Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Endpoint summary

Description of key information

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. The skin irritation potential of the test item was assessed in an in vivo acute dermal irritation / corrosion study (OECD 404) in rabbits. At 72 hours, well-defined erythema was observed at 50-100% test item concentrations, slight erythema was observed at lower concentrations. Slight to barely perceptible oedema was also observed at the high concentrations. All erythema and oedema was reversible, with only flaky skin (white scales) observed during post observation periods, suggesting that treatment may affect transdermal water loss. Performed according to guidelines OECD 404 and GLP, the study is considered reliable without restriction (Klimisch 1). There is no evidence of an inherent capability to irritate the skin, requiring classification or substance specific risk mitigation measures (RMM). 

 

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. Eye irritation potential was evaluated in the in vitro EpiOcular™ Human Cornea Model (OECD 492). Following 30 minute exposure to the undiluted test item, the mean relative absorbance values indicative of cell viability were unaltered (107.0% viability; threshold for irritancy: ≤ 60%), consequently the test item was not considered irritating to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1991 - October 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. In accordance with Annex VI, the in vivo acute dermal irritation / corrosion study (OECD 404) was considered sufficient to fulfil the REACH Annex VII information requirement for skin irritation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: SPF albino rabbits

Remarks:

Mol:Russian

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.6 to 2.8 kg
- Housing: PPL cages (45 x 55 cm) with perforated floor, caged individually
- Diet : ad libitum, feed pellets
- Water: ad libitum, drinking water acidified to pH 2.5 with hydrochloric acid
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 6
- Photoperiod (hrs dark / hrs light): Light was on from 6 to 18 h

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: Ethanol 96% and diethyl phthalate (DEP) in the ratio 1:1 (w/w)

Controls:

yes, concurrent vehicle

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration: 100%, 50% (w/w), 25% (w/w), 10% (w/w) and 5% (w/w)

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): Ethanol 96% and diethyl phthalate (DEP) in the ratio 1:1 (w/w)

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

4

Details on study design:

TEST SITE
- Area of exposure: Dorsal anterior, central and posterior
- % coverage: An area of 10 x 10 cm on the back of each animal was clipped and divided into 6 test sites, each test site with an area of 2.5 x 2.5 cm, with 1 cm between each test site
- Type of wrap if used: Gauze patches, secured with adhesive tape and Scanpor tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with soap and lukewarm water
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS: Skin reactions were assessed 30 min after washing, at 24, 48 and 72 h after application, and at 7 and 14 days after application

SCORING SYSTEM:
- Method of calculation: Erythema and eschar formation and oedema formation were scored from 0 (no effects) to 4 (severe effects). The scores for erythema and oedema formation for the last 3 readings of each rabbit (24, 48 and 72 hours) for each test concentration were obtained separately and divided by 3. The results are the mean scores for erythema and oedema formation of the individual rabbit for each test concentration. The mean scores for erythema and oedema formation in the four rabbits used for each concentration tested were subsequently calculated.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

100% concentration

Time point:

72 h

Score:

1.7

Max. score:

2

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

50% concentration

Time point:

72 h

Score:

1.3

Max. score:

2

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

25% concentration

Time point:

72 h

Score:

0.9

Max. score:

1

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

10% concentration

Time point:

72 h

Score:

0.7

Max. score:

1

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

5% concentration

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

Vehicle

Time point:

72 h

Score:

0.3

Max. score:

1

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

100% concentration

Time point:

72 h

Score:

1.4

Max. score:

2

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

50% concentration

Time point:

72 h

Score:

0.8

Max. score:

2

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

25% concentration

Time point:

72 h

Score:

0.3

Max. score:

1

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

10% concentration

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

5% concentration

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

Vehicle

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Other effects:

A dense layer of white scales was observed in all rabbits at the test sites treated with 50% and 100% concentration on days 7 and 14. In one rabbit, a dense layer of white scales was also observed at 25% concentration on day 7, and scattered white scales were observed at 5% and 10% concentration on days 7 and 14 and at 25% concentration on day 14. The appearance of white scales suggests that the treatment may increase transdermal water loss.

Table 1. Mean individual erythema-eschar and oedema values after 72 hours

Rabbit number	Test concentration	Mean erythema-eschar score	Mean oedema score
1	Vehicle	0.00	0.00
1	5%	0.00	0.00
1	10%	1.00	0.00
1	25%	1.33	1.00
1	50%	2.00	2.00
1	100%	2.00	2.00
2	Vehicle	1.00	0.00
2	5%	0.00	0.00
2	10%	1.00	0.00
2	25%	0.67	0.00
2	50%	1.00	0.00
2	100%	1.67	1.67
3	Vehicle	0.00	0.00
3	5%	0.00	0.00
3	10%	0.67	0.00
3	25%	1.00	0.33
3	50%	1.33	0.67
3	100%	1.67	1.33
4	Vehicle	0.00	0.00
4	5%	0.00	0.00
4	10%	0.00	0.00
4	25%	0.67	0.00
4	50%	1.00	0.67
4	100%	1.33	0.67

Interpretation of results:

GHS criteria not met

Conclusions:

For the vehicle and test item at concentrations at 5, 10, 25, 50 and 100% concentration, the mean erythema scores were 0.3, 0.0, 0.7, 0.9, 1.3 and 1.7, respectively, and the mean oedema scores were 0.0, 0.0, 0.0, 0.3, 0.8 and 1.4, respectively. At 72 hours, well-defined erythema was observed at 50-100% test item concentrations, slight erythema was observed at lower concentrations. Slight to barely perceptible oedema was also observed at the high concentrations. All erythema and oedema was reversible, with only flaky skin (white scales) observed during post observation periods, suggesting that treatment may affect transdermal water loss.

Executive summary:

The skin irritation potential of the test item was assessed in four SPF albino female rabbits, at concentrations of 5, 10, 25, 50 and 100%, in addition to a vehicle control. The back of each rabbit was clipped and divided into 6 test sites, at which 0.5mL of each treatment was applied and secured with a gauze patch. After 4 hours’ exposure, patches were removed and the treated skin was cleaned with soap and water. Skin reactions (erythema and eschar formation and oedema formation) were assessed on a scale from 0 to 4, at 4.5, 24, 48 and 72 hours after application.

 

For the vehicle and test item at concentrations at 5, 10, 25, 50 and 100% concentration, the mean erythema scores were 0.3, 0.0, 0.7, 0.9, 1.3 and 1.7, respectively, and the mean oedema scores were 0.0, 0.0, 0.0, 0.3, 0.8 and 1.4, respectively. At 72 hours, well-defined erythema was observed at 50-100% test item concentrations, slight erythema was observed at lower concentrations. Slight to barely perceptible oedema was also observed at the high concentrations. All erythema and oedema was reversible, with only flaky skin (white scales) observed during post observation periods, suggesting that treatment may affect transdermal water loss.

 

Performed according to guidelines OECD 404 and GLP, the study is considered reliable without restriction (Klimisch 1). There is no evidence of an inherent capability to irritate the skin, requiring classification or substance specific risk mitigation measures (RMM). 

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 February 1984 - 23 March 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

No guideline reported. Some limitations in experimental design (e.g. number of test animals) and reporting (e.g. coverage), however this study is suitable for use as supporting evidence.

Justification for type of information:

The REACH information requirements were revised in to endorse a battery of in vitro assays for skin irritation / corrosion. Whilst Annex VII does not require in vivo testing for skin irritation, all existing available information should be evaluated, including any available animal data, in accordance with Annex VI.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Primary skin Irritation was assessed prior to photosensitivity testing. Guinea pigs were clipped on the dorsal surface and depilatory cream was applied. Approximately two hours following depilation, a 0.05mL aliquot of the test material at concentrations of 10, 30, 50 and 100% was applied.
- Short description of test conditions: The animal room temperature was maintained at 22 ± 3°C. The rate of air exchange was approximately 10 changes per hour. Humidity remained within a range of 45 to 65% relative humidity. The lighting was controlled to give a 12 hour light/dark cycle.
- Parameters analysed / observed: The degree of erythema was assessed on a scale from 0 to 3.

GLP compliance:

yes

Species:

guinea pig

Strain:

Dunkin-Hartley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 to 8 weeks
- Weight at study initiation: 268 to 376 g
- Housing: Solid-floor polypropylene cages with softwood shavings
- Diet: Standard laboratory guinea pig diet, ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 45 to 65% RH
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

not specified

Preparation of test site:

clipped

Vehicle:

other: ethanol

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.05mL
- Concentration (if solution): 10, 30, 50 and 100% in absolute ethanol

Duration of treatment / exposure:

Single application

Observation period:

24 hours

Number of animals:

2

Details on study design:

TEST SITE
- Area of exposure: Dorsal surface

REMOVAL OF TEST SUBSTANCE
- Washing: None

OBSERVATION TIME POINTS: 24 hours after application

SCORING SYSTEM
0: No erythema
1: Scattered mild redness
2: Moderate and diffuse redness
3: Intense redness and swelling

Irritation parameter:

erythema score

Remarks:

10% test material

Basis:

mean

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

erythema score

Remarks:

30% test material

Basis:

mean

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

erythema score

Remarks:

50% test material

Basis:

mean

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

not specified

Irritation parameter:

erythema score

Remarks:

100% test material

Basis:

mean

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

not specified

Irritant / corrosive response data:

Scattered mild redness was observed at 50 and 100% test item concentrations.

Table 1. Primary skin irritation siting test score for reaction observed at test sites

Test material concentration (%)	Guinea pig A	Guinea pig B
10	0	0
30	0	0
50	1	1
100	1	1

Interpretation of results:

study cannot be used for classification

Conclusions:

Exposure to varying concentrations of the test item (50 and 100%) in ethanol, induced scattered mild redness in guinea pigs 24-hours post application. No erythema was observed at lower concentrations of the test item (10 and 30%). There is no evidence of an inherent capability to irritate the skin, requiring classification or substance specific risk mitigation measures (RMM).

Executive summary:

Primary skin irritation was assessed in two Dunkin-Hartley guinea pigs at 10, 30, 50 and 100% test item concentration. Guinea pigs were clipped on the dorsal surface and depilatory cream was applied, approximately two hours later, a 0.05 mL aliquot of the test material was applied. The degree of erythema was assessed 24 hours following application. The test material did not produce erythema at 10 and 30% concentrations. However, scattered mild redness was observed at the two higher concentrations of 50 and 100%. This GLP compliant study is similar to a GPMT, with some limitations in experimental design (e.g. number of test animals) and reporting (e.g. coverage), and considered reliable with restriction (Klimisch 2). The study is suitable for use as supporting evidence.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

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:

16 February 2016 - 28 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The standard information requirements for REACH Annex VII substances for serious eye damage/eye irritation can be satisfied by an assessment of the available in vivo data, physicochemical properties (i.e. acid or alkali reserve) or in vitro eye irritation studies. The EpiOcular™ Eye Irritation Test (OECD 492) is an accepted in vitro test method to detect severe eye irritation/damage (Category 1) and/or the absence of effects (not classified under CLP).

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:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: cornea

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: Eye irritation is defined in Section 3.3.1.1 of Annex I to the CLP regulation as “[…] the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application." According to Annex VII of the REACH Regulation, if new test data are required these must be derived from in vitro methods only. The EpiOcular™ model (OECD 492), using Reconstructed human Cornea-like Epithelium (RhCE), is an accepted in vitro test method to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organised basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. In a pre-validation study, in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.

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 μL
- Concentration (if solution): neat

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2

Details on study design:

METHOD:
- Details of the test procedure used: The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Tissues were incubated with the test item or controls for 30 minutes at standard culture conditions. At the end of the 30 minutes treatment time, the test item or control was removed by extensively rinsing the tissues. After rinsing, the tissues immersed in Assay Medium at room temperature for 12 minutes to remove any test item absorbed into the tissue. The tissues were then incubated in Assay Medium for about 120 minutes at standard culture conditions. The MTT assay was performed after the post-treatment incubation. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

ENVIRONMENTAL CONDITIONS
- RhCE tissue construct used, including batch number: EpiOcular ™ from MatTek Corporation, keratinocyte strain 4F1188, lot numbers 21597 and 23707
- Doses of test chemical and control substances used: 50 μL, neat
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 minute exposure at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH), 12 minute immersion at room temperature, and 120 minute post-treatment incubation at standard culture conditions
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Negative control (deionised water) for direct MTT-reduction test
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm, linearity range not reported
- Description of the method used to quantify MTT formazan: At the end of the post-treatment incubation, tissues were placed into a 24-well plate containing 0.3 mL of MTT solution. The plate was incubated for about 180 minutes at standard culture conditions. After the incubation time, tissues were rinsed and transferred into another plate containing 2.0 mL of isopropanol per well. The plates were sealed and stored overnight at 2-8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken. The extract solution was mixed and two 200 μL aliquots were taken for measurement.

EVALUATION CRITERIA
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The mean viability for the colorant control was calculated and this mean colorant control "viability" value (CC "viability") was subtracted from the relating mean viability of the same test item (TI viability) to determine the colorant control corrected viability (CC corrected viability). The test item was classified regarding the colorant control corrected viability according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Positive control (mean viability 32.0%, relative standard deviation of mean viability 12.8%, range of viabilities 6.90-40.4%, mean absorption 0.538, relative standard deviation of mean absorption 0.258, range of absorbance 0.107-0.849), negative control (mean absorption 1.65, relative standard deviation of mean absorption 0.299, range of absorbance 1.27-2.05); data of 11 studies performed from July 2015 until end of February 2016.
- Complete supporting information for the specific RhCE tissue construct used: Lot number 21597 (tissue viability 1.779 ± 0.054; barrier function 20.25 min; sterile tissue), lot number 23707 (tissue viability 1.918 ± 0.095; barrier function 15.12 min; sterile tissue)
- Reference to historical data of the RhCE tissue construct: Acceptance criteria of tissue functionality and quality (tissue viability (OD 540-570nm) 1.1-3.0; barrier function (ET-50) 12.2=37.5 min; no contamination)
- Positive and negative control means and acceptance ranges based on historical data: The negative control OD must be > 0.8 and < 2.5.
- Acceptable variability between tissue replicates for positive and negative controls: < 20% in the same run
- Acceptable variability between tissue replicates for the test chemical: < 20% in the same run

Irritation parameter:

other: Percent viability

Run / experiment:

Mean

Value:

107

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD was 2.012 to 2.109
- Acceptance criteria met for positive control: The mean relative viability of the positive control 40.4% of the negative control viability
- Range of historical values if different from the ones specified in the test guideline: Historical negative control OD range 1.27-2.05, historical range of viabilities for positive control 6.90-40.4%

Table 1. Absorbance results

Treatment	Replicate	OD_570nm (well 1)	OD_570nm (well 2)	OD_570nm (mean)	OD_570nm (mean of 2 wells, blank corrected)	OD_570nm (mean of 2 tissues, blank corrected)	Relative absorbance (%)	Absolute value of the difference of the relative absorbances (%)	Mean relative absorbance (% of negative control)
Blank	-	0.037	0.036	0.037	0.000	-	-	-	-
Negative control	1	2.012	2.041	2.026	1.990	2.021	98.5	3.1	100.0
Negative control	2	2.068	2.109	2.089	2.052	(see above)	101.5	(see above)	(see above)
Positive control	1	0.846	0.925	0.885	0.849	0.816	42.0	3.2	40.4
Positive control	2	0.805	0.837	0.821	0.784	(see above)	38.8	(see above)	(see above)
Test item	1	2.097	2.052	2.124	2.088	2.166	103.3	7.8	107.2 (without colourant correction); 107.0 (with colourant correction)
Test item	2	2.213	2.350	2.282	2.245	(see above)	111.1	(see above)	(see above)
Additional test with viable tissues without MTT reduction
Blank	-	0.036	0.035	0.036	0.000	-	-	-	-
Negative control	1	0.039	0.040	0.039	0.004	-	100.0	-	-
Test item	1	0.041	0.040	0.040	0.005	-	123.4	-	-

Interpretation of results:

GHS criteria not met

Conclusions:

The EpiOcular™ method (OECD 492) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP), under REACH Annex VII. The viability of test item exposed tissues was 107.0% (threshold for irritancy: ≤ 60%), therefore the test item is not considered to possess eye irritating potential. Conducted according to the aforementioned guidelines and GLP, the EpiOcular™ test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

Eye irritation of the test item was evaluated with the EpiOcular™ Human Cornea Model (OECD 492). Cell viability of a stratified squamous epithelium (cultured from normal, human-derived epidermal keratinocytes), similar to that found in the human cornea, was evaluated using the MTT assay, which measures the conversion of (3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. A 50 µL aliquote of the test item was applied to the tissue for 30 minutes, alongside a negative and positive control. The viability value of the test item exposed tissues was 107.0% (threshold for irritancy: ≤ 60%), therefore the test item is not considered to possess eye irritating potential.

The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 492 and GLP, the study is considered to be reliable without restriction (Klimisch 1).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 January 1984 - 24 February 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Minor restrictions in reporting.

Justification for type of information:

The standard information requirements for REACH Annex VII substances for serious eye damage/eye irritation can be satisfied by an assessment of the available in vivo data, physicochemical properties (i.e. acid or alkali reserve) or in vitro eye irritation studies. Conducted prior to the validation and regulatory acceptance of alternative methods, a reliable (Klimisch 2) and GLP compliant in vivo Draize test was available for the test substance and considered sufficient to fulfil the information requirement as a supporting study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

albino

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 to 16 weeks
- Weight at study initiation: 2.31 to 2.48 kg
- Housing: Individual metal cages
- Diet: Standard laboratory rabbit diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 2°C
- Humidity (%RH): 55 to 60% RH
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: Arachis oil B.P.

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1mL
- Concentration (if solution): 30% in arachis oil

Duration of treatment / exposure:

Single application

Observation period (in vivo):

1, 24, 48, 72 hours after application

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: None

SCORING SYSTEM:

CORNEA
0: No ulceration or opacity
1: Scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible
2: Easily discernible translucent area, details of Iris slightly obscured
3: Nacreous area, no details of iris visible, size of pupil barely discernible
4: Opaque cornea, Iris not discernible through the opacity

IRIS
0: Normal
1: Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive
2: No reaction to light, haemorrhage, gross destruction (any or all of these)

CONJUNCTIVAE
0: Blood vessels normal
1: Some blood vessels definitely hyperaemic (injected)
2: Diffuse, crimson colour, individual vessels not easily discernible
3: Diffuse beefy red

CHEMOSIS
0: No swelling
1: Any swelling above normal (includes nictitating membranes)
2: Obvious swelling with partial eversion of lids
3: Swelling with lids about half closed
4: Swelling with lids more than half closed

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Remarks:

30% test item concentration

Basis:

mean

Time point:

other: 1/24/48/72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

iris score

Remarks:

30% test item concentration

Basis:

mean

Time point:

other: 1/24/48/72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

conjunctivae score

Remarks:

30% test item concentration

Basis:

mean

Time point:

other: 1 h

Score:

1

Max. score:

1

Reversibility:

not specified

Irritation parameter:

conjunctivae score

Remarks:

30% test item concentration

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

chemosis score

Remarks:

30% test item concentration

Basis:

mean

Time point:

other: 1 h

Score:

1

Max. score:

1

Reversibility:

not specified

Irritation parameter:

chemosis score

Remarks:

30% test item concentration

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritant / corrosive response data:

The test material produced minimal ocular Irritation in 3/3 rabbits. Mild conjunctival inflammation, accompanied by slight swelling was observed in all three rabbits one hour after instillation only.

Table 1. Individual daily scores and group total score for ocular irritation

Rabbit number	1					2					3					Total score per tissue
Time after treatment	1 h	24 h	48 h	72 h	7 d	1 h	24 h	48 h	72 h	7 d	1 h	24 h	48 h	72 h	7 d
Cornea	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-	0
Iris	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-	0
Conjunctival redness	1	0	0	0	-	1	0	0	0	-	1	0	0	0	-	3
Conjuctival chemosis	1	0	0	0	-	1	0	0	0	-	1	0	0	0	-	3
Group total score																6

Interpretation of results:

GHS criteria not met

Conclusions:

The test material did not induce ocular irritation in any of the rabbits tested and should not be classified as an eye irritant. Conducted according to OECD TG 405, the study was considered to be reliable with restriction (Klimisch 2), despite minor limitations in reporting.

Executive summary:

Eye irritation was assessed in three albino New Zealand White strain rabbits with 30% test item concentration in arachis oil. An aliquot of 0.1 mL treatment solution was instilled into the conjunctival sac of the right eye of each rabbit, while the left eye remained untreated and was used as the negative control. Assessments of damage/irritation (cornea opacity, iris, conjunctival redness and conjunctival chemosis) were made 1, 24, 48 and 72 hours following treatment. Mild conjunctival inflammation, accompanied by slight swelling was observed in all three rabbits one hour after instillation only. The mean scores were 0, 0, 1 and 1for cornea, iris, conjunctival redness and conjunctival chemosis, respectively. The test material did not induce ocular irritation in any of the rabbits tested and should not be classified as an eye irritant. Conducted according to OECD TG 405, the study was considered to be reliable with restriction (Klimisch 2), despite minor limitations in reporting.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation potential of the test item was assessed in four SPF albino female rabbits, at concentrations of 5, 10, 25, 50 and 100%, in addition to a vehicle control (1991). The back of each rabbit was clipped and divided into 6 test sites, at which 0.5mL of each treatment was applied and secured with a gauze patch. After 4 hours’ exposure, patches were removed and the treated skin was cleaned with soap and water. Skin reactions (erythema and eschar formation and oedema formation) were assessed on a scale from 0 to 4, at 4.5, 24, 48 and 72 hours after application. For the vehicle and test item at concentrations at 5, 10, 25, 50 and 100% concentration, the mean erythema scores were 0.3, 0.0, 0.7, 0.9, 1.3 and 1.7, respectively, and the mean oedema scores were 0.0, 0.0, 0.0, 0.3, 0.8 and 1.4, respectively. At 72 hours, well-defined erythema was observed at 50-100% test item concentrations, slight erythema was observed at lower concentrations. Slight to barely perceptible oedema was also observed at the high concentrations. All erythema and oedema was reversible, with only flaky skin (white scales) observed during post observation periods, suggesting that treatment may affect transdermal water loss. Performed according to guidelines OECD 404 and GLP, the study is considered reliable without restriction (Klimisch 1). There is no evidence of an inherent capability to irritate the skin, requiring classification or substance specific risk mitigation measures (RMM). This study is considered sufficient to fulfil REACH Annex VII information requirements.

 

Primary skin irritation was also assessed in two Dunkin-Hartley guinea pigs with 10, 30, 50 and 100% test item concentrations (1984). Guinea pigs were clipped on the dorsal surface and depilatory cream was applied. Approximately two hours following depilation, a 0.05mL aliquot of the test material was applied. The degree of erythema was assessed at 24 hours. The test material did not produce an erythema response at concentrations up to 30%. This study is reliable with restriction (Klimisch 2) as it was GLP-compliant, however no guideline was reported. There were some limitations in experimental design (e.g. number of test animals) and reporting (e.g. coverage), however this study is suitable for use as supporting evidence to fulfil the REACH Annex VII information requirement.

 

Eye irritation of the test item was evaluated with the EpiOcular™ Human Cornea Model (2016). Cell viability of a stratified squamous epithelium (cultured from normal, human-derived epidermal keratinocytes), similar to that found in the human cornea, was evaluated using the MTT assay, which measures the conversion of (3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. A 50 µL aliquote of the test item was applied to the tissue for 30 minutes, alongside a negative and positive control. The viability value of the test item exposed tissues was 107.0% (threshold for irritancy: ≤ 60%), therefore the test item is not considered to possess an eye irritating potential. The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD 492 and GLP, the study is considered to be reliable without restriction (Klimisch 1) and sufficient to fulfil the REACH Annex VII information requirement as a key study.

 

Eye irritation was assessed in three albino New Zealand White strain rabbits with 30% test item concentration in arachis oil (1984). An aliquot of 0.1 mL treatment solution was instilled into the conjunctival sac of the right eye of each rabbit, while the left eye remained untreated and was used as the negative control. Assessments of damage/irritation (cornea opacity, iris, conjunctival redness and conjunctival chemosis) were made 1, 24, 48 and 72 hours following treatment. Mild conjunctival inflammation, accompanied by slight swelling was observed in all three rabbits one hour after instillation only. The mean scores were 0, 0, 1 and 1for cornea, iris, conjunctival redness and conjunctival chemosis, respectively. The test material did not induce ocular irritation in any of the rabbits tested and should not be classified as an eye irritant. Conducted according to OECD TG 405, the study was considered to be reliable with restriction (Klimisch 2), despite minor limitations in reporting. This study is suitable for use as supporting data.

Justification for classification or non-classification

Two reliable (Klimisch 1 and 2) studies are available to inform the skin irritation / corrosion classification of the test item (1984, 1991). In the key in vivo study, conducted according to OECD 404, mean erythema and oedema scores over 72 hours were ≤2.00 in all four rabbits tested. In the supporting in vivo study (no guideline reported), individual erythema scores over 24 hours were ≤1 in the two guinea pigs tested. Therefore, according to CLP Regulation No 1272/2008, the test item should not be classified as a skin irritant.

 

Two reliable (Klimisch 1 and 2) and GLP-compliant studies are available to inform the eye irritation classification of the test item (1984, 2016). In the key in vitro study, conducted according to OECD 492, the viability value of the test item exposed tissues was 107.0% and the test item is not considered to possess eye irritating potential (threshold for irritancy: ≤60%). In the supporting in vivo study, conducted according to OECD 405, no signs of corneal opacity, iritis, conjunctival redness or conjunctival oedema were observed at 24, 48 and 72 hours after instillation of the test material in 3 of 3 rabbits. According to CLP Regulation No 1272/2008, the test item should not be classified as an eye irritant.