%. No with. + reactions: 2.0. Total no. in groups: 5.0.
Reading:
other: re-challenge - mean of readings 24, 48, 72 h
Group:
test chemical
Dose level:
Intrad.0.3 %, topical 0.3%, challenge conc.5%
No. with + reactions:
3
Total no. in group:
5
Remarks on result:
other: Reading: other: re-challenge - mean of readings 24, 48, 72 h. Group: test group. Dose level: Intrad.0.3 %, topical 0.3%, challenge conc.5%. No with. + reactions: 3.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Table 1.Chloroacetamide-testI: Challenge results, incidence of sensitized animals

Group

 

No. of animals

 

Intradermal conc. %

 

Topical conc. %

 

Challenge conc. 30%

Challenge

Rechallenge

24h

48h

72h

24h

48h

72h

1

5

0

0

0

3

4

3

3

5

2

5

0.003

0.5

2

5

5

4

5

4

3

5

0.01

50

1

2

2

2

1

1

4

5

0.03

0.5

4

3

5

3

3

3

5

4

0.1

50

2

2

4

2

2

2

6

5

0.3

0.5

3

1

5

1

0

0

 

Table 2.Chloroacetamide-testII: Challenge results, incidence of sensitized animals

Group

 

No. of animals

 

Intradermal conc. %

 

Topical conc. %

 

Challenge conc. 5%

Challenge

Rechallenge

24h

48h

72h

24h

48h

72h

1

5

0

0

0

0

0

4

2

4

2

5

0.003

0.3

4

5

3

3

2

4

3

5

0.01

30

2

3

3

0

0

3

4

5

0.03

0.3

3

5

0

3

3

3

5

5

0.1

30

0

3

0

1

1

4

6

5

0.3

0.3

5

5

1

3

3

4

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Chloroacetamide was sensitizing. The results suggest a strong sensitizing potential, as the control animals gave positive reactions at the rechallenge and not at the first challenge. No dose-response relationship could be seen since a very high incidence of responders appeared in the group exposed to the lowest concentration used for intradermal induction. The response in the negative control group at rechallenge indicates that the animals had been sensitized by the application of the first challenge dose.
Executive summary:

Chloroacetamide was tested in 5 dose groups of each 5 guinea pigs and 1 control group of 5 guinea pigs (0.003 -0.3% for intradermal induction; 0.5 -50% for topical induction). The first test was unsuccessful as topical irritation was seen at the site of challenge with 30% chloroacetamide in PEG, in particular in the control group at the examination 48 and 72 hours after challenge.

In the second test the concentrations for topical induction were reduced from 0.5 and 50% to 0.3 and 30% which were well tolerated. The concentration for challenge was reduced from 30% to 5%. The test was positive in all groups, and at rechallenge the response in the control group was even stronger. No dose-response relationship could be seen since a very high incidence of responders appeared in the group exposed to the lowest concentration used for intradermal induction. The response in the negative control group at rechallenge indicates that the animals had been sensitized by the application of the first challenge dose. The results suggest a strong sensitizing potential, as the control animals gave positive reactions at the rechallenge and not at the first challenge.