1,3-diphenyl-2-thiourea

Endpoint summary

Description of key information

DPTU is a human and animal skin sensitizer.

DPTU gave a positive response in a guinea pig maximalisation test (Nakamura 1994) and in a SLNA test (Ikarashi 1994). DPTU is reported to be an allergen associated with its use as a rubber vulcanisation accelerator and in PVC adhesive tape (Freger 1982, Foussereau 1992).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: Magnusson and Kligman J. Invest. Dermatol 1969 ; 52 : 268-276

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A study on guinea pigs was available before the REACH regulation.

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

- Strain: Hartley strain albino
- Sex: female (nulliparous and non-gravid)
- Source, age, weight at study initiation: no data
- preliminary study: no data

Route:

intradermal

Vehicle:

other: ethanol/propylene glycol (20/80)

Concentration / amount:

2, 20, 200, 2000 and 20000 ppm

Route:

epicutaneous, occlusive

Vehicle:

olive oil

Concentration / amount:

250000 ppm

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

other: acetone

Concentration / amount:

0, 2, 20, 200, 2000 ppm

Day(s)/duration:

24h

No. of animals per dose:

10 females per dosed groups
. Negative controls (propylene glycol, petrolatum and acetone undiluted): yes, 1 group of 10 animals

Details on study design:

- Tested concentrations:
. For the induction: 2, 20, 200, 2000 and 20000 ppm were tested by intradermal injection. Each one of these concentrations was associated with a 250 000 ppm-concentration for the topical application. Moreover, a 0 ppm-concentration was tested too in induction stage (by both injection and topical routes).
. For challenge: each concentration used in induction stage was associated with 5 different concentrations in the challenge stage (topical application): 0, 2, 20, 200, 2000 ppm.

- Test procedure: performed almost in accordance with the original procedure of Magnusson and Kligman. So 21 days after the initial intradermal injection, 0.1 mL aliquots of various concentrations of test substance were applied on the flank of each animal for challenge.

- Challenge exposure duration: 24h

- Rechallenge: no

Challenge controls:

no

Positive control substance(s):

yes

Remarks:

dinitrochlorobenzene

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

no induction

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal induction = 2 ppm

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal Induction = 20 ppm

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

other: Challenge 0 ppm (0/10), 2 ppm (1/10), 20 ppm (2/10), 200ppm (4/10), 2000 ppm (4/10)

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal induction= 200 ppm

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: Challenge 0 ppm (0/10), 2 ppm (5/10), 20 ppm (8/10), 200ppm (10/10), 2000 ppm (10/10)

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal indution = 2000 ppm

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: Challenge 0 ppm (0/10), 2 ppm (2/10), 20 ppm (8/10), 200ppm (10/10), 2000 ppm (9/10)

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal induction = 20 000 ppm

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: Challenge 0 ppm (0/10), 2 ppm (9/10), 20 ppm (10/10), 200ppm (9/10), 2000 ppm (9/10)

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

- From the 2 ppm-challenge concentration, the MR as well as the SR,increased with
the challenge concentration, when the induction concentration was held constant.

- The minimum induction concentration of test substance that induces a 
positive response = 20 ppm (because no positive reactions with any
challenge concentrations were found when the induction concentration was 2 ppm).

- The challenge concentration that induces a mean response approximately equal 
to 1.0 among the animals applied with the highest concentration for induction 
= 2 ppm.

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Positive responses were observed in this Guinea pigs maximalisation test, DPTU is considered as a strong skin sensitizer .

Executive summary:

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

In induction phase, guinea pigs were exposed by intradermal injection (0, 2, 20, 200, 2000, 20000 ppm of DPTU) and by topical administration (0 or 250 000 ppm of DPTU). In the challenge phase, animals were exposed by topical administration at: 0, 2, 20, 200, 2000 ppm of DPTU.

No effect was observed in the animals which were not induced and/or challenge with DPTU (negative control).

Below 20 ppm ( induction phase), no cutaneous reactions were observed after the challenge application. In the animals induced by an injection of 20 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 1/10 (2 ppm), 2/10 (20 ppm), 4/10 (200 and 2000 ppm). In the animals induced by an injection of 200 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 5/10 (2 ppm), 8/10 (20 ppm), 10/10 (200 and 2000 ppm). In the animals induced by an injection of 2000 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 2/10 (2 ppm), 8/10 (20 ppm), 10/10 (200 ppm) and 9/10 (2000 ppm). In the animals induced by an injection of 20000 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 9/10 (2 ppm), 10/10 (20 ppm), 9/10 (200 ppm) and 9/10 (2000 ppm). 

 According this guinea pig maximalisation test, DPTU is a strong skin sensitizer .

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: Kimber and Weisenberg (archives of Toxicology 1989 ; 63 : 274-282)

GLP compliance:

not specified

Type of study:

other: LLNA (local lymph node assay) and SLNA (sensitive mouse lymph node assay)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

- Source: Japan SLC (Shizuoa, Japan)
- Age: 6-8 weeks
- Weight at study initiation: no data

Concentration:

SLNA : intradermal injection : 0.2 and 2 %; topical application : 5%
LLNA : 0, 1, 2.5 and 5 % (experiment 1) and 0, 5, 10 and 25 % (experiment 2)

No. of animals per dose:

SLNA : Treated: 3 mice per dose group, Controls: 5 females (untreated)
LLNA : Treated: 3 mice per dose group

Details on study design:

SLNA : Two 25 µl aliquots of DPTU-FCA emulsion were injected intradermally into two sites of the abdominal skin located at both sites of the ventral midline. Five days after injection, 25 µl DPTU in acetone-olive oil (4:1) (AOO) was applied to both sites of each ear daily for 3 consecutive days. The day after the final topical application, auricular lymph nodes were excised, and pooled for each experimental group.

LLNA :
Mice were exposed to 25µl of various concentrations of DPTU in AOO aor AOO alone (control) on each ear for three consecutive days.
Four days following the initial application, the auricular lymph nodes were excised and pooled for each group.
- Application site: on both ears
- Administration frequency: once a day for 3 consecutive days.

CELL PROCESSING:
- Harvesting: On the fourth day, auricular lymph nodes were excised and pooled for each group. A 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 5.10E6 cell/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.

EXAMINATIONS:
3HTdR incorporation was measured by liquid scintillation counting. A stimulation index of LNC proliferation (SIp) was the calculated as follows:
SIp = 3HTdR incorporation (cpm) in test group / 3HTdR incorporation (cpm) in control group.
The increase in LNC number was calculated as follows: SIn = LNC number in treated group / LNC number in control group.
The global stimulation index was calculated according to the following formula: SItotal = SIp x SIn

POSITIVITY CRITERION: A chemical was regarded as a sensitizer if SI total was equal or greater than 3.

NUMBER OF INDEPENDENT EXPERIMENT: 2

Statistics:

no

Key result

Parameter:

SI

Remarks:

SLNA

Value:

11.3

Variability:

Concentration : 0,2%

Key result

Parameter:

SI

Remarks:

SLNA

Value:

32.2

Variability:

Concentration : 2%

Parameter:

SI

Remarks:

LLNA

Value:

0.6

Variability:

Concentration : 1% in AOO

Parameter:

SI

Remarks:

LLNA

Value:

1.1

Variability:

Concentration: 2,5% in AOO

Parameter:

SI

Remarks:

LLNA

Value:

0.7

Variability:

Concentration : 5% in AOO

Parameter:

SI

Remarks:

LLNA

Value:

0.8

Variability:

Concentration : 1% in DMSO

Parameter:

SI

Remarks:

LLNA

Value:

1.1

Variability:

Concentration : 2.5% in DMSO

Parameter:

SI

Remarks:

LLNA

Value:

1.1

Variability:

Concentration : 5% in DMSO

Cellular proliferation data / Observations:

LLNA : SI total were found as < 3, in the 2 experiments at any concentrations. No notable differences were observed between the results obtained with the 2 solvents.
SLNA : The SI total score was found greater than 3. The combined treatment of 2% injection and 5% topical application caused an marked lymph node responses (12.4 Lymph node cells in the control vs 56.0 in treated group) and the highest SI(total) (32.2).

Table 1: Results of the murine local lymph node assay (LLNA)

 

chemical	Conc. (%)	vehicle	LNC^1no. (x10^6)	Si(n)	3HTdR incorporation ^2	SI(p)	SI(total)	classification
DPTU	0	AOO	28.9	-	1.68+/-0.16	-	-	Negative
	1		22.8	0.8	1.58+/-0.36	0.8	0.6
	2.5		25.0	0.9	2.22+/-0.24	1.2	1.1
	5		16.3	0.6	1.74+/-0.18	1.1	0.7
DPTU	0	DMSO	51.7	-	2.23+/-0.14	-	-	Negative
	5		37.4	0.7	2.58+/-0.19	1.2	0.8
	10		38.8	0.8	3.07+/-0.15	1.4	1.1
	25		40.0	0.8	3.18+/-0.14	1.4	1.1

SI(n) = stimulation index of Iymph node cell nu-mber

SI(p= = stimulation index of Iymph node cell proliferation

SI(total)= stimulation index of total lymph node response

3HTdR = [3H]methyl thymidine

AOO = acetone-olive oil (4:1)

DMSO = dimethyl sulfoxide

 

¹LNC = lymph node cells

²Mean cpm +/-SD x 10^3

 

 

Table 2 : Results of the murine local lymph node assay (SLNA)

 

chemical	Intradermal injection Conc. (%)	Topical application – Conc (%)	LNC^1no. (x10^6)	Si(n)	3HTdR incorporation ^2	SI(p)	SI(total)	classification
DPTU	0 (DMSO)	0 (AOO)	12.4	-	2.00+/-0.12	-	-	Positive
	0.2	5	31.1	2.5	9.81+/-0.60	4.9	11.3
	2.0	5	56.0	4.5	16.38+/-1.00	8.2	32.2

SI(n) = stimulation index of Iymph node cell number

SI(p= = stimulation index of Iymph node cell proliferation

SI(total)= stimulation index of total lymph node response

3HTdR = [3H]methyl thymidine

AOO = acetone-olive oil (4:1)

DMSO = dimethyl sulfoxide

 

¹LNC = lymph node cells

²Mean cpm +/-SD x 10^3

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Positive results were obtained with SLNA but negative results with LLNA.
DPTU is considered as a skin sensitizer based on this results.

Executive summary:

 Contact sensitivity of diphenylthiourea (DPTU) was evaluated by a new sensitive mouse lymph node assay (SLNA) and the murine local lymph node assay (LLNA). The results of the SLNA and LLNA. In the LLNA and SLNA, the sensitizing activity was measured as a function of draining lymph node activation following application of the test chemicals.

DPTU was negative in the LLNA. The SLNA successfully detected the sensitivity of this thiourea tested. This result indicated that the SLNA was, in this case, more sensitive than the LLNA for identification of contact allergens.

DPTU is considered as a skin sensitizer based on these results.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

During the years, the allergenic activity of DPTU has been studied in three different animal models: the murine local lymph node assay (LLNA), the sensitive mouse local lymph node assay (SLNA) and the guinea pigs maximalisation test (GPMT). DPTU was identified as a sensitizer in the GMPT and SLNA but was found to be non-sensitising in the LLNA. The predictions of sensitizing potential and the order of the sensitizing capacity of DPTU by the SLNA and the GPMT are very similar. This disparity is most probably due to the differences in the administration of the compound in the LLNA versus the SLNA and the GPMT. In the two latter methods, the test compound is administered intradermally in addition to topical application. Intradermal injections seem to be important to obtain a positive response to DPTU as the penetration of DPTU probably is low when applied topically on intact skin (Samuelsson K. 2011).

 

GPMT test (Nakamura 1994) :

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

In induction phase, guinea pigs were exposed by intradermal injection (0, 2, 20, 200, 2000, 20000 ppm of DPTU) and by topical administration (0 or 250 000 ppm of DPTU). In the challenge phase, animals were exposed by topical administration at: 0, 2, 20, 200, 2000 ppm of DPTU.

No effect was observed in the animals which were not induced and/or challenge with DPTU (negative control).

Below 20 ppm ( induction phase), no cutaneous reactions were observed after the challenge application. In the animals induced by an injection of 20 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 1/10 (2 ppm), 2/10 (20 ppm), 4/10 (200 and 2000 ppm). In the animals induced by an injection of 200 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 5/10 (2 ppm), 8/10 (20 ppm), 10/10 (200 and 2000 ppm). In the animals induced by an injection of 2000 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 2/10 (2 ppm), 8/10 (20 ppm), 10/10 (200 ppm) and 9/10 (2000 ppm). In the animals induced by an injection of 20000 ppm of DPTU, skin sensitisation was observed in all groups of challenged rats : 9/10 (2 ppm), 10/10 (20 ppm), 9/10 (200 ppm) and 9/10 (2000 ppm).

According this Guinea pigs maximalisation test, DPTU is a skin sensitizer.

 

LLNA and SLNA tests (Ikarashi 1994) :

Contact sensitivity of diphenylthiourea (DPTU) was evaluated by a new sensitive mouse lymph node assay (SLNA) and the murine local lymph node assay (LLNA). The results of the SLNA and LLNA were compared with the data of the previous guinea pig maximization test (GPMT).

In the LLNA and SLNA, the sensitizing activity was measured as a function of draining lymph node activation following application of the test chemicals.

DPTU showed negative results in the LLNA. The SLNA successfully detected the sensitivity of this thiourea tested.This result indicated that the SLNA was, in this case, more sensitive than the LLNA for identification of contact allergens.

The predictions of sensitizing potential and the order of the sensitizing capacity of DPTU by the SLNA and the GPMT are very similar.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, DPTU should be classified as a strong skin sensitizer (Skin sens.1A, H317) according to the Regulation (EC) No 1272/2008.

 

Justification: DPTU is positive in the GPMT test (40% at 200 ppm and 2000 ppm of challenge) with a concentration for intradermal induction 0.002% (20 ppm). DPTU is classified in the category 1A according to the Regulation UE n°286/2011 because the incidence of sensitised guinea pigs is higher to 30%, and the concentration for intradermal induction is lower to 0.1%.