2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs.

Endpoint summary

Description of key information

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the skin sensitization of the test chemical 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo) phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8). The studies are as mentioned below:

 

1. In order to study a possible contact allergenic potential of test chemical, three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 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) for three consecutive days. 10 % was the highest technically applicable concentration in the vehicle.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 lymph nodes excised and pooled per group. 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. All treated animals survived the scheduled study period. No clinical signs were observed in any animals of the control group. After the first application, both ears of all test item groups of mice (Groups 2-4) showed black at dosing sites, persisting for the remainder of the in-life phase of the study. No dose-response relationship was observed.Calculation of the EC 3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher. In this study STIMULATION INDICES of 1.8, 0.8 and 1.2 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) , respectively, in acetone/olive oil (4/1 (v/v). The test item was therefore considered to be a non-sensitizer when tested at up to the highest applicable concentration of 10 % (w/v) in acetone/olive oil (4/1 (v/v).

 

2.Human maximization test was carried out in human volunteers for test chemicalto assess its skin sensitizing behavior. In this test, 30 volunteers were exposed to 1% of test chemical in petrolatum and later observed for skin reactions. Since the chemical failed to induce any skin sensitizing effects on human, the test chemical was considered to be not sensitizing on human skin.

 

Thus, based on the above summarized studies for target chemical 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) and its structurally similar read across substances,it can be concluded that the testchemical failed to induce skin sanitization effects and unable to cause skin sensitizing effects. Thus, comparing with the criteria of CLP regulation, 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) can be classified as non-skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

WoE report is based on 2 skin sensitization studies as- WoE-2 and WoE-3.
Skin sensitization of test chemical was determined by performing maximization test on humans and Local Lymph Node Assay on mouse.

GLP compliance:

not specified

Type of study:

other: 1. mouse local lymphnode assay (LLNA) 2. Human maximisation test

Justification for non-LLNA method:

not specified

Specific details on test material used for the study:

- Name of test material : 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs.
- IUPAC name : N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine
- Molecular formula : C30H33N3
- Molecular weight : 435.60 g/mol
- Substance type : Organic
- Physical state : Liquid

Species:

other: 1.Mouse 2. Human

Strain:

other: 1.CBA 2. Not applicable

Sex:

female

Details on test animals and environmental conditions:

1. TEST ANIMALS
- Source: Harlan Netherlands; B.V. Postbus 6174; NL - 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: No data
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 g - 24 g
- Identification: Each cage by unique cage card.
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG,CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 92/04 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen, available ad libitum.
- Acclimation period: 6 days.
- Indication of any skin lesions: No data
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 degC
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
- IN-LIFE DATES: From:09-FEB-2005 To:23-FEB-2005

2. Not specified

Route:

intradermal and epicutaneous

Vehicle:

petrolatum

Concentration / amount:

1%

Day(s)/duration:

No data available

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

1%

Day(s)/duration:

No data available

Adequacy of challenge:

not specified

No. of animals per dose:

2. 30 volunteers

Details on study design:

2.MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:1
- Exposure period: No data available
- Test groups: 30 volunteers
- Control group: No data available
- Site: No data available
- Frequency of applications: No data available
- Duration: No data available
- Concentrations: 1%

B. CHALLENGE EXPOSURE
- No. of exposures:
- Day(s) of challenge: No data available
- Exposure period: No data available
- Test groups: 30 volunteers
- Control group: No data available
- Site: No data available
- Concentrations: 1%

Challenge controls:

2.No data available

Vehicle:

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

Concentration:

1 (Control Group)
Group 2 - 2.5 % (w/v)
Group 3 - 5 % (w/v)
Group 4 - 10 % (w/v)

No. of animals per dose:

1 (Control Group) - 4 animals
Group 2 - 4 animals
Group 3 - 4 animals
Group 4 - 4 animals

Details on study design:

PRE-TEST
In a non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil, (4/1, v/v), dimethylsulfoxide (DMSO) and ehanol/water (7/3, v/v).A suitable vehicle (acetone/olive oil, (4/1, v/v)) was selected and used in the main test.
In a non-GLP animal pre-test in two mice, the test item was tested at four different concentrations: 1 %,2.5 %, 5 % and 10 % (w/v) , on one ear each.
24 hours after a single topical application, the pre-test results determined that 10 % (w/v) was the highest technically applicable concentration while avoiding systemic toxicity and excessive local irritation in the chosen vehicle.

TREATMENT PROCEDURES
TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (Ieft and right) with different test item concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone/olive oil (4/1 (v/v). The application volume, 25 μl, was spread over the entire dorsal surface (Ø - 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 passed briefly over the ear's surface to prevent the loss of any of the test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE*
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration,1 mCi /ml).Five days after the first topical application, all mice were administered with 250 μI of 76.13 μCi/ml 3HTdR (equal to 19.0 μCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR*
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2 (dry ice).
The draining Iymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled Iymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two
times with phosphate buffered saline (approx. 10 ml) the Iymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours 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 'Irga-Safe Plus' scintillation liquid and thoroughly mixed.The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid.The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

1.Positive control results
CALCULATION AND RESULTS OF INDIVIDUAL DATA
The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3H-methylthymidine measured on a ß-scintillation counter.
Test item concentration % (w/v) S.I.
Group 2 5* 2.4*
Group 3 10• 3.6 •
Group 4 25 11.2
EC3 = 7.5 % (w/v)
A clear dose-response relationship was observed.
• This value was used in calculation of EC3.

VIABILITY / MORTALITY
No deaths occurred during the study period.

CLINICAL SIGNS
No clinical signs were observed in any animals of the control group.On the second application day,a slight ear swelling was observed at both dosing sites in all mice of group 4(25%), persisting for the remainder of the in-life phase of the study.One day after the third local application, a slight ear swelling was observed at both dosing sites in all mice of Group2 (5%) and Group3 (10%), persisting for the remainder of the in-life phase of the study.

BODY WEIGHTS
The body wejght of the animals, recorded prior to the 1st application and prior to necropsy,was within the range commonly recorded for animals of this strain and age.

CONCLUSION
A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).
In this study STIMULATION INDICES of 2.4, 3.6 and 11.2 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) , respectively, in acetone:olive oil, 4:1 (vIv).
ALPHA-HEXYLCINNAMALDEHYDE was therefore found to be a skin sensitizer and an EC3 value of 7.5 % (w/v) was derived.

Reading:

1st reading

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

30

Clinical observations:

No signs of sensitization were observed.

Remarks on result:

no indication of skin sensitisation

Parameter:

SI

Value:

1.8

Test group / Remarks:

Group2: 2.5 % (w/v) in acetone/olive oil (4/1, v/v).

Parameter:

SI

Value:

0.8

Test group / Remarks:

Group3: 5 % (w/v) in acetone/olive oil (4/1, v/v).

Parameter:

SI

Value:

1.2

Test group / Remarks:

Group4: 10 % (w/v) in acetone/olive oil (4/1, v/v).

Cellular proliferation data / Observations:

1. CELLULAR PROLIFERATION DATA
The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3H-methylthymidine measured on a ß-scintillation counter.
Test item concentration% (w/v) S.l.
Group 2 2.5 1.8
Group 3 5 0.8
Group 4 10 1.2
No dose-response relationship was observed.
Calculation of the EC3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

EC3 CALCULATION
Calculation of the EC3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

CLINICAL OBSERVATIONS:
No clinical signs were observed in any animals of the control group. After the first application, both ears of all test item groups of mice (Groups 2-4) showed black at dosing sites, persisting for the remainder of the in-life phase of the study.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to neroscopy,was within the range commonly recorded for animals of the strain and age.

VIABILITY / MORTALITY
No deaths occurred during the study period.

2.No signs of sensitization were observed in treated humans.

1.

Positive Control Study

SUMMARY

In order to study a possible contact allergenic potential of ALPHAHEXYLCINNAMALDEHYDE,three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 %(w/v) in acetone:olive oil, 4: 1 (v/v) by topical application to the dorsum of each ear lobe (Ieft and right) for 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-Iabelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. 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 Iymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

All treated animals survived the scheduled study period.

No clinical signs were observed in any animals of the control group.On the second application day,a slight ear swelling was observed at both dosing sites in all mice of group 4 (25%), persisting for the remainder of the in-life phase of the study.One day after the third local application, a slight ear swelling was observed at both dosing sites in all mice of Group2 (5%) and Group3 (10%), persisting for the remainder of the in-life phase of the study.

The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

	Test item concentration % (w/v)	S.I.
Group 2	5*	2.4*
Group 3	10*	3.6*
Group 4	25	11.2
EC3 = 7.5 % (w/v)
A clear dose-response relation was observed. • This value was used in calculation of EC3.

2.Not Specified

Interpretation of results:

other: Not sensitising

Conclusions:

The test chemcial 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) was considered to be not sensitizing to the skin.

Executive summary:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the skin sensitization of the test chemical 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo) phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8). The studies are as mentioned below:

 

1. In order to study a possible contact allergenic potential of test chemical, three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 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) for three consecutive days. 10 % was the highest technically applicable concentration in the vehicle.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 lymph nodes excised and pooled per group. 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. All treated animals survived the scheduled study period. No clinical signs were observed in any animals of the control group. After the first application, both ears of all test item groups of mice (Groups 2-4) showed black at dosing sites, persisting for the remainder of the in-life phase of the study. No dose-response relationship was observed.Calculation of the EC 3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher. In this study STIMULATION INDICES of 1.8, 0.8 and 1.2 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) , respectively, in acetone/olive oil (4/1 (v/v). The test item was therefore considered to be a non-sensitizer when tested at up to the highest applicable concentration of 10 % (w/v) in acetone/olive oil (4/1 (v/v).

 

2.Human maximization test was carried out in human volunteers for test chemicalto assess its skin sensitizing behavior. In this test, 30 volunteers were exposed to 1% of test chemical in petrolatum and later observed for skin reactions. Since the chemical failed to induce any skin sensitizing effects on human, the test chemical was considered to be not sensitizing on human skin.

 

Thus, based on the above summarized studies for target chemical 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) and its structurally similar read across substances,it can be concluded that the testchemical failed to induce skin sanitization effects and unable to cause skin sensitizing effects. Thus, comparing with the criteria of CLP regulation, 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) can be classified as non-skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the skin sensitization of the test chemical 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo) phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8). The studies are as mentioned below:

 

1. In order to study a possible contact allergenic potential of test chemical, three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 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) for three consecutive days. 10 % was the highest technically applicable concentration in the vehicle.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 lymph nodes excised and pooled per group. 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. All treated animals survived the scheduled study period. No clinical signs were observed in any animals of the control group. After the first application, both ears of all test item groups of mice (Groups 2-4) showed black at dosing sites, persisting for the remainder of the in-life phase of the study. No dose-response relationship was observed.Calculation of the EC 3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher. In this study STIMULATION INDICES of 1.8, 0.8 and 1.2 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) , respectively, in acetone/olive oil (4/1 (v/v). The test item was therefore considered to be a non-sensitizer when tested at up to the highest applicable concentration of 10 % (w/v) in acetone/olive oil (4/1 (v/v).

 

2.Human maximization test was carried out in human volunteers for test chemicalto assess its skin sensitizing behavior. In this test, 30 volunteers were exposed to 1% of test chemical in petrolatum and later observed for skin reactions. Since the chemical failed to induce any skin sensitizing effects on human, the test chemical was considered to be not sensitizing on human skin.

 

Thus, based on the above summarized studies for target chemical 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) and its structurally similar read across substances,it can be concluded that the testchemical failed to induce skin sanitization effects and unable to cause skin sensitizing effects. Thus, comparing with the criteria of CLP regulation, 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) can be classified as non-skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the above summarized studies for target chemical 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) and its structurally similar read across substances,it can be concluded that the testchemical failed to induce skin sanitization effects and unable to cause skin sensitizing effects. Thus, comparing with the criteria of CLP regulation, 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs (CAS no.: 92257-28-8) can be classified as non-skin sensitizer.