3-Pentanone, 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-, reaction products with 2-propyn-1-ol

Endpoint summary

Description of key information

The substance is in an LLNA (OECD TG 429) not sensitising

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:

20 September 2016 - 19 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J strain mice were supplied by Janvier, Le Genest-Saint-Isle, France. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant and the acclimatisation period was at least five days. At the start of the study the animals were in the weight range of 21.0 to 25.7 g, and were approximately ten weeks old.

Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment. Free access to tap water and food (pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 18 to 24°C and 40 to 70 %, respectively.

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:

Test item at concentrations of 0.5%, 1% or 2% v/v in vehicle.

No. of animals per dose:

Groups of five mice were treated.

Details on study design:

Weight of evidence analysis:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected.

Preliminary Screening Test:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Initially, Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (2% and 5%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 0.5 %, 1 % or 2 % v/v in acetone/olive oil 4:1. The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). There was no information available regarding the solubility or stability in vehicle. 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 dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to
dosing. 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, techical grade, at a concentration of 5, 10 and 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear. The positive control group served as common control with another study according to the summary of positive control data for the local lymph node assay.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).

Terminal Procedures - Day 6
Termination: After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Preparation of Single Cell Suspension: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Determination of 3HTdR Incorporation: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract back ground and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability Twice daily.

Clinical Observations: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). 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 on Day 6 (prior to termination).

Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the numerical scoring system (see any other information on materials and methods incl. tables). In addition, a description of all other (local) effects was recorded.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not performed.

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, techical grade, 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 SI values calculated for the substance concentrations 0.5, 1 and 2% were 1.0, 1.1 and 1.0, respectively.

Key result

Parameter:

EC3

Remarks:

%

Value:

> 2

Parameter:

other: NOEC %

Value:

> 2

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the test item concentrations 0.5, 1 and 2% were 1.0, 1.1 and 1.0, respectively.
EC3 CALCULATION
There was no indication that the test item elicits a SI ≥ 3 when tested up to and including 2%. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 2%.
CLINICAL OBSERVATIONS/BODY WEIGHTS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Pre-screen Test:

At a concentration of 100%, hunched posture and piloerection were noted for both animals between Days 1 and 4. Very slight (1) to well-defined erythema (2), scaliness and scabs were noted for both animals between Days 3 and 6 and hardened ears were noted on Day 6. At a concentration of 50%, hunched posture and piloerection were noted for both animals on Days 3 and 4. Very slight (1) to well-defined (2) erythema, scaliness and scabs were noted for both animals between Days 2 and 6 and hardened ears were noted on Day 6. At concentrations of 50% and 100%, variations in ear thickness during the observation period were more than 25% from Day 1 pre-dose values. Since severe local effects were noted and signs of systemic toxicity were seen it was decided to treat four additional pre-screen animals at concentrations of 5% and 2%.

At a concentration of 5%, piloerection and/or hunched posture were noted for both animals on Day 3. No erythema was noted. At a concentration of 2%, no erythema or signs of systemic toxicity were noted. At concentrations of 2% and 5%, variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Based on the tolerability of the test item, the highest test item concentration selected for the main study was a 2% concentration.

Interpretation of results:

other: Not sensitising.

Remarks:

According to Regulation (EC) No 1272/2008 and its amendments.

Conclusions:

The SI values calculated for the substance concentrations 0.5, 1 and 2% were 1.0, 1.1 and 1.0, respectively. These results show that the test substance does not elicit a SI ≥ 3. An EC3 has been derived resulted in an EC3 of >2%. A NOEC of >2% is derived. Based on the results of this study, the test substance was considered not to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitization potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay method and GLP principles. The final test concentrations were: 0.5, 1 and 2%.At the concentrations of 50% and 100%irritation was seen and at 5% systemic effects were observed such as piloerection and hunched posture. The substance showed SI values of 1.0, 1.1 and 1.0, respectively. Reliable negative and positive controls were included. There was no indication that the test item elicits a SI ≥ 3 when tested up to and including 2%, and therefore the substance was not considered to be a skin sensitizer. An EC3 of >2% is derived. Based on the results of this study, the substance is not considered a skin sensitiser.

 

Further (in vitro) toxicity testing is not feasible because the substance has a high log Kow, which is out of the applicability domain of the in vitro tests (OECD TG 442C (DPRA), 442D (KeratinoSens) and 442E (h-CLAT)).

 

Information on structural features indicates the substance is not expected to be a direct acting sensitizer as derived from the OECD Toolbox. There is some empirical information that ethers may present some indirect effects at high concentrations. In view of the absence of a suitable analogue a read across is not possible.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitization potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay (LLNA) method and GLP principles. The final test concentrations were: 0.5, 1 and 2%.At the concentrations of 50% and 100%irritation was seen and at 5% systemic effects were observed such as piloerection and hunched posture. The substance showed SI values of 1.0, 1.1 and 1.0, respectively. Reliable negative and positive controls were included. There was no indication that the test item elicits a SI ≥ 3 when tested up to and including 2%, and therefore the substance was not considered to be a skin sensitizer. An EC3 of >2% is derived. Based on the results of this study, the substance is not a skin sensitiser.

Further (in vitro) toxicity testing is not feasible because the substance has a high log Kow, which is out of the applicability domain of the in vitro tests (OECD TG 442C (DPRA), 442D (KeratinoSens) and 442E (h-CLAT)).

 

Information on structural features indicates the substance is not expected to be a direct acting sensitizer as derived from the OECD Toolbox. There is some empirical information that ethers may present some indirect effects at high concentrations. In view of the absence of a suitable analogue a read across is not possible.

Justification for classification or non-classification

In view of the absence of skin sensitisation in the LLNA (OECDTG 429) the substance does not need to be classified for skin sensitisation in accordance with EU CLP (1272/2008 and its amendments).