2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium acetate

Endpoint summary

Description of key information

By applying the weight of evidence approach, the test chemical can be considered to be not sensitizing to skin. Comparing the annotations with criteria of CLP regulation, the test chemical can be classified under the category "Not Classified".

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation, other

Remarks:

in vivo (LLNA and non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Weight of evidence approach based on test chemicals

Justification for type of information:

Weight of evidence approach based on test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on test chemicals

GLP compliance:

not specified

Type of study:

other: Weight of evidence approach based on test chemicals

Species:

other: 1. guinea pigs; 2,3 mouse

Strain:

other: 1. Hartley; 2. CBA/J; 3. BALB/c

Sex:

female

Route:

intradermal

Vehicle:

not specified

Concentration / amount:

Three pairs of injections (0.05 ml each) were administered intradermally into the shaved interscapular area
: Freund’s complete adjuvant (in equal parts water), 0.1% the test chemical, and a mixture of equal parts of the adjuvant and dye

Adequacy of induction:

not specified

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

25% test chemical in petrolatum

Day(s)/duration:

48 hours

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

25% in petrolatum

Adequacy of challenge:

not specified

No. of animals per dose:

1. 10

Details on study design:

The data is based on weight of evidence approach based on various test chemicals

Challenge controls:

The data is based on weight of evidence approach based on various test chemicals

Positive control substance(s):

not specified

Vehicle:

other: 2. dimethylformamide (DMF); 3. saline and dimethylformamide (DMF)

Concentration:

2. 1, 2.5, 5, 10 or 25% (w/v)
3. intradermal and topical applications were 2% and 10% respectively

No. of animals per dose:

2. 28
3. n=3

Details on study design:

The data is based on weight of evidence approach based on various test chemicals

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A chemical was regarded as positive (a sensitizer) if it showed an SI total value of 3 or more.

Reading:

1st reading

Group:

test chemical

No. with + reactions:

0

Clinical observations:

no dermal reactions observed

Remarks on result:

no indication of skin sensitisation

Parameter:

SI

Value:

> 0 - <= 3

Test group / Remarks:

test groups

Remarks on result:

other: not sensitizing

Interpretation of results:

other: not sensitizing

Conclusions:

By applying the weight of evidence approach, the test chemical can be considered to be not sensitizing to skin. Comparing the annotations with criteria of CLP regulation, the test chemical can be classified under the category "Not Classified".

Executive summary:

The skin sensitization potential of the test chemical was assessed based on the available results from the various test chemicals.

The dermal sensitization potential of the test chemical was assessed in guinea pigs using the Kligman – Magnusson procedure.10 female Hartley guinea pigs were used for the study.

Three pairs of injections (0.05 ml each) were administered intradermally into the shaved interscapular area of each guinea pig: Freund’s complete adjuvant (in equal parts water), 0.1% the test chemical, and a mixture of equal parts of the adjuvant and dye. One week later, the sites were shaved again, and a 48-h occlusive patch containing 25% Disperse Black 9 in petrolatum was applied. Two weeks later, an occlusive challenge patch containing 25% test chemical in propylene glycol was applied to the shaved flank of each guinea pig. Sites were evaluated 24, 48, and 72 hours after patch removal.

No signs of dermal sensitization were observed on female guinea pig skin at any time point after challenge exposure.

Hence, the test chemical was considered to be not sensitizing to guinea pig skin.

This is supported by the results of LLNA assay performed on CBA/J female mice according to OECD 429 Guidelines to determine the sensitization potential of the test chemical.

The test material was soluble in dimethylformamide (DMF). This vehicle was selected on the basis of the results from a previous solubility study showing that HC Yellow n° 7 was non-soluble in other recommended vehicles, and that 25% (w/v) in DMF was the maximal practicable concentration. The test substance HC Yellow n° 7, DMF or HCA was applied in dose concentration 1, 2.5, 5, 10 or 25% (w/v) on the ears (25 μL per ear) of the animals for 3 consecutive days designated as days 1, 2 and 3. After 2 days of resting(day 6), mice received a single intravenous injection of triturated methyl thymidine (3H-TdR). Lymph nodes draining the application sites (auricular nodes) were sampled, pooled per group, and the proliferation of lymphocytes was evaluated by measuring the incorporation of 3H-TdR.The values obtained were used to calculate stimulation indices (SI), and the EC3 was estimated (theoretical concentration resulting in a SI of 3). The irritant potential of the test chemical was assessed by measuring ear thickness on days 1, 2, 3 and 6.

25% (v/v) α hexylcinnamaldehyde in DMF was used as positive control.

There were no irritation reactions and no lymphoproliferative responses, and the threshold SI of 3 values was not approached in any of the test chemical groups. Hence, the test chemical was considered to be not sensitizing in mice by LLNA method.

The above results are further supported by a sensitive mouse lymph node assay (SLNA) performed for the detection of contact allergens.

For intradermal injection of chemicals, a mixture of the chemical dissolved in saline with equivalent FCA and an emulsion was prepared for injection. Vehicle used for topical application to ears was DMF. The concentrations of the test chemical for the intradermal and topical applications were 2% and 10% respectively. Groups ofFemale BALB/c strain mice (n=3)were initially injected intradermally with a total of 50 microliters of test chemical-FCA emulsion into two sites of the abdominal skin at both sides of the ventral midline.

Five days after injection, 25 microliters of test chemical in vehicle was applied to both sides of each ear for three consecutive days. Control mice were treated by intradermal injection of vehicle-FCA emulsion into the abdomen and then after 5 days they were exposed to vehicle alone on the ears for three consecutive days. The next day following the final topical application, auricular lymph nodes were excised and pooled for each experimental group.

Control mice were treated by injection of saline-FCA emulsion and topical exposure to DMSO alone. The next day following the final topical application, auricular lymph nodes were excised and LNC suspensions were prepared. The LNCs (5 x lo5 cells/well) were cultured for 24 h and the LNC proliferation was measured by 3HTdR incorporation (cpm), or fluorescence and absorbance by the Alamar Blue assay. Increases in 3HTdR incorporation, fluorescence and absorbance relative to vehicle-treated controls were calculated for each group.

The increases in LNC number and 3HTdR incorporation relative to controls were derived for each experimental group and expressed as SIn and SIP, respectively; SItotal was obtained from SIn, x SIP, which indicates the total lymph node activation induced by the test chemicals. A chemical was regarded as positive (a sensitizer) if it showed an SI total value of 3 or more.

The SItotal value for the test chemical was 0.80.EC3 could not be calculated as the SI total was less than 3. Hence the test chemical was considered to be not sensitizing to the skin of BAlb/c mice in the SLNA assay.

By applying the weight of evidence approach, the test chemical can be considered to be not sensitizing to skin. Comparing the annotations with criteria of CLP regulation, the test chemical can be classified under the category "Not Classified".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitization potential of the test chemical was assessed based on the available results from the various test chemicals.

The dermal sensitization potential of the test chemical was assessed in guinea pigs using the Kligman – Magnusson procedure.10 female Hartley guinea pigs were used for the study.

Three pairs of injections (0.05 ml each) were administered intradermally into the shaved interscapular area of each guinea pig: Freund’s complete adjuvant (in equal parts water), 0.1% the test chemical, and a mixture of equal parts of the adjuvant and dye. One week later, the sites were shaved again, and a 48-h occlusive patch containing 25% Disperse Black 9 in petrolatum was applied. Two weeks later, an occlusive challenge patch containing 25% test chemical in propylene glycol was applied to the shaved flank of each guinea pig. Sites were evaluated 24, 48, and 72 hours after patch removal.

No signs of dermal sensitization were observed on female guinea pig skin at any time point after challenge exposure.

Hence, the test chemical was considered to be not sensitizing to guinea pig skin.

This is supported by the results of LLNA assay performed on CBA/J female mice according to OECD 429 Guidelines to determine the sensitization potential of the test chemical.

The test material was soluble in dimethylformamide (DMF). This vehicle was selected on the basis of the results from a previous solubility study showing that HC Yellow n° 7 was non-soluble in other recommended vehicles, and that 25% (w/v) in DMF was the maximal practicable concentration. The test substance HC Yellow n° 7, DMF or HCA was applied in dose concentration 1, 2.5, 5, 10 or 25% (w/v) on the ears (25 μL per ear) of the animals for 3 consecutive days designated as days 1, 2 and 3. After 2 days of resting(day 6), mice received a single intravenous injection of triturated methyl thymidine (3H-TdR). Lymph nodes draining the application sites (auricular nodes) were sampled, pooled per group, and the proliferation of lymphocytes was evaluated by measuring the incorporation of 3H-TdR.The values obtained were used to calculate stimulation indices (SI), and the EC3 was estimated (theoretical concentration resulting in a SI of 3). The irritant potential of the test chemical was assessed by measuring ear thickness on days 1, 2, 3 and 6.

25% (v/v) α hexylcinnamaldehyde in DMF was used as positive control.

There were no irritation reactions and no lymphoproliferative responses, and the threshold SI of 3 values was not approached in any of the test chemical groups. Hence, the test chemical was considered to be not sensitizing in mice by LLNA method.

The above results are further supported by a sensitive mouse lymph node assay (SLNA) performed for the detection of contact allergens.

For intradermal injection of chemicals, a mixture of the chemical dissolved in saline with equivalent FCA and an emulsion was prepared for injection. Vehicle used for topical application to ears was DMF. The concentrations of the test chemical for the intradermal and topical applications were 2% and 10% respectively. Groups ofFemale BALB/c strain mice (n=3)were initially injected intradermally with a total of 50 microliters of test chemical-FCA emulsion into two sites of the abdominal skin at both sides of the ventral midline.

Five days after injection, 25 microliters of test chemical in vehicle was applied to both sides of each ear for three consecutive days. Control mice were treated by intradermal injection of vehicle-FCA emulsion into the abdomen and then after 5 days they were exposed to vehicle alone on the ears for three consecutive days. The next day following the final topical application, auricular lymph nodes were excised and pooled for each experimental group.

Control mice were treated by injection of saline-FCA emulsion and topical exposure to DMSO alone. The next day following the final topical application, auricular lymph nodes were excised and LNC suspensions were prepared. The LNCs (5 x lo5 cells/well) were cultured for 24 h and the LNC proliferation was measured by 3HTdR incorporation (cpm), or fluorescence and absorbance by the Alamar Blue assay. Increases in 3HTdR incorporation, fluorescence and absorbance relative to vehicle-treated controls were calculated for each group.

The increases in LNC number and 3HTdR incorporation relative to controls were derived for each experimental group and expressed as SIn and SIP, respectively; SItotal was obtained from SIn, x SIP, which indicates the total lymph node activation induced by the test chemicals. A chemical was regarded as positive (a sensitizer) if it showed an SI total value of 3 or more.

The SItotal value for the test chemical was 0.80.EC3 could not be calculated as the SI total was less than 3. Hence the test chemical was considered to be not sensitizing to the skin of BAlb/c mice in the SLNA assay.

By applying the weight of evidence approach, the test chemical can be considered to be not sensitizing to skin. Comparing the annotations with criteria of CLP regulation, the test chemical can be classified under the category "Not Classified".

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results of the experimental studies from the various test chemicals indicate a possibility that the test chemical can be not sensitizing to skin.

Hence, by applying the weight of evidence approach,the test chemical can be considered to be not sensitizing to skin.