3-ethoxy-1,1,5-trimethylcyclohexane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2015 to 12 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 429 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP compliance programme (inspected on June 17, 2015 / signed on September 24, 2015)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-ethoxy-1,1,5-trimethylcyclohexane

EC Number:

266-722-5

EC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Cas Number:

67583-77-1

Molecular formula:

C11H22O

IUPAC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Test material form:

liquid

Details on test material:

- Physical state/Appearance: Clear colorless liquid
- Storage condition of test material: Stored at room temperature in the dark
- Expiration date of the lot/batch: 09 July 2016

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst / The Netherlands.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: ca. 15 air changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 16 December 2015 to 12 January 2016

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25 and 50% in acetone/olive oil 4:1 and 100%

No. of animals per dose:

4 females/dose

Details on study design:

TEST ITEM PREPARATION
For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. 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.

PRE-SCREEN TESTS:
- 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 skin irritation was scored daily and any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
- Ear thickness measurements: The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), 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 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.
Results: No signs of systemic toxicity and no local skin irritation (no increase in mean ear thickness greater than 25%) were noted. 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.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer 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-sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
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). A further group of four mice received the vehicle alone in the same manner. 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 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. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
A single cell suspension of pooled lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The LNC were then washed by adding PBS, pelleted at 1400 rpm for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash. After ca 18 hours with 5% TCA at 4 °C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL scintillation fluid. The 3HTdR incorporation was measured by β scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node).

Positive control substance(s):

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

Results and discussion

Positive control results:

SI for the positive control substance α-Hexylcinnamaldehyde, tech., 85%, was 6.08 considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.91

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

2.09

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

3.27

Test group / Remarks:

100%

Cellular proliferation data / Observations:

DISINTEGRATIONS PER MINUTE/NODE: 2681.20, 2451.46, 5594.38 and 8767.15 for Vehicle, 25, 50 and 100%, respectively

STIMULATION INDEX: 0.91, 2.09 and 3.27 for 25, 50 and 100%, respectively

EC3 CALCULATION: EC3 = 50 + [[(3−2.09)/(3.27−2.09)] x (100−50)] = 89
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 89%.

CLINICAL OBSERVATIONS:
- There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
- 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.

Any other information on results incl. tables

Table 7.4.1/1: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

 

Concentration (% v/v) in acetone/olive oil 4:1	dpm	dpm/node^a	Stimulation Index^b	Result
Vehicle	21449.58	2681.20	NA	NA
25	19611.70	2451.46	0.91	Negative
50	44755.07	5594.38	2.09	Negative
100	70137.23	8767.15	3.27	Positive

 

dpm = Disintegrations per minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

NA = Not applicable

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the test conditions, test item is classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

A study was performed to assess the skin sensitisation potential of test item in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

 

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

 

Disintegrations per minute/node for Vehicle, 25, 50 and 100% were 2681.20, 2451.46, 5594.38 and 8767.15, respectively. Stimulation Indices (S.I.) of 0.91, 2.09 and 3.27 for 25, 50 and 100%, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 89%.

 

SI for the positive control substance 25% hexyl cinnamic aldehyde (HCA), was 6.08 which demonstrates the validity of this study.

 

Under the test conditions, test item is classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.