3,7-dimethylocta-1,6-diene

Endpoint summary

Description of key information

- Skin irritation: Irritating (In vitro episkin, Kr: 1)
- Eye irritation: Not irritating (In vitro human corneal epithelium, Kr: 1); not corrosive (BCOP test, Kr. 1)

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:

from June 22 to July 27, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well conducted and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

other: ECVAM protocol version 1.8 of february 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or test system and environmental conditions:

Three-dimensional human epidermis model, supplied by SkinEthic Laboratories, Nice, France constituted by:
- a collagen type I matrix, coated with type IV collagen
- a differentiated and stratified epidermis model from human keratinocytes, obtained after 13-day culture period.
All biological components of the epidermis and the kit culture medium have been tested for the absence of viruses, bacteria and mycoplasma

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

Duration of treatment / exposure:

15 ± 0.5 min

Number of animals:

3 epidermis/product

Details on study design:

Before the beginning of the study, the non-specific MTT reduction by the test item was checked by incubating MTT solution and 10 µL test item for 3 hours ± 30 minutes and checking the colour of the solution.

Application of the test item and control:
- negative control: 10 µL of PBS+ to each epidermis surface
- positive control: 10 µL of SDS solution at 5% (w/v) in distilled water; after 7 minutes contact time, an intermediate re-spreading is done
- test substance: 10 µL of the test item, as supplied
- contact timepoint for all products: 15 ± 0.5 minutes at room temperature

Rinsing:
- epidermis rinsed thoroughly with 25 mL of PBS+
- remaining product removed with a cotton-bud

Incubation: plates incubated during 42 ± 1 hours in CO2 incubator with maintenance medium

MTT assay:
- epidermis incubated for 3 hours ± 5 minutes in MTT solution in the CO2 incubator
- formazan extraction in 500 µL of acidic isopropanol stored at 4 hours ± 30 minutes at room temperature, protected from light or during 70 ± 5 hours at 4 °C
- measurement of OD at 570 nm

Irritation / corrosion parameter:

other: other: viability % (MTT assay)

Value:

40.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 min. Reversibility: no data. Remarks: ± 5.2. (migrated information)

Irritant / corrosive response data:

No data

Other effects:

No data

Negative control (PBS+): mean OD = 0.821

Positive control (5% SDS): % viability (MTT assay) = 18.7 ± 3.0

Test item: % viability (MTT assay) = 40.9 ± 5.2

Table 1: MTT conversion assay in living epidermis

		OD 1	OD 2	Mean	Standard deviation	Viability %	Mean Viability %	Standard deviation
Negative control	Epidermis 1	0.768	0.808	0.788	0.028	96.0	100	0.044
	Epidermis 2	0.855	0.864	0.860	0.006	104.7
	Epidermis 3	0.819	0.811	0.815	0.006	99.3
Positive control	Epidermis 1	0.124	0.145	0.135	0.015	16.4	18.7	0.030
	Epidermis 2	0.158	0.131	0.145	0.019	17.6
	Epidermis 3	0.191	0.172	0.182	0.013	22.1
Test item	Epidermis 1	0.417	0.352	0.385	0.046	46.8	40.9	0.052
	Epidermis 2	0.325	0.309	0.317	0.011	38.6
	Epidermis 3	0.303	0.309	0.306	0.004	37.3

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, dimethyloctadiene is classified as irritating to the skin in category 2 (H315) according to the CLP Regulation (EC) n° 1272/2008 and as irritating to skin (Xi; R 38) according to the criteria of Annex VI of Directive 67/548/EEC.

Executive summary:

A GLP study conducted in vitro with human epidermis model EPISKIN was performed to assess the irritancy potential of dimethyloctadiene similarly to ECVAM protocol version 1.8 of February 2009.

10 µL of test item was applied directly on epidermis for 15 min on 3 epidermis. Positive control was 10 µL of 5% (w/v) SDS solution and negative control was 10 µL of PBS (each tested on 3 epidermis). MTT conversion assay was peformed to evaluate the percentage of cellular viability of the epidermis.

Positive control had a percentage of viability of 18.7 ± 3.0 and test item 40.9 ± 5.2.

For Dimethyloctadiene, as the percentage of viability was ≤ 50 %, it is considered to be irritating.

Under the test conditions, dimethyloctadiene is classified as irritating to the skin in category 2 (H315) according to the CLP Regulation (EC) n° 1272/2008 and as irritating to skin (Xi; R 38) according to the criteria of Annex VI of Directive 67/548/EEC.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 2010-05-28 to 2010-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well conducted and in compliance with GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD guideline N° 437: Bovine Corneal Opacity and Permeability test method for identifying ocular corrosives and severe irritants

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

other: calf

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes were collected at the slaughter houses of la Talaudière - France
- Age at study initiation: bovine eyes from cattle less than 12 months
- carried in a stable medium (Hanks medium buffered with sodium bicarbonate), at room temperature, and prepared 4 hours maximum after killing the animals

Vehicle:

unchanged (no vehicle)

Remarks:

dimethyloctadiene is tested at 100% (undiluted sample).

Controls:

other: Negative control: 3 eyes treated with sodium chloride at 0.9% (W/V); Positive control: 3 eyes treated with ethanol at 100%.

Amount / concentration applied:

750 µL, undiluted

Duration of treatment / exposure:

10 ± 1 min

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 eyes for each product (test sample, negative and positive controls)

Details on study design:

After the application of the test item on the cornea, the eye was rinsed at least 3 times with nutritive medium with phenol red at 32 ± 1°C.

Irritation parameter:

other: In vitro irritancy score (IVIS)

Run / experiment:

10 min

Value:

ca. 1.1

Remarks on result:

other: SD= +- 0.6

Other effects / acceptance of results:

See table 1

The results of the positive control are in conformity with historical data (within two standard deviations of the historical mean obtained from 31 March to 28 May 2010: 35.7 -67.5).

The results of the opacity and the OD of the negative control are in conformity with criteria of validity of the test.

The results of the OD of fluorescein are in conformity with the criteria of validity of the test.

Results for test item and positive control were corrected by subtracting the values obtained with the negative control, and the in vitro irritancy score IVIS was calculated from the corrected values: IVIS = (OPT2-OPT0)c + 15DOc

Table 1: Results of the BCOP test

Holder N°	Treatment	OPT0	OPT2	OPT2- OPT0	Corrected opacity	DO	Corrected DO	Score OPc+15DOc
70	Negative control	3	3	0	-	0.021	-	-
11		5	5	0	-	0.018	-	-
14		7	7	0	-	0.020	-	-
Mean					-	0.020	-	-
Standard deviation					-	0.002	-	-
67	Ethanol (positive Control)	3	19	16	16.0	2.712	2.692	56.4
15		5	23	18	18.0	2.664	2.644	57.7
66		7	21	16	16.0	2.748	2.728	56.9
Mean					16.7	-	2.688	57.0
Standard deviation					1.2	-	0.042	0.6
55	Dimethyloctadiene	3	4	1	1.0	0.051	0.031	1.5
21		4	5	1	1.0	0.046	0.026	1.4
34		5	5	0	0.0	0.044	0.024	0.4
Mean					0.7	-	0.027	1.1
Standard deviation					0.6	-	0.004	0.6

OPT0: opacity before treatment

OPT2: opacity 2 hours after rinsing

Interpretation of results:

other: IVIS < 55.1: not corrosive or severly irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, Dimethyloctadiene is not classified as corrosive or severly irritant for the isolated bovine cornea, after 10 minutes of contact.

Executive summary:

In an in vitro eye corrosivity and severe irritancy test (BCOP test for Bovine Corneal Opacity and Permeability), performed according to the OECD guideline N°437 and in compliance with GLP, 3 bovine cornea were treated with undiluted Dimethyloctadiene. The test substance was in contact with cornea for 10 minutes and then the eyes were rinsed at least 3 times. Corneal opacity was measured thanks to an opacitometer 2 hours after the treatment. The corneal permeability was quantified by the measurement of the optical density 90 min after the addition of a fluorescein solution to the treated cornea.

Negative control (sodium chloride solution) and positive control (Ethanol) were also used and the results were acceptable for these controls.

The in vitro irritancy score (IVIS) for Dimethyloctadiene was 1.1 ± 0.6. Therefore, Dimethyloctadiene is not classified as corrosive or severly irritant to eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 July 2010 to 17 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well conducted and in compliance with GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Evaluation of the primary ocular irritation of Dimethyloctadiene after application on a human reconstituted corneal epithelium model and quantification of cellular viability by reduction test of MTT (Mosmann, T. 1983) and T50 determination, exposure time that causes 50% of mortality. This reconstituted corneal epithelium model could be consider as the most similar to the human corneal because it is a 3D model.

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Remarks:

dimethyloctadiene is tested at 100% (undiluted sample).

Controls:

other: negative control= untreated epithelium (epithelium in maintenance medium); positive control= Sodium Dodecyl Sulfate (CAS 151-21-3) at 0.5% (W/W) in sterile water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): the test item is applied as supplied (ie. undiluted)

VEHICLE: not applicable

Duration of treatment / exposure:

test item: 10 min, 60 and 180 min
Positive control: 10 and 60 min
Negative control: 180 min

Observation period (in vivo):

at the end of each incubation, the MTT test was performed immediately.

Number of animals or in vitro replicates:

all conditions (test item, negative control and positive control) were performed in duplicate

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of each incubation period rinsing of the epithelia under a jet of PBS (using a washing bottle), by possibly removing the excess product adhering to epithelium using a cotton-wool bud.
- Time after start of exposure: at the end of exposure time (10, 60, or 180 min)

SCORING SYSTEM: not applicable

TOOL USED TO ASSESS SCORE: not applicable

Irritation parameter:

other: T50

Value:

>= 180

None

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, Dimethyloctadiene is not classified as irritating to eyes.

Executive summary:

In an in vitro ocular primary irritation test, performed in compliance with GLP, human reconstructed corneal epithelium was treated with undiluted Dimethyloctadiene. The test substance was in contact with corneal epithelium for 10, 60 or 180 minutes and then the epithelia were rinsed.

MTT conversion assay was peformed to evaluate the percentage of cellular viability of the epithelium in order to caculate the T50, which is the time when 50% of the cells are dead.

Negative control (maintenance medium) and positive control (Sodium dodecyl sulfate) were also used and the results were acceptable for these controls.

The T50 for Dimethyloctadiene calculated from 3 contact time points was higher than 180 minutes. Therefore, under the test conditions, Dimethyloctadiene is not classified as irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

An in vitro study was conducted with the human epidermis model EPISKIN and identified as the Key study (Maillet, Episkin, 2010). This GLP study was performed to assess the irritancy potential of dimethyloctadiene similarly to ECVAM protocol version 1.8 of February 2009.

10 µL of test item was applied directly on epidermis for 15 min on 3 epidermis. Positive control was 10 µL of 5% (w/v) SDS solution and negative control was 10 µL of PBS (each tested on 3 epidermis). MTT conversion assay was peformed to evaluate the percentage of cellular viability of the epidermis. Positive control had a percentage of viability of 18.7 ± 3.0 and test item 40.9 ± 5.2. As the percentage of cell viability was ≤ 50 %, the test item is considered to be irritating.

Eye irritation:

In an in vitro eye corrosivity and severe irritancy test (BCOP test for Bovine Corneal Opacity and Permeability), performed according to the OECD guideline N°437 and in compliance with GLP, dimethyloctadiene was applied for 10 minutes to the epithelial surface of 3 bovine cornea, mounted in specially designed corneal holders. After rinsing and 2-h incubation, the corneal opacity and permeability were determined. They were combined to calculate thein vitroirritancy score (IVIS). IVIS for dimethyloctadiene was 1.1±0.6. Opacity and permeability values of the negative control (sodium chloride at 0.9%) as IVIS of the positive control (ethanol at 100% – 57±0.6) were in conformity with the criteria of validity of the test. As IVIS was < 55.1, dimethyloctadiene is not classified as corrosive or severe irritant.

An in vitro study was conducted and identified as the Key study (Maillet, Human reconstructed Corneal epithelium, 2010). The undiluted test substance was in contact with corneal epithelium for 10, 60 or 180 minutes and MTT conversion assay was peformed to evaluate the percentage of cellular viability of the epitheliumin order to caculate the T50, which is the time when 50% of the cells are dead. As the T50 calculated for dimethyloctadiene was higher than 180 minutes, it is not considered as irritating to eyes.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

As the percentage of cell viability foun in an in vitro skin irritation test using EPISKIN model is ≤ 50 %, dimethyloctadiene is classified as irritating to skin in category 2 (H315) according to the CLP Regulation (EC) N° (1272-2008) and as irritating to skin (Xi; R 38) according to the criteria of Annex VI to the Directive 67/548/EEC.

As dimethyloctadiene was not found corrosive in a BCOP test and had a T50 value higher than 180 min in an in vitro human recontrusted corneal epithelium test, it is not classified as irritating to eyes according to the Annex VI of Directive 67/548/EEC and according to the CLP Regulation (EC) n° 1272/2008.