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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:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
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 group
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.