Pregna-1,4-diene-3,20-dione, 9-bromo-6-fluoro-11-hydroxy-16-methyl-21-[(1-oxopentyl)oxy]-, (6.alpha.,11.beta.,16.alpha.)-

Endpoint summary

Description of key information

Bromhydrin-valerate is neither corrosive nor irritant to the skin in tests using reconstructed human epidermis (Wingenroth, 2017a+b). In vitro studies with bromhydrin-valerate reveal no potential to serious eye damage (BCOP - Gmelin, 2016) or to irritant effects to the eyes (EpiOcular - Leidenfrost, 2017).

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:

key study

Study period:

Aug 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: Human Skin Model Test)

Version / remarks:

26 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Bromhydrinvalerat
- Lot/batch No.: UN100EK
- Purity: 96.9 %
- Expiry date: 26 Dec 2016

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA:
- Corrosivity potential of test materials is predicted from the cell viabilities obtained after 3 min and 60 min treatment compared to the negative control. A chemical is classified "corrosive" (sub-category 1A) if the cell viability after 3 min treatment is decreased by more than 50 %. If cell viability after 3 min exposure is ≥ 50 %, while it is below 15 % after 60 min exposure the substance is also classified as corrosive, but sub-category 1 B/1 C. If cell viability after 3 min exposure is ≥ 50 % and after 60 min exposure ≥ 15 %, the substance is classified as non-corrosive.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Reliability of the test was previously confirmed by interlaboratory validation


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure of the test item the inserts were washed carefully in PBS.
- Observable damage in the tissue due to washing: Not reported.


NUMBER OF REPLICATE TISSUES: triplicates

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (plus 50 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 8N KOH

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

cell viability after 3 min [%]

Value:

108.61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

cell viability after 60 min [%]

Value:

104.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Tabular summary of the results   

Sample No.	Test item	Time (min)	OD mean *	Std Dev	% Viability
1 - 3	Negative control NaCl 0.9 %	60	2.32	0.07	100.00
4 - 6	Positive control 8N KOH	60	0.03	---	1.48
7 - 9	Bromhydrinvalerat	60	2.42	0.06	104.30
10 - 12	Negative control NaCl 0.9 %	3	2.32	0.05	100.00
13 - 15	Bromhydrinvalerat	3	2.52	0.04	108.61

* 6 values

Interpretation of results:

GHS criteria not met

Conclusions:

Bromhydrinvalerat (100% a.i.) was not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion) (adopted July 26, 2016), Bromhydrinvalerat (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 and 60 minutes in triplicates. 50 μL of 0.9% NaCl were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 25 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

 

After 3 and 60 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Cytotoxicity (Corrosivity) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

The positive (8NKOH) and negative (0.9% NaCl) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

 

The relative mean tissue viability obtained after 3 minutes treatment with Bromhydrinvalerat compared to the negative control tissues was 108.61% and after 60 minutes 104.30%. Since the mean relative tissue viabilities for the test substance were above 50%, Bromhydrinvalerat is identified to be not corrosive.

 

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

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:

commercially available test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany)
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION
- The optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA
- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

cell viability after 20 min [%]

Value:

106.24

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

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

Table 1: Tabular summary of the results    

Sample No.	Test item	OD mean *	Std Dev	% Viability
1 - 3	Negative control NaCl 0.9 %	1.95	0.05	100.00
4 - 6	Positive control SDS 5 %	0.04	0.03	2.01
7 - 9	Bromhydrinvalerat	2.08	0.07	106.24

* 6 values

Interpretation of results:

GHS criteria not met

Conclusions:

A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439. After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 106.24 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, bromhydrinvalerat is considered to have no skin irritation category as defined in the UN GHS.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), Bromhydrinvalerat (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 20 minutes in triplicates. 30 μL of 0.9% NaCl were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 30 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

 

After 20 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

The positive (5% SDS) and negative (0.9% NaCl) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

 

The relative mean tissue viability obtained after 20 minutes treatment with Bromhydrinvalerat compared to the negative control tissues was 106.24%. Since the mean relative tissue viability for the test substance was above 50%, Bromhydrinvalerat is identified to be not irritating.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 2016

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

Deviations:

yes

Remarks:

analytical determination of stability and homogeneity of the test item in the vehicle was not performed

GLP compliance:

yes (incl. QA statement)

Species:

other: isolated cornea from eyes of slaughtered cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Extraction: Staff of the slaughterhouse
- Transport: 1L containers with 500 mL HSS and 1 % penicillin/streptomycin solution; transport of the containers in coolers on ice
- Time interval prior to initiating testing: 1 day
- Indication of any existing defects or lesions in ocular tissue samples: Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
- Indication of any antibiotics used: 1 % penicillin / streptomycin solution
- Selection and preparation of corneas: For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed in a cornea holder with the endothelial side on the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C). Following 1 hour in the incubator, the MEM medium was aspirated and the chambers were refilled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± standard deviation were selected for the actual test and assigned to the test groups.
The numbers of the corneas selected were documented in an appropriate protocol. The holders were labeled with a new serial number for the further testing.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

750 μL per cornea

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

- PREPARATION OF CORNEAS: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin I streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded. On the next day (day of experiment) the containers with the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours. For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 %penicillin I streptomycin solution and 1 % FBS. Each cornea was placed in a cornea holder with the endothelial side on the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C).
- SELECTION OF CORNEAS FOR APPLICATION: Following 1 hour in the incubator, the MEM medium was aspirated and the chambers were refilled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± standard deviation were selected for the actual test and assigned to the test groups. The numbers of the corneas selected were documented in an appropriate protocol. The holders were labeled with a new serial number for the further testing.
- APPLICATION OF THE TEST MATERIAL AND INCUBATION: Immediately before application, the medium was aspirated from the anterior chamber. 750 µL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently. The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 4 hours. After the exposure, the formulations were aspirated from the anterior chamber. The corneas treated with the vehicle control, the negative and the positive control were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. The corneas treated with the test item were rinsed at first with com oil and then also at least 3 times with phenol red containing MEM. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers.
- DETERMINATION OF OPACITY: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). The validation of the opacitometer was carried out under the study number T8082017. Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- DETERMINATION OF PERMEABILITY: The medium in anterior chamber of each holder was replaced by 1 ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 oc (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA- Reader (Bio-Tek EL 808, Software Gen5).
- CALCULATION AND EVALUATION OF IN VITRO IRRITANCY SCORE (IVIS): All parameters and the IVIS values were calculated by using Microsoft Excel. The validation of the Excel file was carried out under the study number T0082019.
The opacity values were calculated by applying the following formulae:
1) Opacity= (Io/I-0.9894)/0.0251
2) Opacity change = opacity after application - opacity before application
3) Corrected opacity change= opacity change- mean opacity change NC
4) Mean opacity = mean of all corrected opacity changes per group
The permeability values were calculated by applying the following formulae:
1) OD49o change OD490 value - mean blank value OD490
2) Corrected OD490 change= OD490 change- mean OD490 change NC
3) Mean OD490 =mean of all corrected OD490 changes per group
Calculation of In Vitro Irritancy Score (IVIS):
1) IVIS per cornea= corrected opacity change+ (15 x corrected OD490 change)
2) IVIS per group mean of IVIS values per cornea in a group

Io = single value of the measurement of empty holder with medium but without cornea, measured l-2days before the experiment;
I = individual value of each opacity measurement before and after application 0.9894/0.0251 is a constant, which is required for calculation.

- DECISION CRITERIA: The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
IVIS <= 3 (No category), IVIS > 3 - <= 55 (No prediction can be made / No Category 1) or IVIS > 55 (Category 1)

Irritation parameter:

in vitro irritation score

Run / experiment:

4 hrs

Value:

8.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Tabular in vitro irritancy scores (IVIS)

	Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group mean	SD
Vehicle control	1	- 2.1	0.008	- 1.9	- 2.0	1.0
(0.9 % NaCl)	2	- 1.1	0.007	- 1.0
	3	- 3.1	0.007	- 3.0
Positive control	4	83.3	1.629	107.7	94.1	13.5
(20 % Imidazole)	5	65.2	1.028	80.7
	6	77.4	1.107	94.0
Test item	7	16.6	0.001	16.6	8.4	7.1
(20 % Bromhydrinvalerat)	8	4.1	0.001	4.1
	9	4.6	0.000	4.6

No potential for serious eye damage was concluded from the study, as the IVIS was below 55 for the test item.

Interpretation of results:

study cannot be used for classification

Remarks:

Based on OECD TG 437 and the experimental conditions reported the test item cannot be classified.

Conclusions:

Bromhydrinvalerat was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. The epithelial surface of the corneas was exposed to 750 µL of the test substance formulated in physiological saline. Measurement of corneal opacity and permeability after a 4 hours exposure followed by a post-treatment incubation of 90 minutes revealed an in vitro irritation score (IVIS) of 8.4, well below the threshold for classification of serious eye damage (IVIS <= 55). The positive (20 % imidazole) and vehicle (physiological saline solution) controls confirmed the validity of the test. Thus, under the conditions of this test bromhydrinvalerat was characterized by having no potential to seriously damage the eye.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Bromhydrinvalerat (20% a.i.) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013.

 

The corneae were incubated with the test substance and controls for 4h at 32°C ± 1°C. After rinsing with phenol red containing MEM. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

 

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 8.4.

 

The positive control (20% imidazole) increased the opacity and permeability of the corneae (mean in vitro score 94.1) corresponding to a classification as corrosive to the eye. With the negative control (0.9% NaCl) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score -2.0).

 

Since the mean in vitro irritancy score of the test substance was > 3, the eye irritation potential of Bromhydrinvalerat cannot be determined in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

The outcome of the test is “No prediction can be made.”

 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human cornea-like epithelium (RhCE)

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHOD AND CONSIDERATIONS REGARDING APPLICABILITY :
The EpiOcular™ eye irritation test (EIT) follows international OECD test guidelines. The EIT measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation).

DESCRIPTION OF THE CELL SYSTEM USED, INCL. CERTIFICATE OF AUTHENTICITY AND THE MYCOPLASMA STATUS OF THE CELL LINE :
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The model is standardized and commercially available. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier (MatTek Corporation, Slovakia).

ENVIRONMENTAL CONDITIONS :
The environmental conditions in the incubator were standardized as follows:
Incubator temperature: 37 +/- 2° C
CO2 gas concentration: 5 %
Humidity: maximum
All incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode, Germany).


- RhCE tissue or hCE cell construct used, including batch number: The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s
medium (Part#: OCL-200, OCL -212; Lot No.: 23746; Testing date: 08 Nov 2016).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg per tissue insert in duplicate
- Concentration (if solution): neat test item

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 μL methyl acetate per tissue insert in duplicate
- Concentration (if solution): neat
- Lot/batch no. (if required): 091916MHB
- Purity: 99.0 %

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

tissue inserts were used in duplicate for test item, negative and positive control

Details on study design:

RhCE TISSUE CONSTRUCT USED, INCLUDING BATCH NUMBER :
EpiOcular RhCE tissue supplied by MatTek Corporation, Lot No. 23746

PRE-CHECK FOR POTENTIAL OPTICAL INTERFERENCES OF THE TEST ITEM :
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control must be conducted.

PROCEDURE FOR KILLED CONTROL OR COLOR CONTROL :
Killed control and color control not required.

DESCRIPTION OF THE METHOD USED TO QUANTIFY MTT FORMAZAN :
For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C). The tissues were incubated for 180 ± 10 min. under standard culture conditions. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 μl). Data acquisition and evaluation were performed with the software "Gen5" (Bio-Tek).

ASSAY ACCEPTANCE CRITERIA :
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Irritation parameter:

mean percent tissue viability 

Value:

100.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS :
- Visible damage on test system: none

ACCEPTANCE OF RESULTS :
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

According to the conducted pre-check the test item was demonstrated to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore a killed control and color control had to be conducted.

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro study for assessing ocular irritation properties of the test item bromhydrinvalerat was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %. The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. The final mean percent tissue viability recorded for the test item is 100.2 %. According to the results of this study bromhydrinvalerat was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Executive summary:

An in vitro study for assessing ocular irritation properties of the test item bromhydrinvalerat was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular^TM. The EpiOcular^TM 

In this study conducted according to OECD test guideline 492 (adopted July 28, 2015), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular™, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

The test item was applied topically to the EpiOcular™ tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After an 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

 

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

 

The test item showed no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol thus, killed control and color control were not required.

 

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was 100.2%.

 

The controls confirmed the validity of the study. The mean relative tissue viability (% negative control) of the positive control was < 50% (21.1%).

In this study under the given conditions the test item showed no irritant effects.

According to the results of this study bromhydrinvalerat was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation

In a dermal irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion) (adopted July 26, 2016), Bromhydrinvalerat (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 and 60 minutes in triplicates. 50 μL of 0.9% NaCl were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 25 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 3 and 60 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Cytotoxicity (Corrosivity) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (8NKOH) and negative (0.9% NaCl) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 3 minutes treatment with Bromhydrin-valerat compared to the negative control tissues was 108.61% and after 60 minutes 104.30%. Since the mean relative tissue viabilities for the test substance were above 50%, Bromhydrin-valerat is identified to be not corrosive.

 

To exclude a dermal irritation potential a dermal irritation study was performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015). Bromhydrin-valerat (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 20 minutes in triplicates. 30 μL of 0.9% NaCl were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 30 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 20 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (0.9% NaCl) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 20 minutes treatment with Bromhydrin-valerat compared to the negative control tissues was 106.24%. Since the mean relative tissue viability for the test substance was above 50%, Bromhydrin-valerat is identified to be not irritating.

 

Eye corrosion/irritation

The presented in vitro study was performed to assess the corneal irritation and damage potential of Bromhydrin-valerat (20% a.i.) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013.

The corneae were incubated with the test substance and controls for 4h at 32°C ± 1°C. After rinsing with phenol red containing MEM. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 8.4.

The positive control (20% imidazole) increased the opacity and permeability of the corneae (mean in vitro score 94.1) corresponding to a classification as corrosive to the eye. With the negative control (0.9% NaCl) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score -2.0).

Since the mean in vitro irritancy score of the test substance was > 3, the eye irritation potential of Bromhydrin-valerat cannot be determined in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report. The outcome of the test is “No prediction can be made.”

 

To further exclude an eye irritation potential an in vitro study for assessing ocular irritation properties of the test item bromhydrin-valerat was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™.

In this study conducted according to OECD test guideline 492 (adopted July 28, 2015), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular™, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

The test item was applied topically to the EpiOcular™ tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After an 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest. The test item showed no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol thus, killed control and color control were not required.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was 100.2%.

The controls confirmed the validity of the study. The mean relative tissue viability (% negative control) of the positive control was < 50% (21.1%). In this study under the given conditions the test item showed no irritant effects.

According to the results of this study bromhydrin-valerat was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Based on these results eye irritation and corrosion can be excluded because the IVIS detected in the BCOP assay was far below the threshold for classification in category 1.

Justification for classification or non-classification

Based on the study results of in vitro skin and eye irritation/corrosion tests a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.