Registration Dossier

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Administrative data

Description of key information

In the study the test item suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5 and 10% (w/v).

The animals (4 female mice/dose grouop) did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.71, 1.32 and 2.26 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in propylene glycol, respectively. The results obtained with the positive control confirmed the validity of the test.

The test item was not a skin sensitiser in this assay. Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 AUG 2006 to 15 AUG 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study (OECD TG 429)
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to Chemikaliengesetz and Directive 88/320/EEC
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 7-8 weeks (beginning of acclimatization)
- Weight at study initiation: mean: 19.2 g (17.1-20.8)
- Housing: individually, Makrolon Type I cages
- Diet: pelleted standard diet (Harlan Winkelmann, Borchen), ad libitum
- Water: tap water, ad libitum
- Acclimatization: yes (acclimatization period not given)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3
- Humidity (%): 30-85%
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs


Vehicle:
propylene glycol
Concentration:
0%, 2.5%, 5%, 10% (w/v)
No. of animals per dose:
4 females per dose group
2 females in the pre-test
Details on study design:
RANGE FINDING TESTS:
- non GLP
- Compound solubility: 10% (w/v) suspension in propylene glycol was the highest technically applicable concentration; higher concentrations could also not be achieved with other vehicles
- Irritation: no irritation effects were observed at these concentrations after a single application
- Lymph node proliferation response: no data


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1: exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index
2: data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression


TREATMENT PREPARATION AND ADMINISTRATION:
- individual preparation of weight volume dilutions using a magnetic stirrer as homogenizer
- test item preparations were made freshly before each dosing occasion
- 25 µl were spread over the entire dorsal surface of each ear lobe once daily for three consecutive days
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- calculation of mean values and standard deviations for body weight
Positive control results:
Stimulation indices of 2.30, 4.31 and 11.21 were determined with the positive control substance at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil (4+1). An EC3 value of 6.7% (w/v) was calculated.
Key result
Parameter:
SI
Value:
2.26
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
17.1
Test group / Remarks:
2,5%
Remarks on result:
other: Stimulation indices were all below 3. The following SI were calculated: 2.5% test item: 1.71 5% test item: 1.32 10% test item: 2.26
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
There was a dose dependent increase in the dpm, which were measured for the pooled lymph nodes of each treatment group (8 lymph nodes per dose group): Background: 17.56 or 20.36 dpm Control group: 3102.24 dpm 2.5% test item: 5291.91 dpm 5% test item: 4100.26 dpm 10% test item: 6990.34 dpm
Key result
Parameter:
SI
Value:
1.32
Test group / Remarks:
5%

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. The body weight of the animals was within the normal range. Calculation of the EC3 value was not performed, because no test concentration produced a SI of 3 or higher.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test substance was not sensitising in this LLNA in concentrations up to 10% (w/v) in propylene glycol, the highest technically achievable concentration.
Executive summary:

In the study the test item suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5 and 10% (w/v).

The animals (4 female mice/dose grouop) did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.71, 1.32 and 2.26 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in propylene glycol, respectively. The results obtained with the positive control confirmed the validity of the test.

The test item was not a skin sensitiser in this assay. Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March and April 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
according to the method described by Buehler
Qualifier:
according to guideline
Guideline:
other: method described by Buehler
Version / remarks:
E.V. Buehler, delayed contact hypersensitivity in the guinea pig, Arch. Dermatol. 91, 1965, S. 171 - 177
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The substance has been tested comparable to the OECD Guideline 406, Bühler method before the LLNA test model was selected to be the preferred test system under the REACH Regulation.
Species:
guinea pig
Strain:
other: Hsd/Poc:DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Porcellus, Firgrove Farm, Heathfield, Sussex, UK
- Age at study initiation: "young adults"
- Weight at study initiation: 403-537g
- Housing: individually ind stainless steel cages
- Diet: RGP Diet, ad libitum
- Water: ad libitum
- Acclimation period: at least six days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 25-30
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 60% (w/v)
Challenge: 3%, 10%, 30%, 60% (w/v)
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 60% (w/v)
Challenge: 3%, 10%, 30%, 60% (w/v)
No. of animals per dose:
10 animals in the control group
20 animals in the test group
Details on study design:
RANGE FINDING TESTS:
- Induction stage: Concentrations of 30% and 60% (highest achievable concentration) (w/v) were applied to the flanks of two females (one concentration per animal) in a similar fashion as described in the main study. Yellow staining of the application site prevented assessment of erythema. In the absence of any other signs of irritation, the 60% (w/v) preparation was used in the main study.
- Challenge stage: Concentrations of 3%, 10%, 30% and 60% (highest achievable concentration) (w/v) in corn oil were applied to each of two females as described in the main study. Application sites were stained yellow by the test material, preventing assessment of erythema.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: two weeks
- Test groups: 400 mg of 60% (w/v) preparation in corn oil
- Control group: 0.4 ml of vehicle
- Site: scapular region
- Frequency of applications: 7-day intervals
- Duration: 6 hrs


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: two weeks after the final induction exposure
- Exposure period: one day
- Duration: at least 6 hrs
- Site: both flanks
- Concentrations: 3%, 10%, 30%, 60% (w/v) in corn oil, appr. 200 mg, animals received four patches with different concentrations.
- Evaluation: 24 and 48 hrs after removal of the dressings


OTHER: Yellow discoloration of the application sites prevented assessment of erythema. Therefore, skin samples were submitted for histopathological examination.
- skin samples from the challenge sites of test and control animals and untreated skin were examined
- fixation in Bouin's solution and wax,
- staining with haematoxylin and eosin
- light microscope
Challenge controls:
- none
Positive control substance(s):
yes
Remarks:
hexylcinnamaldehyde
Positive control results:
The positive control substance stained the skin of all test animals yellow during the induction phase, but this did not prevent the assessment of irritation. Signs of slight irritation were seen in the test animals during the induction phase. There were no signs of irritation in any of the control animals.

After challenge with an undiluted sample of the positive control substance scattered mild redness was seen in seven out of twenty animals (scattered mild redness in 7/20 animals at first reading after 24 hours; in 5/20 animals at second reading after 48 hours). No reactions were observed in the control animals. The net percentage response was calculated to be 35%.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
3%, 10%, 30%, 60%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
no effects
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
no effects
Remarks on result:
positive indication of skin sensitisation

Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction: A predominantly minimal to slight inflammatory reaction with acanthosis and inflammatory cell infiltration was recorded in approximately equal proportions in skin samples from control and test animals treated with 3%, 10% or 30% (w/v) preparations of the test material in corn oil. There was considered to be no significant difference in the frequency of this effect between test and control samples. Following application of the 60% (w/v) preparation in corn oil only a very minor effects was observed in the test group.

Additional investigations were performed on skin irritation potential of corn oil, the vehicle used in this study, under Buehler challenge conditions. There were no skin reactions visible on clinical examination. Histopathological findings were similar to those reported in this test with the test item.

Interpretation of results:
GHS criteria not met
Remarks:
regulation (EC) no 1272/2008
Conclusions:
The test item is non-sensitising under the conditions of the test.
Executive summary:
The sensitisation potential of the test item was assessed in female guinea pig (20 animals in the test and 10 animals in the control group) using a method based on that described by Buehler (1965). Following challenge with 60%, 30%, 10% and 3% w/v preparations of the test material in corn oil, all application sites were stained yellow preventing assessment of erythema. On this basis skin samples from all application sites and untreated sites were excised and fixed in Bouin's fixative. Skin samples from all the challenge sites, together with samples of untreated skin were processed and examined using light microscopy.

Histopathological examination revealed a background level of minimal irritation to the skin for all the concentrations used at challenge. There was considered to be no significant difference in the skin response between test and control animals. In a positive control study, challenge of previously induced guinea pigs with an undiluted preparation of hexylcinnamaldehyde elicited a moderate skin sensitisation response, confirming the validity of the assay.

Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
March and April 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
according to the method described by Buehler
Justification for type of information:
Please ee read across document in Chapter 13
Reason / purpose for cross-reference:
read-across source
Positive control results:
The positive control substance stained the skin of all test animals yellow during the induction phase, but this did not prevent the assessment of irritation. Signs of slight irritation were seen in the test animals during the induction phase. There were no signs of irritation in any of the control animals.

After challenge with an undiluted sample of the positive control substance scattered mild redness was seen in seven out of twenty animals (scattered mild redness in 7/20 animals at first reading after 24 hours; in 5/20 animals at second reading after 48 hours). No reactions were observed in the control animals. The net percentage response was calculated to be 35%.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
3%, 10%, 30%, 60%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
no effects
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
no effects
Remarks on result:
positive indication of skin sensitisation

Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction: A predominantly minimal to slight inflammatory reaction with acanthosis and inflammatory cell infiltration was recorded in approximately equal proportions in skin samples from control and test animals treated with 3%, 10% or 30% (w/v) preparations of the test material in corn oil. There was considered to be no significant difference in the frequency of this effect between test and control samples. Following application of the 60% (w/v) preparation in corn oil only a very minor effects was observed in the test group.

Additional investigations were performed on skin irritation potential of corn oil, the vehicle used in this study, under Buehler challenge conditions. There were no skin reactions visible on clinical examination. Histopathological findings were similar to those reported in this test with the test item.

Interpretation of results:
GHS criteria not met
Remarks:
regulation (EC) no 1272/2008
Conclusions:
The test item is non-sensitising under the conditions of the test.
Executive summary:
The sensitisation potential of the test item was assessed in female guinea pig (20 animals in the test and 10 animals in the control group) using a method based on that described by Buehler (1965). Following challenge with 60%, 30%, 10% and 3% w/v preparations of the test material in corn oil, all application sites were stained yellow preventing assessment of erythema. On this basis skin samples from all application sites and untreated sites were excised and fixed in Bouin's fixative. Skin samples from all the challenge sites, together with samples of untreated skin were processed and examined using light microscopy.

Histopathological examination revealed a background level of minimal irritation to the skin for all the concentrations used at challenge. There was considered to be no significant difference in the skin response between test and control animals. In a positive control study, challenge of previously induced guinea pigs with an undiluted preparation of hexylcinnamaldehyde elicited a moderate skin sensitisation response, confirming the validity of the assay.

Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 09 AUG 2006 to 15 AUG 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study (OECD TG 429)
Justification for type of information:
Please, see read across document in chapter 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to Chemikaliengesetz and Directive 88/320/EEC
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 7-8 weeks (beginning of acclimatization)
- Weight at study initiation: mean: 19.2 g (17.1-20.8)
- Housing: individually, Makrolon Type I cages
- Diet: pelleted standard diet (Harlan Winkelmann, Borchen), ad libitum
- Water: tap water, ad libitum
- Acclimatization: yes (acclimatization period not given)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3
- Humidity (%): 30-85%
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs


Vehicle:
propylene glycol
Concentration:
0%, 2.5%, 5%, 10% (w/v)
No. of animals per dose:
4 females per dose group
2 females in the pre-test
Details on study design:
RANGE FINDING TESTS:
- non GLP
- Compound solubility: 10% (w/v) suspension in propylene glycol was the highest technically applicable concentration; higher concentrations could also not be achieved with other vehicles
- Irritation: no irritation effects were observed at these concentrations after a single application
- Lymph node proliferation response: no data


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1: exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index
2: data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression


TREATMENT PREPARATION AND ADMINISTRATION:
- individual preparation of weight volume dilutions using a magnetic stirrer as homogenizer
- test item preparations were made freshly before each dosing occasion
- 25 µl were spread over the entire dorsal surface of each ear lobe once daily for three consecutive days
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- calculation of mean values and standard deviations for body weight
Positive control results:
Stimulation indices of 2.30, 4.31 and 11.21 were determined with the positive control substance at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil (4+1). An EC3 value of 6.7% (w/v) was calculated.
Key result
Parameter:
SI
Value:
2.26
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
17.1
Test group / Remarks:
2,5%
Remarks on result:
other: Stimulation indices were all below 3. The following SI were calculated: 2.5% test item: 1.71 5% test item: 1.32 10% test item: 2.26
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
There was a dose dependent increase in the dpm, which were measured for the pooled lymph nodes of each treatment group (8 lymph nodes per dose group): Background: 17.56 or 20.36 dpm Control group: 3102.24 dpm 2.5% test item: 5291.91 dpm 5% test item: 4100.26 dpm 10% test item: 6990.34 dpm
Key result
Parameter:
SI
Value:
1.32
Test group / Remarks:
5%

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. The body weight of the animals was within the normal range. Calculation of the EC3 value was not performed, because no test concentration produced a SI of 3 or higher.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test substance was not sensitising in this LLNA in concentrations up to 10% (w/v) in propylene glycol, the highest technically achievable concentration.
Executive summary:

In the study the test item suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5 and 10% (w/v).

The animals (4 female mice/dose grouop) did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.71, 1.32 and 2.26 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in propylene glycol, respectively. The results obtained with the positive control confirmed the validity of the test.

The test item was not a skin sensitiser in this assay. Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No classification

The test item did no cause sensitising effects in a LLNA in mice.