Octahydro-4,7-methano-1H-indenecarbaldehyde

Endpoint summary

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:

The study was conducted between 31 January 2012 and 16 February 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Skin Sensitisation: Local Lymph Node Assay method and in compliance with GLP. Reliability 2 is assigned because of read across.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five 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 eight to twelve weeks old.

The animals were individually 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 Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70 %, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve 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:

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

Concentration:

Undiluted test item or the test item at concentrations of 50 % or 25 % v/v in acetone/olive oil 4:1

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Preliminary Screening Test
Using available information regarding the 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 irritation was scored daily according to the erythema scores. 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 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. This vehicle was chosen as it produced the most suitable formulation at the required concentration according to the vehicle determination record. 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. 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.
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 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
The vehicle control group and the positive control group served as common controls with Project number 41200064 according to the summary of positive control data for the local lymph node assay.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 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.

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 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 beta 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.
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 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.

Parameter:

SI

Remarks on result:

other: The following SI values were derived at 25, 50 and 100%: 9.28, 12.75 and 15.41. Based on these results the EC3 was extrapolated by regression to 7.14. A NOEC was not derived in this study.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Interpretation of Results The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non‑sensitiser". For chemicals, which the lowest concentration tested resulted in a stimulation index of greater than 3, an EC3 value was extrapolated from the two lowest doses utilized. The extrapolated EC3 value was calculated by log‑linear interpolation between the two points on a plane where the c-axis represents the dose level and theg‑axis represents the stimulation index. The point with the higher stimulation index was denoted (a, b) and the point with the lower stimulation index was denoted (c, d). The formula for the extrapolated EC3 value was as follows: EC3= 2^ {log2(c) + (3-d)/(b-d)] x [log2(a)-log2(c)]} a= mid concentration giving a stimulation index >3 b = actual stimulation index caused by ‘a’ c = lowest concentration giving a stimulation index of >3 d = actual stimulation index caused by ‘c’ The radioactive disintegrations per minute per animal and the stimulation index were recorded. 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 provided.

No signs of systemic toxicity or excessive irritation indicated by anequal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema was noted on both ears on Days 2 and 3. 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:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be a sensitiser under the conditions of the test and resulted in an EC3 of 7.14%.

Executive summary:

The skin sensitisation potential of the test substance, TM 11-213 has been tested according to OECD TG 429: Local Lymph Node Assay" method. At 25, 50 and 100% the substance showed SI values of 15.41, 12.75 and 9.28, respectively. At these concentrations also ear thickness was increased with 8% at the 100% test substance application indicating sligh irritation of the test substance but unlikely to have influenced the result of the study. An EC3 has been derived using extrapolation of the data and resulted in an EC3 of 7.14%. A NOEC could not be derived.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The read across justification to use the LLNA data of Aquaflora for Melozone is presented below.

Melozone and its sensitising properties using EC3 from the LLNA of Aquaflora

Introduction and hypothesis for the read across

For Melozone a guinea pig maximization test (OECD TG 406), showing skin sensitisation, is available. In view of absence of irritation at intradermal concentration of 1%, the intradermal irritation concentration is > 1%. For deriving a NOEC for sensitisation (for safety assessment) read across to Aquaflora is used. 

This additional information is used in accordance with Article 13 of REACH where it is said thatlacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, SARs, grouping and read-across.

Hypothesis:Substance target has a similar LLNA EC3based on analogue information.

Target and Source chemical(s):The information on substance target and the analogue information from substance source are presented in the data matrix.

Purity / Impurities:

Melozone has one main constituent and several minor constituents, which are stereoisomers. The all have the same backbone but the position of the alkyl chain with the functional group on the pentyl ring can differ.

Aquaflora consist of two isomers with a similarity resulting in circa 90% composition. It has one impurity of with the same alkyl chain and functional group. Only one methyl group is on the hexyl-ring.

These slight differences in purity and impurities do not influence the reactivity because the backbone and the alkyl chain with the aldehyde group will have similar reactivity.

Analogue justification

According REACH Annex XI an analogue approach and structural alert information is used to refine the available testing information. Melozone is tested in an OECD TG 406 where it is shown to be a sensitiser and only a qualitative potency indication can be derived from this GPMT test.

For the structural related substance Aquaflora a well conducted LLNA test (OECD TG 429), with an EC3 7.14% is available presenting a quantitative potency value.

Structural similarities and differences:Melozone and Aquaflora are cyclic alkyl aldehydes. Both have a backbone with a fused hexyl and pentyl-ring, and the hexyl ring being bridged with one C-atom. To this backbone an alkyl chain is attached. In Melozone this alkyl chain is attached to the pentyl ring and in Aquaflora this is attached to the hexyl ring. This difference in the spot of attachment is not influencing the reactivity. The alkyl aldehyde is the functional group. The structural difference between these two substances is that the alkyl group is a methyl group in Melozone and an ethyl group in Aquaflora. 

Toxicological aspects of the reactivity: There are two indications that Aquaflora has a slightly higher reactivity. Aquaflora is a skin irritant and Melozone is not, but this may also be due to differences in the test methods. The reactivity can be estimated with the pKa value. Though the pKa of the aldehydes could not be calculated (pKa > 14), for the respective acids a pKa was calculated (the acids being the first oxidation product). The pKa of Melozone-acid is 4.82 and of Aquaflora-acid is 4.75 (using SPARC on line calculator) presenting similar but slightly higher reactivity of Aquaflora (the lower the pKa the more reactive). This indicates that Aquaflora likely has a slightly higher skin sensitization potential. When comparing the physico-chemical properties of Melozone and Aquaflora it can be seen that they have similar log Kows (3.9 and 3.5, respectively). Aquaflora would be expected to show a slightly higher log Kow in view of its additional methyl group but this may be due to some experimental error. The higher water solubility of Melozone does reflect its shorter alkyl chain. The log Kows and water solubilities indicate that these substances will be absorbed by the skin to a similar extent and are not expected to influence the EC3 in the LLNA.

Remaining uncertainties:The shorter alkyl chain of Melozone compared to Aquaflora is not expected to present a difference in the EC3 and if anything Aquaflora is expected to have a slightly lower EC3 than Melozone and therefore using the Aquaflora EC3 this will be a conservative approach.

Conclusions per endpoint for C&L and dose descriptor

Melozone has skin sensitising properties in GPMT test. In view of absence of irritation at intradermal concentration of 1%, the intradermal irritation concentration is > 1%. For deriving a NOEC for sensitisation (for safety assessment) read across to Aquaflora is used. Aquaflora has an EC3 of

7.14%, therefore Melozone has an EC3 of 7.14.

Melozone will be classified and labelled for sensitisation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008: 1B and H317.

 

Data matrix for Melozone the target and Aquaflora the source substance for deriving a quantitative value for skin sensitisation.

Name	Melozone	Aquaflora
Structure
Cas no	30772-79-3	1339119-15-1
Smiles	O=CC2CCC3C1CCC(C1)C23	O=CCC1CC2CC1C3CCCC23
	C11H16O	C13H20O
REACH	Annex VII	Annex VII
Molecular weight	178	190
Physico-chemical properties
Appearance s	Liquid	liquid
Melting point (˚C)	-20	-20
Vapour pressure at 25˚C(Pa)	2.92	6
Water solubility	625	59.6
Log Kow	3.9	3.45
Human Health toxicity
Skin irritation	Not irritant (OECD TG 404)	Irritant (OECD TG 439)
Skin sensitisation	Sensitiser (OECD TG 406, GPMT)	Sensitiser: EC3 7.14 (OECD TG 429)
pKa of the respective-acid as an indicator for reactivity (the lower the pKa the more reactive is the substance)	4.82	4.75
Ames	Negative	Negative

 

Migrated from Short description of key information:
The skin sensitisation potential of the substance was investigated by performing a guinea pig maximisation test (GPMT) in accordance with OECD TG 406. A concentration of 1% was used for the intradermal induction, 10% for the topical (epidermal) induction and 10% for the challenge. In view of the intradermal irritation concentration of > 1% and 19/20 positive reacting animals during challenge the substance is a skin sensitiser. The GPMT test is not suitable for potency estimation and therefore the LLNA data of Aquaflor an analogue of Melozone is used as key study. Aquaflora has been tested in an LLNA (OECD TG 429) showing an EC3 of 7.14%.

Justification for selection of skin sensitisation endpoint:
The result of this study is reliable and adequate for covering this endpoint.

Justification for classification or non-classification

According to the criteria outlined in Annex VI of 67/548/EEC (DSD) and Annex I of 1272/2008/EC (CLP), the substance needs to be classified as a skin sensitizer. The skin sensitisation potential of the substance was investigated by performing a guinea pig maximisation test in accordance with OECD TG 406. The intradermal irritation concentration was > 1% and 19/20 animals were sensitised. The GPMT test is not suitable for potency estimation and therefore the LLNA data of Aquaflor, an analogue of Melozone, is used as key study. Aquaflora has been tested in an LLNA (OECD TG 429) showing an EC3 of 7.14%. According to the CLP criteria the Melozone needs to be classified as sensitiser 1B: H317.