Registration Dossier

Administrative data

Description of key information

The registered substance was assessed for sensitizing properties in a Local Lymph Node Assay according to OECD guideline 429 at concentrations of 6.25%, 12.5% and 25% (v/v), The stimulation indices were 2.3, 2.6 and 7.1 at the concentrations of 6.25%,12.5% and 25%, respectively. The EC3 value was calculated to be 13.61%.Consequently, according to OECD 429 solutions or preparations containing more than 13.61% 2,6-Di-tert-butyl-4-nonylphenol are expected to have a stimulation index of > 3 and are therefore considered as dermal sensitisers.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted: July 22, 2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
EPA 712-C-03-197, March 2003
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Delivered by the sponsor; Batch: 222375101
- Expiration date of the lot/batch: 02 May 2018
- Purity test date: 02 September 2016
-Arrival of the Test item: 13 September 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature



Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands. The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: full-barrier maintained
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 18-22 g
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding.
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice.
- Water (e.g. ad libitum): Free access to tap water, Sulphur acidified to a pH value of approx.. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals).
- Acclimation period: Adequate acclimatization period (at least five days under laboratory conditions)
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 12/12
- Photoperiod (hrs dark / hrs light): At least 10x/hour
- IN-LIFE DATES:
Experimental Starting date: 10 October 2016
Experimental Completion Date: 16 November 2016
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
main test: 6.25%, 12.5% and 25% (v/v)
prescreentest: 12%, 25%, 50% and 100%
No. of animals per dose:
5 mice/group main test
10 mice/prescreen test (2/dose)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item was found to be 100%.
The maximum technically applicable concentration of the test item in the vehicle was found to be 25% in AOO.
Due to the solubility properties of the test item the vehicle AOO (4:1 (v/v) acetone/olive oil) was used.
- Irritation: In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for local irritation at the application site. Both ears were observed for erythema. and scored (see furhter)
- Systemic toxicity: The mice were observed daily for any clinical signs of systemic toxicity. Body weights were recorded pre-test and prior to termination
- Ear thickness measurements: Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by ear swelling of ≥25%.
- Erythema scores: Excessive local irritation was indicated by an erythema score ≥ 3.
No erythema: 0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema): 4

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
The animals were randomly selected suing the validated departmental computerized system E-WorkBook (version 9.4.0, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).
- Name of test method: Local Lymph Node Assay-LLNA
- Criteria used to consider a positive response: A substance is regarded as a ‘sensitiser’ in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine– incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.
-Topical Application
Each mouse was treated by topical application of 25µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.
-Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.
-Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx.. 1 mL 5% TCA at approx.. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
-Determination of Incorporated 3H-methyl thymidine
The 3H-methyl thymidine – incorporation was measured in a β-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: Phenylenediamine in AOO
Statistics:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine – incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
Positive control results:
A shared positive control was performed concomitantly, using 5 animals.
1% phenylenediamine in AOO was used as positive-control substance.
The positive-control substance Phenylenediamine in AOO exceeded the stimultion index of 3 confirming the reliability of the test.
Parameter:
SI
Value:
2.3
Test group / Remarks:
6.25% main test
Parameter:
SI
Value:
2.6
Test group / Remarks:
12.5% main test
Parameter:
SI
Value:
7.1
Test group / Remarks:
25% main test
Key result
Parameter:
EC3
Remarks:
%
Value:
13.61
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine – incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
One of the three tested concentrations exceeded the stimulation index of 3.
The stimulation index at a concentration of 6.25% was 2.3.
The stimulation index at a concentration of 12.5% was 2.6.
The stimulation index at a concentration of 25% was 7.1.

EC3 CALCULATION
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 13.61%

CLINICAL OBSERVATIONS
All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2g throughout the study.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
One of the three tested concentrations exceeded the stimulation index of 3.
The stimulation index at a concentration of 6.25% was 2.3.
The stimulation index at a concentration of 12.5% was 2.6.
The stimulation index at a concentration of 25% was 7.1.
The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 13.61%.
Consequently, according to OECD 429 solutions or preparations containing more than 13.61% 2,6-Di-tert-butyl-4-nonylphenol are expected to have a stimulation index of > 3 and are therefore considered as dermal sensitisers.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item 2,6-Di-tert-butyl-4-nonylphenol has obligatory labelling requirement for skin sensitization and is classified into Category 1B.
Executive summary:

Based on the results of the prescreen test the test item 2,6-Di-tert-butyl-4-nonylphenol was assessed for sensitizing properties at concentrations of 6.25%, 12.5% and 25% (v/v), each diluted with AOO 4:1 (v/v), 4 parts acetone and 1 part olive oil in female CBA/CaOlaHsd mice. 5 Mice per dose group were tested in the main test.

Prior to the application and once daily thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested. AOO (4:1 (v/v) acetone/olive oil) was used as vehicle due to the solubility properties of the test item. Positive controls are performed periodically.1% phenylenediamine in AOO was used as positive-control substance.

Topical Application

Each mouse was treated by topical application of 25µL of the selected solution to the entire dorsal surface of each ear.

Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine

Five days after the first topical application all mice were dosed with 20µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension

Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.

After the final wash each pellet was resuspended in approx.. 1 mL 5% TCA at approx.. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H-methyl thymidine

The 3H-methyl thymidine – incorporation was measured in a β-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine – incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.

A substance is regarded as a ‘sensitiser’ in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine– incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

One of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of 6.25% was 2.3.

The stimulation index at a concentration of 12.5% was 2.6.

The stimulation index at a concentration of 25% was 7.1.

All animals showed the expected weight development, which includes a weight loss of up to 2g throughout the study.

All animals survived throughout the test period without showing any clinical signs. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 13.61%

Consequently, according to OECD 429 solutions or preparations containing more than 13.61% 2,6-Di-tert-butyl-4-nonylphenol are expected to have a stimulation index > 3 and are therefore considered to be dermal sensitisers.

According to Commission regulation (EU) No 286/2011 as well as GHS, the test item 2,6-Di-tert-butyl-4-nonylphenol has obligatory labelling requirement for skin sensitization and is classified into Category 1B.

The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Based on the results of the prescreen test the test item 2,6-Di-tert-butyl-4-nonylphenol was assessed for sensitizing properties at concentrations of 6.25%, 12.5% and 25% (v/v), each diluted with AOO 4:1 (v/v), 4 parts acetone and 1 part olive oil in female CBA/CaOlaHsd mice. 5 Mice per dose group were tested in the main test.

Prior to the application and once daily thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested. AOO (4:1 (v/v) acetone/olive oil) was used as vehicle due to the solubility properties of the test item. Positive controls are performed periodically.1% phenylenediamine in AOO was used as positive-control substance.

Topical Application

Each mouse was treated by topical application of 25µL of the selected solution to the entire dorsal surface of each ear.

Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine

Five days after the first topical application all mice were dosed with 20µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension

Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.

After the final wash each pellet was resuspended in approx.. 1 mL 5% TCA at approx.. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H-methyl thymidine

The 3H-methyl thymidine – incorporation was measured in a β-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine – incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.

A substance is regarded as a ‘sensitiser’ in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine– incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

One of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of 6.25% was 2.3.

The stimulation index at a concentration of 12.5% was 2.6.

The stimulation index at a concentration of 25% was 7.1.

All animals showed the expected weight development, which includes a weight loss of up to 2g throughout the study.

All animals survived throughout the test period without showing any clinical signs. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 13.61%

Consequently, according to OECD 429 solutions or preparations containing more than 13.61% 2,6-Di-tert-butyl-4-nonylphenol are expected to have a stimulation index > 3 and are therefore considered to be dermal sensitisers.

According to Commission regulation (EU) No 286/2011 as well as GHS, the test item 2,6-Di-tert-butyl-4-nonylphenol has obligatory labelling requirement for skin sensitization and is classified into Category 1B.

The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to CLP the test item 2,6-Di-tert-butyl-4-nonylphenol has obligatory labelling requirement for skin sensitization and is classified into Category 1B.