Registration Dossier

Administrative data

Description of key information

in vitro studies;

OECD 442C - dpra

170494

Technically not feasible, study cancelled

OECD 442D - keratinosens

170495

Negative

OECD 442E -hClat

170496

Positive

Based on this a conclusion cannot be drawn. therefore an LLNA was performed. The substance is not a sensitizer based on LLNA results.

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:
Experimental start date 20 September 2017 Experimental completion date 17 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: 2,2'-[propane-1,3-diylbis(oxy)]bis(3",5,5"-tri-tert-butyl-5'-methyl-1,1':3',1"-terphenyl-2’-ol)
Batch: 1502501005
CAS number: 1042662-40-7
Purity: 98.1%
Physical state / Appearance: Sponsor description - solid powder, white, odorless Envigo description - white powder
Expiry date: 01 February 2025
Storage conditions: room temperature in the dark
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
other: butanone
Concentration:
Preliminary Screening Test: 25 μL of the test item, 10% w/w in butanone.
Main Test: 25 μL of the test item, 10%, 5% or 2.5% w/w in butanone.
No. of animals per dose:
Preliminary Screening Test: 1 mouse.
Main Test: Five mice at each concentration.
Details on study design:
Test Item Preparation and Analysis
For the purpose of the study, the test item was freshly prepared as a solution in butanone. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preliminary Screening Test
Using available information regarding the systemic toxicity potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at a maximum attainable concentration of 10% w/w in butanone, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the following scale:
Scale for Erythema
Observation Score
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

Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in butanone. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 μL of the positive control item, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 15% v/v in butanone, was applied to the dorsal surface of each ear.
Local skin irritation was scored daily according to the above scale. The thickness of each ear was measured and recorded pre and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control item or positive control item (Day 6) all mice were injected via the tail vein with 0.25 mL (250 μL) of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
BodyWeights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting.
The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)
Positive control results:
The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in butanone, thus, demonstrating the sensitivity and reliability of the test system.
Parameter:
SI
Value:
1.15
Test group / Remarks:
2.5% w/w in butanone
Parameter:
SI
Value:
0.96
Test group / Remarks:
5% w/w in butanone
Parameter:
SI
Value:
1.99
Test group / Remarks:
10% w/w in butanone
Cellular proliferation data / Observations:
Preliminary Screening Test
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in butanone.

Main Test
In the initial study, the concurrent positive control produced a stimulation index of 2.25 (below the ≥3 cut-off value) and therefore did not demonstrate the sensitivity and reliability of the test system. For this reason, the study was repeated at the same concentrations. The initial study was considered not to be valid and as such has not been reported.

Clinical Observations and Mortality Data
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Estimation of the Proliferative Response of Lymph Node Cells

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment Group

Concentration

Stimulation Index

Result

Test Item

2.5%w/win
butanone

1.15

Negative

5%w/win
butanone

0.96

Negative

10%w/win
butanone

1.99

Negative

Positive
Control Item

15% v/v in
butanone

6.42

Positive

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
The test item does not meet the criteria for classification according to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures or the criteria for classification according to the Globally Harmonized Classification System.
The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in butanone, thus, demonstrating the sensitivity and reliability of the test system.
Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in butanone at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with butanone alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 15% v/v in butanone.

In the initial study, the concurrent positive control failed and therefore did not demonstrate the sensitivity and reliability of the test system. For this reason, the initial study was considered to be invalid and therefore was repeated at the same concentrations.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment Group

Concentration

Stimulation Index

Result

Test Item

2.5%w/win
butanone

1.15

Negative

5%w/win
butanone

0.96

Negative

10%w/win
butanone

1.99

Negative

Positive
Control Item
*

15% v/v in
butanone

6.42

Positive

*=   In the initial study, the concurrent positive control produced a stimulation index of 2.25 (below the≥3 cut‑off value) and therefore did not demonstrate the sensitivity and reliability of the test system. For this reason, the study was repeated at the same concentrations. The initial study was considered not to be valid and as such has not been used for reporting purposes.

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

The test item does not meet the criteria for classification according to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures or the criteria for classification according to the Globally Harmonized Classification System.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in butanone, thus, demonstrating the sensitivity and reliability of the test system.

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

The substance is not a sensitizer based on LLNA results.