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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:
2001-02-09 to 2001-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. The study has been conducted before the new annex of the REACH Regulation entered into force. Moreover, clear indication for strong skin sensitisation was observed in this study, thus allowing potency estimation. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium dicyanoamide
EC Number:
217-703-5
EC Name:
Sodium dicyanoamide
Cas Number:
1934-75-4
Molecular formula:
C2HN3.Na
IUPAC Name:
sodium dicyanamide
Test material form:
solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Microbiological status of animals: unknown
- Age at study initiation: approximately eight to twelve weeks
- Weight at study initiation: 308 to 378 g
- Housing: singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 09 Feb 2001 To: 18 March 2001

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Intradermal induction: 5 % of the test item, in distilled water
Dermal induction: 25 % of the test item, in distilled water
Day(s)/duration:
intradermal: day 0; dermal: day 7 (for 48 h)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25% w/w in distilled water
Day(s)/duration:
day 21 (for 24 hours)
Adequacy of challenge:
highest non-irritant concentration
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
10 % w/w in distilled water
Day(s)/duration:
day 21 (for 24 hours)
Adequacy of challenge:
other: to ensure that the maximum non-irritant concentration was used
No. of animals per dose:
Number of animals in the test group: 10
Number of animals in the negative control group: 5
Number of animals in the dose range finding study: 6
Details on study design:
RANGE FINDING TESTS:

Selection of concentration for intradermal induction
Four animals were treated intradermally (0.1 mL/injection site) with a concentration of 1 %, 5%, 10% and 25% w/w of the test item in distilled water. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 hours and 7 days after injection.

Selection of concentration for topical induction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant ten days earlier) were treated with four preparations of the test material (75%, 50%, 25% and 10% w/w in distilled water). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.

Selection of concentration for topical challenge
Four preparations of the test material (50%, 25%, 10% and 5% w/w in distilled water) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal Application (day 0)
- No. of exposures: 3 pairs of intradermal injections of 0.1 mL
- Test groups:
a) - a 1:1 mixture (v/v) FCA/ distilled water
b) - a 5% formulation of the test material in distilled water
c) - a 5% formulation of the test material in a 1:1 mixture (v/v) FCA/ distilled water
- - Control groups:
a) - a 1:1 mixture (v/v) FCA/ distilled water
b) - distilled water
c) – distilled water in a 1:1 mixture (v/v) FCA/ distilled water

Site: shoulder region
- Frequency of applications: single application

Topical Application (day 7)
A filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), saturated with the test material formulation (25% w/w in distilled water) was applied to the clipped skin and held in contact with an occlusive dressing for 48 hours.
Control Group: A patch was fully loaded with 0.5 mL of the vehicle and applied to the test area and held in contact by an occlusive dressing for 48 hours.

B. CHALLENGE EXPOSURE (day 21)
A square filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), saturated with the test material formulation at the maximum non-irritant concentration (25% w/w in distilled water) was applied to the shorn right flank of each animal and held in contact with an occlusive dressing for 24 hours. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 10% w/w in distilled water was similarly applied to a skin site on the left shorn flank.
After 24 hours, the dressing was carefully removed and discarded. The challenge sites were swabbed with cotton wool soaked in distilled water to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.

Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified.
Challenge controls:
The test material at a concentration of 10%w/w in distilied water was similarly applied to a skin site on the left shorn flank.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole, a-Hexylcinnamaldehyde. The recent reliability checks were performed between 1998 and 2001 (positive control data for 6 studies documented in report).

Results and discussion

Positive control results:
The sensitisation rate after application of the positive control substance Mercaptobenzothiazole (5 -10 % in arachis oil BP) was between 90 and 100 %, the sensitisation rate after application of the positive control substance a-Hexylcinnamaldehyde (5 % in arachis oil BP) was between 40 and 50 % confirming the liability of the test system.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
discrete or patchy to moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
discrete or patchy to moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
discrete or patchy erythema
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:
test chemical
Dose level:
10%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
discrete or patchy to moderate and confluent erythema
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
discrete or patchy to moderate and confluent erythema
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5

Any other information on results incl. tables

Intradermal Sighting Test - Summary of Results

Animal

Identification

Time of Observation

Concentration of Test Material
% (w/w)

Grade of Erythema at Injection Sites

Evidence of System ic Toxicity

 

A

24 hours

48 hours

72 hours

7 days

1

1

0

0

0

None

None

None

None

 

B

24 hours

48 hours

72 hours

7 days

5

2

1

0

0

None

None

None

None

 

C

24 hours

48 hours

72 hours

7 days

10

2E

2E

2E

*

None

None

None

*

 

D

24 hours

48 hours

72 hours

7 days

25

NE

NE

NE

*

None

None

None

*

 

E=Eschar N=Dark grey/black-coloured necrosis*=Animal killed for humane reasons due to severity of dermal reactions

 

 

Topical Sighting Test for Induction Application (48-Hour Exposure) - Individual Skin Reactions

Animal

Identification

Concentration of Test Material
% (w/w)

Skin Reactions (Hours After Removal of Patches)

1

24

48

ER

Oe

Other

ER

Oe

Other

ER

Oe

Other

E

75

50

25

10

3

3

2

2

2

1

1

1

BL

-

-

-

2

2

2

1

2

2

1

0

StCf

StCf

Cf

-

2

2

1

0

2

2

0

0

StCf

StCf

Cf

-

F

75

50

25

10

2

2

2

2

1

1

0

0

-

-

-

-

2

2

2

1

1

0

0

0

-

-

-

-

1

1

0

0

0

0

0

0

-

-

-

-

Er=Erythema, Oe=Oedema

 

BI=Blanching of the skin

Cf=Crust formation

- =No other reactions noted

St=Hardened dark brown / black-coloured scabs

 

Topical Sighting Test for Challenge Application (24-Hour Exposure) - Individual Skin Reactions

Animal

Identification

Concentration of Test Material
% (w/w)

Skin Reactions (Hours After Removal of Patches)

1

24

48

ER

Oe

Other

ER

Oe

Other

ER

Oe

Other

G

50

25

10

5

2

2

2

1

0

0

0

0

-

-

-

-

1

0

0

0

0

0

0

0

-

-

-

-

0

0

0

0

0

0

0

0

-

-

-

-

H

50

25

10

5

2

2

2

1

0

0

0

0

-

-

-

-

1

0

0

0

0

0

0

0

-

-

-

-

0

0

0

0

0

0

0

0

-

-

-

-

Er=Erythema, Oe=Oedema

- =No other reactions noted

 

Intradermal Induction - Individual Skin Reactions

Group

Animal Number

 

Grade of Erythema at Observation Site

24 Hours

48 Hours

 

Left Side

Right Side

Left Side

Right Side

 

Test

1

2

3

4

5

6

7

8

9

10

2

2

2

2

1

2

2

2

2

2

2

1

2

2

2

1

2

2

2

2

2

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

1

Control

11

12

13

14

15

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

 

 

Topical Induction - Individual Skin Reactions

Group

Animal Number

 

Skin Reactions (Hours After Removal of Dressing)

1 hour

24 hours

ER

Oe

Other

ER

Oe

Other

Test

1

2

3

4

5

6

7

8

9

10

2

2

2

2

2

2

2

2

2

2

0

1

0

0

0

1

0

1

1

1

BS

BS

BS

BS

BS

BS

BS

BS

BS

BS

2

2

2

2

2

2

2

2

2

2

0

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

-

-

Control

11

12

13

14

15

1

1

1

1

1

0

0

0

0

0

BS

BS

BS

BS

-

0

0

0

0

0

0

0

0

0

0

-

-

-

-

-

Er=Erythema, Oe=Oedema

Bs=Bleeding from intradermal injection sites

- =No other reactions noted

 

Individual Bodyweights and Bodyweight Gains

Bodyweight increases of the guinea pigs in the test group between Day 0 and Day 24 were comparable to those noted in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study, sodium dicyanoamide (a.i. 97.5 %) is a strong dermal sensitiser (90 % positive reactions at 5 % intradermal induction dose). The response in the GPMT assay was > 60% and the substance was not tested at ≤ 1 % intradermal induction dose in the guinea pig maximisation test. Although the criteria for classification to subcategory 1B are fulfilled, the classification for subcategory 1A cannot be excluded and therefore the substance is classified as a Category 1 skin sensitiser.
Executive summary:

In a dermal sensitisation study according to OECD TG 406, 1992) with Sodium dicyanoamide (a.i. 97.5 %) in distilled water, young adult Dunkin-Hartley guinea pigs were tested using the Maximization test method. Positive control substances were mercaptobenzothiazole with a sensitisation rate of 90 - 100 % and α-hexylcinnamaldehyde with a sensitisation rate of 40 – 50%.

Mild to moderate skin reactions were observed after intradermal induction (5 %) and dermal induction (25 %) in the test substance animals. After challenge exposure (25 %) positive responses (discrete or patchy to moderate and confluent erythema) were noted in test group animals at the 24 and 48-hour observations.

Transient challenge reactions (discrete or patchy erythema) were noted at the challenge sites of two control group animals at the 24-hour observations. No skin reactions were observed in control animals at the 48-hour observation time. Therefore the sensitisation rate was 90 %.  

In this study sodium dicyanoamide gave a positive response in a guinea pig maximisation test with 90 % responding at 5 % intradermal induction dose. The response in the GPMT assay was > 60% and the substance was not tested at ≤ 1 % intradermal induction dose in the guinea pig maximisation test. Although the criteria for classification to subcategory 1B are fulfilled, the classification for subcategory 1A cannot be excluded and therefore the substance is classified as a Category 1 skin sensitiser.

In this study, sodium dicyanoamide (a.i. 97.5 %), is a strong dermal sensitiser.