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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-18 to 2017-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Oct-7-en-1-ol
EC Number:
236-122-8
EC Name:
Oct-7-en-1-ol
Cas Number:
13175-44-5
Molecular formula:
C8H16O
IUPAC Name:
oct-7-en-1-ol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Chemical name: Oct-7-en-1-ol (7-OEA)
- CAS no.: 13175-44-5
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / OEA-162736NC
- Expiration date of the lot/batch: not stated
- Molecular weight: 128.213 g/mol
- Purity: 94.4%

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from a single donor
Source strain:
other: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue
Details on animal used as source of test system:
TEST KIT
- Source: MatTex Corporation
- Type: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue
- Lot no.: 25765

Cell culture media:
- Assay medium (MatTex Corporation)
- Lot no.: 041217CMHA
- Vehicle / postive controls: deionised water / 8N KOH (Wako Pure Chemical)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C, humidified incubator
- CO2 (%): 5%

IN-LIFE DATES: 18 April 2017 to 19 April 2017
Vehicle:
unchanged (no vehicle)
Details on test system:
Pre-experimental checks:
To check the non-specific MTT-reducing capability of the test article 50 µL of the test article was mixed with 1 mL MTT (1 mg MTT/mL) medium and incubated for 1 hour and observed visually after stirring.

Nylon mesh compatibility was also assessed, using the same methodology as outlined above. The nylon mesh was evaluated microscopically.

Procedure:
- Pre-incubation:
One 6-well plate was prepared for each 3 culture insert and the medium (0.9 mL) was added to each well of the plate and pre-incubated for 60 minutes in a CO2 incubator.

- Skin corrosion test:
3 and 60 minute exposures were conducted. 50 uL of the test article, vehicle (distilled water) and positive (8N KOH) controls were applied onto each tissue surface at 45 second intervals. Duplicate tissues were used for each dose level. After the exposure, a nylon mesh was placed on each tissue surface to spread the substances over the tissue surface. The 45 second interval allowed sufficient time for both application and washing procedures at the end of the exposure.

After the initial dosing of the 60 minute application, the plate was placed into the incubator for the remainder of the exposure period was reached.
At the end of the exposure period tissues were washed with PBS to remove any residual test article. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed into a new 24 well plate filled with 300 uL/well of fresh medium.

Cytotoxicity analysis (MTT):
Following the post-incubation period, the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium (1 mg/mL) and further incubated for 3 h, under the same conditions as previously stated.

After the 3 h MTT incubation period tissues, MTT medium was removed and 2 mL of 2-propanol was added to the tissue inserts to allow formazan extraction from the tissues. Plates were placed into a plastic bag and extraction was performed at room temperature for 2 h or more using a plate shaker.

The 200 uL of the extract were transferred into a 96-well plate and the optical density was measured at 570 nm without reference wavelength in a plate spectrophotometer. 2-propanol was used as a blank.

Cell viability was calculated as follows:
Cell viability (%) = (OD of each tissue / Mean OD of the negative control group) x 100

Tissue binding test was carried using the same procedure as described above, with the exception of medium without MTT was used. After the measuring of OD, the staining ratio was calculated by the following formula:
Staining ratio (%) = (OD of each tissue [without MTT] / Mean OD of the negative control group [without MTT]) x 100
Control samples:
yes, concurrent vehicle
Amount/concentration applied:
50 uL of test article
Duration of treatment / exposure:
single exposure / 3 and 60 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
2 replicates/dose

Test animals

Species:
other:
Details on test animals or test system and environmental conditions:
n/a, in vitro test system used

Test system

Amount / concentration applied:
n/a, in vitro test system used
Duration of treatment / exposure:
n/a, in vitro test system used
Observation period:
n/a, in vitro test system used
Number of animals:
n/a, in vitro test system used
Details on study design:
n/a, in vitro test system used

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
ca. 106
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
no indication of corrosivity
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
ca. 8.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
positive indication of corrosivity
Other effects / acceptance of results:
refer to "Any other information on results incl. tables"

Any other information on results incl. tables

Formulation analysis:

None conducted.

 

Pre-experiment:

The mixture of test article and MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.

 

The mixture of the test article and aqua destain showed no colouring detectable by unaided eye-assessment

 

Skin corrosion:

The test article showed corrosivity effects during the prolonged exposure (60 minutes) following STEP 1 analysis. The mean relative tissue viability was >50% (106%) after the 3 minute exposure, but <15% (8.6%) after the 60 minute treatment (+ 42 hour post-incubation). In accordance with STEP 2 of the judgement criteria, as tissue viability was <25% following the 3 minute exposure, 7-Octen-1-ol is deemed to be corrosive to skin, Category 1A.

 

The positive control viability was <15% (1.6%) after the 60 minute treatment, following a 42 hour post-incubation therefore confirming that corrosivity was detected with the test system.

 

The controls confirmed the validity of the study. The mean OD550for the 3 minute and 60 minute negative control groups were 1.725 and 1.799, respectively.

 

Table CA 7.3.1/02-1: Summary of in vitro EPIDERM corrosivity result following application of Oct-7-en-1-ol

Parameter

Negative control

Oct-7-en-1-ol

Positive control

3 minute exposure

OD 570 nm

1.725

1.820

0.148

Mean relative tissue viability (%) ±CV

100 ±6.5

106 ±1.1

8.6 ±12.3

60 minute exposure

OD 570 nm

1.799

0.154

0.028

Mean relative tissue viability (%) ±CV

100 ±1.3

8.6 ±18.9

1.6 ±0.0

 

Deficiencies:

None.

 

Conclusion

Under the conditions of this study the Oct-7-en-1-ol showed corrosion effects. The mean relative tissue viability was>50% (106%) after the 3 minute exposure, but <15% (8.6%) after the 60 minute treatment (+42 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, 7-Octen-1-ol is classified as Skin corrosion, Category 1A.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
Under the conditions of this study the Oct-7-en-1-ol showed corrosion effects. The mean relative tissue viability was >50% (106%) after the 3 minute exposure, but <15% (8.6%) after the 60 minute treatment (+42 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-en-1-ol is classified as Skin corrosion, Category 1B.
Executive summary:

The potential of the test article to induce skin corrosion was analysed using the three-dimensional human skin model EPIDERMTM comprising a reconstructed epidermis with a functional stratum corneum. In the present study 7-Octen-1-ol was applied topically to the EPIDERMTM tissue for 3 and 60 minutes followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay. Corrosivity potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with distilled water. The positive control, 8N potassium hydroxide viability was <20% following a 60 minute exposure, thereby confirming that corrosivity could be detected in this test system. The test article showed corrosivity effects during the prolonged exposure (60 minutes). The mean relative tissue viability was >50% (106%) after the 3 minute exposure, but <15% (8.6%) after the 60 minute treatment (+ 42 hour post-incubation). In accordance with STEP 2 of the judgement criteria, as tissue viability was <25% following the 3 minute exposure, 7-Octen-1-ol is deemed to be corrosive to skin, Category 1A. Under the conditions of this study the Oct-7-en-1-ol showed corrosion effects. The mean relative tissue viability was>50% (106%) after the 3 minute exposure, but <15% (8.6%) after the 60 minute treatment, following a 42 hour post-incubation. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-en-1-ol is classified as Skin corrosion, Category 1B.