Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

Endpoint summary

Description of key information

The test substance is sensitiser to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2006 to 08 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Substance name: C.I. Reactive Yellow 174
Item No.: 1897214 from synthesis: 631178/174
Stability: 19.01.2010
Storage: at room temperature

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen.
- Age at study initiation: 6-12 weeks
- Housing: The animals were kept in groups in Macrolon-cages on Lignocel bedding
- Diet: Feeding ad libitum, ssniff R/M-H, 10 mm VI534-000 complete diet for rats/mice, totally-pathogen-free (TPF)
- Water: Free access to tap water (drinking water, municipal residue control, microbiol. controlled periodically)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Vehicle:

other: AOO (3+1 (v/v) Acetone/Olive Oil).

No. of animals per dose:

5

Positive control substance(s):

other: P-Phenylenediamine (CAS 106-50-3, Sigma GmbH, purity >98 %) at a concentration of 1 % (w/v) in AOO (3+1 (v/v) Acetone/Olive Oil) served as positive control.

Positive control results:

Mean ear thickness of the positive control group was 0.19 and 0.20 mm at Day 1 and Day 6, respectively. While the mean weights of the lymph nodes was 5.5 mg. The stimulation of the positive control (Phenylenediamine) at a concentration of 1 % was 9.9.

Parameter:

SI

Value:

7.8

Test group / Remarks:

Concentration level 15 %

Parameter:

SI

Value:

5.5

Test group / Remarks:

Concentration level 9 %

Parameter:

SI

Value:

3.3

Test group / Remarks:

Concentration level 3 %

Parameter:

SI

Value:

4.2

Test group / Remarks:

Concentration level 1 %

Four concentrations were chosen to gain a wide spectrum for the test design: Due to results of a solubility test and in consultation with the sponsor, the test item was assayed at concentrations of 15 %, 9 %, 3 % and 1 % (w/v). Additionally a positive control for verifying the functionality of the current test run was carried along.

The vehicle was AOO (3+1 (v/v) Acetone/Olive Oil). Stability of the test item in the vehicle was proven. Each mouse was treated by topical application of the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with ³H-methyl thymidine. Directly prior to the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured. This is to exclude irritating properties of the test item, which may lead to false positive results.

 

Mean Ear thickness at:                                 day 1                          day 6

of the 15 % group was                                 0.21 mm                     0.22 mm

of the 9 % group was                                   0.21 mm                     0.21 mm

of the 3 % group was                                   0.20 mm                     0.21 mm

of the 1 % group was                                   0.22 mm                     0.22 mm

of the negative control group was                     0.20 mm                     0.20 mm

of the positive control group was                      0.19 mm                     0.20 mm

 

Approximately 5 hours after ³H-methyl thymidine-injection all mice were sacrificed and the draining "auricular lymph nodes" were excised and weighed individually.

The mean weights of the lymph nodes

for the 15 % group was 4.7 mg

for the 9 % group was 4.5 mg

for the 3 % group was 3.7 mg

for the 1 % group was 3.9 mg

for the negative control-group was 2.6 mg

for the positive control-group was 5.5 mg

 

A single cell suspension of the lymph node cells for each animal was prepared. The ³H-methyl thymidine - incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal.

 

The proliferative response of lymph node cells was calculated as the ratio of ³H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

The stimulation index at a concentration 15, 9, 3 and 1 % was 7.8, 5.5, 3.3 and 4.2 respectively.

The stimulation of the positive control (Phenylenediamine) at a concentration of 1 % was 9.9.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study. At the daily clinical observation the animals did not show any visible clinical symptoms.

Interpretation of results:

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

Conclusions:

FAT 40224 (C.I. Reactive Yellow 174) has shown skin sensitizing properties in LLNA study in mice.

Executive summary:

The LLNA study in CBA female mice was carried out with FAT 40224 according to OECD guideline 429. Five female mice were used per concentration. Four concentrations were chosen to gain a wide spectrum for the test design: Due to results of a solubility test and in consultation with the sponsor, the test item was assayed at concentrations of 15 %, 9 %, 3 % and 1 % (w/v). Additionally a positive control for verifying the functionality of the current test run was carried along. The vehicle was AOO (3+1 (v/v) Acetone/Olive Oil). Stability of the test item in the vehicle was proven. Each mouse was treated by topical application of the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with ³H-methyl thymidine. Directly prior to the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured. This is to exclude irritating properties of the test item, which may lead to false positive results.

 

Mean Ear thickness at:                                   day 1                          day 6

of the 15 % group was                                       0.21 mm                     0.22 mm

of the 9 % group was                                         0.21 mm                     0.21 mm

of the 3 % group was                                         0.20 mm                     0.21 mm

of the 1 % group was                                         0.22 mm                     0.22 mm

of the negative control group was                     0.20 mm                     0.20 mm

of the positive control group was                      0.19 mm                     0.20 mm

 

Approximately 5 hours after ³H-methyl thymidine-injection all mice were sacrificed and the draining "auricular lymph nodes" were excised and weighed individually.

The mean weights of the lymph nodes

for the 15 % group was 4.7 mg

for the 9 % group was 4.5 mg

for the 3 % group was 3.7 mg

for the 1 % group was 3.9 mg

for the negative control-group was 2.6 mg

for the positive control-group was 5.5 mg

 

A single cell suspension of the lymph node cells for each animal was prepared. The ³H-methyl thymidine - incorporation was measured in a ßcounter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal. The proliferative response of lymph node cells was calculated as the ratio of ³H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

The stimulation index at a concentration 15, 9, 3 and 1 % was 7.8, 5.5, 3.3 and 4.2 respectively.

The stimulation of the positive control (Phenylenediamine) at a concentration of 1 % was 9.9.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study. At the daily clinical observation the animals did not show any visible clinical symptoms.

The EC3 value could not be calculated as the stimulation indices of all concentrations were above 3. This finding was confirmed by the second endpoint, the weight of the lymph nodes, as all of the test groups showed increased lymph node weights compared to the control group. Based on the study results, FAT 40224 (C.I. Reactive Yellow 174) has shown skin sensitizing properties in LLNA study in mice under the given experimental conditions.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 August 1986 to 19 September 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

non GLP test

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
TZ 2382/8
- Expiration date of the lot/batch:
May 1987

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility of the test substance in the solvent/dispersant/vehicle/test medium:
solubility in water >100 g/l

Species:

guinea pig

Strain:

other: Pirbright White Strain (Tif: DHP)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY LTD. Tierfarm, 4334 Sisseln, Switzerland
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 305 to 416 g
- Housing: The animals were housed individually in Macrolon cages (Type3).
- Diet: The animals received ad libitum standard guinea pig pellets - NAFAG No.846, Gossau SG.
- Water: Fresh water, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours light cycle day

Route:

intradermal

Vehicle:

other: NaCl

Concentration / amount:

1 % / 0.1 ml

Day(s)/duration:

Day 1

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

other: Vaseline

Concentration / amount:

approx. 0.4 g paste of 30 %

Challenge
Dose of application : approx. 0.2 g paste of 10 % FAT 40224/B in vaseline.

Day(s)/duration:

Day 8

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

other: vaseline

Concentration / amount:

10 % / 0.2g

Day(s)/duration:

Day 21

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10/sex

Details on study design:

A. INDUCTION EXPOSURE
The induction was a two-stage operation. First, intradermal injections (into the neck region); second, closed patch exposure over the injection sites one week later.
First Induction intradermal application:
Three pairs of intradermal injections (0.1 mL per injection) were made simultaneously into the shaved neck of the guinea pigs as follows:
- adjuvant and saline (1:1)
- test compound FAT 40224/B in NaCl
- test compound FAT 40224/B in the adjuvant saline mixture
Concentration of FAT 40224/B in NaCl and adjuvant mixture: 1 %.

Second Induction, epidermal application
One week later FAT 40224/B was incorporated in vaseline and applied on a filter paper patch to the neck of the animals (patch 2 x 4 cm; occluded administration for 48 hours). The application sites were pretreated the day before with 10 % sodium lauryl sulfate (open application). Dose of application: approx. 0.4 g paste of 30 % FAT 40224/B in vaseline

B. CHALLENGE EXPOSURE
Two weeks after the epidermal induction application the animals were tested on the flank with FAT 40224/B in vaseline and the vehicle alone (patch 2 x 2 cm; occluded administration for 24 hours). Dose of application: approx. 0.2 g paste of 10 % FAT 40224/B in vaseline. The concentration of the test compound for the induction and challenge periods were determined on separate animals.

A control group was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound (at least 10 animals) to control the maximal subirritant concentration of the test compound in adjuvant treated animals.

Twenty four hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings. The sensitizing potential of FAT 40224/B was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading; Hours after challenge: 24.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading; Hours after challenge: 48.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

17

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading; Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 17.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

17

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading; Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 17.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

not measured/tested

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Remarks on result:

not measured/tested

Interpretation of results:

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

Conclusions:

FAT 40224/B showed an extreme grade of skin-sensitizing (contact allergenic) potential in albino guinea pigs.

Executive summary:

In a skin sensitization study, performed according to OECD guideline 406, Pirbright White strain guinea pigs were treated with FAT 40224/B. The test was performed on 10 male and 10 female animals per group. The induction was a two-stage operation. For the first induction three pairs of intradermal injections (0.1 ml per injection) were made simultaneously into the shaved neck of the guinea pigs as follows: 1) adjuvant and saline (1:1), 2) test substance in NaCl, 3) test substance in the adjuvant saline mixture. The concentration of test substance in NaCl and adjuvant mixture was 1 %. Second induction was performed one week later by epidermal application over the injection sites. The test substance was incorporated in vaseline and applied on a filter paper patch to the neck of the animals (occluded administration for 48 hours). The application sites were pretreated the day before with 10 % sodium lauryl sulfate (open application). Dose of application was approx. 0.4 g paste of 30 % test substance in vaseline. Two weeks after the epidermal induction application the animals were tested on the flank with test substance in vaseline and the vehicle alone (occluded administration for 24 hours), approx. 0.2 g paste of 10 % test substance in vaseline. A control group was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound (at least 10 animals). Twenty four hours and 48 hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. Under the experimental conditions employed, 85 % of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings. The test substance is, therefore, classified as an extreme sensitizer in albino guinea pigs according to the grading of Magnusson and Kligman.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In a key skin sensitization study, performed according to OECD guideline 406, guinea pigs were treated with test substance or vehicle. The induction was a two-stage operation. For the first induction three pairs of intradermal injections (0.1 mL per injection) were made into the shaved neck of the guinea pigs as follows: 1) adjuvant and vehicle, 2) test substance in saline, 3) test substance in the adjuvant vehicle mixture. The concentration of test substance in vehicle and adjuvant mixture was 1 %. Second induction was performed one week later by, epidermal application over the injection sites. The test substance was applied on a filter paper patch to the injection sites of the animals (administration for 48 hours). The dose of application was approximately 30 % test substance in the first study (CG 1986) and 25% in the second study (RCC 1988). Two weeks after the epidermal induction application the animals were tested on the flank with test substance (10 %) and the vehicle alone, 24 hour administration. Twenty four hours after removing the dressings, the challenge reactions were graded. A second evaluation was made 48 hours after removing the dressings. A second challenge was performed in study two (RCC 1988) equal to that described for the first challenge with the exception that the flanks were changed. Under the experimental conditions employed, 17 out of 20 animals of the test group showed skin reactions 24 and 48 hours after removing the dressings in study one (CG 1986). However, in the second study (RCC 1988) nine respectively eight animals from the test group (20 animals) showed positive reactions after the 24 and 48 hours reading and after the second challenge twelve respectively eleven animals showed positive reactions after the 24 and 48 hours reading out of the 20 animals tested. The allergenic potency of the test article is considered to be of a moderate grade following the grading of Magnusson and Kligman.

In LLNA study, the stimulation index at a concentration 15, 9, 3 and 1 % was 7.8, 5.5, 3.3 and 4.2 respectively. The stimulation of the positive control (Phenylenediamine) at a concentration of 1 % was 9.9. All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study. At the daily clinical observation the animals did not show any visible clinical symptoms.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings in the skin sensitisation study, the test substance should be classified as Skin Sensitiser 1A according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.