Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-11-14 to 2012-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Remarks:
Read-accross with a study conduct with the Sepisol fast yellow MG-DPG
Reference
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 2012-11-14 to 2012-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The basic structures of the target and the source substances are the same: the target substance is the source substance which is substituted on the phenylamine cycles by 2 methyl groups or 1 ethyl group (see Figure I in the document attached).
Remark: the target and the source substances are manufactured by the same manufacturer, within the same facilities, and the anionic starting raw material used for the synthesis is identical.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance (Sepisol fast yellow MG-DPG) and the target substance (Sepisol fast yellow MG-F) share the same impurities and a very close physicochemical properties profil ((see table 3 of the attached document)


3. ANALOGUE APPROACH JUSTIFICATION
All the physicochemical properties were determined thank to same method for the 2 sources, and their profile is similar (see attached document). The structural differences in the substitution of the diphenylamine cycles do not significantly influence the physicochemical properties. However, some trends in the physicochemical parameters due to the substitution of the cycles can be observed : for example, while a clear and sharp melting point is observed at 160°C for the source substance (mono-constituent), a softening point with no clear melting is observed at 130°C for the target substance (UVCB). It is consistent with the fact that when substances with close melting point are mixed together, the mixture has a lowered melting point and a broadened melting range.
The solubility of the target substance is lower than the solubility of the source substance, which is consistent with the increase of its molecular weight. The partition coefficient has been calculated from the measured solubilities of the test item in octanol and in water separately: the one of the target substance is higher than the one of the source substance (higher molecular weight so lower solubility and high solubility in octanol) but it remains under the cut-off value of 4.

Comparison of the results of the acute toxicity test performed on the source and the target substance are detailled in the attached document: both have a LD50 between 300 mg/kg and 2000 mg/kg and are classification acute toxicity oral cat. 4.

4. DATA MATRIX
See the attached document.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
: Use of the lymph node cell count for measurement of the cell proliferation instead of the determination of DNA synthetis (3H-thymidine incorporation into the lymph node cells).
Principles of method if other than guideline:
Lymph node cell count is a more direct measurement of cell proliferation than determination of DNA synthesis and therefore considered an appropriate parameter for evaluation of cell proliferation in the assay. In this case, the cut-off value is much lower and it is fixed at 1.4 times increase of stimulation index (instead of 3 for the 3H-thymidine method). This is understandable by the facts that cell count indices have:
- Lower individual variance compared to 3H-thymidine incorporation
- Lower maximum stimulation indices compared to radioactive labelling.
GLP compliance:
yes (incl. certificate)
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: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 9 weeks old
- Nulliparous, non pregnant
- Weight at study initiation: 18.9 to 22.5 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet : Food (A04, SAFE) ad libitum
- Water : Tap water from public distribution system (microbiolohical and chemical analysis every 6 months) ad libitum
- Acclimation period: At least 5 days under stabling and nutritional conditions identical to those of the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 15 changes /hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
Vehicle:
dimethylformamide
Concentration:
Test item diluted at concentrations of 50% (v/v), 25% (v/v) and 10% (v/v) in the dimethylformamide (DMF).
No. of animals per dose:
4 animals per dose + 4 animals for the vehicle control (vehicle alone) : Pooled lymph nodes approach.

(An additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As no problem occurred, the lymph nodes of the additional mouse were not collected and only the data concerning the 4 selected animals of each group were used in the study)
Details on study design:
RANGE FINDING TESTS
- Concentration: 25 µL of the test item diluted at 50% in DMF to the dorsal surface of each ear for three consecutive days (D1, D2 and D3).
- Irritation: Daily examination. Ear thickness recorded on D1, D3 and D6.
- Lymph node proliferation response: Not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Pooled approach with the lymph node cell count method.
- Criteria used to consider a positive response: In order to assess any posible irritant effect 1) Ear thickness measurements of the right ear of each animal (vehicle and treated groups), by a micrometer were performed on D1, D3 and D6; 2) Any local reactions (irritation reaction or any other observation) were recorded. 3) On D6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed and the two lymph nodes per mouse were weighed. In fact, possible irritancy may be involved in false positive lymphoproliferative responses.

In order to assess the skin sensisation of the test item, animals were anaesthetised on D6. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. The proliferation response of lymph node cells was expressed as the stimulation index :

SI = Cell count of treated group / Cell count of control group

The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. The irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI<1.4 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the appropriate concentration of the test item (10% and 25% , 50% or the vehicule) was applied to the dorsal surface of each ear, using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. The application was repeated on days 2 and 3. On D6, the draining auricular lymph nodes from mouses were excised and pooled for each experimental group after anaesthetisia. Then, a single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS containing 0.5% BSA into a 6-well multiwell plate. 10 µL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counted using a cell counter. For the run, the lower size selected was 5 µM and the upper size selected was 15 µm (the average size of a lymphocyte is 8 µm).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
CURRENT POSITIVE CONTROL STUDY

Groups Test item Cell count/groups Stimulation Index Result
concentration (x10^6 cells/mL) (S.I.)
1 AOO 25.02 n.a. n.a.
2 5% 31.11 1.24 Negative
3 10% 45.01 1.80 Positive
4 25% 68.08 2.72 Positive

The EC1.4 value = 6.43%

In conclusion, in view of these results, under these experimental conditions, the positive control must be classified R43 "may cause sensitisation by skin contact" in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This substance must be characterised by the symbol "Xi" and the warning label "Irritant". In accordance with the regulation (EC) n°1272/2008, the test item must be classified in category 1 "Skin sensitisation" sub category 1B. The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
SI = Cell count of treated group / Cell count of control group. It's calculated by the pooled approach Group 2 (10%), SI = 2.10 (positive) Group 3 (25%), SI = 2.75 (positive) Group 4 (50%), SI = 3.54 (positive) The EC1.4 > 2% (cannot be determined)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The disintegrations per minute wasn't performed. The cell counting deviation was used: Group 1 (DMF) = 25.83 x E6 cells/mL Group 2 (10%) = 54.13 x E6 cells/mL Group 3 (25%) = 70.94 x E6 cells/mL Group 4 (50%) = 91.52 x E6 cells/mL

Cell count, stimulation index and calculation of EC1.4

Groups

Test item

Cell count/groups (x106cells/mL)

Stimulation Index (SI)

Results

EC1.4value

1

DMF

25.83

n.a

n.a

n.a

2

10%

54.13

2.10

Positive

n.a

3

25%

70.94

2.75

Positive

4

50%

91.52

3.54

Positive

 

 

The EC1.4 value is determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.4-fold threshold, according the equation:

EC1.4= c + [(1.4 – d) / (b – d)] x (a – c)

a= the lowest concentration giving stimulation index > 1.4

b= the actual stimulation index caused by a.

c= the highest concentration failing to produce a stimulation index of 1.4

d= the actual stimulation index caused by c.

As there is no concentration failing to produce a stimulation index of 1.4, it is not possible to determine any EC1.4

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In view of these results, under these experimental conditions, the test item must be classified as a skin sensitiser. In, accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45, this item must be characterised by the symbol "Xi" and the warning label "Irritant" with the risk sentence R43 "May cause sensitisation by skin contact". In accordance with the regulation with the Regulation EC N° 1272/2008, the test item must be classified in category 1 "Skin sensitisation". The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.
Executive summary:

The test was performed to assess the skin sensitisation potential of the test item Sepisol Fast Yellow MG-DPG in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

A preliminary study was performed using one mouse, treated daily (on D1, D2 and D3) with 25 µL per ear of the test item diluted at 50% in dimethylformamide (DMF). After a daily examination until D6, any signs of toxicity or excessive local irritation observed during this period were recorded. Therefore, the concentration of 50% was chosen as the highest concentration for the main study.

For the main test, 3 groups of four animals, were treated for three consecutive days (D1, D2 and D3) with 25 µL per ear of the test item diluted in dimethylformamide (DMF) at concentrations of 10%, 25% and 50% (v/v). A further group of four animals was treated with DMF.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according the OECD guideline N° 429 dated 22 july 2010 and the test method B.42 of the council regulation N° 440/2008.

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Slight dryness was noted on day 6 in all animals treated at 10% and 25% (4/4) and on days 3 and 4 at 50% (4/4). An orange coloration and remaining test item was noted in all animals (4/4) treated at 10% and 25% between day 2 and day 6 and in all animals treated at 50% (4/4) between day 2 and day 5.

Alopecia was noted in all animals (4/4) treated at 50% on day 6.

No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%.

Therefore, the test item must be considered as not excessively irritant at these three concentrations.

The stimulation index (SI) calculated by pooled approach was 2.10, 2.75 and 3.54 for the treated groups at 10%, 25% and 50% respectively.

The EC1.4  cannot be determined in this study.

In view of these results, under these experimental conditions, the test item Sepisol Fast Yellow MG-DPG must be classified as a skin sensitiser. In accordance with the criteria for classification, packaging and labelling of dangerous substances and preparation of the E.E.C. Directives 67/548, 2001/59 and 99/45. This item must be characterised by the symbol "Xi" and the warning label "Irritant" with the risk sentence R43 "May cause sensitisation by skin contact".

In accordance with the Regulation EC N°1272/2008, the test item must be classified in category 1 "Skin sensitisation" . The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
: Use of the lymph node cell count for measurement of the cell proliferation instead of the determination of DNA synthetis (3H-thymidine incorporation into the lymph node cells).
Principles of method if other than guideline:
Lymph node cell count is a more direct measurement of cell proliferation than determination of DNA synthesis and therefore considered an appropriate parameter for evaluation of cell proliferation in the assay. In this case, the cut-off value is much lower and it is fixed at 1.4 times increase of stimulation index (instead of 3 for the 3H-thymidine method). This is understandable by the facts that cell count indices have:
- Lower individual variance compared to 3H-thymidine incorporation
- Lower maximum stimulation indices compared to radioactive labelling.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Sepisol Fast Yellow MG-DPG.
- Substance type: Organic salt
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 9 weeks old
- Nulliparous, non pregnant
- Weight at study initiation: 18.9 to 22.5 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet : Food (A04, SAFE) ad libitum
- Water : Tap water from public distribution system (microbiolohical and chemical analysis every 6 months) ad libitum
- Acclimation period: At least 5 days under stabling and nutritional conditions identical to those of the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 15 changes /hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Test item diluted at concentrations of 50% (v/v), 25% (v/v) and 10% (v/v) in the dimethylformamide (DMF).
No. of animals per dose:
4 animals per dose + 4 animals for the vehicle control (vehicle alone) : Pooled lymph nodes approach.

(An additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As no problem occurred, the lymph nodes of the additional mouse were not collected and only the data concerning the 4 selected animals of each group were used in the study)
Details on study design:
RANGE FINDING TESTS
- Concentration: 25 µL of the test item diluted at 50% in DMF to the dorsal surface of each ear for three consecutive days (D1, D2 and D3).
- Irritation: Daily examination. Ear thickness recorded on D1, D3 and D6.
- Lymph node proliferation response: Not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Pooled approach with the lymph node cell count method.
- Criteria used to consider a positive response: In order to assess any posible irritant effect 1) Ear thickness measurements of the right ear of each animal (vehicle and treated groups), by a micrometer were performed on D1, D3 and D6; 2) Any local reactions (irritation reaction or any other observation) were recorded. 3) On D6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed and the two lymph nodes per mouse were weighed. In fact, possible irritancy may be involved in false positive lymphoproliferative responses.

In order to assess the skin sensisation of the test item, animals were anaesthetised on D6. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. The proliferation response of lymph node cells was expressed as the stimulation index :

SI = Cell count of treated group / Cell count of control group

The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. The irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI<1.4 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the appropriate concentration of the test item (10% and 25% , 50% or the vehicule) was applied to the dorsal surface of each ear, using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. The application was repeated on days 2 and 3. On D6, the draining auricular lymph nodes from mouses were excised and pooled for each experimental group after anaesthetisia. Then, a single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS containing 0.5% BSA into a 6-well multiwell plate. 10 µL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counted using a cell counter. For the run, the lower size selected was 5 µM and the upper size selected was 15 µm (the average size of a lymphocyte is 8 µm).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
CURRENT POSITIVE CONTROL STUDY

Groups Test item Cell count/groups Stimulation Index Result
concentration (x10^6 cells/mL) (S.I.)
1 AOO 25.02 n.a. n.a.
2 5% 31.11 1.24 Negative
3 10% 45.01 1.80 Positive
4 25% 68.08 2.72 Positive

The EC1.4 value = 6.43%

In conclusion, in view of these results, under these experimental conditions, the positive control must be classified R43 "may cause sensitisation by skin contact" in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This substance must be characterised by the symbol "Xi" and the warning label "Irritant". In accordance with the regulation (EC) n°1272/2008, the test item must be classified in category 1 "Skin sensitisation" sub category 1B. The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
SI = Cell count of treated group / Cell count of control group. It's calculated by the pooled approach Group 2 (10%), SI = 2.10 (positive) Group 3 (25%), SI = 2.75 (positive) Group 4 (50%), SI = 3.54 (positive) The EC1.4 > 2% (cannot be determined)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The disintegrations per minute wasn't performed. The cell counting deviation was used: Group 1 (DMF) = 25.83 x E6 cells/mL Group 2 (10%) = 54.13 x E6 cells/mL Group 3 (25%) = 70.94 x E6 cells/mL Group 4 (50%) = 91.52 x E6 cells/mL

Any other information on results incl. tables

Cell count, stimulation index and calculation of EC1.4

Groups

Test item

Cell count/groups (x106cells/mL)

Stimulation Index (SI)

Results

EC1.4value

1

DMF

25.83

n.a

n.a

n.a

2

10%

54.13

2.10

Positive

n.a

3

25%

70.94

2.75

Positive

4

50%

91.52

3.54

Positive

 

 

The EC1.4 value is determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.4-fold threshold, according the equation:

EC1.4= c + [(1.4 – d) / (b – d)] x (a – c)

a= the lowest concentration giving stimulation index > 1.4

b= the actual stimulation index caused by a.

c= the highest concentration failing to produce a stimulation index of 1.4

d= the actual stimulation index caused by c.

As there is no concentration failing to produce a stimulation index of 1.4, it is not possible to determine any EC1.4

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In view of these results, under these experimental conditions, the test item must be classified as a skin sensitiser. In, accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45, this item must be characterised by the symbol "Xi" and the warning label "Irritant" with the risk sentence R43 "May cause sensitisation by skin contact". In accordance with the regulation with the Regulation EC N° 1272/2008, the test item must be classified in category 1 "Skin sensitisation". The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.
Executive summary:

The test was performed to assess the skin sensitisation potential of the test item Sepisol Fast Yellow MG-DPG in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

A preliminary study was performed using one mouse, treated daily (on D1, D2 and D3) with 25 µL per ear of the test item diluted at 50% in dimethylformamide (DMF). After a daily examination until D6, any signs of toxicity or excessive local irritation observed during this period were recorded. Therefore, the concentration of 50% was chosen as the highest concentration for the main study.

For the main test, 3 groups of four animals, were treated for three consecutive days (D1, D2 and D3) with 25 µL per ear of the test item diluted in dimethylformamide (DMF) at concentrations of 10%, 25% and 50% (v/v). A further group of four animals was treated with DMF.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according the OECD guideline N° 429 dated 22 july 2010 and the test method B.42 of the council regulation N° 440/2008.

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Slight dryness was noted on day 6 in all animals treated at 10% and 25% (4/4) and on days 3 and 4 at 50% (4/4). An orange coloration and remaining test item was noted in all animals (4/4) treated at 10% and 25% between day 2 and day 6 and in all animals treated at 50% (4/4) between day 2 and day 5.

Alopecia was noted in all animals (4/4) treated at 50% on day 6.

No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%.

Therefore, the test item must be considered as not excessively irritant at these three concentrations.

The stimulation index (SI) calculated by pooled approach was 2.10, 2.75 and 3.54 for the treated groups at 10%, 25% and 50% respectively.

The EC1.4  cannot be determined in this study.

In view of these results, under these experimental conditions, the test item Sepisol Fast Yellow MG-DPG must be classified as a skin sensitiser. In accordance with the criteria for classification, packaging and labelling of dangerous substances and preparation of the E.E.C. Directives 67/548, 2001/59 and 99/45. This item must be characterised by the symbol "Xi" and the warning label "Irritant" with the risk sentence R43 "May cause sensitisation by skin contact".

In accordance with the Regulation EC N°1272/2008, the test item must be classified in category 1 "Skin sensitisation" . The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.