Reaction mass of (Z)-1-chloro-1-propene and 2-chloropropane and 2-chloropropene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to relevant recognized method

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Draft Proposal for a new Guideline: In Vitro Skin Irritation: REconstructed Human Epidermitis (RhE) Test Method

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission regulation (EC) N° 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 "In Vitro Skin Irritation: Reconstructed Human Epidermitis Model Test"

Deviations:

no

Principles of method if other than guideline:

The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test substance by assessment of its effect on a three dimensional human epidermis model.

The test consists of topical application of Reaction mass containing mainly 2-chloropropene on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test substance and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-chloropropene

EC Number:

209-187-5

EC Name:

2-chloropropene

Cas Number:

557-98-2

Molecular formula:

C3H5Cl

IUPAC Name:

2-chloroprop-1-ene

Constituent 2

Reference substance name:

2-chloropropane

EC Number:

200-858-8

EC Name:

2-chloropropane

Cas Number:

75-29-6

Molecular formula:

C3H7Cl

IUPAC Name:

2-chloropropane

Constituent 3

Reference substance name:

1-chloropropene

EC Number:

209-675-8

EC Name:

1-chloropropene

Cas Number:

590-21-6

IUPAC Name:

1-chloroprop-1-ene

Details on test material:

Identification Reaction mass containing mainly 2-chloropropene
Molecular formula C3H5Cl
Molecular weight 76.53
CAS Number 557-98-2
Description Clear light yellow to brown liquid (determined at NOTOX)
Batch RBA100301A
Purity Minimum 60.0%
Test substance storage In refrigerator (2-8°C) in the dark
Stability under storage conditions Stable
Expiry date 08 March 2011 (allocated by NOTOX, 1 year after receipt of the test substance)

Study specific test substance information:
General information Extremely flammable
Test substance handling If necessary, handling after sampling will be performed on dry ice in order to keep the test substance liquefied

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

Test system:
EPISKIN Standard ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 10-EKIN-020).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Source
SkinEthic Laboratories, Nice, France.

Environmental conditions:
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 76 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.7- 36.0°C) and humidity (with a maximum of 4%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Ten µl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 µl PBS (negative control) and 3 tissues with 10 µl 5% SDS (positive control) respectively. The positive control was
re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Duration of treatment / exposure:

Exposure of 15 minutes

Observation period:

Incubation for 42 hours

Details on study design:

Test for reduction of MTT by the test substance:
Reaction mass containing mainly 2-chloropropene has been tested previously for possible direct MTT reduction in the Skin corrosion test using EpiDerm as a skin model (NOTOX project 492886). The MTT solution colour was not turned blue / purple and a blue / purple precipitate was not observed. Reaction mass containing mainly 2-chloropropene did not interact with MTT.

Cell viability measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol. Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan Spectrum (Thermo Labsystems).

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme(s):
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity.
Multiskan spectrum version 1.00 (Thermo labsystems, Breda, The Netherlands) for optical density measurement.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: cell viability

Value:

84

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Remarks: Score correspond to the relative mean tissue viability compared to the negative control tissues (%). (migrated information)

In vivo

Irritant / corrosive response data:

The mean absorption at 570 nm measured after treatment with Reaction mass containing mainly 2-chloropropene and controls are presented in Table 1.

Table 2 shows the mean tissue viability obtained after 15 minutes treatment with Reaction mass containing mainly 2-chloropropene compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Reaction mass containing mainly 2-chloropropene compared to the negative control tissues was 84%. Since the mean relative tissue viability for Reaction mass containing mainly 2-chloropropene was above 50% Reaction mass containing mainly 2-chloropropene is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 7%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix II). The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table1           Mean absorption in thein vitroskin irritation test with Reaction mass containing mainly 2-chloropropene

 

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.889	0.879	0.883	0.884	±	0.005
Test substance	0.765	0.745	0.727	0.746	±	0.019
Positive control	0.053	0.062	0.068	0.061	±	0.008

 

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Test substance = Reaction mass containing mainly 2-chloropropene

 

In this table the values are corrected for background absorption. Isopropanol was used to measure the background absorption.

 

Table2           Mean tissue viability in thein vitro skin irritation test with Reaction mass containing mainly 2-chloropropene

 

	Mean tissue viability (percentage of control)
Negative control	100
Reaction mass containing mainly 2-chloropropene	84
Positive control	7

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other:

Conclusions:

Finally, it is concluded that this test is valid and that Reaction mass containing mainly 2-chloropropene is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

This report describes the ability of Reaction mass containing mainly 2-chloropropene to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM^TM)). The possible skin irritation potential of Reaction mass containing mainly 2-chloropropene was tested through topical application for 15 minutes.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch RBA100301A of Reaction mass containing mainly 2-chloropropene was a clear light yellow to brown liquid with a purity of 60.0% (minimum). Reaction mass containing mainly 2-chloropropene was applied undiluted (10 µl) directly on top of the skin tissue. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Reaction mass containing mainly 2-chloropropene compared to the negative control tissues was 84%. Since the mean relative tissue viability for Reaction mass containing mainly 2-chloropropene was above 50% after 15 minutes treatment Reaction mass containing mainly 2-chloropropene is considered to be non-irritant.

 

The positive control had a mean cell viability after 15 minutes exposure of 7%. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

 

Finally, it is concluded that this test is valid and that Reaction mass containing mainly 2-chloropropene is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.