Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine

Endpoint summary

Description of key information

The skin sensitization potential of test chemical was assessed in various experimental studies conducted on human subjects and animals. Based on the available data for the test chemical and supporting studies, it can be concluded that the test chemical is unable to cause skin sensitization and thus can be considered as not sensitizing. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “not classified”.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation, other

Remarks:

LLNA and 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

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 3 skin sensitization studies as- WoE-2, WoE-3 and WoE-4. Skin sensitization of test chemical was determined by performing patch tests on humans and animals.

GLP compliance:

not specified

Type of study:

other: 1.mouse local lymphnode assay (LLNA) 2.modified Landsteiner test 3.Human maximisation test

Species:

other: 1.mouse 2.guinea pig 3.Human

Strain:

other: 1.CBA/CaOlaHsd

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

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:

no data

No. with + reactions:

0

Clinical observations:

no skin sensitization effects were observed

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

30

Clinical observations:

No skin sensitizing effects 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.

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.No skin sensitizing effects were observed in treated guinea pigs.

3.No skin sensitizing effects were observed.

Interpretation of results:

other: Not sensitising

Conclusions:

The test chemical was considered to be not sensitizing to the skin on the basis of summarized studies.

Executive summary:

Data available for the read across chemicals has been reviewed to determine the skin sensitization potential of the test chemical. The studies are as mentioned below:

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. The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. 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 chemical 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).

Another modified Landsteiner test was conducted on guinea pigs to determine the skin sensitization potential of test chemical.Negative skin effects were observed when the guinea pigs were exposed to the test chemical. Hence under the test condition, the test chemical was considered to be not sensitizing on skin of guinea pigs.

The above results were further supported by the Human maximization test was carried out in human volunteers for test chemical to 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.

Based on the above summarized studies for target chemical and its structurally and functionally similar read across substances,it can be concluded that the test chemical is unable to cause skin sensitization and considered as non-skin sensitizer. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Data available for the read across chemicals has been reviewed to determine the skin sensitization potential of the test chemical. The studies are as mentioned below:

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. The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. 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 chemical 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).

the above study was further supported by a modified Landsteiner test was conducted on guinea pigs to determine the skin sensitization potential of test chemical.Negative skin effects were observed when the guinea pigs were exposed to the test chemical. Hence under the test condition, the test chemical was considered to be not sensitizing on skin of guinea pigs.

 

The above results were further supported by the Human maximization test was carried out in human volunteers for test chemical to 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.

Based on the above summarized studies for target chemical and its read across substances,it can be concluded that the test chemical is unable to cause skin sensitization and considered as non-skin sensitizer. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin sensitization potential of test substance and its read across substances were observed in various studies. From the results obtained from these studies it is concluded that the chemical is not likely to cause skin sensitization and hence can be classified as non-skin sensitizer.