Oleonitrile

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

09 March 2010 - 23 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles. Read-across from structural identical substance with on avarage a somewhat shorter aliphatic chain length.

Qualifier:

according to guideline

Guideline:

other: OECD No. 406 (1992) "Skin Sensitization" EC No 440/2008; B6: "Skin Sensitization: Guinea-Pig Maximization Test (GPMT)" EPA OPPTS 870.2600 (2003) “Skin Sensitization” JMAFF Guidelines (2000) including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

GPMT has been demonstrated in scientific literature to be effective at determining skin sensitization. LLNA has been demonstrated to deliver false positives when testing surfactants to which the tested substance is a member of.
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Roberts, D.W., Schultz, T.W. and Api, A.M., 2016. Chemical applicability domain of the Local Lymph Node Assay (LLNA) for skin sensitisation potency. Part 3. Apparent discrepancies between LLNA and GPMT sensitisation potential: False positives or differences in sensitivity?. Regulatory Toxicology and Pharmacology, 80, pp.260-267.
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Ball, N., Cagen, S., Carrillo, J.C., Certa, H., Eigler, D., Emter, R., Faulhammer, F., Garcia, C., Graham, C., Haux, C. and Kolle, S.N., 2011. Evaluating the sensitization potential of surfactants: integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach. Regulatory Toxicology and Pharmacology, 60(3), pp.389-400.
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Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: approx. 6-7 weeks old
- Weight at study initiation:
- Housing: Group housing of maximally 5 animals per labeled cage (74 cm x 54 cm x 25 cm height) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France).

- Diet (e.g. ad libitum): Complete breeding diet for guinea pigs (SSNIFF® MS-Z, V2273; SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 – 20.7ºC
- Humidity (%): 23 - 95%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 09 March 2010 To: 23 April 2010

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

intradermal inductie: 50%
epidermal inductie: 100%
Challange: 0 and 100%
Re-challange: 50 and 100%

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

intradermal inductie: 50%
epidermal inductie: 100%
Challange: 0 and 100%
Re-challange: 50 and 100%

No. of animals per dose:

Experimental group: 10 females.
Control group: 5 females.

Details on study design:

RANGE FINDING TESTS:

Series of test substance concentrations were tested (0.5/1/2/5/10/20/50/100%).
The test system and procedures were identical to those used during the main study, unless otherwise specified.
The four animals selected were between 4 and 9 weeks old. No body weights were determined.

MAIN STUDY
A. INDUCTION EXPOSURE
-Day 1
The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 50% concentration.
C) A 1:1 w/w mixture of the undiluted test substance and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).

Day 3
The dermal reactions caused by the intradermal injections were assessed for irritation.

Day 7
The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

Day 8
The 10% SDS treated area between the injection sites was treated with 0.5 mL of a 100% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.

The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

B. CHALLENGE EXPOSURE
-Day 21
One flank of all animals was clipped and treated by epidermal application of a 100% test substance concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.

The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

Day 28 A re-challenge was conducted approximately one week after the first challenge, to clarify the results in the first challenge. The contralateral flank of all animals was similarly treated. All animals were treated epidermally with 0.05 mL of each of the following test substance concentrations, 100% and 50% on a clipped flank, using Square chambers, attached to Micropore tape and secured with Coban elastic bandage.
The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

After termination, animals were sacrificed by intra peritoneal injection of pentobarbital (Euthesate®; Sanofi Sante B.V., Maassluis, The Netherlands).

In life examinations:
Mortality/Viability: Twice daily
Toxicity: At least once daily.

Body weights: Prior to start and at termination of the study.

Irritation:
Skin reactions were graded according to the following numerical scoring systems. Furthermore, a description of all other (local) effects was recorded.
To facilitate scoring, the epidermally treated skin-areas were clipped at least 3 hours before the 48-hour reading of these areas in the preliminary irritation study and challenge phase.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamicaldehyde

Positive control results:

Reliability test:

Was performed not more than 6 months previously. Similar procedures were used in the reliability test and in this study.

The skin reactions observed in seven experimental animals in response to the 20% test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 70 per cent to the 20% concentration.

From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitizing potential of a substance in a Maximization type of test.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

3

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 3.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

other: control

Dose level:

0

No. with + reactions:

1

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: 0. No with. + reactions: 1.0. Total no. in groups: 5.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 2.0. Total no. in groups: 10.0.

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 10.0.

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 10.0.

Grading Irritation Reactions*:

Erythema and eschar formation:

No erythema..........................................................................................................................0

Slight erythema (barely perceptible).................................................................................1

Well-defined erythema.........................................................................................................2

Moderate erythema...............................................................................................................3

Severe erythema (beet redness) to slight eschar formation (injuries in depth).......4

 

Oedema formation:

No oedema...................................................................................................................... ........0

Slight oedema (barely perceptible).............................................................................. .......1

Well-defined oedema (edges of area well-defined by definite raising)........................2

Moderate oedema (raised approximately 1 millimeter)...................................................3

Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure)..................................................................................................................................4

(*. Intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis.)

 

                                        

Grading Challenge Reactions:

No visible change..................................................................................................................0

Discrete or patchy erythema................................................................................................1

Moderate andfluent erythema..............................................................................................2

Moderate erythema and swelling.......................................................................................3

Intense erythema and swelling...........................................................................................4

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The skin reactions that were confirmed in the second challenge in one (of the ten) experimental animals in response to a 100% test substance concentration consisted of a grade 1 reaction (discrete or patchy erythema), and was considered indicative of sensitization, based on the absence of any response in the control animals. These results indicate a sensitization rate of 10 per cent.

It was discussed with the sponsor that “According to the guidelines and if the results indicate a sensitization rate of >0% and <30% an extension of the study with another set of 10 experimental and 5 control animals is strongly recommended. A definitive classification will then be based on the results of the total of 20 experimental and 10 control animals“. It was decided not to do this and terminated the study.

Executive summary:

PRELIMINARY IRRITATION STUDY

 

The results of the intradermal injections and epidermal exposures for the selection of suitable test substancecentrations for the main study are described in Table 1.

 

Based on the results, the test substancecentrations selected for the main study were a 50%centration for the intradermal induction and a 100%centration for the epidermal induction exposure.

No evident signs of irritation were observed to the highest test substancecentration epidermally tested. Therefore, the test site of all animals was treated with 10%approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction.

A 100% test substancecentration was selected for the challenge phase.

 

MAINSTUDY

The skin effects caused by the intradermal injections and epidermal exposure during the induction phase are reported. All animals showed considerable necrosis following intradermal injections of test substance during the induction phase.

Subsequent epidermal induction exposure on day 10 resulted to reaction of grade 1 or 2 in 60% of the animals, although such treatment has shown not to result to dermal effects in the preliminary irritation study. The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the treatment.

 

First Challenge

Skin reactions of grade 1 were observed in one control animal and in three experimental animals in response to the 100% test substancecentration.

Because of the FCA treatment, the threshold for irritation could be possibly lowered for subsequent dermal exposure, explaining de response following epidermal induction, and the (slight) response in treated animals on this first challenge. In order to obtain a clearer response, a second challenge was performed.

Second challenge

Since it could not be decided whether the substance is a sensitizer or not, a sed challenge was performed one week later. The animals were then treated with a 100% and a 50% test substancecentration. Skin reactions of grade 1 were observed in one experimental animal and scaliness in another in response to the 100% test substancecentration. No skin reactions were evident in the control animals.

 

No mortality occurred and no symptoms 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 astrols over the study period.

The skin reactions that were confirmed in the second challenge in one (of the ten) experimental animals in response to a 100% test substance concentration were considered indicative of sensitization, based on the absence of any response in the control animals. These results

indicate a sensitization rate of 10 per cent.

Formally, OECD guideline indicates that in cases of doubt (defined as all results showing response between 0 and 30%) additional 10 animals and 5 controls should be used. However, it was considered that additional animals would not lead to more positive reactions reaching to an overall average of ≥ 30%, and in view of animal welfare considerations it was therefore deicded that there is no need for testing on additional animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

These results from Coconitrile can be regarded as valid for Oleylnitrile as the tested substance is actually the same: They share the same chemical structure, and only differ in the average length of the alkyl chain. Aspects of sensitization are related to possible dermal penetration, and subsequent reactivity and protein binding (needed for haptenisation) which are properties that are relative independent to actual chain length. If anything, it is assumed that shorter chain lengths are possibly somewhat more bio-available, and thus the testing of Coconitrile might represent a worse case situation compared to Oleylnitrile.

Migrated from Short description of key information:

A GPMT study with Coconitrile showed no concern for sensitisation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

GPMT study on coconitrile performed under GLP and according to OECD guideline 406 resulted to a weak reaction in only 10% of the animals. Accordingly, no classification is required for dermal sensitisation. 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.

Migrated from Short description of key information:

A GPMT study with Coconitrile showed no concern for sensitisation.

Justification for classification or non-classification

A standard study to evaluate the sensitsing potential of coconitrile indicated that no classification is required for skin sensitisation. As such tests are also positive when tested with respiratory sensitisers, the non-sensitisng results of this tests also indicate that the substance is also not a respiratory sensitiser.

These results from Coconitrile can be regarded as valid for Oleylnitrile as the tested substance is actually the same:They share the same chemical structure, and only differ in the average length of the alkyl chain. Aspects of sensitization are related to possible dermal penetration, and subsequent reactivity and protein binding (needed for haptenisation) which are properties that are relative independent to actual chain length. If anything, it is assumed that shorter chain lengths are possibly somewhat more bio-available, and thus the testing of Coconitrile might represent a worse case situation compared to Oleylnitrile.