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Description of key information

Two key studies were completed for irritancy.  Both studies were in-vitro assays (per EC and OECD guidelines and under GLP) with clearly negative results reported.

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:
2014-06-17 to 2014-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study described in this report has been correctly reported and was conducted in compliance with: The Organization for Economic Co-operation and Development (OECD) Principles of Good Laboratory Practice (GLP) (as revised in 1997) ENV/MC/CHEM (98) 17.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes
Species:
other: in vitro human skin model
Details on test animals and environmental conditions:
Human Skin Model
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
15 minutes
Observation period:
42 hour
Number of animals:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls.
Details on study design:
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 (1-10).
The test consists of topical application of Bis-Tes (dried) 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.

The test was performed on a total of 3 tissues per test substance together with negative and positive
controls. Twenty five 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 25 µl PBS (negative control) and 3 tissues
with 25 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 and moved to a new well 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.

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 approximately 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
68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in
duplicate with the TECAN Infinite® M200 Pro Plate Reader.
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.

Remarks on result:
other:
Irritation / corrosion parameter:
other: other: mean relative tissue viability
Value:
ca. 103
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 mins. Remarks: expressed as % . (migrated information)
Irritant / corrosive response data:
Bis-Tes (dried) is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Other effects:
Bis-Tes (dried) was checked for possible direct MTT reduction by adding the test substance to MTT
medium. Because no colour change was observed it was concluded that Bis-Tes (dried) did not
interact with MTT.

The positive control had a mean cell viability after 15 minutes exposure of 35%. The absolute mean
OD570 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 8%, indicating that the test system functioned properly.

Mean tissue viability in the in vitro skin irritation test with Bis-Tes (dried)(expressed as % of control)

negative control 100%

Bis-Tes (dried): 103%

Postive control 35%

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It is concluded that this test is valid and that Bis-Tes (dried) is non-irritant in the in vitro skin
irritation test under the experimental conditions described in this report.
Executive summary:

In vitro skin irritation test with Bis-Tes (dried) using a human skin model.

This report describes the ability of Bis-Tes (dried) to induce skin irritation on a human three

dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation

potential of Bis-Tes (dried) 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 209-3 of Bis-Tes (dried) was a pale yellow liquid. Bis-Tes (dried) was applied undiluted (25 µl)

directly on top of the skin tissue for 15 minutes. After a 42 hour post-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 Bis-Tes (dried) compared to the

negative control tissues was 103%. Since the mean relative tissue viability for Bis-Tes (dried) was

above 50% after 15 minutes treatment Bis-Tes (dried) is considered to be non-irritant.

The positive control had a mean cell viability of 35% after 15 minutes exposure. 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 8%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Bis-Tes (dried) is non-irritant in the in vitro skin

irritation test under the experimental conditions described in this report.

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:
28 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study described in this report has been correctly reported and was conducted in compliance with: The Organization for Economic Co-operation and Development (OECD) Principles of Good Laboratory Practice (GLP) (as revised in 1997) ENV/MC/CHEM (98) 17.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes
Species:
other: bovine cornea
Details on test animals or tissues and environmental conditions:
Bovine eyes were used as soon as possible after slaughter on the same day.

Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's
Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse
employee as soon as possible after slaughter.

Transport: Eyes were collected and transported in physiological saline in a suitable container
under cooled conditions.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 C.
Vehicle:
unchanged (no vehicle)
Controls:
other: positive and negative controls
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µl
Duration of treatment / exposure:
10 ± 1 minutes at 32± 1˚C
Observation period (in vivo):
none
Details on study design:
REMOVAL OF TEST SUBSTANCE
-After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium

SCORING SYSTEM: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

TOOL USED TO ASSESS SCORE: . Opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation
period with sodium fluorescein.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 10 minutes
Score:
ca. 1
Max. score:
2
Reversibility:
fully reversible within: 10 mins
Remarks on result:
other: BCOP
Irritant / corrosive response data:
The corneas treated with Bis-Tes (dried) showed opacity values ranging from 0 to 2 and permeability
values ranging from 0.001 to 0.002. The corneas were clear after the 10 minutes of treatment with BisTes
(dried). No pH effect of the test substance was observed on the rinsing medium. Hence, the in
vitro irritancy scores ranged from 0.0 to 2.0 after 10 minutes of treatment with Bis-Tes (dried).
Other effects:
The negative control responses for opacity and permeability were less than the upper limits of the
laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 126
and was within the historical positive control data range. It was therefore
concluded that the test conditions were adequate and that the test system functioned properly.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It is concluded that this test is valid and that Bis-Tes (dried) is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.
Since Bis-Tes (dried) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

Screening for the eye irritancy potential of Bis-Tes (dried) using the Bovine Corneal Opacity and

Permeability test (BCOP test).

This report describes the ocular irritation properties of Bis-Tes (dried) on an isolated bovine cornea.

The possible ocular irritancy of Bis-Tes (dried) was tested through topical application for

10 ± 1 minutes.

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

guidelines.

Batch 209-3 of Bis-Tes (dried) was a pale yellow liquid. The test substance was applied as it is

(750 µl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the

laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 126

and was within the historical positive control data range. It was therefore concluded that the test

conditions were adequate and that the test system functioned properly.

Bis-Tes (dried) did not induce ocular irritation through both endpoints, resulting in a mean in vitro

irritancy score of 1.0 after 10 minutes of treatment.

Finally, it is concluded that this test is valid and that Bis-Tes (dried) is not irritant or corrosive in the

Bovine Corneal Opacity and Permeability test under the experimental conditions described in this

report.

Since Bis-Tes (dried) induced an IVIS ≤ 3, no classification is required 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

Justification for selection of skin irritation / corrosion endpoint:
IN VITRO SKIN IRRITATION TEST WITH BIS-TES (DRIED) USING A HUMAN SKIN MODEL : OECD 439

Justification for selection of eye irritation endpoint:
SCREENING FOR THE EYE IRRITANCY POTENTIAL OF BIS-TES (DRIED) USING THE BOVINE CORNEAL OPACITY AND PERMEABILITY TEST (BCOP TEST); OECD 437

Justification for classification or non-classification

In vitro skin irritation test with Bis-Tes (dried) using a human skin model. This report describes the ability of Bis-Tes (dried) to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The study procedures described in this report were based on the most recent OECD and ECguidelines. It is concluded that this test is valid and that Bis-Tes (dried) is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Screening for the eye irritancy potential of Bis-Tes (dried) using the Bovine Corneal Opacity and

Permeability test (BCOP test). It is

concluded that this test is valid and that Bis-Tes (dried) is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Since Bis-Tes (dried) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.