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

Endpoint summary

Description of key information

Skin sensitisation: Sensitising Cat 1B based on read-across from Beta-Irone, which was tested in an LLNA study according to OECD TG 429.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

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)

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.

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

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.

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.

Key result

Parameter:

EC3

Remarks:

%

Value:

3.6

Parameter:

SI

Value:

1.52

Remarks on result:

other: 0.1% test group

Parameter:

SI

Value:

1.77

Remarks on result:

other: 1% test group

Parameter:

SI

Value:

6.03

Remarks on result:

other: 10% test group

Parameter:

SI

Value:

6.75

Remarks on result:

other: 25% test group

Parameter:

SI

Value:

7.36

Remarks on result:

other: 50% test group

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.

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Rationale for reliability incl. deficiencies:

other: Read-across from guideline study

Justification for type of information:

A read-across justification is provided as an attached document.

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

EC3

Remarks:

%

Value:

3.6

Parameter:

SI

Value:

1.52

Remarks on result:

other: 0.1% test group

Parameter:

SI

Value:

1.77

Remarks on result:

other: 1% test group

Parameter:

SI

Value:

6.03

Remarks on result:

other: 10% test group

Parameter:

SI

Value:

6.75

Remarks on result:

other: 25% test group

Parameter:

SI

Value:

7.36

Remarks on result:

other: 50% test group

Parameter:

SI

Value:

15.44

Remarks on result:

other: 100% test group

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

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

Conclusions:

The test substance was considered to be a skin sensitiser based on the results of the source substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation is assessed based on read-across from beta-Irone to Methyl Ionone Alpha Extra / Beta. The executive summary of the source information is presented below followed by the read-across rationale.

Beta-Irone, skin sensitisation

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.

The skin sensitising properties ofMethyl Ionone Alpha Extra / Betausing read across from Beta-Irone (CAS# 79-70-9).

 

Introduction and hypothesis for the analogue approach

Methyl Ionone Alpha Extra / Beta is a reaction mass resulting in a multi-constituent substance belonging to the group of Ionones. These Ionones consist of a cyclohexene ring to which a C5 alkene chain is attached with a conjugated ketone bond. This substance has 2 major constituents of whichMethyl ionone alpha extrahas the higher (7779-30-8) andMethyl ionone beta(127-43-5)the lower concentration. There are five minor ones (present < 5%). For this substance no experimental skin sensitisation information is available. In accordance with Article 13 of REACH, lacking information can be generated by means other than experimental testing, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the skin sensitising properties of Methyl Ionone Alpha Extra / Beta the analogue approach is selected because for one closely related analogue, Beta-Irone (CAS# 79-70-9) LLNA information is available, which can be used for read across.

Hypothesis: Methyl Ionone Alpha Extra / Beta has the same skin sensitizing potential as Beta-Irone.

Available information: For Beta-Irone, the skin sensitisation potential 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. The study is reliable (Klimisch 1). 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 1B under the conditions of the test.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including physico-chemical properties and available toxicologicalinformation.

Purity / Impurities

The constituents of Methyl Ionone Alpha Extra / Beta are presented in the data matrix including the ones between 1 and 10%. All are considered in the read across approach.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue selection: For Methyl Ionone Alpha Extra / Beta the substance Beta-Ironecan be used as an analogue because both have a reactive alpha, beta conjugated ketone group which are known to be sensitisers. In addition, for Beta-Irone an EC3 from an LLNA test is available. Ionone Methyl, - an LLNA was not available and for read across. The LLNA result of Beta-Irone was considered more applicable for read across and more conservative. 

Structural similarities and differences: Methyl Ionone Alpha Extra / Beta and Beta-Irone share the same backbone and functional group. They both have a similar cyclohexene ring with an alkene chain which contains an alpha-beta conjugated ketone. They both have also a conjugation with a double bond in the ring. The difference is that Methyl Ionone Alpha Extra / Beta has two unreactive methyl group left of the alkene chain, while Beta-Irone has them at the right hand side. This will not affect the electrophilicity of the conjugated ketone bond.

Skin absorption:Methyl Ionone Alpha Extra / Beta has the same molecular weight and similar log Kow compared to Beta-Irone and both will have similar skin absorption potential.

Skin sensitisation reactivity: Methyl Ionone Alpha Extra / Beta and Beta-Irone both have the conjugated ketone as the functional group including a conjugation with the double bone in the ring. The attachment of the methyl groups are one carbon away from this conjugation in both substances and will therefore not affect the reactivity.

Uncertainty of the prediction: There are no other remaining uncertainties other than discussed previously. For addition support of the read across, the Nexus 2.2 EC3 predictions for both substances are 3.8 (Cas no. 7779-30-8) and 3.5 (Cas no 127-43-5), respectively. The closest analogue (61% similarity) has an EC3 of 4.6% (Data not shown).

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix below.

Conclusions on skin sensitisation for hazard and risk assessment

ForMethyl Ionone Alpha Extra / Betano skin sensitisation information is available. Read-across is used to fill this data gap. When using read-across, the result derived should be applicable for C&L and/or risk assessment, and be presented with adequate and reliable documentation. This documentation is presented in the current text. For the analogueBeta-Irone, reliable data is available from an LLNA study from which an EC3 of 3.6% is derived. This result can directly be used for Methyl Ionone Alpha Extra / Beta.

Final conclusion for hazard and risk assessment:Methyl Ionone Alpha Extra / Beta has an EC3 of 3.6% and is a skin sensitiser 1B.

 

Data matrix supporting the read across toMethyl Ionone Alpha Extra / Beta fromBeta-Irone for sensitisation

Substance	Methyl Ionone Alpha Extra / Beta			Beta-Irone
		Const. Major	Const. Minor
	Target			Source
Chemical name	Reaction mass	Methyl ionone alpha extra	Methyl ionone beta	-
Chemical structures	Key structures in next two cells
% in product	>=40-<=100	30-77	10-50	Available in the test report
CAS	Not appl.	7779-30-8	127-43-5	79-70-9
EC No	907-713-4	231-926-5	204-843-7	201-220-1
REACH regist.	2018	NA	NA	NA
MW	(206)	206	206	206
Phys-chem*
Appearance	Liquid	Liquid	(Liquid)	Liquid
VP (Pa)	1.27 (exp.)	0.87	1.35	1.65
WS (mg/L)	46.6 (exp.)	3.33	2.58	2.98
Log Kow	4.7-4.9 (exp.)	4.8	4.9	4.8
Human health
Skin sens.: EC3 in an LLNA	3.6% (Read across)			3.6% (OECD TG 429)

ECHA site visited 30^thApril 2018; NA=Not Available; Regist=Registered; * Physico-chemical properties are calculated with EpiSuite unless stated otherwise (exp.); RA = read-across

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results, the substance needs to be classified as Skin sensitizer Category 1B, and shall be labelled as ‘H317: May cause an allergic skin reaction’, according to EU CLP (EC 1272/2008, and its amendments).