Reaction mass of 4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide and 4-[[4-(aminocarbonyl)phenyl]azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

Endpoint summary

Description of key information

PR112

In an OECD guideline and GLP compliant LLNA, Pigment Red 112 (containing <0.01% Naphtol AS-D) was found to be not a skin sensitiser when tested at 0, 0.5, 10 and 20% in DMSO.

PO38

The test substance was not sensitising when tested in the  LLNA at concentrations up to 10 % (w/v) in propylene glycol, the highest technically achievable concentration.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2007 - 25 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

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 Chemikaliengesetzt and OECD Principles 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
-strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 8-9 weeks (beginning of treatment)
- Weight at study initiation: 18.6 g to 22.0 g
- Housing: single caging, Makrolon Type I, with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethyl sulphoxide

Concentration:

0, 0.5, 10 and 20% (w/v)

No. of animals per dose:

5 animals per treatment group, 5 animals in the control group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
The highest test item concentration, which could be technically used was a 20 % suspension in DMSO. In other vehicles a higher concentration could not be suspended or the test substance was not considered stable.
- Irritation:
No irritation was observed in a pretest of an earlier LLNA with Pigment Red 112.
- Lymph node proliferation response: not evaluated


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual (5 animals/group) LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser 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:
Five female mice were treated with different concentrations of the test item and vehicle alone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed 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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

not required

Positive control results:

Test item concentration %(w/v) Group Measurement DPM DPM-BG a) Number of lymph nodes DPM per lymph node b) S.I.
- BG I 51 - - - -
- BG II 24 - - - -
- CG 1 5421 5384 8 672.9
5 TG 1 6985 6948 8 868.4 1.29
10 TG 2 16340 16303 8 2037.8 3.03
25 TG 3 34048 34011 8 4251.3 6.32
BG = Background (1 ml 5% trichloroacetic acid) in duplicate
CG = Control Group (Vehicle: acetone:olive oil (4+1))
TG = Test Group
S.I. = Stimulation Index
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

EC3 = (a-c) [(3-d)/(b-d)] + c = 9.9%(w/v)

Parameter:

SI

Remarks on result:

other: The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Results expressed as mean per group Group -- DPM per lymph node -- SI Vehicle -- 1715.4 -- 1.00 0.5% -- 981.4 -- 1.16 10% -- 1974.1 -- 1.15 20% -- 1164.8 -- 0.68

No deaths occurred during the study period.

Due to the intense violett colour of the test item local irritation reactions such as ear redness could not be detected. However, no swelling of the ears was observed. Body weights were within the normal range.

Conclusions:

67/548/EEC: Skin irritation: no classification warranted
1272/2008/EC: Skin irritation: no classification warranted
In an OECD guideline and GLP compliant LLNA, Pigment Red 112 (containing <0.01% Naphtol AS-D) was found to be not a skin sensitiser when tested at 0, 0.5, 10 and 20% in DMSO.

Executive summary:

In the study the test item suspended in dimethylsulfoxide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 10, 20%. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Redness of the ear skin could not be observed due to the colour of the test item. In this study Stimulation Indices (S.I.) of 1.16, 1.15, 0.68 were determined with the test item at concentrations of 0.5, 10, 20% in dimethylsulfoxide, respectively. The test item was not a skin sensitiser in this assay.

Reference 2

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:

17 October 2007 - 25 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Justification for type of information:

See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 210 (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:

according to German Chemikaliengesetzt and OECD Principles 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
-strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 8-9 weeks (beginning of treatment)
- Weight at study initiation: 18.6 g to 22.0 g
- Housing: single caging, Makrolon Type I, with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethyl sulphoxide

Concentration:

0, 0.5, 10 and 20% (w/v)

No. of animals per dose:

5 animals per treatment group, 5 animals in the control group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
The highest test item concentration, which could be technically used was a 20 % suspension in DMSO. In other vehicles a higher concentration could not be suspended or the test substance was not considered stable.
- Irritation:
No irritation was observed in a pretest of an earlier LLNA with Pigment Red 112.
- Lymph node proliferation response: not evaluated


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual (5 animals/group) LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser 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:
Five female mice were treated with different concentrations of the test item and vehicle alone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed 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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

not required

Positive control results:

Test item concentration %(w/v) Group Measurement DPM DPM-BG a) Number of lymph nodes DPM per lymph node b) S.I.
- BG I 51 - - - -
- BG II 24 - - - -
- CG 1 5421 5384 8 672.9
5 TG 1 6985 6948 8 868.4 1.29
10 TG 2 16340 16303 8 2037.8 3.03
25 TG 3 34048 34011 8 4251.3 6.32
BG = Background (1 ml 5% trichloroacetic acid) in duplicate
CG = Control Group (Vehicle: acetone:olive oil (4+1))
TG = Test Group
S.I. = Stimulation Index
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

EC3 = (a-c) [(3-d)/(b-d)] + c = 9.9%(w/v)

Parameter:

SI

Remarks on result:

other: The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Results expressed as mean per group Group -- DPM per lymph node -- SI Vehicle -- 1715.4 -- 1.00 0.5% -- 981.4 -- 1.16 10% -- 1974.1 -- 1.15 20% -- 1164.8 -- 0.68

No deaths occurred during the study period.

Due to the intense violett colour of the test item local irritation reactions such as ear redness could not be detected. However, no swelling of the ears was observed. Body weights were within the normal range.

Conclusions:

67/548/EEC: Skin irritation: no classification warranted
1272/2008/EC: Skin irritation: no classification warranted
In an OECD guideline and GLP compliant LLNA, Pigment Red 112 (containing <0.01% Naphtol AS-D) was found to be not a skin sensitiser when tested at 0, 0.5, 10 and 20% in DMSO.

Executive summary:

In the study the test item suspended in dimethylsulfoxide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 10, 20%. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Redness of the ear skin could not be observed due to the colour of the test item. In this study Stimulation Indices (S.I.) of 1.16, 1.15, 0.68 were determined with the test item at concentrations of 0.5, 10, 20% in dimethylsulfoxide, respectively. The test item was not a skin sensitiser in this assay.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 May 2007 to 23 May 2007

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

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, Horst, The Netherlands
- Age at study initiation: 7-12 weeks (beginning of acclimatisation)
- Weight at study initiation: 18.9 g (18.0 g - 21.0 g)
- Housing: individually, Makrolon Type I with mesh top
- Diet: pelleted standard diet, ad libitum
- Water: tap water (ad libitum)
- Acclimation period: yes (acclimatization period not given)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-96 % (this deviation from the study plan did not affect the validity of the study)
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Vehicle:

propylene glycol

Concentration:

0 %, 2.5 %, 5 %, 10 %

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) was the highest concentration at which the test item could be suspended in propylene glycol so that an applicable test item formulation was obtained. Higher concentrations could also not be achieved with other vehicles.
- Irritation: Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected.
- 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 2.43, 4.07 and 4.88 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.

Parameter:

SI

Remarks on result:

other: Stimulation indices were all below 3. The following SI were calculated: 2.5 % test item: 1.28 5 % test item: 1.30 10 % test item: 0.78

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The DPM were measured for the pooled lymph nodes of each treatment group (8 lymph nodes per dose group): Background I: 21.62 dpm Background II: 33.29 dpm Control group: 1430.00 dpm 2.5 % test item: 1824.51 dpm 5 % test item: 1850.46 dpm 10 % test item: 1119.37 dpm

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. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. 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 Criteria used for interpretation of results: EU

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 1.28, 1.30 and 0.78 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.

 

Reference 4

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 02 May 2007 to 23 May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 429)

Justification for type of information:

See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 210 (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

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, Horst, The Netherlands
- Age at study initiation: 7-12 weeks (beginning of acclimatisation)
- Weight at study initiation: 18.9 g (18.0 g - 21.0 g)
- Housing: individually, Makrolon Type I with mesh top
- Diet: pelleted standard diet, ad libitum
- Water: tap water (ad libitum)
- Acclimation period: yes (acclimatization period not given)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-96 % (this deviation from the study plan did not affect the validity of the study)
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Vehicle:

propylene glycol

Concentration:

0 %, 2.5 %, 5 %, 10 %

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) was the highest concentration at which the test item could be suspended in propylene glycol so that an applicable test item formulation was obtained. Higher concentrations could also not be achieved with other vehicles.
- Irritation: Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected.
- 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 2.43, 4.07 and 4.88 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.

Parameter:

SI

Remarks on result:

other: Stimulation indices were all below 3. The following SI were calculated: 2.5 % test item: 1.28 5 % test item: 1.30 10 % test item: 0.78

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The DPM were measured for the pooled lymph nodes of each treatment group (8 lymph nodes per dose group): Background I: 21.62 dpm Background II: 33.29 dpm Control group: 1430.00 dpm 2.5 % test item: 1824.51 dpm 5 % test item: 1850.46 dpm 10 % test item: 1119.37 dpm

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. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. 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 Criteria used for interpretation of results: EU

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 1.28, 1.30 and 0.78 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.

 

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, as no adverse effects were observed.