Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

Endpoint summary

Description of key information

The substance was found to be non-irritant when tested in in-vitro human skin and rabbit eyes models according to OECD guidelines 439 and 437 respectively.

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:

23 October 2009 --- 9 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiskinTM Model Kit (0.38 cm2 tissues)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: several
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: no data
- Wavelength: 540 nm
- Filter: no data
- Filter bandwidth: no data

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
SCORING SYSTEM:
- Optical density (OD) was measured at 540 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank
- mean cOD Positive Control = mean ODPC - mean ODblank
DECISION CRITERIA
Mean relative viability is = or < 50%: category 2 (H315)
Mean relative viability is > 50% : no category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amount/concentration applied
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 +/- 2 mg
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution):
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution): 5% in distilled water

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells

Number of replicates:

3

Species:

other: in vitro test

Details on test animals or test system and environmental conditions:

The test was conducted in accordance with the Standard Operating Procedure " In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2009)" supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM) and the
OECD Guideline For The Testing of Chemicals Draft proposal for a new guideline In Vitro Skin Irritation (OECD 2008).

The test involves the application of the test substance for 15 minutes to the EPISKIN threedimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm2. The EPISKIN kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.
The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability is determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Time point: 15 min exposure + 42h expression

Value:

117

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

9.6%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Assay validity:

The mean absorbance of the triplicate negative control values was 0.7 which was above the minimum acceptance value of 0.6. The standard deviation (SD) of the % viability was 12.0 which was below the maximum acceptance value of 18.

The mean % viability of the positive control was 9.60 ± 4.10 of the negative control. These were below the maximum acceptance values of 30% viability and SD of 18.

Table 7.3.1/1 : Episkin results

Sample	Tissue viability as percentage of mean OD negative control				Prediction
	Replicate tissues			Mean +/- SD
	a	b	c
Negative control	86.3	109	105	100 +/- 12.0	Not applicable
Positive control	8.00	6.50	14.3	9.60 +/-4.10	Irritant
Test Substance	115	113	123	117 +/- 5.40	Non irritant

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is considered to be non-irritant to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test substance using the EPISKIN^TMreconstructed human epidermis model (in vitro assay). The test procedure was equivalent to the one described in the OECD guideline 439 and the study was performed in compliance with Good Laboratory Practice..

Preliminary tests were performed to detect the ability of the test substance to directly reduce MTT as well as its colouring potential. following these preliminary tests, triplicate tissues (Episkin epidermis surface area of 0.38 cm²) were treated with 10 mg of the test item as a powder for an exposure period of 15 minutes. The tissues were wetted with 5 µL of distilled water prior to application of the test substance. At the end of the exposure period each tissue was rinsed to remove residual test substance before incubating for 42 hours at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the post-exposure incubation period each tissue was taken for MTT-loading (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm with acidified isopropanol solution as a blank.

Negative (sterile Dulbecco's Phosphate Buffered Saline (DPBS) with magnesium and calcium) and positive controls (5% sodium dodecyl sulphate (SDS) in distilled water) were used in the study. The mean absorbance of the triplicate negative control values was 0.7 which was above the minimum acceptance value of 0.6. The standard deviation (SD) of the % viability was 12.0 which was below the maximum acceptance value of 18. The mean % viability of the positive control was 9.60 +/- 4.10 of the negative control. This was below the maximum acceptance values of 30% viability and SD of 18. Therefore the study was considered as valid.

The relative mean viability of the test item treated tissues was 117 +/- 5.40 % after the 15-minute exposure period. As the mean viability was above 50% after MMTT reduction, the result met the criteria for a non-irritant response.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine cornea

Details on test animals or tissues and environmental conditions:

Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the local slaughterhouse.
Age: not provided.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to laboratory: the eyes were transported to the laboratory at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].
Upon arrival at the laboratory, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were rinced in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature.
The eyes were used 4 hours 16minutes after slaughter.

(Pre)Incubation T°C: 32°C

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/w)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): Sigma S8776, Lot batch 128K2337

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

90-min for tPermeability measurement

Number of animals or in vitro replicates:

3 cornea per treatment groups

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the corneas washed, at least three times or until the wash medium (MEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on the top of the holder. The test substance required eight washes.
- Time after start of exposure: 4 hours

SCORING SYSTEM / TOOL USED TO ASSESS SCORE:
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Irritation parameter:

in vitro irritation score

Value:

-0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

133

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

-1

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

90.3

Irritation parameter:

fluorescein leakage

Value:

0.004

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

0.018

Positive controls validity:

valid

Remarks:

2.83

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
No notable opaque spots or irregularities were observed on the three test substance and negative control-treated corneas.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (Negative control: opacity = 0.000 +/- 1.00; permeability = 0.018 +/- 0.008.)
- Acceptance criteria met for positive control: yes ( IVIS = 133 +/- 2.60 The positive control IVIS was within the range of historical mean +/- 2 SD: 112.8 - 164.3).

Table 7.3.2/2: Individual and Mean Corneal Opacity and Permeability Measurements

 

Treatment	Cornea Number	Opacity				Permeability (OD)		In vitro Irritancy Score (IVIS)
		Pre-Treatment	Post-Treatment	Change Post -Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	5	6	1		0.022
	14	5	4	-1		0.024
	16	5	5	0		0.009
	Mean			0.000		0.018
Positive Control	2	6	99	93	93.000	2.475	2.457	129.850
	3	6	100	94	94.000	2.635	2.617	133.250
	4	4	88	84	84.000	3.420	3.402	135.025
	Mean				90.333		2.825	132.7
Test Item	8	6	4	-2	-2.000	0.022	0.004	-1.945
	9	6	8	2	2.000	0.033	0.015	2.220
	10	6	3	-3	-3.000	0.013	-0.005	-3.080
	Mean				-1.000		0.004	-0.9

OD= Optical density

 

Interpretation of results:

GHS criteria not met

Conclusions:

the test substance is considered as not requiring classification for eye irritation or serious eye damage.

Executive summary:

A study was performed to assess the ocular irritancy potential of the test substance to the isolated bovine cornea. The study was conducted according to the OECD guideline No. 437 and in compliance with the principles of Good Laboratory Practice .Negative (Sodium chloride 0.9%) and positive (imidazole purity 99.7%) control items were tested concurrently. 

The test substance was mixed using a mortar and pestle. The 0.9% sodium chloride solution was added to achieve the desired concentration of 20%. Then 750 µL of the diluted test substance was applied on each cornea (3 corneas/treatment condition). The corneas were incubated for 4 hours at 32°C. Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea washed.

The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined to generate an In Vitro Irritancy Score (IVIS). 

To consider the study as valid, the positive control should elicit an In Vitro Score that falls within two standard deviations of the historical mean for the laboratory, which was the case; IVIS of imidazole was found to be 132.7 +/- 2.60. Furthermore, the negative control mean opacity change value should be<2.0 and the permeability mean value<0.1, which was the case as opacity of sodium chloride 0.9% was 0.000 +/- 1.00 and the permeability was 0.018 +/- 0.008.

 

 IVIS of the test item was determined to be -0.90 +/- 2.80, which was below the cut-off value of 3 and thus indicating that the test substance was not requiring classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two in vitro tests were available to assess the skin and eye irritation potential of the test substance.

The skin irritation potential was assessed using the EPISKIN ^TM reconstructed human epidermis model. The test procedure was equivalent to the one described in the OECD guideline 439 and the study was performed in compliance with the principles of Good Laboratory Practice. The test substance and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, tissues were rinsed and incubated for 42 hours at 37°C. The cell viability was then assessed by means of the colourimetric MTT reduction assay. The relative mean viability of the tissues treated with the test substance was 117% with a standard deviation of 5.4%. As the mean relative viability was above 50%, The test substance was therefore considered to be non-irritant to the skin (Hubbard, 2010a).

The non-ocular irritant potential of the registered substance was assessed using a BCOP (Bovine Corneal opacity and permeability) assay. This study was conducted in compliance with OECD Guideline No. 437 and the principles of Good Laboratory Practices. Corneas obtained from freshly slaughtered calves were used as in vitro model. The test substance, and both negative and positive controls were applied in triplicate on corneas and incubated for 4 hours. Before the treatment, a first opacity measurement was performed using an opacitometer.

At the completion of the treatment period, the test item was removed and a second opacity measurement was then performed. After this measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. After incubation, the optical density of the solution from the posterior chamber was measured in order to determine the permeability of the cornea.

The In Vitro Irritancy Score (IVIS) of the test item was -0.90 +/- 2.80. As the test item induced an IVIS below 3, it was considered as not requiring classification for eye irritation or serious eye damage (Hubbard, 2010b).

.

Justification for classification or non-classification

Based on the results from in vitro studies, the substance is not classified as a skin or eye irritant according to regulation EC No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.