3-(isodecyloxy)propiononitrile

Endpoint summary

Description of key information

Profiling and QSARs indicate a low risk for sensitisation, and due to use as intermediate in industrial and professional setting only, exposures are limited. There are no reports on incidents of sensitisation to ethernitrile available.

Read-across to a GPMT study on coconitrile performed under GLP and according to OECD guideline 406 indicated that no classification is required for dermal sensitisation.

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:

15 March 2018 - 01 May 2018

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:

July 2012

Deviations:

no

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)

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

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)

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.

Key result

Parameter:

SI

Value:

1.2

Variability:

0.3

Test group / Remarks:

0.5%

Key result

Parameter:

SI

Value:

1.4

Variability:

0.3

Test group / Remarks:

1%

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The following in vitro/in chemico studies have been performed asrecommended as part of weight of evidence of the evaluation for skin sensitization potential of Ehernitrile-C10i:

(a) molecular inter­ action with skin proteins – DPRA (OECD 442C)

(b) inflammatory response in keratinocytes -KeratinoSens™Assay (OECD 442D)

(c) activation of dendritic cells - U-Sens™ (OECD 442E)

 

DPRA:

The reactivity of Ehernitrile-C10i towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was evaluated in the Direct Peptide Reactivity Assay (DPRA) in a GLP compliant study according to the most recent OECD 442C guideline. After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. All validation parameters were within the acceptability criteria for the DPRA.

In the cysteine reactivity assay Ehernitrile-C10i showed 42.6% SPCC depletion while in the lysine reactivity assay the test item showed 14.8% SPCL depletion. As the average depletion of cysteine and lysine containing peptides is above 6.38% depletion (threshold for positive result), the results are considered positiveandEhernitrile-C10iwas classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

 

KeratinoSens:

Ehernitrile-C10i was tested for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in aGLP compliantstudy according to the most recent OECD 442D guideline. Two independent experiments were performed which both passed the acceptance criteria for the positive control Ethylene dimethacrylate glycol and showing a dose response relation, and the average coefficient of for the negative (solvent) control DMSO was below 20%.

Ehernitrile-C10i showed toxicity (IC30 values of 83 µM and 75 µM and IC50 values of 98 µM and 89 µM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.23-fold and 1.25-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations≥1000 µM.

In conclusion, Ehernitrile-C10i is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

 

U-Sens:

Ehernitrile-C10i wasevaluated forthe ability to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens) assay, performedin a GLP compliant study according to the most recent OECD 442Eguideline.

Three independent experiments were performed.All experimentspassed the acceptance criteria and inall experimentsthe positive and negative control were considered valid.

The test item showed only toxicity in the first experiment (CV70 value of 31 µg/mL).A biologically relevant, induction of the CD86 activity (EC150 values of 14 µg/mL, 17 µg/mL and 19 µg/mL in experiment 1, 2 and 3, respectively) was measured in all experiments. In experiment 1 the first induction of the CD86 activity above 150% was observed at the highest non-cytotoxic concentration and therefore classified as non-conclusive. The test item is classified as Positive in the U-Sens assay in experiment 2 and 3 since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% comparedto the vehicle control.

 

LLNA:

Ehernitrile-C10i concentrations selected for the main study were based on the results of a pre-screen test, indicating that at 2% the ear thickness increase was borderline, with only one ear of one animal on day 6 higher than the (25%) threshold at 28%.

In the main study, three groups of five female mice were treated with concentrations of 0, 0.5, 1 or 2% w/w in Acetone/Olive oil (4:1 v/v) on three consecutive days, by open application on the ears. 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.

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.

The SI values calculated for the test item concentrations 0.5, 1 and 2% were 1.2, 1.4 and 2.6, respectively. Consequently it is concluded that Ethernitrile 10 was non-sensitizing when tested up to 2% in the LLNA.

GPMT:

There is GPMT data based on read-across to Coconitrile.Support for read-across is added to respective study summary.

Coconitrile was evaluated for skin sensitization in a GPMT, performed under GLP and according to OECD guideline 406. Based on results from preliminary irritation testing, induction consisted of concentration of 50% for intradermal injections, and 100% for topical applications 24 hours following 10% SDS application to induce irritation.All animals showed considerable necrosis following intradermal injections of test substance during the induction phase.

Subsequent challenge in 10 animals was done with 100% applications. This resulted to a weak reaction in only one of the animals (10%). Considering such a minimal reaction to 100% challenge dose on a substance that causes irritation in rabbits (OECD 404), following a high intradermal induction dose of 50%, it was considered that testing in a further group of 10 test animals would not be ethical. Concluded was that Coconitrile is not sensitizing to skin.

 

QSAR:

The profiling (QSAR Toolbox v.4.2) of Ethernitrile-C10i indicates that no alerts are found for protein binding, thiol reactivity is not expected, and that the structure is not represented among the categories of high, moderate or low reactivity in DPRA (direct peptide reactivity assays) for either cysteine or lysine depletion. Additionally, the molecular structure of the ethernitrile does not contain toxicophores indicating a concern for sensitization, and also read across to data available on structurally related Coconitrile (and alkylnitriles in general) do not indicate a concern.

The automated workflow in QSAR Toolbox for skin sensitization for Ethernitrile-C10i and Dodecanenitrile concludes to ‘Non sensitizer’ for both structures.

Information from QSARs (Reports attached):

- DEREK (Derek Nexus 6.0.1): Predicted to be non-sensitiser, with no misclassified or unclassified features.

- VEGA (Skin Sensitization model (CAESAR) 2.1.6): Predicts sensitizer. However with low reliability, with indication that structure is outside the Applicability Domain. Also the reported similar compounds show structural large differences, despite indicated high similarity and concordance. Actually, no nitrile is present in the dataset!

- TOPKAT_Skin_Sensitization_None_vs_Sensitizer (3DS/BIOVIA -ADMET Toxicity Prediction (Extensible)): predicts non-sensitizer.

- Danish (Q)SAR Database (April 2018): Negative predictions for Battery, CASE Ultra, Leadscope and SciQSAR, with only two exception for CASE Ultra with two ‘Inconclusive’ results, both with the comment ‘outside applicability domain’.

 

There are no reports on incidences of sensitisation from industrial production and use of the substance.

 

Interpretation of data:

The ‘2-out-of-3’ prediction model for the results of the three in vitro assays would suggest thatEhernitrile-C10i poses a skin sensitisation hazard on the basis of positive results for both DPRA and U-Sens. However, these results are unexpected as profiling and QSARs show no suggestion of possible sensitisation hazards. The subsequent performed in vivo LLNA study did not conclude to a positive result when tested up to 2% on the ears of mice.

Also available Guinea Pig Maximization Test (GPMT) used in read-across, was negative.

Literature evaluation provides supportive information that these nitriles result to false positive in vitro results with only the keratinoSens as negative against a negative LLNA:

- Urbisch et al.(2015) evaluated results from in vitro assays for sensitization to both LLNA results and available human data. They mention nitriles among the group of substances for which no reaction mechanism (no alert) is assigned, and further the only nitrile for which data is available for the comparison (3-phenoxypropiononitrile, CAS 3055-86-5) as false positive for the ‘2-out-of-3’ approach, where only the keratinoSens was negative, but the LLNA was clear negative.

- Kern et al. (2010) published a database of available reliable LLNA data for over 300 chemicals to serve as primary resource for comparative analyses in the evaluation of alternative testing methods. Among the substances in this database are three nitriles that all three are negative in LLNA (Geranyl nitrile, CAS 5146-66-7; Octanenitrile, CAS 124-12-9; and 3-phenoxypropiononitrile, CAS 3055-86-5 (also mentioned inUrbisch et al.2015)The information fromUrbisch et al.2015 and Kern et al. 2010 are fully in line with the obtained results on Ethernitrile-C10i and in support that the obtained positive results in DPRA and U-Sens assays are reflecting false positive results.

 

References:

-        Urbisch D et al., 2015, Assessing skin sensitization hazard in mice and men using non-animal test methods. Reg.Toxol.Pharm. 71, 337-351.

-        Kern PS et al., 2010, Local Lymph Node Data for the Evaluation of Skin Sensitization Alternatives: A Second Compilation.Dermatitis 21(1), 8-32

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Ethernitrile-C10i has a low vp pressure and its use is limited to industrial settings that do not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur.

Available data based on read-across indicate no concerns for skin sensitisation. Additionally, information from profiling for expected protein interaction and QSARs for sensitisation, also indicate non-sensitising properties.

 

As chemical respiratory sensitisers also elicit positive results in predictive tests for contact sensitisation, the negative outcome for dermal sensitisation is also predictive for non respiratory sensitisation of the substance.

Justification for classification or non-classification

Weight-of-evidence evaluation of all available data does not indicate that there is a need for classification.