Registration Dossier

Administrative data

Description of key information

The skin sensitisation potential of 1,3 -dibutylthiourea was evaluated in three types of test: GMPT, LLNA and SLNA.

DBTU showed positive reactions with GPMT and SLNA, but negative with LLNA. Based on the weight-of-evidence, DBTU is considered to be a strong sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no detailed data about induction conditions
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
Method: Magnusson and Kligman J. Invest. Dermatol 1969 ; 52 : 268-276
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An old GPMT was available in the publication. No new study was performed for skin sensitisation endpoint for REACH regulation.
Test material information:
Composition 1
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
no data
Route:
intradermal and epicutaneous
Vehicle:
other: olive oil (intradermal induction), petrolatum (topical induction) and acetone (challenge)
Concentration / amount:
Induction / intradermal injection : 0, 2, 20, 200, 2000 ppm
Induction / topical : 0, 250 000 ppm
Challenge / topical : 0, 0.2, 2, 20, 200, 2000 ppm
Route:
epicutaneous, open
Vehicle:
other: olive oil (intradermal induction), petrolatum (topical induction) and acetone (challenge)
Concentration / amount:
Induction / intradermal injection : 0, 2, 20, 200, 2000 ppm
Induction / topical : 0, 250 000 ppm
Challenge / topical : 0, 0.2, 2, 20, 200, 2000 ppm
No. of animals per dose:
10 per group
Details on study design:
Challenge : On day 22, all the animals received a topical open application of 0.1 ml of the test substance in their right flank (while their left flank received the vehicle).
- Clinical examinations: no
- Mortality: no
- Body weight: no
- Necropsy: no
Challenge controls:
no
Positive control substance(s):
no
Reading:
1st reading
Group:
negative control
Dose level:
no induction
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No effects with a challenge of 0 - 0.2 - 2 - 20 - 200 and 2000 ppm
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
Intradermal induction of 2 ppm
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No effects at challenge concentrations (0 - 0.2 - 2 - 20 - 200 and 2000 ppm)
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
intradermal induction of 20 ppm
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No effects at challenge concentration of 0 - 0.2 - 2 and 20 ppm
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
intradermal induction of 20 ppm
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
at challenge concentrations of 200 ppm
Reading:
1st reading
Group:
test group
Dose level:
intradermal Induction of 20 ppm
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
At challenge concentration of 2000 ppm
Reading:
1st reading
Group:
test group
Dose level:
Interdermal induction of 200 ppm
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No effects at challenge concentration of 0 - 0.2 - 2 - 20 ppm
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
Intradermal concentration of 200 ppm
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
at challenge concentration of 200 ppm
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
Intradermal induction of 200 ppm
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
At challenge concentration of 2000 ppm
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
Intradermal induction of 2000 ppm
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No effects at challenge concentrations of 0 - 0.2 and 2 ppm
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
Intradermal induction of 2000 ppm
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
at challenge concentration of 20 ppm
Reading:
1st reading
Group:
test group
Dose level:
Intradermal induction of 2000 ppm
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
At challenge concentration of 200 ppm
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Group:
test group
Dose level:
Intradermal induction of 2000 ppm
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
At challenge concentration of 2000 ppm
Remarks on result:
positive indication of skin sensitisation

No redness was observed in the control animals (in animals not induced but challenged, and in animals not induced and not challenged).

In the test groups (induced and challenged), a positive effect (redness in more 30% of animal) was observed at the induction concentration of 200 ppm (0.02%) at 200 and 2000 ppm of challenge doses (60 and 80% respectively).

This positive effect was confirmed at the induction concentration of 2000 ppm.

DBTU is a skin sensitizer according to this GPMT test.

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
According this Guinea pigs maximalisation test, DBTU is a skin sensitizer .
Executive summary:

A guinea pigs maximalisation test was performed according to the Magnuson and Kligman method with DBTU.

In induction phase, guinea pigs were exposed by intradermal injection (0, 2, 20, 200, 2000 ppm of DBTU) and by topical administration (250 000 ppm of DBTU).

In the challenge phase, animals were exposed by topical administration at : 0, 0.2, 2, 20, 200 or 2000 ppm of DBTU.

No redness was observed in the control animals (in animals not induced but challenged, and in animals not induced and not challenged).

In the test groups (induced and challenged), a positive effect (redness in more 30% of animal) was observed at the induction concentration of 200 ppm (0.02%) at 200 and 2000 ppm of challenge doses (60 and 80% respectively).

This positive effect was confirmed at the induction concentration of 2000 ppm.

DBTU is a skin sensitizer according to this GPMT test.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
The assay was performed according to the method of Kimber and Weisenberger (1989), with minor modifications (Ikarashi et al. 1992).
GLP compliance:
not specified
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
- Source: Japan SLC Inc (Shizuoka, Japan)
- Age: 6-8 weeks
- Weight at study initiation: no data
Vehicle:
other: dichloromethane
Concentration:
- Volume administered: 25 µl
- Tested concentrations: 0, 10, 25 and 50 %
- Application site: on both ears
- Administration frequency: once a day for 3 consecutive days.
No. of animals per dose:
- 3 tested groups: 3 mice per dose group
- Control group: vehicle alone (untreated mice)
Details on study design:
- Harvesting: On the day following the final application, draining auricular lymph nodes were excised, pooled for each group and weighed. A single-cell suspension of lymph node cells (LNC) was then prepared.
- Washing: After having been washed once with Hank's balanced salt solution, total cell number was determined.
- Resuspension: at a concentration of 5.10^6 cells/ml, in RPMI 1640 culture medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum.
- Radio-labelling: 200 µl of LNC suspension were seeded in 96-well culture plates (5 wells/group) and cultured with 0.5 µCi of tritiated methylthymidine (3HTdR) for 24h at 37°C. LNC proliferation was measured: the data are mean 3HTdR (tritiated methylthymidine) incorporation from 5 culture wells.
Parameter:
SI
Value:
1.4
Test group / Remarks:
10%
Parameter:
SI
Value:
1.6
Test group / Remarks:
25%
Parameter:
SI
Value:
0.9
Test group / Remarks:
50%
Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
DBTU faided to produce positive LLNA responses, the test substance was not predicted to be sensitizer.
Executive summary:

The Murine local lymph node assay was performed according to the method of Kimber and Weisenberger (1989), with minor modifications (Ikarashi et al. 1992).

Groups of mice (n=3) were exposed to 25 µl of various concentrations of test chemical in AOO or AOO alone (control) on each ear for three consecutive days. 4 days following the initial application, the auricular lymph nodes were axcised and pooled for each group. Lymph node cells (LNC) suspensions were prepared, and then LNC number and 3HTdR incorporation into LNCs were measured. The increases in LNC number and increase in 3HTdR incorporation (SI) relative to controls were derived for each test group, and the SI was calculated. A chemical was regarded as a sensitizer if it scored an SI of 3 or more.

DBTU was tested at the concentrations of 10, 25, 50% in dichloromethane. The Simulation index obtained were lower than 3 : 1.4, 1.6, 0.9 respectively.

Indeed, DBTU faided to produce positive LLNA responses, it is not a skin sensitizer.

Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
The Sensitive lymph node assay (SLNA) was carried out according to the method described by Ikarashi et al. (1993).
GLP compliance:
not specified
Type of study:
other: sensitive lymph node assay (SLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
- Source: Japan SLC Inc (Shizuoka, Japan)
- Age: 6-8 weeks
- Weight at study initiation: no data
Vehicle:
other: cf Test conditions
Concentration:
- Volume administered: 25 µl
- For intradermal injection: a water-in-oil emulsion was prepared from a mixture of dimethyl sulfoxide (DMSO) containing the test substance, FCA and saline at a ratio of 1:4:5. Two 25-µl aliquots of test substance-FCA emulsion were injected intradermally, at various concentrations, into two sites of the abdominal skin located at both sides of the ventral midline.
- Five days after injection, 25 µl test chemical in Acetone-olive oil (AOO, 4:1) was topically applied to both sides of each ear, daily for three consecutive days.
- Control mice were initially treated by intradermal injection of DMSO-FCA-saline emulsion into the abdomen and after 5 days, the mice were exposed to AOO alone on the ears for three consecutive days.
- Tested concentrations: 0, 0.2 and 2 % in DMSO for the intradermal injection, associated respectively with the following concentrations, 0, 5 and 5 % in AOO, for the topical application.
No. of animals per dose:
9
- 3 tested groups: 3 mice per dose group
- Control group: vehicle alone (untreated mice
Details on study design:
- Harvesting: The day after the final topical application, auricular lymph nodes were excised and pooled for each group. A single-cell suspension of lymph node cells (LNC) was then prepared by mechanical disaggregation.
- Washing: After having been washed once with Hank's balanced salt solution, LNC were counted
- Resuspension: at a concentration of 5.10^6 cells/ml, in RPMI 1640 culture medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum.
- Radio-labelling: 200 µl of LNC suspension were seeded in 96-well culture plates (5 wells/group) and cultured with 0.5 µCi of tritiated methylthymidine (3HTdR) for 24h at 37°C.
Parameter:
SI
Remarks on result:
other: The combined treatment of 2% injection and 5% topical application induced the highest SI total score, that was found greater than 3, and the 3HTdR incorporation doubled compared to the negative control.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on this SLNA, DBTU is considered to be skin sensitizer.
Executive summary:

The Sensitive lymph node assay (SLNA) was carried out according to the method described by Ikarashi et al. (1993). Three mice per group were used. For intradermal induction of test chemical, a water-in-oil emulsion was prepared from a mixture of DMSO containing the chemical, FCA and saline at a ration of 1:4:5. Two 25 µl aliquots of test chemical-FCA emulsion were injected intradermally into two sites of the abdominal skin located at both sides of the ventral midline. Five days after injection, 25µl test chemical in acetone-olive oil (4:1) (AOO) was applied to both sides of each ear daily for 3 consecutive days. Control mice were initially treated by intradermal injection of DMSO-FCA-saline emulsion into the abdomen and, after 5 days, the mice were exposed to AOO alone on the ears for 3 consecutive days. The day after the final topical application, auricular lymph nodes were excised, and pooled for each experimental group. The stimulation index of each concentration was determined using radiolabelling.

DBTU was tested at the following concentrations: 0.2 and 2% in AOO. At 0.2%, the SI was 1.1. DBTU had a SI score of 4.1 with 2% intradermal injection, indicating a positive response (SI >3).

Based on this SLNA, DBTU is considered to be skin sensitizer.

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

GPMT study (Nakamura 1994)

A guinea pigs maximalisation test was performed according to the Magnuson and Kligman method with DBTU.

In induction phase, guinea pigs were exposed by intradermal injection (0, 2, 20, 200, 2000 ppm of DBTU) and by topical administration (250 000 ppm of DBTU).

In the challenge phase, animals were exposed by topical administration at : 0, 0.2, 2, 20, 200 or 2000 ppm of DBTU.

 No redness was observed in the control animals (in animals not induced but challenged, and in animals not induced and not challenged).

In the test groups (induced and challenged), a positive effect (redness in more 30% of animal) was observed at the induction concentration of 200 ppm (0.02%) at 200 and 2000 ppm of challenge doses (60 and 80% respectively).

This positive effect was confirmed at the induction concentration of 2000 ppm.

DBTU is a skin sensitizer according to this GPMT test.

LLNA (Ikarashi 1994) :

The Murine local lymph node assay was performed according to the method of Kimber and Weisenberger (1989), with minor modifications (Ikarashi et al. 1992).

Groups of mice (n=3) were exposed to 25 µl of various concentrations of test chemical in AOO or AOO alone (control) on each ear for three consecutive days. 4 days following the initial application, the auricular lymph nodes were axcised and pooled for each group. Lymph node cells (LNC) suspensions were prepared, and then LNC number and 3HTdR incorporation into LNCs were measured. The increases in LNC number and increase in 3HTdR incorporation (SI) relative to controls were derived for each test group, and the SI was calculated. A chemical was regarded as a sensitizer if it scored an SI of 3 or more.

DBTU was tested at the concentrations of 10, 25, 50% in dichloromethane. The Simulation index obtained were lower than 3 : 1.4, 1.6, 0.9 respectively.

Indeed, DBTU faided to produce positive LLNA responses, it is not a skin sensitizer.

SLNA (1994) :

The Sensitive lymph node assay (SLNA) was carried out according to the method described by Ikarashi et al. (1993). Three mice per group were used. For intradermal induction of test chemical, a water-in-oil emulsion was prepared from a mixture of DMSO containing the chemical, FCA and saline at a ration of 1:4:5. Two 25 µl aliquots of test chemical-FCA emulsion were injected intradermally into two sites of the abdominal skin located at both sides of the ventral midline. Five days after injection, 25µl test chemical in acetone-olive oil (4:1) (AOO) was applied to both sides of each ear daily for 3 consecutive days. Control mice were initially treated by intradermal injection of DMSO-FCA-saline emulsion into the abdomen and, after 5 days, the mice were exposed to AOO alone on the ears for 3 consecutive days. The day after the final topical application, auricular lymph nodes were excised, and pooled for each experimental group. The stimulation index of each concentration was determined using radiolabelling.

DBTU was tested at the following concentrations: 0.2 and 2% in AOO. At 0.2%, the SI was 1.1. DBTU had a SI score of 4.1 with 2% intradermal injection, indicating a positive response (SI >3).

Based on this SLNA, DBTU is considered to be skin sensitizer.

With this results, it was confirmed that SLNA was more sensitive than LLNA for thiourea.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the key study (GPMT), 1,3 -dibutylthiourea should be classified as strong skin senitizer (Skin sens. 1A, H317) according to the regulation EC.N°1272/2008.

Justification: DBTU was positive in the GPMT test (60% at 200 ppm of challenge, and 80% at 2000 ppm) with an concentration for intradermal induction of 0.02% (200 ppm). DBTU is classified in the category 1A according to the Regulation UE n°286/2011 because the incidence sensitised guinea pigs is higher to 30% , and the concentration for intradermal induction is lower to 0.1%.