Registration Dossier

Administrative data

Description of key information

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be sensitizing to skin. Thus it can be further classified under the category “Skin Sensitizer 1” as per CLP regulation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (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 various test chemicals
Justification for type of information:
Weight of evidence approach based on various test chemicals
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 various test chemicals
Principles of method if other than guideline:
Weight of evidence approach based on various test chemicals. The study 2,3 are referred as study 1,2
GLP compliance:
not specified
Type of study:
other: Weight of evidence approach based on various test chemicals
Species:
mouse
Strain:
CBA
Sex:
female
Vehicle:
other: 1. acetone in olivve oil (4:1); 2. acetone
Concentration:
1. 25µl
2. 25µl
No. of animals per dose:
1. groups of mice
2. Five animals/ dose
Details on study design:
The data is based on the weight of evidence approach based on various test chemicals
Positive control substance(s):
not specified
Parameter:
EC3
Value:
> 3
Test group / Remarks:
test group
Remarks on result:
other: positive sensitizer
Cellular proliferation data / Observations:
The EC3 value was estimated to be greater than 3
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be sensitizing to skin. Thus it can be further classified under the category “Skin Sensitizer 1” as per CLP regulation.
Executive summary:

The skin sensitization potential of the test chemical was assessed based on the information available for its various test chemicals. The results are summarized below:

The skin sensitizing potential of the test chemical was assessed by using the Mouse Local Lymphnode Assay.

 

The LLNA was conducted on groups of CBA mice (7 -12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl or to an equal volume of relevant vehicle (Acetic acid in olive oil (4:1))only. Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group.

A substance was classified skin sensitizer, if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3) The approach to estimation of the relative skin sensitization potential is 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.

The EC3 of the test chemical was calculated to be 5.8.

Based on the EC3 value, the test chemical was considered to be sensitizing to mice skin

This is supported by the results of other study to evaluate the utility of the LLNA assay to determine the contact sensitization potential of the test chemical. 1, 5, 10% test chemical in acetone was used as test concentrations. 

Female CBA/J mice 8-9 weeks old were used for the study. 12.5 microliters of test material or vehicle applied to each side of both ears (25 microliter total/ear). Five animals per test group were used. Eighteen to 24 hr after the fourth induction treatment, 20 microCi of [methyl-[3H]thymidine ([3H]TdR; 5.0 curies/mmol sp act, Amersham Corp., IL), in 0.25 ml of phosphate-buffered saline (PBS; GIBCO, NY), was injected into the tail vein of each mouse. The mice were euthanized 5 hr after [3H]TdR injection. The bilateral auricular lymph nodes were excised and pooled for each mouse.

A single cell suspension was prepared from the lymph nodes of each mouse by gently rubbing the nodes through a nylon mesh filter (100 micrometer pore size, The Spectra Co., CA). The cell suspensions were washed with 10 ml PBS, re-suspended in 1 ml PBS and a 20-/11 sample of the cell suspension taken for cell number determination using an automated cell counter (Coulter Model ZM; Coulter Electronics, Inc., FL).Following a second wash in PBS, the cell pellet was re- suspended in 3 ml of 5% trichloroacetic acid (TCA; Sigma) and left overnight (- 18 hr) at 0-4°C.

The samples were centrifuged, the supernatant was decanted, and the pellet was re-suspended in 2 ml of 5% TCA. The cells were transferred to vials containing 10 ml liquid scintillation cocktail (Ready Safe; Beckman Instruments, CA). The ‘H disintegrations per minute (dpm) were determined by counting for 5 to 10 min on a liquid scintillation counter (Beckman Model LS5000TD, Beckman Instruments, Inc., CA).

A chemical was considered positive (a sensitizer) in the local lymph node assay if two criteria were met. First, exposure to at least one concentration of the chemical resulted in a 2-fold or greater increase in [3H]TdR (expressed as dpm)incorporation compared to vehicle-treated control mice.

Second, this mean dpm value was statistically different from vehicle-treated mice (p > 0.0 1). Test materials that failed to cause a greater than a 2-fold elevation of [3H]TdR incorporation were regarded as negative in the local lymph node assay. For ranking purposes, a chemical was considered in the moderate to strong sensitization category if it demonstrated a >30-fold increase in [3H]TdR incorporation over vehicle-treated mice. A chemical in the 2- to 30-fold range of increased [3H]TdR incorporation was classified as a weak to- moderate sensitizer.

The mean dpm values for 1%,5% and 10% test chemical in acetone were 0.0467,0.0476 and 0.0949 respectively. The test chemical yielded a greater than 2- fold increase in its proliferative response, but the mean dpm value was not significantly different from the vehicle-treated animals.

Hence, the test chemical can be considered to be sensitizing to skin.

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be sensitizing to skin. Thus it can be further classified under the category “Skin Sensitizer 1” as per CLP regulation.

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

The skin sensitization potential of the test chemical was assessed based on the information available for its various test chemicals. The results are summarized below:

The skin sensitizing potential of the test chemical was assessed by using the Mouse Local Lymphnode Assay.

 

The LLNA was conducted on groups of CBA mice (7 -12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl or to an equal volume of relevant vehicle (Acetic acid in olive oil (4:1))only. Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group.

A substance was classified skin sensitizer, if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3) The approach to estimation of the relative skin sensitization potential is 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.

The EC3 of the test chemical was calculated to be 5.8.

Based on the EC3 value, the test chemical was considered to be sensitizing to mice skin

This is supported by the results of other study to evaluate the utility of the LLNA assay to determine the contact sensitization potential of the test chemical. 1, 5, 10% test chemical in acetone was used as test concentrations. 

Female CBA/J mice 8-9 weeks old were used for the study. 12.5 microliters of test material or vehicle applied to each side of both ears (25 microliter total/ear). Five animals per test group were used. Eighteen to 24 hr after the fourth induction treatment, 20 microCi of [methyl-[3H]thymidine ([3H]TdR; 5.0 curies/mmol sp act, Amersham Corp., IL), in 0.25 ml of phosphate-buffered saline (PBS; GIBCO, NY), was injected into the tail vein of each mouse. The mice were euthanized 5 hr after [3H]TdR injection. The bilateral auricular lymph nodes were excised and pooled for each mouse.

A single cell suspension was prepared from the lymph nodes of each mouse by gently rubbing the nodes through a nylon mesh filter (100 micrometer pore size, The Spectra Co., CA). The cell suspensions were washed with 10 ml PBS, re-suspended in 1 ml PBS and a 20-/11 sample of the cell suspension taken for cell number determination using an automated cell counter (Coulter Model ZM; Coulter Electronics, Inc., FL).Following a second wash in PBS, the cell pellet was re- suspended in 3 ml of 5% trichloroacetic acid (TCA; Sigma) and left overnight (- 18 hr) at 0-4°C.

The samples were centrifuged, the supernatant was decanted, and the pellet was re-suspended in 2 ml of 5% TCA. The cells were transferred to vials containing 10 ml liquid scintillation cocktail (Ready Safe; Beckman Instruments, CA). The ‘H disintegrations per minute (dpm) were determined by counting for 5 to 10 min on a liquid scintillation counter (Beckman Model LS5000TD, Beckman Instruments, Inc., CA).

A chemical was considered positive (a sensitizer) in the local lymph node assay if two criteria were met. First, exposure to at least one concentration of the chemical resulted in a 2-fold or greater increase in [3H]TdR (expressed as dpm)incorporation compared to vehicle-treated control mice.

Second, this mean dpm value was statistically different from vehicle-treated mice (p > 0.0 1). Test materials that failed to cause a greater than a 2-fold elevation of [3H]TdR incorporation were regarded as negative in the local lymph node assay. For ranking purposes, a chemical was considered in the moderate to strong sensitization category if it demonstrated a >30-fold increase in [3H]TdR incorporation over vehicle-treated mice. A chemical in the 2- to 30-fold range of increased [3H]TdR incorporation was classified as a weak to- moderate sensitizer.

The mean dpm values for 1%,5% and 10% test chemical in acetone were 0.0467,0.0476 and 0.0949 respectively. The test chemical yielded a greater than 2- fold increase in its proliferative response, but the mean dpm value was not significantly different from the vehicle-treated animals.

Hence, the test chemical can be considered to be sensitizing to skin.

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be sensitizing to skin. Thus it can be further classified under the category “Skin Sensitizer 1” as per CLP regulation.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be sensitizing to skin. Thus it can be further classified under the category “Skin Sensitizer 1” as per CLP regulation.