Methoxycyclododecane

Endpoint summary

Description of key information

skin irritation (WoE OECD 439, OECD 431, and OECD 402): irritating
eye irritation (OECD 405): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 April - 23 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

July 27, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional subcategory 1B and 1C) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Kit
- Tissue batch number: 23338

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, for exposure of 3 ± 0.5 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 times, no defined volume
- Modifications to validated SOP: There was the following alteration from the MatTek test protocol: Concerning exposure conditions (chapter 3.6.2): Only the 6-well plates for the 60 ± 5 minutes exposure period was placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) during treatment, the wells for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not prove to be a MTT reducer and the test item did not dye water.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (Cat. 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (Cat. 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: Controls for MTT assay

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL (79.4 μL/cm2)

Duration of treatment / exposure:

3 min and 1 h

Duration of post-treatment incubation (if applicable):

3 h MTT incubation, 67.5 h extraction

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

94.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

16.2 %

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure

Value:

91.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

10.7

Remarks on result:

other: on indication of corrosion

Interpretation of results:

other:

Remarks:

not GHS cat. 1

Conclusions:

The test substance was determined to be non-corrosive in the in vitro human skin model test with EpiDerm.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16-20 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 22 July 2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

other: human

Strain:

other: EpiSkin

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: EpiSkin
- Lot No.: 11-EKIN-024

TEST METHOD
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 12-well plates containing prewarmed assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1.5 °C, 5% ± 0.5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5% ± 0.5%
- Humidity: maximum

Type of coverage:

open

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with the vehicle served as negative controls, positive controls were exposed to 5% sodium lauryl sulphate (SLS)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL

Duration of treatment / exposure:

15 ± 1 min at 37°C at 5 ± 0.5%

Observation period:

Not applicable

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with phosphate buffered saline.
- Post-treatment incubation period: 42 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 42 h after the incubation period. Therefore, tissues were incubated in 300 µL prewarmed MTT solution for 3 h at 37 ± 1.5 °C and 5 ± 0.5% CO2. After aspiration of the MTT solution, tissues were washed 3 times in PBS followed by tissue drying. Extraction of the formazan product was carried out in 0.5 mL isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (distilled water). Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

31.7

Remarks on result:

other:

Remarks:

Basis: other: mean values of positive controls (5% SLS). Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

24.9

Remarks on result:

other:

Remarks:

Basis: other: mean values of test item. Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Table 1: Results after treatment

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	0.738	0.751	0.758	0.749	98.6 100.3 101.2	1.3	100.0
Positive Control	15 min	0.248	0.263	0.201	0.237	33.1 35.2 26.8	4.4	31.7
Test item	15 min	0.160	0.248	0.152	0.187	21.3 33.2 20.3	7.1	24.9

* Mean of two replicate well after blank correction

** relative absorbance per tissue [rounded values]: (100 x (absorbance tissue)) / (mean absorbance negative control))

*** relative absorbance per treatment group [rounded values]: (100 x mean absorbance test item) / (mean absorbance negative control)

Interpretation of results:

other: classification according to CLP (category 2 or 1) required.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In the skin irritation study in vitro, the treatment with the test substance resulted in a cell viability of 24.9%. This value is below the threshold for irritancy of <= 50% and thus the test substance requires classification (CLP Cat. 1 or 2).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-09 Nov 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Feb 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Jan 1997

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Kißlegg, Germany
- Weight at study initiation: 2.4 - 3.0 kg
- Housing: during a pre-period of at least one week and throughout the experiment the rabbits were caged individually in PPO cages (floor area: 2576 cm²) with perforated floor.
- Diet: Altromin 2123, Lage, Lippe, Germany, ad libitum
- Water: ad libitum


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

Vehicle:

unchanged (no vehicle)

Controls:

other: The right eye remained untreated and served as control

Amount / concentration applied:

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

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

Reading time points: 1, 24, 48, 72 hours and 7 days after treatment

Number of animals or in vitro replicates:

4 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, the treated eye was rinsed with 20 mL 0.9% sodium chloride solution
- Time after start of exposure: 24 hours

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of 4 animals

Time point:

other: mean after 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 4 animals

Time point:

other: mean after 24, 48 and 72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

of 4 animals

Time point:

other: mean after 24, 48 and 72 h

Score:

1.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 4 animals

Time point:

other: mean after 24, 48 and 72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

One hour after application of the test material one animal showed some conjunctival vessels definitely injected, some swelling above normal and discharge with moistening of the lids and hairs just adjacent to lids. A diffuse, crimson red conjunctiva, individual vessels were not easily discernible, obvious swelling with partial eversion of lids and discharge with moistening of the lids and hairsjust adjacent to lids were seen in three animals.
After 24 and 48 hours 2 animals showed some conjunctival vessels definitely injected. A diffuse, crimson red conjunctiva, individual vessels were not easily discernible, and some swelling above normal were observed in 2 animals.
After 72 hours all animals showed some conjunctival vessels definitely injected.
On Day 7 after application of the test material all animals were free of any signs of eye irritation.

Table: Scores for ocular lesions

Rabbit #	Time	conjunctivae		iris	cornea
		redness	swelling
1	1 h	1	1	0	0
	24 h	1	0	0	0
	48 h	1	0	0	0
	72 h	1	0	0	0
	7 d	0	0	0	0
	average	1.0	0.0	0.0	0.0
2	1 h	2	2	0	0
	24 h	1	0	0	0
	48 h	1	0	0	0
	72 h	1	0	0	0
	7 d	0	0	0	0
	average	1.0	0.0	0.0	0.0
3	1 h	2	2	0	0
	24 h	2	1	0	0
	48 h	2	1	0	0
	72 h	1	0	0	0
	7 d	0	0	0	0
	average	1.67	0.67	0.0	0.0
4	1 h	2	2	0	0
	24 h	2	1	0	0
	48 h	2	1	0	0
	72 h	1	0	0	0
	7 d	0	0	0	0
	average	1.67	0.67	0.0	0.0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the mean scores over 24, 48 and 72 h for conjunctival erythema, chemosis, cornea and iris determined after single ocular instillation of the neat test substance to 4 female rabbits, the test substance is not expected to be irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Hazard assessment is based on the weight of evidence from an in vitro skin irritation study according to OECD 439, the in vitro skin corrosion study and an acute dermal toxicity study according to OECD 402.

Justification for selection of eye irritation endpoint:
There is only one study available.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

In the skin irritation study in vitro, the treatment with the test substance resulted in a cell viability of 24.9%. This value is below the threshold for irritancy of <= 50% and thus the test substance requires classification (CLP Cat. 1 or 2).

In the skin corrosion study in vitro, the treatment with the test substance resulted in a viability of 94.6 % after 3 min and 91.9 % after 1 h incubation, indicating that the test substance has no corrosive effects on skin (not CLP Cat. 1).

In the acute dermal toxicity study in rats, crust formation (as a sign of irritation) was noted in 2/10 animals two and three days after dosing. No other signs of dermal irritation were noted and the scores for erythema and edema recorded daily after patch removal up to 14 days were 0. Thus taking into account the results of the acute dermal toxicity study in the rat for skin reactions, the test substance is found to be non-corrosive.

In conclusion, based on the weight of evidence approach, the test substance is considered irritating and meets the classification criteria as Skin Irrit. 2; H315 according to Regulation (EC) 1272/2008.

The available data on eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.