1H-Imidazole, hydrobromide (1:1)

Endpoint summary

Description of key information

Skin irritation/corrosion: not irritating (OECD 439, G





Skin irritation: not irritating (OECD 439, GLP); eye irritation: not irritating (OECD 437, GLP)

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:

2015-12-17 to 2016-01-25

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:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: humans

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

other: Dulbecco's phosphate buffered saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 23308
- Delivery date: 2016-01-19

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (24 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were rinsed with DPBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with fresh assay medium. Tissues were incubated for nearly 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2, RH 95%. After incubation the inserts were transferred into new plates containing fresh medium. Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2, RH 95%. The complete incubation time was nearly 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT/ EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new plates and immersed into extractant solution (isopropanol). Tissues were completely covered from both sides and the plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 69.5 hours without shaking in the refrigerator in the dark.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken and the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. The optical density was determined with a spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter bandwidth: 1 nm

TEST FOR COLOUR INTERFERENCE
Prior to the start of the test, the test item’s colour interference potential was evaluated. 25 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C. Deionized water was used as negative control.

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results, the ability of the test item to directly reduce MTT was assessed. For this purpose, approx. 25 mg of the test item were added to 1 mL of MTT solution (resulting: 1 mg/mL). This mixture was incubated in the dark at 37 ± 1.5 °C for 60 minutes. An untreated MTT/DMEM-Solution was used as negative control. If the colour of the MTT turned blue/purple, the test item is persumed to have reduced MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (OD test item/ OD mean of negative control) x 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritauion potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS
- Lot no.: 23308

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% Sodium Lauryl Sulfate (SLS) solution

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

75.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Colour interference with MTT: colour of test item/water mixture did not change during the incubation period compared with the colour of the pure test item, and the colour of the test item was not intensive (beige). Therefore, an additional test with one viable tissue (entire test procedure, but with DPBS addition instead of MTT addition) for data correction was not required and consequently not performed.
- Direct-MTT reduction: the colour did not turn blue/purple and the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues for data correction did not have to be performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (1.946, 1.845, and 1.881 (mean: 1.891)) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3 % (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 7.0% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%).
Please refer to the field "Any other information on results incl. tables" below

HISTORICAL DATA

Positive Control		Negative Control [OD570]
Mean Viability	4.64 %	Mean Absorption	1.74
Rel. Standard Deviation	11.2 %	Rel. Standard Deviation	8.68 %
Range of Viabilities	4.00 % - 5.90 %	Range of Absorbance	1.48– 1.98
Mean Absorption	0.0803
Rel. Standard Deviation	12.6 %
Range of Absorbance	0.066- 0.097

Data of 31 studies performed from July 2015 until March 2016

Table 1: Results after treatment with Imidazole hydrobromide and the controls  

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Mean Rel. Absorbance [% of Negative Control]***
Negative control	60 min	1.946	1.845	1.881	1.891	100.0
Positive control	60 min	0.079	0.084	0.080	0.081	4.3
Test item	60 min	1.507	1.330	1.426	1.421	75.2

*       mean of three replicate wells 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 (absorbance(test item/ positive control))/ (mean absorbance(negative control))

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.

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:

2015-12-14

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:

2013-07-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details

Version / remarks:

April 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Specific details on test material used for the study:

- Storage condition of test material: at room temperature

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were transported to the laboratory in Hank’s Buffered Salt Solution (HBSS) supplemented with streptomycin / penicillin at cooled temperature.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes

Vehicle:

other: 0.9% (w/v) NaCl in deionised water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20 % suspension (w/v) in vehicle

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

PREPARATION OF CORNEAS
- each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium (MEM, supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum).
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
- all eyes were carefully examined macroscopically for defects before removing the cornea. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- each corneae with a value of the basal opacity > 7 was discarded.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- after the incubation time, the test item or control items were rinsed off with saline.
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
- permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometer (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 0.5% (w/v) sodium fluorescein solution in HBSS
- corneae were incubated in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, mixed and the optical density at 490 nm was determined with the spectrophotometer.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values, which is determined from each individual treatment and positive control cornea.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437 (please refer to table 1 in the field "Any other information on material and methods incl. tables" below).

DECISION CRITERIA
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- with the negative control neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.45).
- the positive control caused clear opacity on the corneae (mean IVIS = 104.53) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Please refer to the field "Any other information on results incl. tables" below.

Table 1: Results after 240 minutes incubation time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative Control	0	0.33	0.084	0.075	1.26	1.45
	1		0.082		2.23
	0		0.058		0.87
Positive Control	113.67*		-0.009*		113.54	104.53
	108.67*		-0.013*		108.48
	91.67*		-0.007*		91.57
Imidazole hydrobromide	-0.33*		-0.014*		-0.54	0.00*
	-1.33*		-0.017*		-1.58
	-1.33*		-0.021*		-1.64

*corrected values

** negative value was set to zero

Table 2: Historical data

	Positive Control	Negative Control
Mean IVIS	116.59	1.41
Standard Deviation IVIS	9.20	0.39
Range of IVIS	98.49 – 128.43	0.88 – 2.04
Mean Opacity t240min	113.4	0.23
Standard Deviation Opacity t240min	11.57	0.28
Range of Opacity t240min	92.67 – 140.33	0.00 – 0.67
Mean Permeability	0.245	0.146
Standard Deviation Permeability	0.354	0.213
Range of Permeability	0.015 – 1.438	0.055 – 0.750

Values of 18 studies with solid test items performed from February 2015 (calendar week 7) until October 2015

Interpretation of results:

GHS criteria not met

Conclusions:

Relative to the negative control, the substance did not cause an increase of the corneal opacity or permeability. In conclusion, according to the current study and under the experimental conditions reported, the substance is not irritating to the eye (the calculated IVIS = 0.00). A substance with an in vitro irritation score ≤ 3 does not require classification for eye irritation and serious eye damage according to OECD 437 (2013). According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is not irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation

The substance was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The substance was not oberved to be irritating to the eye in a reliable in vitro eye irritation study according to OECD 437.

Justification for classification or non-classification

Skin irritation

The substance does not possess an skin irritating potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance does not possess an eye irritation potential according to an in vitro OECD 437 test and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.