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Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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.
Qualifier:
equivalent or similar to
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
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
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
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)
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.
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 group
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 group
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 group
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 group
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 group
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 group
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 group
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 group
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 group
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 group
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.

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

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.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a key Magnusson & Kligman maximization study in Dunkin-Hartley Guinea-pigs (Nordic Council of Ministers, 1996) Chloroacetamide was tested in 5 dose groups of each 5 guinea pigs and 1 control group of 5 guinea pigs (0.003, 0.01, 0.03, 0.1 and 0.3% for intradermal induction; 0.5 and 50% for topical induction). The control group was treated with Freund's adjuvant only. seven days later, Chloroacetamide was applied without adjuvant to the skin of the experimental animals as a 0.3 or 30% solution in a 1:7 polyethylene glycol/water mixture and left for 48 hours under occlusive cover. The controls received the vehicle only. After another 12 days, the sensitisation reaction was induced in all groups, including the controls, with the aid of a 5% Chloroacetamide solution in a 1:7 polyethylene glycol/water mixture by exposing the shorn skin to the solution for 24 hours under occlusive cover. The results were assessed 24, 48 and 72 hours after removal of the dressing. Rechallenge was carried out in the same way 7 days after the first challenge. Irrespective of the Chloroacetamide concentrations used, 60 to 100% of the Chloroacetamide-treated animals showed a positive response after the initial challenge (controls: 0/5). At rechallenge, the initial challenge proved to have induced sensitisation in the controls, with 80% of the animals showing a positive reaction. The response in the negative control group at rechallenge indicates that the animals had been sensitised by the application of the first challenge dose.The results suggest a strong sensitising potential, as the control animals gave positive reactions at the rechallenge and not at the first challenge.

Supporting studies in Pirbright Guinea-pigs were performed according to a modified Magnussen & Kligman method (IBR 1985a; IBR 1985b). The guinea pigs were induced by intradermal (with FCA), dermal (in white vaseline) and intradermal (in FCA, diluted in oleum arachidis) treatment, and challenged by dermal treatment. For the induction, the sample was diluted 9% in aqua dest; for the challenge phase the sample was applied as a 3%, 1% and 0.3% dilution in aqua dest. According to this method Chloroacetamide was considered to cause no delayed contact hypersensitivity. In a supporting study (Prof. Schulz, 1984), female guinea pigs were treated during 4 weeks by open epicutaneous administration of 3% resp. 1% solution of Chloroacetamide in a vehicle consisting of Methylcellosolve 45%, Tween 80 10% and Water 45% for 4 weeks (5 days/week) on the shaved flanks. Challenged with 0.2% Chloroacetamide solution after 10 days did not elicit allergic reactions.

Finally, a supporting study in guinea pigs according to the modified Buehler test (IBR, 1981) with a formulation containing 70% Chloroacetamide and 30% sodium benzoate did not show a sensitising effect.

These supporting studies with largely negative results do not satisfy the requirements of the testing guidelines which have become common standard in recent years, and have considerable methodological flaws. For instance, the concentrations of chloroacetamide employed to induce and trigger the allergic reaction, generally, were considered too low.

Migrated from Short description of key information:
In a key in vivo study the test results suggest a strong sensitising potential for Chloroaectamide. No dose-response relationship could be seen since a very high incidence of repsonders appeared in the low dose group.
Three supporting in vivo studies with largely negative results, do not satisfy the requirements of the testing guidelines which have become common standard in recent years, and have considerable methodological flaws. For instance, the concentrations of chloroacetamide employed to induce and trigger the allergic reaction, generally, were too low. Hence, it cannot be concluded that chloroacetamide cannot be demonstrated to have a sensitising potential in animals.
In a disregarded in vivo study no sensitising effects could be proven, but the test substance had a different formulation (70% act. ingr.) and the test concentrations were low (0.3%).
Weight of evidence for the sensitizing properties of Chloroacetamide is the fact that the test is not needed because the active substance is classified as a sensitiser according to Annex I of Directive 67/548/EEC.

Justification for selection of skin sensitisation endpoint:
Key study; the study was conducted according to valid methods. Therefore it is considered relevant, reliable and adequate for classification.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

The potential of Chloroacetamide to cause contact allergies in humans is addressed in a large number of case reports and reprots of positive patch tests in different groups of patients and healthy volunteers. The frequency of skin sensitisation occurence in the patinet groups varies within the range oof 0.3 to 2.8%. Patients sensitive fo formaldehyde could also develop sensitivity to Chloroacetamide.

Justification for classification or non-classification

Chloroacetamide was classified as a sensitiser according to Annex I of Directive 67/548/EEC: according the EU labelling regulations Commision Directive 93/21/EEC symbol 'Xi' and risk phrase R 43 'MAY CAUSE SENSITISATION BY SKIN CONTACT'; according to CLP regulation (No. 1272/2008 of 16 December 2008), Category 1 classification is proposed with signal word 'WARNING' and hazard statement H317 'MAY CAUSE AN ALLERGIC SKIN REACTION'.