Dipotassium 4,4'-bis[6-anilino-4-[bis(2-hydroxyethyl)amino-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

data is from safety assessment reports

Data source

Reference

Reference 1

Reference Type:

secondary source

Title:

SCREENING-LEVEL HAZARD CHARACTERIZATION CHEMICAL CATEGORY NAME Stilbene Fluorescent Brighteners

Author:

U.S. Environmental Protection Agency

Year:

1992

Bibliographic source:

U.S. Environmental Protection Agency,Hazard Characterization Document,2012

Reference 2

Reference Type:

secondary source

Title:

Contact hypersensitivity to FAT 65´019/H in albino guinea pigs. Maximisation-test

Author:

Ciba Specialty Chemicals Inc.

Year:

1989

Bibliographic source:

RCC Project 247825. Study reported from RCC to Ciba-Geigy AG

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Principles of method if other than guideline:

To assess the skin sensitization potential of disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate (CAS No: 4193-55-9) in Dunkin-Hartley guinea pigs.

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

EC Number:

224-073-5

EC Name:

Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Cas Number:

4193-55-9

Molecular formula:

C40H44N12O10S2.2Na

IUPAC Name:

disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Test material form:

solid

Details on test material:

- Name of the test material: disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Common Name: Blankophor BA
- Molecular Formula: C40H44N12O10S2.2Na
- Molecular Weight: 960.9578 g/mol
- Substance type: Organic
- Physical State: Solid

Specific details on test material used for the study:

- Name of the test material: disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Common Name: Blankophor BA
- Molecular Formula: C40H44N12O10S2.2Na
- Molecular Weight: 960.9578 g/mol
- Substance type: Organic
- Physical State: Solid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Test animals:
Age: Males were 7 week old
Weights: 359-454 g,
Age: Females were 8 week old
Weights: 390-450 g at pretest.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: bi-distilled water

Concentration / amount:

a) Freund complete adjuvant mixed 50:50 with water,
b) test item 1% in bi-destilled water,
c) 50:50 mixture of a) and b).

Day(s)/duration:

1 week

Adequacy of induction:

not specified

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: petrolatum oil

Concentration / amount:

15 %

Day(s)/duration:

48 hours

Adequacy of induction:

not specified

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: petrolatum oil

Concentration / amount:

10 %

Day(s)/duration:

48 hours

Adequacy of challenge:

not specified

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: petrolatum oil

Concentration / amount:

10 %

Day(s)/duration:

48 hours

Adequacy of challenge:

not specified

No. of animals per dose:

Treatment group: 10 males and 10 females
Control: 5 males and 5 females

Details on study design:

RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures:1
- Exposure period: no data
- Test groups: 20
- Control group: 10
- Site: scapular area
- Frequency of applications:1
- Duration: 7 days
- Concentrations: 3 pairs of intradermal injections .
a) Freund complete adjuvant mixed 50:50 with water,
b) test item 1% in bi-destilled water,
c) 50:50 mixture of a) and b).

A.2. INDUCTION EXPOSURE: DERMAL
- No. of exposures:1
- Exposure period: 48 hours
- Test groups: 20
- Control group: 10
- Site: - scapular area
Frequency of applications: 1
- Duration: 48 hours
- Concentrations: 15 % in petrolatum oil


B. CHALLENGE EXPOSURE
- No. of exposures:1
- Day(s) of challenge: 2 weeks later
- Exposure period: 24 hours
- Test groups: 20
- Control group: 10
- Site: The flanks of the animals
- Concentrations: 10 % in petrolatum oil)
- Evaluation (hr after challenge): 24 and 48 hours after challenge application

C.RECHALLENGE EXPOSURE
Rechallenge was performed as for the first challenge two weeks after the first challenge. The flanks of the animals were changed and controls were treated with vehicle only on the left flank.

Challenge controls:

concurrent vehicles were used.

Positive control substance(s):

yes

Remarks:

Formaldehyde

Study design: in vivo (LLNA)

Statistics:

Mean values with standard deviation.
Exact Fischer-test
p-values were calculated from 24 hours values of treatment and control group.

Results and discussion

Positive control results:

In a separate experiment using the same guinea pig strain clear positive results were obtained in formaldehyde treated animals after the epidermal challenge application. For the induction period a 10 % dilution of HCHO (Fluka AG; article No. 4003, 37 % solution) in bi-distilled water, and for the challenge procedure a 5 % dilution of HCHO was used. HCHO posess a strong skin sensitizing (contact allergenic) potential in Dunkin-Hartley albino guinea pigs.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1 %

Total no. in group:

20

Clinical observations:

Injection sites 1 and 3 were found showing erythema and edema,necroses from day 10-32 and exfoliation from day 33 to 39

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1 %. Total no. in groups: 20.0. Clinical observations: Injection sites 1 and 3 were found showing erythema and edema,necroses from day 10-32 and exfoliation from day 33 to 39.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15 %

Total no. in group:

20

Clinical observations:

erythema and edema from day 2 to 8.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15 %. Total no. in groups: 20.0. Clinical observations: erythema and edema from day 2 to 8..

Results 3

Reading:

other: 1st challenge

Hours after challenge:

168

Group:

test chemical

Dose level:

10%

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

erythema was observed

Remarks on result:

other: Reading: other: 1st challenge. . Hours after challenge: 168.0. Group: test group. Dose level: 10%. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: erythema was observed.

Results 4

Reading:

other: 1st challenge

Hours after challenge:

192

Group:

test chemical

Dose level:

10%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

erythema was observed

Remarks on result:

other: Reading: other: 1st challenge. . Hours after challenge: 192.0. Group: test group. Dose level: 10%. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: erythema was observed.

Results 5

Reading:

rechallenge

Hours after challenge:

336

Group:

test chemical

Dose level:

10 %

No. with + reactions:

0

Clinical observations:

no allergenic potency

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 336.0. Group: test group. Dose level: 10 % in petrolatum oil. No with. + reactions: 0.0. Clinical observations: no allergenic potency.

In vivo (LLNA)

Cellular proliferation data / Observations:

After the first challenge application with 10 % test item in petrolatum oil a mild allergenic potency was concluded. After a second challenge application no allergenic potency
of the test article diluted to 10 % in petrolatum oil was observed when the results were interpreted according to Magnusson-Kligman.

Any other information on results incl. tables

SENSITIZING EFFECTS

-Control group: No positive reactions observed.

-Test group: First challenge: erythema was observed in 5/20 animals at the 24 hours reading and in 3/20 animals at 48 hours inspection when treated with a 10 % test item concentration.

Second challenge: no findings in any animal.

VIABILITY / MORTALITY: No death occurred during the test period.

LOCAL SYMPTOMS

-Control group: application sites 1 and 3 were found showing erythema and edema from day 2 to 8; necroses from day 10-32 and exfoliation from day 33 to 39 (termination of test). Injection site 2 showed erythema and edema from day 2 to 4. The treatment with SDS is therefore obsolete.

-Test group: i.c. injection sites 1 and 3 were found showing erythema and edema from day 2 to 8; necroses from day 10-32 and exfoliation from day 33 to 39 (termination of test).

Injection site 2 showed erythema and edema from day 2 to 8. The treatment with SDS is therefore obsolete. Epidermal application area for induction showed discoloration from day 10 to 14. Discoloration was observed on first challenge application area from day 23 to 32 and on second challenge application area from day 37 to 39 (termination of study).

No observations were made on day 9 because animals were treated semi occlusively.

Syptoms: No systemic syptoms were observed during study time.

Body weights:

During acclimatization: males No 267 (-6.5 g) and 270 (-6.4 g)and female 280 (-7.9 g) experienced weight loss.

During treatment: females No 277 (contr. group; -9.0 g) and 289 (treatment group; -43.3 g) lost weight.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitizing

Conclusions:

After the first challenge application with 10 % test chemical in petrolatum oil a mild allergenic potency was concluded. After a second challenge application no allergenic potency of the test article diluted to 10 % in petrolatum oil was observed. Therefore the chemical disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate (CAS No: 4193-55-9) was considered to be not sensitizing in Dunkin-Hartley guinea pigs according to Magnusson-Kligman test.

Executive summary:

A Maximization test of chemical disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate (CAS No: 4193-55-9) was conductedin Dunkin-Hartley guinea pigs(male and female) to assess the degree of skin sensitization caused by the chemical in accordance with Directive 84/449/EEC, B.6 "Acute toxicity (skin sensitization)".

 

Induction was performed by intradermal injections and later by topical application in the scapular area region. Intradermal induction was performed by 3 pairs of intradermal injections consist of a) Freund complete adjuvant mixed 50:50 with water, b) test item 1% in bi-destilled water, c) 50:50 mixture of a) and b). A control group was treated accordingly without the test item.

 

Epicutaneous induction was made one week after the induction with 15 % chemical in petrolatum oil or vehicle only. The dressings were left in place for 48 hours. Skin reactions (edema, erythema) were read immediately following removal of bandages and 24 and 48 hours later.

 

Challenge was made two weeks after the epidermal induction for test item (10 % in petrolatum oil) and vehicle controls. The dressings were removed after 24 hours and oedema and erythema assessed immediately as well as 24 and 48 hours later. Rechallenge was performed as for the first challenge two weeks after the first challenge. The flanks of the animals were changed .Controls were treated with vehicle only on the left flank.

 

The formaldehyde was used as positive control in a separate experiment using the same guinea pig strain with 10 % dilution of HCHO in bi-distilled water for induction period a, and a 5 % dilution of HCHO for the challenge procedure which possessed a strong skin sensitizing (contact allergenic) potential in Dunkin-Hartley albino guinea pigs.

 

The reactions were scored according to the Draize score system. Allergenicity was evaluated from the 24 hours readings and the percentage of animals sensitized.Mean values with standard deviation. Exact Fischer-test p-values were calculated from 24 hours values of treatment.

 

 After the first challenge application with 10 % test chemical in petrolatum oil a mild allergenic potency was concluded. After a second challenge application no allergenic potency of the test article diluted to 10 % in petrolatum oil was observed. No death occurred during the test period.Therefore the chemical disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate (CAS No: 4193-55-9)was considered to be notsensitizing in Dunkin-Hartley guinea pigs.