Reaction mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2012 - 16 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The LLNA was performed before the REACH regulation came into force requesting in vitro skin sensitisation information first (October, 2016). This information is used for read-across to Reaction mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

4-(2,5,6,6-tetramethyl-1-cyclohexen-1-yl)-3-buten-2-one

EC Number:

201-220-1

EC Name:

4-(2,5,6,6-tetramethyl-1-cyclohexen-1-yl)-3-buten-2-one

Cas Number:

79-70-9

Molecular formula:

C14H22O

IUPAC Name:

4-(2,5,6,6-tetramethylcyclohex-1-en-1-yl)but-3-en-2-one

Test material form:

liquid

Specific details on test material used for the study:

These results show that the test substance did elicit a SI ≥ 3 and an EC3 of 3.6% is derived. The test substance was considered to be a sensitiser under the conditions of the test.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: no data
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 15 to 23g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Free access to food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water: Free access to mains tap water
- Acclimation period: at least five days
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 01 June 2012 To: 16 October 2012

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Undiluted test item or the test item at concentrations of 0.1%, 1%, 10%, 25%, 50% and 100% v/v in vehicle

No. of animals per dose:

Groups of five mice were treated per dose.

Details on study design:

Preliminary Screening Test:
Using available information regarding the systemic toxicity/irritancy 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 undiluted test item 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. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 to 3 and Days 1 to 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 sensitisation.

Main Test
Test Item Administration:
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local 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. The concentrations used were based on those recommended in the test guideline but were selected without confirmation from the Sponsor. A further group of five mice received the vehicle alone in the same manner.

Ear Thickness Measurement:
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose and post dose on Day 1 and on Days 2 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 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 sensitisation.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of sterile phosphate buffered saline (PBS) containing tritiated 3H-methyl thymidine (3HTdR:80μCi/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.
Local Skin Irritation Observations: Local skin irritation was scored daily.
Bodyweights: The bodyweight 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- micron mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project 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 ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty 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 by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks:

%

Value:

3.6

Results 2

Parameter:

SI

Value:

1.52

Remarks on result:

other: 0.1% test group

Results 3

Parameter:

SI

Value:

1.77

Remarks on result:

other: 1% test group

Results 4

Parameter:

SI

Value:

6.03

Remarks on result:

other: 10% test group

Results 5

Parameter:

SI

Value:

6.75

Remarks on result:

other: 25% test group

Results 6

Parameter:

SI

Value:

7.36

Remarks on result:

other: 50% test group

Results 7

Parameter:

SI

Value:

15.44

Remarks on result:

other: 100% test group

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the test item concentrations 0.1, 1, 10, 25, 50 and 100% were 1.52, 1.77, 6.03, 6.75, 7.36 and 15.44, respectively.

EC3 CALCULATION
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 3.6%.

CLINICAL OBSERVATIONS
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Very slight erythema was noted on the ears of the test animals at concentrations of 10, 25, 50 and 100%. No visual local skin irritation was noted in the remaining animals.

BODY WEIGHTS
One animal treated with the test item at a concentration of 0.1% v/v in acetone/olive oil 4:1 and one vehicle control animal (initial test) showed a greater than expected bodyweight loss. Bodyweight changes of the remaining test animals between Day 1 and Day 6 were comparable to those observed in the remaining corresponding control group animals over the same period.

Any other information on results incl. tables

Pre-screen Test:

No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema was noted on both ears on Days 2 to 4.

Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

Applicant's summary and conclusion

Interpretation of results:

other: Skin sensitiser, category 1B

Remarks:

according to EU CLP (EC No. 1272/2008, and its amendments).

Conclusions:

The SI values calculated for the test item concentrations 0.1, 1, 10, 25, 50 and 100% were 1.52, 1.77, 6.03, 6.75, 7.36 and 15.44, respectively. These results show that the test substance did elicit a SI ≥ 3. An EC3 is derived of 3.6%. The test substance was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. The test substance was tested at 0.1, 1, 10, 25, 50 and 100%. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Very slight erythema was noted on the ears of the test animals at concentrations of 10, 25, 50 and 100%. No visual local skin irritation was noted in the remaining animals. The SI values calculated for the test item concentrations 0.1, 1, 10, 25, 50 and 100% were 1.52, 1.77, 6.03, 6.75, 7.36 and 15.44, respectively. Reliable negative and positive controls were included. These results show that the test substance did elicit a SI ≥ 3 and an EC3 of 3.6% is derived. The test substance was considered to be a sensitiser under the conditions of the test.