Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin sensitisation:

Structure analogue pigment red 122 (nano form): not sensitising (OECD 406)

Structure analogue pigment violet 19 (nano form): not sensitising (OECD 429)

Structure analogue pigment red 209 (nano form): not sensitising (OECD 429)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2 NOV 2004 to 24 NOV 2004
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:
according to German Chemical Law and OECD Principles of GLP
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: 8-12 weeks (beginning of acclimatization)
- Weight at study initiation: mean: 18.9 +/- 2.0 g
- 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 (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 20 % (w/v)
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS:
- non GLP
- Compound solubility: 10% (w/v) suspension in acetone:olive oil (4:1 v/v) was the highest technically applicable concentration; higher concentrations could also not be achieved with other vehicles
- Irritation: irritation effects could not be determined due to the intense red colour of the test item. No ear swelling was 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
- the application volume of 25 µl was 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
- statistical evaluation of the dose-response relationship
Positive control results:
Stimulation indices of 2.0, 3.0 and 4.9 were determined in a separate test with the positive control substance at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil. An EC3 value of 9.9% (w/v) was calculated.

Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
20%
Remarks on result:
other: 5%: 2.110%: 1.820%: 1.6
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
10%
Remarks on result:
other: 5%: 2.110%: 1.820%: 1.6
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
5%
Remarks on result:
other: 5%: 2.1 10%: 1.8 20%: 1.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Background: 0; 4.7 (duplicate) Control: 2584.5 (2.582.2 without background) 5%: 5397.8 (5395.5 without background) 10%: 4542.4 (4540.1 without background) 20%: 4183.7 (4184.4 without background)

No deaths occurred during the study period.

The body weight of the animals, recorded prior to the 1stapplication and prior to necropsy,

was within the range commonly recorded for animals of this strain and age.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the colour of the test item, a redness of the ears could not be analysed. However, no signs of ear swelling occurred.  

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:
Under the conditions of this LLNA-assay, the test substance was not sensitising.
Executive summary:

In the study the test item dissolved in acetone:olive oil, 4:1 (v/v) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed in CBA mice (4 females per group) using test item concentrations of 5, 10 and 20% (w/v). The animals 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 2.1, 1.8 and 1.6 were determined with the test item at concentrations of 5, 10 and 20% (w/v) in acetone:olive oil, 4:1 (v/v), respectively. A EC3 value could not be determined. The test item was not a skin sensitiser under the conditions tested. The positive control substance was sensitising (EC value 9.9%), thus confirming the validity of the tests system.

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:
11 July 1995 to 4 September 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with OECD Guideline 406 and EU Method B.6
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
In vivo information already in existence and available to use.
Species:
guinea pig
Strain:
other: Pirbright-White
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
-Strain specifics: Hoe: DHPK (SPFLac)
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Weight at study initiation: mean 385 g (range: 352-431 g)
- Housing: in groups of five animals in Makrolon type 4 cages in fully air-conditioned rooms
- Diet (e.g. ad libitum): standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: not necessary (breeding at identical conditions)
- Randomisation: randomisation scheme 95.0421

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light):12/12
Route:
intradermal and epicutaneous
Vehicle:
other: Freund's Adjuvant, semi-liquid paraffin and petrolatum
Concentration / amount:
Intradermal induction on day 1:
1% in semi-liquid paraffin, injection site 2
1% in 50% Freund's Adjuvant, injection site 3
(50% Freund's Adjuvant injected without test item at injection site 1)
Dermal induction on day 8:
25% in petrolatum (0.5 g applied)
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
Dermal challenge on day 22:
5% in petrolatum (0.5 g applied)
Adequacy of challenge:
not specified
No. of animals per dose:
10 animals in the treatment group
5 animals in the control group
(in case of a questionable result additional animals will be tested to give a total of 20 test and 10 control animals)

6 animals in the group for determination of a primary non-irritant concentration
3 animals in the group for determination of tolerance of intradermal injections
5 animals in the escort group
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 intradermal injections and 1 dermal application
- Exposure period: intradermal injections on day 1 and dermal application on day 8 (occlusive, bandage removed after 48 h)
- Test groups: 3 sites (50% Freund's Adjuvant only, test substance in semi-liquid paraffin and test substance in 50% Freund's Adjuvant) per injection,test substance in petrolatum for dermal application
- Control and escort group: 3 sites (50% Freund's Adjuvant, semi-liquid paraffin and 50% Freund's Adjuvant) per injection, petrolatum for dermal application
- Site: dorsal area measuring 2 x 4 cm in the vicinity of the shoulders for intradermal injections, dermal application covered the same area where the intradermal injections had been placed
- Frequency of applications and duration: see above
- Concentrations: see above

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: occlusive for 24 h (removal of bandage on day 23)
- Test groups: test substance
- Control group: test substance
- Site: L flank
- Concentrations: see above
- Evaluation (hr after challenge): 48 h and 72 h
Challenge controls:
5 animals treated with the test substance (5% in petrolatum, 0.5 g applied)
Positive control substance(s):
no
Group:
positive control
Remarks on result:
other: Positive ontrols were not included in the study but regularly tested in separate studies at the time of performance of this study
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs of intoxication
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs of intoxication.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs of intoxication
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs of intoxication.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 5.0.

No effects on body weight gains or clinical signs were observed. The intradermal injection with Freund's adjuvant (with and without test substance) caused severe edema as well as indurations and encrustations. The application sites treated with Freund's adjuvant without the test substance showed severe erythema; the sites treated with the test substance showed slight edema. The injection sites treated with the test substance in Freund's adjuvant and in the vehicle were discolored pink, therefore, erythema formation was not assessable. Intradermal injections of the vehicle alone exhibited very slight erythema and edema. Due to these strong irritation reactions of the skin, treatment with 10% sodium dodecylsulfate was not done on day 7. Irritation readings during induction phase

Dermal Induction treatment (day 15)

Escort group animals 16 17 18 19 20

1. reading (48 hours)

Erythema 0 0 0 0 0  

Edema 0 0 0 0 0

Pink discoloration x x x x x  

2. reading (72 hours)

Erythema 0 0 0 0 0  

Edema 0 0 0 0 0

Pink discoloration x x x x x  

After the removal of the patches at day 10, severe erythema (only in the control group) and oedema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with Freund's Adjuvant. Additionally, the treated skin was disoloured pink in the animals of the treatment group, therefore erythema formation was not assessable. The application sites treated with the test substance in the vehicle showed slight oedema and indurations. No signs of irritation were observed at the application sites treated with the vehicle alone.

Skin readings after challenge treatment (day 29)

Control animals No. 1 2 3 4 5

Treatment animals No. 6 7 8 9 10 11 12 13 14 15

1. reading (48 hours)

Control animals

Erythema 0 0 0 0 0  

Edema 0 0 0 0 0

Pink discoloration x x x x x  

Treatment animals

Erythema 0 0 0 0 0 0 0 0 0 0

Edema 0 0 0 0 0 0 0 0 0 0

Pink discoloration x x x x x x x x x x

2. reading (72 hours)

Control animals

Erythema 0 0 0 0 0  

Edema 0 0 0 0 0

Pink discoloration x x x x x  

Treatment animals

Erythema 0 0 0 0 0 0 0 0 0 0

Edema 0 0 0 0 0 0 0 0 0 0

Pink discoloration x x x x x x x x x x

Although the skin surface was discolored light pink, possible erythema formation was assessable. No signs of irritation were observed in the control and the treatment group 24 and 48 hours after removal of the occlusive bandage.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In a guinea pig maximisation test, the test item caused no skin reaction in the ten animals of the treatment group or in the five control animals. Based on the results of this study the test item showed no evidence for sensitising properties. The test substance is considered relevant and adequate to assess skin sensitisation properties of Pigment Red 122.
Executive summary:

Testing for sensitizing properties of the test item was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN. Intradermal induction was performed using 1 % test item in semi-liquid paraffin. Dermal induction was carried out with 25 % test item in petrolatum. Challenge treatment were carried out with 5 % test item in petrolatum. The challenge treatment caused no skin reaction in the ten animals of the treatment group or in the five control animals. Based on the results of this study the test item showed no evidence for sensitizing properties.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).
Reason / purpose for cross-reference:
read-across source
Positive control results:
Stimulation indices of 2.0, 3.0 and 4.9 were determined in a separate test with the positive control substance at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil. An EC3 value of 9.9% (w/v) was calculated.

Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
20%
Remarks on result:
other: 5%: 2.1; 10%: 1.8; 20%: 1.6
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
10%
Remarks on result:
other: 5%: 2.1; 10%: 1.8; 20%: 1.6
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
5%
Remarks on result:
other: 5%: 2.1 10%: 1.8 20%: 1.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Background: 0; 4.7 (duplicate) Control: 2584.5 (2.582.2 without background) 5%: 5397.8 (5395.5 without background) 10%: 4542.4 (4540.1 without background) 20%: 4183.7 (4184.4 without background)

No deaths occurred during the study period.

The body weight of the animals, recorded prior to the 1stapplication and prior to necropsy,

was within the range commonly recorded for animals of this strain and age.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the colour of the test item, a redness of the ears could not be analysed. However, no signs of ear swelling occurred.  

Calculation of the EC3 value was not performed, because no test concentration produced a SI of 3 or higher.

Interpretation of results:
GHS criteria not met
Conclusions:
The toxicity potential of Pigment Red 202 is assessed based on analogue approaches. Under the conditions of this LLNA-assay, the test substance was not sensitising.
Executive summary:

In the study the test item dissolved in acetone:olive oil, 4:1 (v/v) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed in CBA mice (4 females per group) using test item concentrations of 5, 10 and 20% (w/v). The animals 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 2.1, 1.8 and 1.6 were determined with the test item at concentrations of 5, 10 and 20% (w/v) in acetone:olive oil, 4:1 (v/v), respectively. A EC3 value could not be determined. The test item was not a skin sensitiser under the conditions tested. The positive control substance was sensitising (EC value 9.9%), thus confirming the validity of the tests system.

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
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).
Reason / purpose for cross-reference:
read-across source
Species:
guinea pig
Strain:
other: Pirbright-White
Sex:
female
Group:
positive control
Remarks on result:
other: Positive ontrols were not included in the study but regularly tested in separate studies at the time of performance of this study
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs of intoxication
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs of intoxication.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs of intoxication
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs of intoxication.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Interpretation of results:
GHS criteria not met
Conclusions:
The toxicity potential of Pigment Red 202 is assessed based on analogue approaches.
In a guinea pig maximisation test, the test item caused no skin reaction in the ten animals of the treatment group or in the five control animals. Based on the results of this study the test item showed no evidence for sensitising properties. The test substance is considered relevant and adequate to assess skin sensitisation properties of Pigment Red 122.
Executive summary:

Testing for sensitizing properties of the test item was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN. Intradermal induction was performed using 1 % test item in semi-liquid paraffin. Dermal induction was carried out with 25 % test item in petrolatum. Challenge treatment were carried out with 5 % test item in petrolatum. The challenge treatment caused no skin reaction in the ten animals of the treatment group or in the five control animals. Based on the results of this study the test item showed no evidence for sensitizing properties.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31 JUL 2002 to 14 AUG 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 429)
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
; Remark: it is mentioned that the study is based also on OECD TG406
GLP compliance:
yes (incl. QA statement)
Remarks:
according to Swiss legislation of Good Laboratory Practice
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands
- Age at study initiation: 7-12 weeks (beginning of acclimatisation)
- Weight at study initiation: 17.3-21.3 g (beginning of acclimatisation)
- Housing: 4 per cage, Makrolon type -3 cages
- Diet (ad libitum): Pelleted standard Kliba 3433, batch no. 34/02 mouse maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland)
- Water (ad libitum): tap water
- Acclimation period: from 31 JUL 2002 to 6 AUG 2002, under test conditions after health examination


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 5, 10%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Irritation: To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in 2 mice with concentrations of 1 %, 2 . 5 %, 5 % and 1 0% (w/v) (pretest excluded from Statement of Compliance).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per Iymph node (DPM/NODE) and as the ratio of 3HTdR into lymph node cells of test Iymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM/NODE values were determined, mean scintillation¬background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1 %, 5 % and 10 °/0 (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (diameter 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Five days after the first topical application, all mice were administered with 250 µI of79.87 pCi/ml3H-methyl thymidine (3HTdR, equal to 20.0 µCi3HTdR) by intravenous injection via a tail vein.
Approximately five hours after treatment with3HTdR all mice were euthanized byintraperitoneal injection of VETANARCOL at a dose of at least 2 ml/kg body weight (equivalent to 324 mg sodium pentobarbitone/kg body weight).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymphnode cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing three times with phosphate buffered saline (approx.10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C overnight for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background3HTdR levels were also measured in two ml-aliquots of 5 % trichloroacetic acid.The ß-scintillation counter expresses3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

TREATMENT PREPARATION AND ADMINISTRATION: The preparations were made shortly before each dosing. The test item was placed into a volumetric flask an a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no data
Positive control results:
Stimulation indices of 2.9, 2.6 and 7.1 were determined with the positive control substance at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). Alpha-hexylcinnamaldehyde showed an allergenic potency when tested at concentration of 25 % (w/v). Based on these results a EC3 value of 11.3% (w/v) was calculated.

Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
1%
Remarks on result:
other: 1%: 2.4 5%: 2.0 10%: 1.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Background: 7, 8 (duplicate) control: 1593 (1585 without background) 1%: 3735 (3727 without background) 5%: 3178 (3170 without background) 10%: 2567 (2559 without background)

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of this LLNA-assay the test item was not sensitising.
Executive summary:

Three groups of four female CBA mice each were treated with the test item at concentrations of 1 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular Iymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. In this study stimulation indices (S.I.) of 2.4, 2.0 and 1.6 were determined with the test item at concentrations of 1%, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v).

Based on these criteria, the test item was found to be non sensitizing. Therefore no classification is required 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
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).
Reason / purpose for cross-reference:
read-across source
Positive control results:
Stimulation indices of 2.9, 2.6 and 7.1 were determined with the positive control substance at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). Alpha-hexylcinnamaldehyde showed an allergenic potency when tested at concentration of 25 % (w/v). Based on these results a EC3 value of 11.3% (w/v) was calculated.

Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
1%
Remarks on result:
other: 1%: 2.4 5%: 2.0 10%: 1.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Background: 7, 8 (duplicate) control: 1593 (1585 without background) 1%: 3735 (3727 without background) 5%: 3178 (3170 without background) 10%: 2567 (2559 without background)

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The toxicity potential was assessed based on analogue approach.
Under the conditions of this LLNA-assay the test item was not sensitising.
Executive summary:

Three groups of four female CBA mice each were treated with the test item at concentrations of 1 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular Iymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. In this study stimulation indices (S.I.) of 2.4, 2.0 and 1.6 were determined with the test item at concentrations of 1%, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v).

Based on these criteria, the test item was found to be non sensitizing. Therefore no classification is required 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

Testing for sensitizing properties of sturcure analogues was performed according to the method of MAGNUSSON & KLIGMAN and LLNA. The challenge treatment caused no skin reaction. Based on the results of this study the test item showed no evidence for sensitizing properties.