[2-(1-ethoxyethoxy)ethyl]benzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 September 2016 - 08 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

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)

Test material

Test material information

1

Reference substance name:

[2-(1-ethoxyethoxy)ethyl]benzene

EC Number:

219-868-9

EC Name:

[2-(1-ethoxyethoxy)ethyl]benzene

Cas Number:

2556-10-7

Molecular formula:

C12H18O2

IUPAC Name:

[2-(1-ethoxyethoxy)ethyl]benzene

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/J strain mice were supplied by Janvier, Le Genest-Saint-Isle, France.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: approximately ten weeks old
- Weight at study initiation: 19.3 to 24.7 g
- Housing: Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment. 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
- Diet (e.g. ad libitum): ad libitum (pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days
- Other: On receipt the animals were randomly allocated to cages.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Undiluted test item or the test item at concentrations of 2%, 10% or 30% v/v in vehicle.

No. of animals per dose:

Groups of five mice were treated.

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: soluble up to the maximum required concentration
- 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).
- Systemic toxicity: 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.
- Ear thickness measurements: 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.
- Other information: Initially, two test item concentrations were tested; a 30% 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, two additional animals were treated in a similar manner with an intermediate concentration (50%) at a later stage.The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-11 weeks (at initiation of treatment) and 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.

MAIN STUDY
- Test Item Administration: Groups of five mice were treated with the undiluted test item or the test item at concentrations of 2 %, 10 % or 30 % 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. 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.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA

TREATMENT PREPARATION AND ADMINISTRATION:
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 solutions 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.

Controls:
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.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6 (prior to termination).

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, techical grade, gave a Stimulation Index 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:

%

Remarks on result:

other: EC3 was not reached

Results 2

Parameter:

SI

Value:

0.9

Test group / Remarks:

2%

Remarks on result:

other: 2%

Results 3

Parameter:

SI

Value:

1.1

Test group / Remarks:

10%

Remarks on result:

other: 10%

Results 4

Parameter:

SI

Value:

1.3

Test group / Remarks:

30%

Remarks on result:

other: 30%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the test item concentrations 2, 10 and 30% were 0.9, 1.1 and 1.3, respectively.
EC3 CALCULATION
There was no indication that the test item elicits an SI ≥ 3 when tested up to 30%, therefore Hyacinth body was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 30%.
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.
No erythema was observed either in any of the animals.

Any other information on results incl. tables

Estimation of the Proliferative Response of Lymph Node Cells

Treatment Group	Concentration	Stimulation Index	Result
Test Item	2%v/vin acetone/olive oil4:1	0.9	Negative
	10%v/vin acetone/olive oil4:1	1.1	Negative
	30% v/v in acetone/olive oil4:1	1.3	Negative
Positive Control ItemÅ	25%v/vin acetone/olive oil4:1	4.3	Positive

Å =     Positive control group served as a common control with Project number 513924.

PRE-SCREEN TEST

Body Weights, Skin Reactions and Clinical Signs

TS^1 (%)	animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
		bw (g)^2	erythema^3		erythema		erythema		erythema		erythema		erythema		bw (g)
			left	right	left	right	left	right	left	right	left	right	left	right
30	1	22.9	0	0	1	1P	1	1HP	0	0HP	0	0	0	0s	22.8
	2	21.2	0	0	1	1P	1	1HP	0	0HP	0	0	0	0s	21.2
100	3	22.2	0	1	1	1	1	1	0	1	0	0	0	0s	23.3
	4	22.9	1	1	2	1	1	1	0	0	0	0	0	0	24.5
Additional pre-screen test:
50	5	22.3	1	1	1	1	1	1P	1	1P	0	1	0	0	22.3
	6	20.9	1	1	1	1	1	1	1	1	1	1	1	1	19.9

¹  TS = test item (% w/w).

²  Body weight (grams).

³  Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):

    0 = No erythema

    1 = Very slight erythema (barely perceptible)

    2 = Well-defined erythema

s Scaliness

H Hunched posture

P         Piloerection

Ear Thickness Measurements

TS^1(%)	Animal	Day 1		Day 3				Day 6
		left	right	left		right		left		right
		(mm)	(mm)	(mm)	%^2	(mm)	%^2	(mm)	%^2	(mm)	%^2
30	1	0.225	0.225	0.225	0	0.220	-2	0.230	2	0.230	2
	2	0.230	0.230	0.230	0	0.225	-2	0.240	4	0.235	2
100	3	0.220	0.225	0.220	0	0.225	0	0.240	9	0.240	7
	4	0.220	0.220	0.225	2	0.225	2	0.230	5	0.230	5
Additional pre-screen test:
50	5	0.225	0.220	0.235	4	0.235	7	0.235	4	0.235	7
	6	0.220	0.220	0.230	5	0.235	7	0.240	9	0.235	7

Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters.

¹  TS = test item (% w/w).

²  Percent increase compared to Day 1 pre-dose value.

PRE-SCREEN TEST

At 100%, hunched posture and/or piloerection were noted for both animals between Days 2 and 4. Very slight to well-defined erythema was noted for both animals between Days 1 and 4 and scaliness was noted for animal on Day 6.

At 50%, piloerection was noted for both animals on Days 3 and 4. Very slight erythema was noted for both animals between Days 1 and 6.

At 30%, no signs of systemic toxicity were noted. Very slight erythema was noted for both animals on Days 2 and 3.

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for all animals.

Based on the systemic tolerability of the test item, the highest test item concentration selected for the main study was a 30% concentration.

MAIN STUDY

Body Weights and Skin Reactions

group	TS^1 (%)	animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			bw (g)^2	erythema^3		erythema		erythema		erythema		erythema		erythema		bw (g)
				left	right	left	right	left	right	left	right	left	right	left	right
1	0	1	21.3	0	0	0	0	0	0	0	0	0	0	0	0	20.5
		2	22.8	0	0	0	0	0	0	0	0	0	0	0	0	22.2
		3	24.1	0	0	0	0	0	0	0	0	0	0	0	0	22.7
		4	23.3	0	0	0	0	0	0	0	0	0	0	0	0	22.0
		5	20.5	0	0	0	0	0	0	0	0	0	0	0	0	20.5
2	2	6	23.2	0	0	0	0	0	0	0	0	0	0	0	0	21.3
		7	19.8	0	0	0	0	0	0	0	0	0	0	0	0	19.5
		8	19.3	0	0	0	0	0	0	0	0	0	0	0	0	19.4
		9	24.7	0	0	0	0	0	0	0	0	0	0	0	0	24.9
		10	20.3	0	0	0	0	0	0	0	0	0	0	0	0	21.6
3	10	11	22.2	0	0	0	0	0	0	0	0	0	0	0	0	23.0
		12	22.9	0	0	0	0	0	0	0	0	0	0	0	0	23.4
		13	24.0	0	0	0	0	0	0	0	0	0	0	0	0	25.9
		14	24.2	0	0	0	0	0	0	0	0	0	0	0	0	23.8
		15	21.5	0	0	0	0	0	0	0	0	0	0	0	0	22.5
4	30	16	22.0	0	0	0	0	0	0	0	0	0	0	0	0	22.6
		17	21.0	0	0	0	0	0	0	0	0	0	0	0	0	22.5
		18	24.1	0	0	0	0	0	0	0	0	0	0	0	0	23.4
		19	22.6	0	0	0	0	0	0	0	0	0	0	0	0	22.2
		20	22.8	0	0	0	0	0	0	0	0	0	0	0	0	22.3

¹  TS = test item (% w/w).

²  Body weight (grams).

³  Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):

           0 = No erythema

Individual Disintegrations per Minute and Stimulation Indices Main test:

Treatment Group	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle acetone/olive oil 4:1 Å	1	611	725 (±51)	N/A	N/A
	2	745
	3	698
	4	662
	5	911
Test Item 2% v/vin acetone/olive oil 4:1	6	201	635 (±215)	0.9	Negative
	7	1049
	8	377
	9	1253
	10	296
Test Item 10% v/vin acetone/olive oil 4:1	11	662	780 (±50)	1.1	Negative
	12	870
	13	855
	14	652
	15	863
Test Item  30% v/v in acetone/olive oil 4:1	16	271	909 (±196)	1.3	Negative
	17	1246
	18	940
	19	1365
	20	722
Positive Control Item 25% v/v in acetone/olive oil4:1 Å	4-1	no data	2566 (±300)	4.3	Positive
	4-2	no data
	4-3	no data
	4-4	no data
	4-5	no data

dpm=Disintegrations per minute

N/A=  Not applicable

EC3 CALCULATION

There was no indication that the test item elicits a SI ≥ 3 when tested up to 30%, Hyacinth body was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 30%.

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. No erythema was observed either in any of the animals.

Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Applicant's summary and conclusion

Interpretation of results:

other: Not a skin sensitiser

Remarks:

based on EU CLP criteria (EC 1272/2008 and its updates)

Conclusions:

The SI values calculated for Hyacinth body concentrations 2, 10 and 30% were 0.9, 1.1 and 1.3, respectively. These results show that Hyacinth body does not elicit a SI ≥ 3. An EC3 has been derived resulted in an EC3 of >30%. A NOEC of >30% is derived. Based on the results of this study it is concluded that Hyacinth body is not a skin sensitiser.

Executive summary:

The skin sensitisation potential of Hyacinth body was tested according to OECD TG 429: Local Lymph Node Assay method. As this is a guideline study performed under GLP conditions, the study was assigned a Klimisch 1 rating. The substance was tested up to a maximum concentration of 30% due to expected systemic toxicity at higher levels, based on the results of the pre-screen test, which showed systemic effects like piloerection at 50% and 100% and hunched posture, erythema and scaliness at 100%. Three concentrations were used: 2, 10 and 30%. At 2, 10 and 30% the substance showed SI values of 0.9, 1.1 and 1.3, respectively. Reliable negative and positive controls were included. 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. No erythema was observed either in any of the animals. There was no indication that the test item elicits a SI ≥ 3 when tested up to and including 30%, and therefore the substance was not considered to be a skin sensitizer. An EC3 value could not be derived and is therefore expected to be higher than 30%. The same applies to the NOEC. Based on the results of this study, Hyacinth body is not considered to be skin sensitising.