N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Endpoint summary

Description of key information

Structure analogue Pigment Red 242 (nano form): not sensitising (LLNA)

Structure analogue Pigment Red 144 (bulk form): not sensitising (LLNA)

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:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD 429 compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Vehicle:

dimethylformamide

Concentration:

2.5, 5, and 10%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
a pretest was performed in two mice on three consecutive days.
- Compound solubility: The data showed that the highest test item concentration, which could be technically used was a 10% solution. Higher concentrations could not be achieved with other vehicles (e.g. acetone:olive oil, methyl ethyl ketone, propylene glycol, DMSO, or ethanol:water).
- Irritation: Irritation of the ear skin could not be observed, due to the colour of the test item.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: 3fold increase of S.I.

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 2.5, 5, and 10% (w/v) in dimethylformamide. The application volume, 25 µl, was spread over the entire dorsal surface ( 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).

Positive control substance(s):

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

Positive control results:

EC3 = 6.7%

Parameter:

SI

Value:

ca. 2.37

Test group / Remarks:

Concentration 10%

Parameter:

SI

Value:

ca. 1.43

Test group / Remarks:

Concentration 5%

Parameter:

SI

Value:

ca. 1.53

Test group / Remarks:

Concentration 2.5%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Control: 5703 2.5%: 8735 5%: 8128 10%: 13466

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. Due to the colour of the test item iritation of the ear skin could bot be observed at the mid and high dose.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant study

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
- Source: Harlan Netherlands, NL - 5960 AD Horst
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: mean 18.5g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:

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

IN-LIFE DATES: From: 2004-11-03 To: 2004-11-09

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

7.5, 15 and 30%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The data showed that the highest test item concentration, which can be used was a 30 % suspension in acetone:olive oil, 4:1 (viv). In DMF or DMSO only up to 20 % could be suspended
- Irritation: In a non-GLP conform pre-test in !wo mice, test item concentrations of 5, 10, 20, and 30 % (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application. (Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected" No swelling of the ears was observed.)
- Lymph node proliferation response: not measured in the range-finder

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation
- Criteria used to consider a positive response: 3fold increase in Stimulation index compared to vehicle control

TREATMENT PREPARATION AND ADMINISTRATION: Substance was suspended in vehicle on the day of treatment.

Positive control substance(s):

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

Statistics:

Included for body weight and SI

Positive control results:

performed in April 2004, a-Hexylcinnamaldehyde in Acetone/olive oil. EC3 = 6.3%

Parameter:

SI

Value:

ca. 0.5

Test group / Remarks:

Concentration 30%

Parameter:

SI

Value:

ca. 0.7

Test group / Remarks:

Concentration 15%

Parameter:

SI

Value:

ca. 0.8

Test group / Remarks:

Concentration 7.5%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per lymph node: Control: 521 7.5% = 424 15% = 348 30% = 259

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

modifications as described in publications of Ulrich et al. 2001; Ehling et al. 2005; Ehling et al. 2005. Modification includes the endpoint different from that described in original guideline (non radioactive measuring of cell proliferation).

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic: BALB/CBYJICO
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 17.9 to 21.5 g
- Housing: in groups of maximum six in macrolon cages
- Diet (e.g. ad libitum): pelleted standard diet for experimental animals, ad libitum
- Water (e.g. ad libitum): drinking tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol

Concentration:

0.1%, 1%, 10%

No. of animals per dose:

6

Details on study design:

RANGE FINDING TESTS:
The highest concentration 10% (maximum technically practicable concentration) was administered to three animals to assess a possible systemic toxicity. The route of administration was same as in the main study. During pilot experiment no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were found out in all three animals.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA

Criteria used to consider a positive response:
The following thresholds were determined by Ulrich (2007) from analysis of historical data:
Ear weight index: 1.05; lymph node weight: 1.2; lymph node cell count: 1.3
1. Values which exceed these thresholds were considered positive
- when a statistically significant increase in one of the parameters occurs and a clear concentration-dependence can be derived
- or with no statistical significance, but a clear concentration-dependence.
2. Values which are below these thresholds were considered positive
- when a statistical significant occurs in one of the parameters together with a cIear concentration dependence.

TREATMENT PREPARATION AND ADMINISTRATION: The volume of the application form was constant in all groups of animals - 25 µL of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette (losses caused by draining from the ear must be minimized).

Positive control substance(s):

other: Dinitrochlorbenzene (DNCB)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. At first the global comparison of all three values of the concentration groups with vehicle control was performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.

Positive control results:

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and lymph node hyperplasia, which was in congruence with his expected mode of action as a contact allergen.

Parameter:

SI

Value:

ca. 1.22

Test group / Remarks:

Concentration 10%

Parameter:

SI

Value:

ca. 1.02

Test group / Remarks:

Concentration 1%

Parameter:

SI

Value:

ca. 1.05

Test group / Remarks:

Concentration 0.1%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: not applicable

The animals exposed to test substance showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of all groups in comparison to the vehicle control. There were no clinical observations attributable to the treatment with test substance.

The test substance showed a tendency to increase ear weight at the highest dose level but not paired with lymph node hyperplasia. Residues of the test substance on the ears caused this increased weight.

Summary of results

Group	LN weight		LN cell count		Ear weight
	Mean (mg)	Index	Mean (10e6/mL)	Index	Mean (mg)	Index
NC	4.60	1.00	7.03	1.00	22.90	1.00
PC	14.78*	3.21*	29.70*	4.22*	27.55*	1.20*
0.1%	4.85	1.05	5.87	0.83	23.38	1.02
1%	4.68	1.02	6.42	0.91	23.80	1.04
10%	5.60	1.22 *	8.87	1.26	27.75*	1.21*
Figures with asterisk = values statistically significant on probability level 0.05 (Mann-Whitney test)
Figures with cross = values exceeding thresholds
NC - Negative control group
PC - Positive control group

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

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

Justification for type of information:

Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Positive control results:

EC3 = 6.7%

Parameter:

SI

Value:

ca. 2.37

Test group / Remarks:

Concentration 10%

Parameter:

SI

Value:

ca. 1.43

Test group / Remarks:

Concentration 5%

Parameter:

SI

Value:

ca. 1.53

Test group / Remarks:

Concentration 2.5%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Control: 5703 2.5%: 8735 5%: 8128 10%: 13466

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. Due to the colour of the test item iritation of the ear skin could bot be observed at the mid and high dose.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Reference 5

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:

Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Positive control results:

performed in April 2004, a-Hexylcinnamaldehyde in Acetone/olive oil. EC3 = 6.3%

Parameter:

SI

Value:

ca. 0.5

Test group / Remarks:

Concentration 30%

Parameter:

SI

Value:

ca. 0.7

Test group / Remarks:

Concentration 15%

Parameter:

SI

Value:

ca. 0.8

Test group / Remarks:

Concentration 7.5%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per lymph node: Control: 521 7.5% = 424 15% = 348 30% = 259

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Reference 6

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:

Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Positive control results:

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and lymph node hyperplasia, which was in congruence with his expected mode of action as a contact allergen.

Parameter:

SI

Value:

ca. 1.22

Test group / Remarks:

Concentration 10%

Parameter:

SI

Value:

ca. 1.02

Test group / Remarks:

Concentration 1%

Parameter:

SI

Value:

ca. 1.05

Test group / Remarks:

Concentration 0.1%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: not applicable

The animals exposed to test substance showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of all groups in comparison to the vehicle control. There were no clinical observations attributable to the treatment with test substance.

The test substance showed a tendency to increase ear weight at the highest dose level but not paired with lymph node hyperplasia. Residues of the test substance on the ears caused this increased weight.

Summary of results

Group	LN weight		LN cell count		Ear weight
	Mean (mg)	Index	Mean (10e6/mL)	Index	Mean (mg)	Index
NC	4.60	1.00	7.03	1.00	22.90	1.00
PC	14.78*	3.21*	29.70*	4.22*	27.55*	1.20*
0.1%	4.85	1.05	5.87	0.83	23.38	1.02
1%	4.68	1.02	6.42	0.91	23.80	1.04
10%	5.60	1.22 *	8.87	1.26	27.75*	1.21*
Figures with asterisk = values statistically significant on probability level 0.05 (Mann-Whitney test)
Figures with cross = values exceeding thresholds
NC - Negative control group
PC - Positive control group

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Reference 7

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 conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.