9-Decenamide, N,N-dimethyl-

Endpoint summary

Description of key information

Skin irritation
GLP in vitro study performed according to OECD TG 439 using the substance itself indicates that the substance is irritating to skin. Supported by the results of an in vivo study performed according OECD Guideline No. 404 and EEC Guideline B.4 using the read-across substance N,N-Dimethyldecan-1-amide (CAS No. 14433-76-2).
Eye irritation
GLP in vivo study performed according to OECD TG 405 and EC Method B.5 using for the read-across substance, N,N-Dimethyldecan-1-amide (CAS No. 14433-76-2) indicates that the substance is irritating to eyes. An in vitro study performed according to OECD TG 437 using the substance itself indicated that the substance was not corrosive/ a severe eye irritant, but no further conclusion on classification could be made.
Respiratory irritation
Based on findings seen in an acute inhalation toxicity study performed according to OECD TG 403 using the read-across substance N,N-dimethyldecan-1 -amide, mixture with N,N-dimethyloctan-1 -amide (CAS No. 67359 -57 -3)  the substance is considered to be a respiratory irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January 2014 to 24 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD 439 test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

Species:

human

Strain:

other: Epidermal skin model

Type of coverage:

other: Direct application to epidermis model

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 uL per well

Duration of treatment / exposure:

60 minutes

Observation period:

42+/-2 hours

Number of animals:

3 wells per treatement

Details on study design:

The test substance was tested in one valid definitive trial. After the overnight incubation for 18±3 hours, the 6-well plates containing the EpiDerm™ tissues were removed from the incubator and placed at room temperature for at least 5 minutes prior to dosing. The EpiDerm™ tissues were treated in triplicate with the test substance for 60±1 minutes. Thirty microliters of the test substance were applied to each of three tissues at 1 minute intervals per tissue. A nylon mesh was placed gently over the dose to spread the test substance. A sterile rod was used to flatten the mesh over the test article applied onto the tissues. The EpiDerm™ tissues were tested in triplicate with the positive or negative control for 60±1 minutes. Thirty microliters of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control administration onto the tissue, a nylon mesh was placed gently over the dose to spread the negative and positive controls. The plates with dosed tissues were kept in the laminarflow hood until the last tissue was dosed. After the last tissue was dosed, all of plates were transferred to the incubator for 35±1 minutes at standard culture conditions. After 35 minutes, all of the plates were removed from the incubator, placed into the laminar flow hood and kept at room temperature until the exposure period was completed for the first dosed tissue.After 60±1 minutes of test or control substance exposure, the tissues were rinsed with sterile, CMF-DPBS by filling and emptying the tissue insert 15 times. A stream of CMF-DPBS was directed onto the tissue surface. For the test and control substances where a mesh was used, the mesh was carefully removed with forceps (if necessary) after the 5th rinse. After the removal of the mesh, the rinsing procedure of the tissue continued for 10 times. After the 15th rinse, each of the 3 inserts per treatment group (test article, positive and negative control) was completely submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of CMF-DPBS and specifically assigned for each treatment group; this procedure was repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMF-DPBS was decanted. The bottoms of the tissue inserts were blotted on sterile paper towels and the inserts were transferred to new 6-well plates containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The tissue surface was carefully blotted with sterile cotton-tipped applicators to remove any excess moisture, and the tissue surface was visually observed for residual test substance using a dissecting scope. The tissues were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 42±2 hours. After an initial 24±1 hours of incubation, the 6-well plates were removed from the incubator and the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh Maintenance Medium warmed to approximately 37ºC. The tissues were placed back into the incubator at standard culture conditions for an additional 18±1 hours for the remainder of the 42±2 hour post-treatment expression
incubation.
A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) was thawed and diluted in warm MTT Addition Medium to produce a 1.0 mg/mL solution no more than two hours before use. Three hundred microliters of the MTT solution were added to each designated well of a pre-labeled 24-well plate. After the total 42±2 hours post-exposure expression incubation, the 6-well plates were removed from the incubator. Each tissue was blotted on a sterile paper towel and transferred to an appropriate well containing 0.3 mL of MTT solution. The 24-well MTT plates were incubated at standard culture conditions for 3±0.1 hours. After the 3±0.1 hour incubation, the EpiDerm™ tissues were submerged, gently swirled, and rinse media decanted in a beaker containing approximately 150 mL of CMF-DPBS three times. The tissue was then blotted on absorbent paper, cleared of excess liquid, and transferred to a prelabelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plate was covered with parafilm and shaken for at least 2 hours at room temperature to extract the MTT. At the end of the extraction period, the insert was gently agitated up and down in its extractant well. The tissues were pierced with forceps to allow the extract to flow back into the well from which the insert was removed, and the Millicell® inserts were discarded. The extract solution was mixed (homogenized by pipetting up and down three times) and two x 200 µL aliquots were transferred to the appropriate wells of a 96-well plate. Two hundred µL of isopropanol were added to the wells designated as blanks. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader with the AUTOMIX function selected.

Irritation / corrosion parameter:

other: other: EpiDerm result

Value:

3.26

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 48 hours. Max. score: 100.0. Reversibility: no data. (migrated information)

Assay date	IIVS Test Substance No.	Sponsor's Designation	Conc (w/v)	Mean Viability (%)	Skin irritation prediction	pH
22/01/2014	13AN40	Test Substance	Neat	3.26	Irritant	5.0
22/01/2014	Positive Control	SLS	5%	3.27	Irritant	NA

Interpretation of results:

Category 2 (irritant)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based upon the results of this assay, the test substance is predicted to be irritating.

Executive summary:

Introduction

The test substance was tested in one valid definitive assay to determine the identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label). The laboratory phase of the study was conducted from 20 January 2014 to 24 January 2014 at the Institute for In Vitro Sciences, Inc.

Methods

The EpiDerm Skin Kit (MatTek Corporation) was used in a procedure that followed the methods of the OECD 439 test guideline. The epidermis model were exposed to the test substance for 60 minutes before washing. After a further 42 hours the viability of the cells was measured using MTT. Vehicle and positive (SLS) controls were included and each sample was tested in triplicate.

Results

The mean OD570 of the negative control, CMF-DPBS, was 2.042. The mean viability of the positive control, 5% SLS, was 3.27%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was <18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid.

The test substance was not observed to interact with the nylon mesh, and therefore a nylon mesh was used to aid in the spreading of the test substance after dosing the EpiDerm™ tissues.

The test substance was not observed to directly reduce MTT in the absence of viable cells. Although, the test substance was observed to change the MTT solution yellow.

The mean viability of the cells exposed to the test substance was 3.26%, which is less than 50% and therefore indicates that the substance is an irritant.

Conclusion

Based upon the results of this assay , the test substance was predicted to be irritating.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD guideline, GLP, well documented. Reliability reduced for read-across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: Mol: Russian

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Mollegaard Breeding and Research Centre A/S
- Weight: 2.4-2.6kg
- Housing: animal room 2, filtered Air, ppo cages
- Diet (e.g. ad libitum): ad libitum / Altromin 2123 , Altromin , Lage , Germany
- Water (e.g. ad libitum): ad libitum (acified, HCl)
- Acclimation period: at least one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3°C
- Humidity (%): 55±15%
- Air changes (per hr): 10times/h
- Photoperiod (hrs dark / hrs light):12h each

Vehicle:

unchanged (no vehicle)

Controls:

other: right eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1ml

Observation period (in vivo):

1h, 24h, 48h, 72h, 7d, 14d, 21d

Number of animals or in vitro replicates:

3

Irritation parameter:

cornea opacity score

Remarks:

corne opacity

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 1

Max. score:

4

Reversibility:

fully reversible within: 21 days in 2 of 3 animals

Remarks on result:

other: When Fluorescein has been applied only the highest score for degree of corneal opacity from three readings are included

Irritation parameter:

iris score

Remarks:

iris lesion

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 0

Max. score:

2

Remarks on result:

other: normal reaction observed

Irritation parameter:

conjunctivae score

Remarks:

redness of conjunctiva

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 2.6

Max. score:

3

Reversibility:

fully reversible within: 21 days in 2 of 3 animals

Irritation parameter:

chemosis score

Remarks:

oedema of conjunctiva (chemosis)

Basis:

mean

Remarks:

three animals

Time point:

other: mean from 24h, 48h and 72h reading

Score:

<= 2.8

Max. score:

4

Reversibility:

fully reversible within: 21 days in 2 of 3 animals

Interpretation of results:

other: Category 2

Remarks:

Criteria used for interpretation of results: other: EU GHS EC 1272/2008

Conclusions:

Eye irritant potential

Executive summary:

The eye irritant effect of SAT 970 419 (N,N-Dimethyldecan-1-amide) was investigated according to the method recommended in the OECD Guideline No. 405, "Acute Eye Irritation/Corrosion", Feb. 1987, and EEC Guideline B.5 "Acute Toxicity (Eye Irritation)", 29.12.1992.

Three female albino rabbits were exposed to 0.1 ml of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours as well as 7, 14 and in two animals 21 days after dosing.

Moderate to severe signs of eye irritation were observed among the rabbits.

The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated:

Cornea opacity 1.0

Iris lesion 0.0

Redness of conjunctiva 2.6

Oedema of conjunctiva 2.8

After 14 days all animals showed hairless areas around the eye. Treatment related adverse eye reactions were fully reverse within 14 (1 animal) and 21 days.

Based on the result of this study the test substance is classified as eye irritant

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin Irritation

The skin irritation potential of the substance was assessed using the EpiDerm Skin Kit (MatTek Corporation) in a procedure that followed the methods of OECD TG 439 [Costin, 2014]. The epidermis model was exposed to the test substance for 60 minutes before washing. After a further 42 hours the viability of the cells was measured using MTT. Vehicle and positive (SLS) controls were included and each sample was tested in triplicate. The mean OD570 of the negative control, CMF-DPBS, was 2.042. The mean viability of the positive control, 5% SLS, was 3.27%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was <18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid. The test substance was not observed to interact with the nylon mesh, and therefore a nylon mesh was used to aid in the spreading of the test substance after dosing the EpiDerm™ tissues. The test substance was not observed to directly reduce MTT in the absence of viable cells, although, the test substance was observed to change the MTT solution yellow. The mean viability of the cells exposed to the test substance was 3.26%, which is less than 50% and therefore indicates that the substance is an irritant.

The results of this study are supported by the results of an in vivo study performed according OECD Guideline No. 404 and EEC Guideline B.4 using the read-across substance N,N-Dimethyldecan-1-amide (CAS No. 14433-76-2) [Hoyer, 1997]. Three female albino rabbits were exposed to the test article at two skin sites on the back. After 4 hours of exposure the test article was removed and the skin was examined 1, 24, 48 and 72 hours as well as 7, 14 and 21 days after termination of exposure. Moderate to severe skin reactions were observed. Under the experimental conditions described in this report, the mean score for erythema was 2.7 (2.00, 3.00, 3.00) and for oedema 2.8 (2.00, 3.33, 3.00). After 21 days rests of scales on both sides were observed in all animals.

Eye Irritation

The eye corrosion/irritation potential of the substance was assessed using the BCOP method according to OECD TG 437 [Wilt, 2014]. Five corneas were treated with either the test substance, a negative control substance or a positive control substance. Based on changes in corneal opacity and permeability (relative to the control corneas), an in vitro score was determined. The test substance has an in vitro score of 30.8 and the positive control had an in vitro score of 42.2. The results of the positive control fell within two standard deviations of the historical mean (within a range of 39.2 to 63.6), the assay was considered valid. On the basis of these results the substance is considered to be moderately irritating, but as the IVIS lies in the range of >3; <=55, no prediction may be made regarding the classification of the substance.

In vivo data are available for the read-across substance, N,N-Dimethyldecan-1-amide (CAS No. 14433-76-2). The acute eye irritant effect of N,N-Dimethyldecan-1-amide was investigated according to OECD TG 405 and EC Method B.5 [Hoyer, 1997].Three female albino rabbits were exposed to 0.1 ml of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours as well as 7, 14 and in two animals 21 days after dosing. Moderate to severe signs of eye irritation were observed among the rabbits. The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated: Cornea opacity 1.0; Iris lesion 0.0; Redness of conjunctiva 2.6; Oedema of conjunctiva 2.8.After 14 days all animals showed hairless areas around the eye. Treatment related adverse eye reactions were fully reverse within 14 (1 animal) and 21 days.

Respiratory Irritation

A study for acute inhalation toxicity was conducted on a read-across substance N,N-dimethyldecan-1 -amide, mixture with N,N-dimethyloctan-1 -amide (CAS No. 67359-57-3) in accordance with OECD TG 403 [Pauluhn, 1991]. Five SPF-bred Wistar rats were exposed (head/nose only) to 118.5; 586.4; 2007.6 and 3550.7 mg/m3 (analytical determined).Rats subjected to a concentration of 119 mg/m3 air tolerated the exposure without signs occurring. Rats exposed to 586 mg/m3 air exhibited a transient reddening of the nose on the day of exposure and reduced motility. Rats subjected to the maximum tested concentration (3551 mg/m3 air; nebulisation of the undiluted test article) exhibited persistent signs causally linked to an irritation of the respiratory tract (slower breathing, serous nasal discharge, dyspnea, stridor, hypothermia). 3551 mg/m3 air was the range at which mortality started for male rats (1 of 5 died). The results of this study show that the respirable test article aerosol had relatively low acute inhalation toxicity in the rat. The acute potential hazard of the respiratory tract is attributed to the potency of the test substance aerosol to act as a mucosa irritant. LC50 inhalation (aerosol) Rat (exposure: 4 h) > 3551 mg/m3 air.

It must also be noted that such a high concentration of 3551 mg/m3 (analytically determined) is created by a nominal concentration of 50000 mg/m3

Justification for selection of skin irritation / corrosion endpoint:
Klimisch grade 1 OECD 439 study conducted according to GLP.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Effects on respiratory irritation: irritating

Justification for classification or non-classification

Skin irritation:

The results of the in vitro skin irritation test indicate that a classification of 'Category 2 skin irritant' (DSD Xi, R38) is appropriate.

Eye irritation:

The results of the in vitro eye irritation test indicate that no prediction on classification can be made on the basis of this study alone. However, taking into account the result of the in vivo study on the read-across substance N,N-Dimethyldecan-1-amide (CAS No. 14433-76-2) a classification of 'Category 2 eye irritant' (DSD Xi R36) is appropriate.

Respiratory irritation:

On the basis of the effects seen in the acute inhalation study performed with the read across substance N,N-dimethyldecan-1 -amide, mixture with N,N-dimethyloctan-1 -amide (CAS No. 67359-57-3) a classification of STOT SE3 is appropriate.