Registration Dossier

Administrative data

Description of key information

Since a positive response was observed in the challenge exposure at 10% challenge exposure, the test chemical was re-tested using a 1% induction exposure and challenge concentrations of 1.0%, 0.5%, and 0.25%.

The test chemical produced a positive reaction at 10% challenge exposure, also in the retest at 1% challenge concentration, but no reactions were noted at 0.5 and 0.25% challenge concentrations.

Hence, it was considered that the test chemical was positive sensitizer in guinea pigs.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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:
data from handbook or collection of data
Justification for type of information:
data is from peer reviewed journals
Qualifier:
according to
Guideline:
other: Buehler test a modification of the Buehler and the Klecak method for open epicutaneous testing (OET)
Principles of method if other than guideline:
Skin sensitisation of the test chemical was evaluated using a modification of the Buehler and the Klecak method for open epicutaneous testing (OET) in a guinea pig model
GLP compliance:
not specified
Type of study:
open epicutaneous test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Route:
epicutaneous, open
Vehicle:
propylene glycol
Concentration / amount:
0.1 ml of test chemical [10% in propylene glycol]
Day(s)/duration:
3 times weekly(Monday, Wednesday , Friday) for 3 weeks
Adequacy of induction:
not specified
No.:
#1
Route:
epicutaneous, open
Vehicle:
propylene glycol
Concentration / amount:
10%, 5%,2.50% in propylene glycol
Day(s)/duration:
single
Adequacy of challenge:
not specified
No. of animals per dose:
10 albino guinea pigs
Details on study design:
Details on study design
RANGE FINDING TESTS: no data available

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:3 times weekly(Monday, Wednesday , Friday)
- Exposure period:3 weeks
- Test groups: Yes
- Control group:No
- Site:Left flank shaved albino guinea pigs
- Frequency of applications:3 times weekly
- Duration:3 weeks
- Concentrations:0.1 ml of 10 % test chemical in propylene glycol

B. CHALLENGE EXPOSURE
- No. of exposures:Once
- Day(s) of challenge:After 2 weeks of rest period from induction exposure
- Exposure period: No data available
- Test groups: Yes
- Control group: No
- Site:Right flank of shaved guinea pig
- Concentrations:10,5 and 2.5 % of the induction concentration
- Evaluation (hr after challenge):24-hrs

OTHER: All test sites were graded for erythema and edema 24 and 48 hours post-application using a four-point ordinal scale
0 = no reaction,
1 -- slight reaction,
2 = moderate reaction,
3 = severe reaction
Challenge controls:
no data available
Positive control substance(s):
yes
Remarks:
0.5% of 2,4-dinitrochlorobenzene (DNCB) in ethanol was included for both the induction and challenge phases
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10% in propylene glycol
Total no. in group:
10
Clinical observations:
80% animals exhibited a positive allergic reactions
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5% in propylene glycol
Total no. in group:
10
Clinical observations:
70% animals exhibited a positive reaction
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2.50% in propylene glycol
Total no. in group:
10
Clinical observations:
40% animals exhibited a positive reaction
Remarks on result:
positive indication of skin sensitisation

Table 1: Allergic contact dermatitis of dyes

CAS number

Chemical class

Induction concentration in propylene glycol

Challenge concentration in propylene glycol*

10%

5%

2.5%

99-57-0

nitroaminophenol

10%

+(80%)

+(70%)

+(20%)

 

Table 2: Dose-response of dyes eliciting positive response at 10% concentration

CAS number

Chemical class

Induction concentration in propylene glycol

Challenge concentration in propylene glycol*

1%

0.5%

0.25%

99-57-0

nitroaminophenol

1%

+(30%)

Negative

Negative

*results expressed as negative or positive (+) with percent of animals in the group demonstrating an allergic reaction in parenthesis

Interpretation of results:
other: sensitizing
Conclusions:
Since a positive response was observed in the challenge exposure at 10% challenge exposure, the test chemical was re-tested using a 1% induction exposure and challenge concentrations of 1.0%, 0.5%, and 0.25%.
The test chemical produced a positive reaction at 10% challenge exposure, also in the retest at 1% challenge concentration, but no reactions were noted at 0.5 and 0.25% challenge concentrations.
Hence, it was considered that the test chemical was positive sensitizer in guinea pigs.
Executive summary:

A modified Buehler and Klecak method for open epicutaneous testing[OET] was performed to assess the sensitization potential of the test chemical.

The test chemical was tested at an induction concentration of 10% and challenge concentrations of 10.0%, 5.0%, and 2 .5% in propylene glycol.

During the induction phase, the left flanks of ten albino guinea pigs were shaved and the dye test material applied three times weekly (Monday, Wednesday, Friday) for three consecutive weeks. Each animal received 0.1 ml of the dye test material over a 1.8-cm circular area. Following the induction period, the guinea pigs entered the challenge phase. The challenge phase began after a two-week rest period when the right flank of each guinea pig was shaved and exposed to three different dye test material concentrations in propylene glycol(10.0%, 5.0%, and 2 .5%). Twenty-four hours after the last induction and challenge application, the animals were depilated to clearly observe dermal reactions.

All test sites were graded for erythema and edema 24 and 48 hours post-application using a four-point ordinal scale.A positive control of 0.5% 2,4-dinitrochlorobenzene(DNCB) in ethanol was included for both the induction and challenge phases.

Since a positive response was observed in the challenge exposure at 10% challenge exposure, the test chemical was re-tested using a 1% induction exposure and challenge concentrations of 1.0%, 0.5%, and 0.25%.

The test chemical produced a positive reaction at 10% challenge exposure, also in the re-test at 1% challenge concentration, but no reactions were noted at 0.5 and 0.25% challenge concentrations.

Hence, it was considered that the test chemical was positive sensitizer in guinea pigs.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin Sensitization

In various studies, the test chemical been investigated for potential to cause dermal sensitization to a greater or lesser extent. The studies are based on in vivo experiments in guinea pigs, mice for the test chemicals. The results are summarized as follows:

A modified Buehler and Klecak method for open epicutaneous testing[OET] was performed to assess the sensitization potential of the test chemical.

The test chemical was tested at an induction concentration of 10% and challenge concentrations of 10.0%, 5.0%, and 2 .5% in propylene glycol.

During the induction phase, the left flanks of ten albino guinea pigs were shaved and the dye test material applied three times weekly (Monday, Wednesday, Friday) for three consecutive weeks. Each animal received 0.1 ml of the dye test material over a 1.8-cm circular area. Following the induction period, the guinea pigs entered the challenge phase. The challenge phase began after a two-week rest period when the right flank of each guinea pig was shaved and exposed to three different dye test material concentrations in propylene glycol(10.0%, 5.0%, and 2 .5%). Twenty-four hours after the last induction and challenge application, the animals were depilated to clearly observe dermal reactions.

All test sites were graded for erythema and edema 24 and 48 hours post-application using a four-point ordinal scale.A positive control of 0.5% 2,4-dinitrochlorobenzene(DNCB) in ethanol was included for both the induction and challenge phases.

Since a positive response was observed in the challenge exposure at 10% challenge exposure, the test chemical was re-tested using a 1% induction exposure and challenge concentrations of 1.0%, 0.5%, and 0.25%.

The test chemical produced a positive reaction at 10% challenge exposure, also in the re-test at 1% challenge concentration, but no reactions were noted at 0.5 and 0.25% challenge concentrations.

Hence, it was considered that the test chemical was positive sensitizer in guinea pigs.

This is supported by the results of a mouse local lymphnode assay(LLNA) performed as per OECD 429 Guidelines to evaluate the sensitization potential of the test chemical. Groups of female CBA mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25µl of the test material or to an equal volume of relevant vehicle only. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.The approach to estimation of the relative skin sensitization potential was based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value. A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3).

The relative potency index of the test chemical was calculated to be 0.2.

Based on the relative potency index, the test chemical was considered to be a strong sensitizer.

The above results are further supported by a similar LLNA study performed as per OECD 429 Guidelines to assess the dermal sensitization potential of the test chemical. Groups of female CBA mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25µl of the test material or to an equal volume of relevant vehicle only. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group. The approach to estimation of the relative skin sensitization potential was based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value. A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3).

The relative potency index of the test chemical was calculated to be 0.05.

Based on the relative potency index, the test chemical was considered to be an extreme sensitizer.

Considering the results of the in vivo studies, the test chemical can be considered to be sensitizing to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Skin Sensitizer 1”.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Available studies for the test chemical indicate a very strong possibility that the test chemical has the potential to cause moderate sensitization to skin. Hence, the test chemical can be considered to be a skin sensitizer.