3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Endpoint summary

Description of key information

The skin irritation/corrosion potential of the test substance, Pinyl Nitrile was assessed according to the following guideline studies:

OECD Test Guideline 431 using a Reconstructed Human EpiDermis (RHE): The test item was considered to be non-corrosive to the skin.

OECD Test Guideline 404 using the Acute Dermal Irritation/Corrosion Method: The test item produced a primary irritation index of 0.0 and was classified as non irritant irritant to rabbit skin according to the Draize classification scheme.

The eye irritation potential of the test substance, Pinyl Nitrile was assessed according to the following guideline studies:

OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability (BCOP) Assay (2016): In Vitro irritancy score for the test item was 0.1, not requiring classification according to UN GHS or EU CLP

OECD Test Guideline 405 Acute Eye Irritation/Corrosion method: The test item does not meet the criterial for classification according to UN GHS or EU CLP.

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:

The study was conducted between 23 March 2016 and 25 March 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: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

dentification: ES421 Pinyl Nitrile

SOURCE OF TEST MATERIAL
- Appearance/physical state: white solid
- Source and lot/batch No.of test material: SM15077102
- Expiration date of the lot/batch: 22 July 2016
- Purity: 98.08%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C, in the dark

The test item was warmed to 45 °C in a water bath prior to dosing.

Test system:

human skin model

Remarks:

EpiDerm™ Reconstructed Human Epidermis Model Kit

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Supplier MatTek

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System: EpiDerm™ Reconstructed Human Epidermis Model Kit

Supplier: MatTek
Date received: 22 March 2016
EpiDermTM Tissues (0.63cm2) lot number: 23325
Assay Medium lot number: 031716TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

Study Design

Pre-Test Procedure

Assessment of Direct Test Item Reduction of MTT

MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT. The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze- killed tissues. This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues. Freeze-killed tissues were prepared by placing untreated EPIDERMTM tissues in an empty 12-well plate and storing in a freezer (−14 to −30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature. In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main Test

Pre-Incubation
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required. After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period. When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated. After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

Data Evaluation

Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = mean OD562 of test item/mean OD562 of negative control x 100

The test item was shown to directly reduce MTT and freeze-killed tissues were employed, the results of the MTT assay were therefore corrected as follows:
True viability = mean OD tvt−(OD tkt−OD ukt)
OD = optical density at 562 nm
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues

If direct reduction by the test item is greater than 30% of the negative control value, additional steps must be taken into account or the test item may be considered incompatible with this test system. If direct reduction by the test item is less than 30% of the negative control value, the mean OD of the test item treated killed control may be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

Classification of corrosivity potential is based on relative viabilities for both exposure times. Refer to 'Any other information on materials and methods incl. tables'.

Quality Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

Negative Control: The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.

Positive Control: Potassium Hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is <15%.

Coefficient of Variation: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

NEGATIVE CONTROL
Identification: Sterile distilled water
Batch: 3011529
Purity: Not supplied
Expiry Date: 01 February 2017
Storage Conditions: Room temperature

POSITIVE CONTROL
Information as provided by the Supplier.
Identification: 8.0 N Potassium Hydroxide
Batch: SLBJ7763V
Purity: 8.0N
Expiry Date: 05 June 2016
Storage Conditions: Room temperature

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 117.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item was considered to be non-corrosive to the skin.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

ca. 120.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item was considered to be non-corrosive to the skin.

Test Item, Positive Control Item and Negative Control Item

 

Mean OD₅₆₂values and viabilities for the negative control, positive control and test item are given below:

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.609	1.467	0.202	13.7	100*
		1.324
	60 Minutes	1.945	1.781	0.233	13.1
		1.616
Positive Control	3 Minutes	0.057	0.061	0.005	na	4.1
		0.064
	60 Minutes	0.064	0.067	0.004	na	3.8
		0.070
Test Item	3 Minutes	1.753	1.728	0.035	2.0	117.8
		1.703
	60 Minutes	1.970	2.153	0.258	12.0	120.9
		2.335

O=   Optical density

*=    The mean % viability of the negative control tissue is set at 100%

na=  Not applicable

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	4.1	117.8
60 minute	100*	3.8	120.9

*The mean viability of the negative control tissues is set at 100%

Quality Criteria

 

The mean OD₅₆₂for the negative control treated tissues was 1.467 for the 3-Minute exposure period and 1.781 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.8% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:

other: The test item was considered to be non-corrosive to the skin.

Conclusions:

The test item, ES421 Pinyl Nitrile, was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item, ES421 Pinyl Nitrile, using the EpiDerm™ Human Skin Model (after OECD Guideline for the Testing of Chemicals No. 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method, 28 July 2015) after treatment periods of 3 and 60 minutes with ES421 Pinyl Nitrile. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD₅₆₂). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	4.1	117.8
60 minute	100*	3.8	120.9

*The mean viability of the negative control tissues is set at 100%

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 August 2016 and 26 August 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 404 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 28 July 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: ES421 Pinyl Nitrile

SOURCE OF TEST MATERIAL
- Appearance/physical state: white solid
- Source and lot/batch No. of test material: SM15077102
- Expiration date of the lot/batch: 22 July 2017
- Purity: 98%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.99 or 3.62 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study. The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

not specified

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

At each test site a quantity of 0.5 g of the test item, moistened sufficiently with 0.5 mL of distilled water, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch.

Duration of treatment / exposure:

Application: 4 hours.

Observation period:

Approximately 1, 24, 48 and 72 hours after application.

Number of animals:

One male one female.

Details on study design:

Study Design
On the day before the test each of a group of two rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study. On the day of the test a suitable test site was selected on the back of each rabbit. At each test site a quantity of 0.5 g of the test item, moistened sufficiently with 0.5 mL of distilled water, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period. Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the scale shown under 'Any other information on materials and methods incl. tables'. Any other skin reactions and clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Data Evaluation

The scores for erythema and edema at the 24 and 72 Hour readings were totaled for the two test rabbits (8 values) and this total was divided by 4 to give the primary irritation index of the test item. The test item was classified according to the following scheme devised by Draize, J.H. (1959) (refer to 'Any other information on materials and methods incl. tables'). If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable. The results were also evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals and to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

References
Draize, J.H. (1959) "Dermal Toxicity" In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the United States, Austin, Texas, p. 46–59.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Skin Reactions

The individual scores for erythema/eschar and edema are given below. No evidence of skin irritation was noted during the study.

Skin Reaction	Observation Time (following patch removal)	Individual Scores		Total
		Rabbit Number and Sex
		75523Female	75524Male
Erythema/Eschar Formation	Immediately	0	0	(0 )
	1 Hour	0	0	( 0 )
	24 Hours	0	0	0
	48 Hours	0	0	( 0 )
	72 Hours	0	0	0
Edema Formation	Immediately	0	0	( 0 )
	1 Hour	0	0	( 0 )
	24 Hours	0	0	0
	48 Hours	0	0	( 0 )
	72 Hours	0	0	0
Sum of 24 and 72‑Hour Readings (S)          :             0
Primary Irritation Index (S/4)                         :             0/4 = 0.0
Classification                                                     :             NON‑IRRITANT

(   ) =    Total values not used for calculation of primary irritation index

 

Body Weight

Individual body weights and body weight change are given below. Both animals showed expected gain in body weight during the study.

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
75523Female	3.62	3.64	0.02
75524Male	2.99	3.04	0.05

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item, ES421 Pinyl Nitrile, produced a primary irritation index of 0.0 and was classified as non irritant irritant to rabbit skin according to the Draize classification scheme. The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals or to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit.

Results

A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced no evidence of skin irritation. 

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:

The study was conducted on 01 April 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

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:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Eyes from adult cattle (typically 12 to 60 months old)

Strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Duration of treatment / exposure:

Exposure period: 32 ± 1 ºC for 10 minutes.

Duration of post- treatment incubation (in vitro):

At 32 ± 1 ºC for 120 minutes

Number of animals or in vitro replicates:

Not applicable

Irritation parameter:

in vitro irritation score

Value:

0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No category. Not requiring classification to UN GHS or EU CLP.

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 05 September 2016 Experimental completion date 22 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: ES421 Pinyl Nitrile
Batch: SM15077102
Purity: 98%
Physical state / Appearance: white solid
Expiry Date: 22 July 2017
Storage Conditions: approximately 4 °C in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 3.09 or 3.78 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL of the test item, which was found to weigh approximately 90 mg

Duration of treatment / exposure:

8 hours

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.

A volume of 0.1 mL of the test item, which was found to weigh approximately 90 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made.

Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Annex 4.

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Any clinical signs of toxicity, if present, were also recorded.

Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

cornea opacity score

Basis:

other: Animals 1 and 2

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

other: no effects observed

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

other: Animals 1 and 2

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

other: no effects observed

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

other: Animals 1 and 2

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

Female

Time point:

24 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

Female

Time point:

48 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

Female

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed at 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

Male

Time point:

24 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

Male

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks:

48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

Male

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24 Hour observation and in one treated eye at the 48 Hour observation.
One treated eye appeared normal at the 48 Hour observation and the other treated eye appeared normal at the 72 Hour observation.

Other effects:

Both animals showed expected gain in body weight during the study.

Appendix1     Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	75520Female				75535Male
	IPR= 0				IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	1	1	0	2	1	0	0
B = Chemosis	1	1	0	0	1	1	0	0
C = Discharge	1	0	0	0	2	0	0	0
Score (A + B + C) x 2	8	4	2	0	10	4	0	0
Total Score	8	4	2	0	10	4	0	0

 

Appendix2     Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
75520Female	8	4	2	0
75535Male	10	4	0	0
Group Total	18	8	2	0
Group Mean Score	9.0	4.0	1.0	0.0

 

Appendix3     Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
75520Female	3.78	3.80	0.02
75535Male	3.09	3.11	0.02

 

IPR=Initial pain reaction

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals or to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 48‑Hour observation and the other treated eye appeared normal at the 72‑Hour observation.

Conclusion

The test item produced a maximum group mean score of9.0and was classified as amildirritant (Class4on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandraclassification system.

The test item does not meet the criteria for classification according to theGlobally Harmonized Systemof Classification and Labelling of Chemicals or to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Acute Dermal Irritation

Substances can be allocated to one of two skin irritation/corrosion categories based on the skin irritation/corrosion route according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the classification, Labelling and Packaging of Substances and Mixtures.

Skin irritation/corrosion is assessed based on effects noted during testing with classification being determined by the severity of the effects.

A test substance is considered to be corrosive if up to 4 hours after application it produces irreversible damage to the skin. Classification is further sub-divided based on the severity of the corrosive effects.

A test substance is considered to be irritant if up to 4 hours after application it produces reversible damage to the skin.

Two studies were carried out, one in vitro and one in vivo, to determine the skin irritation/corrosion potential of the test material Pinyl Nitrile.

The in vitro study indicated that the substance was not considered corrosive.

The in vivo testing provided results indicating that the test material produced no irritancy.

Based on the results of the in vivo study carried out according to OECD 404, it can be concluded that the test material Pinyl Nitrile does not need to be classified for skin irritation/corrosion based on GHS criteria.

Eye Irritation

Substances can be allocated to one of two serious eye damage/irritation categories based on the serious eye damage/irritation route according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the classification, Labelling and Packaging of Substances and Mixtures.

Serious eye damage/irritation is assessed based on effects noted during testing with classification being determined by the severity of the effects.

A test substance is considered to cause serious eye damage after application to the anterior surface of the eye if it produces irreversible tissue damage and/or serious physical decay in vision.

A test substance is considered to be an irritant if after application to the anterior surface of the eye it produces changes that are fully reversible within 21 days.

Two studies were carried out, one in vitro and one in vivo, to determine the eye irritation potential of the test material Pinyl Nitrile.

The in vitro study indicated that the substance was not considered an irritant.

The in vivo testing indicated that the test item does not meet the criterial for classification according to UN GHS and EU CLP.