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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:
19 June 2017 - 28 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
other: EC No 440/2008 Part B. Skin Sensitization: Guinea-Pig Maximization Test (GPMT)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF)
Version / remarks:
November 2000
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig Maximization test was selected since the DEREK assessment was positive, the DPRA assay was negative and the KeratinoSensTM assay was inconclusive for skin sensitization for the test item. Based on these tests, no conclusion on skin sensitization could be drawn as the test item needs to be metabolized before binding to proteins. Since a positive h-CLAT assay does not give information on potency and a negative h-CLAT may be false negative due to insufficient metabolizing capacity, in vivo testing was recommended. According to DEREK, dibutylthiourea (and other dialkylthiourea) was positive in a GPMT test, but not in an LLNA test. These compounds are believed to exhibit low skin penetration and as such the GPMT (intradermal) is considered to be a more sensitive test than the LLNA test.

Test material

Constituent 1
Chemical structure
Reference substance name:
Tributyl-2-thiourea
EC Number:
219-350-2
EC Name:
Tributyl-2-thiourea
Cas Number:
2422-88-0
Molecular formula:
C13H28N2S
IUPAC Name:
1,1,3-tributylthiourea
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Tri butyl thiourea
- Appearance: Clear pale to medium brown liquid
- Storage conditions: At room temperature
Specific details on test material used for the study:
- pH (1% in water, indicative range): 5.0-4.6
- Specific gravity/density: 0.943 g/cm^3
- Solubility in vehicle: soluble
- Stability in vehicle: stable

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 4 weeks old
- Weight at study initiation: 245-286 g
- Housing: up to 5 animals of the same dosing group together in Noryl cages containing sterilized sawdust and equipped with water bottles.
- Diet: complete maintenance diet for guinea pigs, ad libitum; hay, at least twice a week.
- Water: municipal tap water (periodically analyzed for contaminants), ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no, animals in poor health were not assigned to the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 47-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 20 June 2017 To: 28 July 2017

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
20%
Day(s)/duration:
3 days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100%
Day(s)/duration:
48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100%
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Treatment group: 10
Control group: 5
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test item concentrations to be used in the main study. Four animals were used and four test item concentrations were tested: 100%, 50%, 20% and 10%.
- Intradermal injection: two animals received two different concentrations (10, 20, 50 or 100%) in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.
- Epidermal application: Two different concentrations were applied (0.5 mL each) to each of four animals to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage. Animals used for previous intradermal application received the two loweest concentrations.
After 24 hours, the dressing was removed and the skin cleaned of residual test item using water. The treated skin areas were assessed for irritation 24 and 48 hours after removal of the dressings.

The test system and procedures were identical to those used during the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal: on day 1, three pairs of injections (one 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection, one test item at a 20% concentration and one 1:1 w/w mixture of the test item (40%) and Freunds' Complete Adjuvant.
- On day 7, the skin was pretreated with 10% SDS
- On day 8, one epidermal exposure with a test item concentration of 100%
- Exposure period: 3 days for intradermal injections, 48 hours for epidermal application
- Control group: five control animals were treated in the same way but instead of the test item, the vehicle was administered.
- Site: scapular region (clipped)
- Frequency of applications: once, semiocclusive dressing
- Concentrations: 20% for the intradermal injections, 100% for the epidermal application

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Days of challenge: 21 days
- Exposure period: 24 hours
- Test groups: ten animals were treated by epidermal application of 100% test item concentration and the vehicle (0.1 mL each) using Patch Test Plasters. The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
- Control group: five control animals were treated in the same way but instead of the test item, the vehicle was administered.
- Site: flank (clipped)
- Concentrations: 100%
- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
On day 28 (approx. one week after the first challenge), a re-challenge was conducted to clarify the results in the first challenge. The contralateral flank of all animals was similarly treated.
Positive control substance(s):
yes
Remarks:
Alpha- Hexylcinnamaldehyde (20%)

Results and discussion

Positive control results:
The reliability check with the positive control was perfomed in July 2017.
After 24 hours of the challenge exposure: out of 10 animals, 8 animals had skin reactions graded score 2 and 2 animals had skin reactions graded score 1
After 48 hours of the challenge exposure: out of 10 animals, 7 animals had skin reactions graded score 2 and 3 animals had skin reactions graded score 1
From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitizing potential of a test item in a Maximization type of test.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
other: Not conducted
Hours after challenge:
0
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
0

Any other information on results incl. tables

Table 1 Results of the preliminary irritation study

Skin reactions after intradermal injection

Animal Number

Test item Concentration %

24 Hours after injection

48 Hours after injection

Erythema

Necrosis
(Ø mm)

Erythema

Necrosis
(Ø mm)

28

100

 

2

 

3

50

2

 

 

1

29

20

2

 

1

 

10

2

 

1

 

 

Skin reactions after epidermal exposure

Animal Number

Test item Concentration %

24 Hours after exposure

48 Hours after exposure

Erythema

Oedema

Erythema

Oedema

26

100

0

0

0

0

50

0

0

0

0

27

100

0

0

0

0

50

0

0

0

0

28

20

0

0

0

0

10

0

0

0

0

29

20

0

0

0

0

10

0

0

0

0

Grading erythema: 0 = No erythema; 1 = Slight erythema (barely perceptible); 2 = Well-defined erythema

Grading Oedema: 0 = No oedema

Table 2 Induction readings

Control Animals

Animal Number

Intradermal injections (readings Day 3)

Epidermal exposure
(readings Day 10)

1:1 Mixture of FCA and water for injection

Vehicle

1:1 Mixture of FCA and vehicle

Vehicle

Erythema

Signs of necrosis
(Ø mm)

Erythema

Signs of necrosis
(Ø mm)

Erythema

Signs of necrosis
(Ø mm)

Erythema

Oedema

31

3

 

2

 

3

 

0

0

32

3

 

0

 

3

 

0

0

33

3

 

1

 

1

 

0

0

34

2

 

2

 

2

 

0

0

35

3

 

0

 

3

 

0

0

Experimental Animals

Animal Number

Intradermal injections (readings Day 3)

Epidermal exposure
(readings Day 10)

1:1 Mixture of FCA and water for injection

20% test item concentration

1:1 Mixture of FCA and
a 40% concentration

100% test item concentration

Erythema

Signs of necrosis
(Ø mm)

Erythema

Signs of necrosis
(Ø mm)

Erythema

Signs of necrosis
(Ø mm)

Erythema

Oedema

36

3

 

2

 

3

 

2

0

37

3

 

1

 

2

 

2

0

38

3

 

2

 

3

 

1

0

39

3

 

1

 

3

 

1

0

40

3

 

1

 

3

 

1

0

41

3

 

1

 

3

 

1

0

42

3

 

2

 

3

 

2

0

43

3

 

1

 

3

 

1

0

44

3

 

1

 

3

 

1

0

45

3

 

1

 

3

 

1

0

FCA = Freunds' Complete Adjuvant

Grading erythema: 0 = No erythema; 1 = Slight erythema (barely perceptible); 2 = Well-defined erythema; 3 = Moderate erythema

Grading Oedema: 0 = No oedema

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not sensitizing according to Regulation (EC) 1272/2008
Conclusions:
In the guinea pig maximization test, Tri butyl thiourea cause no skin hypersensitivity in the experimental animals in response to a 100% test item concentration in the (re-)challenge phase (sensitization rate of 0%). Based on these results, the test substance is not classified as skin sensitizing according to the GHS and Regulation (EC) 1272/2008.