3-(isodecyloxy)propiononitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2018 - 01 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 22 January 2018

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-(isodecyloxy)propiononitrile

EC Number:

264-840-1

EC Name:

3-(isodecyloxy)propiononitrile

Cas Number:

64354-92-3

Molecular formula:

C13H25NO

IUPAC Name:

3-(decylalkyl-(branched)oxy)-propiononitrile

Test material form:

liquid

Details on test material:

- Physical appearance: hazy light yellow liquid
- Name of test material (as cited in the report): Propanenitrile, 3-(isodecyloxy)-
- Purity: 100% UVCB
- Purity test date: 23 May 2017
- Batch no.: 1576742
- Expiry date of batch: 1 May 2018
- Storage conditions: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks old
- Weight at study initiation: 18.7 to 22.3 g
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), provided ad libitum
- Water: municipal tap-water, freely available
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 23°C
- Humidity (%): 40 to 51%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 29 March 2018 To: 30 April 2018

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Pre-screen test: 2, 5, 10, 25, 50 and 100%
Main study: 0, 0.5, 1 and 2%

No. of animals per dose:

5 animals per dosing group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: the vehicle was selected on the basis of maximizing solubility which was based on trial preparations performed at the test facility. No further information on the solubility of the test item was available.

At a 100%, 50%, 25%, 10% and 5% test item concentration, variation in ear thickness during the observation period were more than 25% from day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria.
- Systemic toxicity: no signs of systemic toxicity at a 2% test item concentration
- Ear thickness measurements: there was no clear increase in ear thickness exceeding the threshold with only one ear of one animal exceeding the 25% threshold at one time point.
- Erythema scores: at a test item concentration of 2%: 0 for both animals at all time points measured. For more details see table 1 in 'more information on results'.

MAIN STUDY : Three groups of five animals were treated with one test item concentration per group (0.5, 1 and 2%). One group of five animals was treated with the vehicle.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: if the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
- Induction (days 1, 2 and 3): both ears were topically treated with 25 μL test item per ear.
- Excision of the nodes (day 6): each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection and the draining lymph node of each ear was excised.
- Tissue processing for radioactivity (day 6): 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 (maze size: 200 μm, diameter: ± 1.5 cm). 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.
- Radioactivity measurements (day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail 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 background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

OBSERVATIONS:
- Mortality: twice daily
- Clinical observations: once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
- Bodyweight: individually on day 1 (predose) and 6 (prior to necropsy).

ANALYSIS:
A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

Positive control substance(s):

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

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamaldehyde resulted in an EC3 value of 19.2% (calculated using linear interpolation).
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%. This indicates that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.2

Variability:

0.3

Test group / Remarks:

0.5%

Results 2

Key result

Parameter:

SI

Value:

1.4

Variability:

0.3

Test group / Remarks:

1%

Results 3

Key result

Parameter:

SI

Value:

2.6

Variability:

0.5

Test group / Remarks:

2%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : see table 2
The majority of auricular lymph nodes were considered normal in size, except for the nodes in two animals treated at 0.5% and all animals treated at 2%, which were considered enlarged.

DETAILS ON STIMULATION INDEX CALCULATION :
Mean DPM/animal values for the experimental groups treated with test item concentrations 0.5, 1 and 2% were 757, 875 and 1611 DPM, respectively. The mean DPM/animal value for the vehicle control group was 625 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 1.2, 1.4 and 2.6, respectively.

EC3 CALCULATION : The test item did not elicit a SI ≥ 3 when tested up to 2% in this study and therefore an EC3 value >2% was established.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

OTHER: No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Any other information on results incl. tables

Table 1 Pre-Screen Test: Erythema Scores

TS^a (%)	animal	Day 1		Day 2		Day 3		Day 4		Day 5		Day 6
		erythema^b		erythema		erythema		erythema		erythema		erythema
		left	right	left	right	left	right	left	right	left	right	left	right
100	3	0	0	0	0h	1	1h	1	1	0s	0s	0sk	0sk
	4	0	0h	1	0h	1	1h	1	1	0s	0s	0sk	0sk
50	1	0	0	1	1	1	1	1	1	0	1	0s	0s
	2	0	0	1	1h	1	1	1	1	1	1	0s	0s
25	7	0	0	1	1	2	2	2	2	1	1s	0s	0s
	8	0	0	1	1	2	2	2	2	1s	1s	0s	0s
10	5	0	0	0	0	1	1	1	1	1	1s	0s	0s
	6^c	0	0	0	0	1	1	1	1	1	1	0s	0s
5	11	0	0	0	0	1	1	0	0	0	0	1s	1s
	12	0	0	0	0	1	1	0	1	0	0	1s	1s
2	9	0	0	0	0	0	0	0	0	0	0	0	0
	10	0	0	0	0	0	0	0	0	0	0	0	0

h. hunched posture, s. Scaliness, k. Scabs.

^a  TS = test item (% w/w).

^b  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

^c  Animal had a scab above the right eye from Day 3 onwards, this caused the eye to be partially closed.

Table 2: Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

group	TS^a (%)	animal	Size nodes^b		DPM^c/ animal	mean DPM ± SEM^d			mean SI ± SEM
			left	right
1	0	1	n	n	471	625	±	118	1.0	±	0.2
		2	n	n	1067
		3	n	n	383
		4	n	n	626
		5	n	n	578
2	0.5	6	n	n	399	757	±	161	1.2	±	0.3
		7	+	n	688
		8	n	n	581
		9	+	+	1350
		10	n	n	769
3	1	11	n	n	930	875	±	212	1.4	±	0.3
		12	n	n	615
		13	n	n	728
		14	n	n	1660
		15	n	n	440
4	2	16	+	+	2688	1611	±	320	2.6	±	0.5
		17	+	+	1493
		18	+	+	1612
		19	+	+	673
		20	+	+	1588

^a  TS = test item (% w/w).

^b  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).

^c   DPM= Disintegrations per minute.

^d   SEM = Standard Error of the Mean.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) No. 1272/2008

Conclusions:

The results of an LLNA study, performed according to OECD guideline 429 and GLP principles, indicate that Propanenitrile, 3-(isodecyloxy)- did not elicit a SI ≥ 3 when tested up to 2%.

Executive summary:

The objective of this study was to evaluate whether Propanenitrile, 3-(isodecyloxy)- induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in the study plan. The study was carried out based on the guidelines described in:

• OECD, Section 4, Health Effects, No.429 (2010),

• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test item concentrations selected for the main study were based on the results of a pre-screen test. The 100%, 50%, 25%, 10% and 5% test item concentrations did not meet the selection criteria. At a 2% test item concentration there was no clear increase in ear thickness exceeding the threshold. Based on the other data, this concentration was considered suitable for use in the main study with inclusion of ear thickness measurements in the main study.

 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 0.5, 1 or 2% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

No erythema was observed in any of the animals. Variation in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for all animals.

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.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in two animals treated at 0.5% and all animals treated at 2%, which were considered enlarged.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 0.5, 1 and 2% were 757, 875 and 1611 DPM, respectively. The mean DPM/animal value for the vehicle control group was 625 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 1.2, 1.4 and 2.6, respectively.