Reaction mass of 2-[[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]methyl]oxirane and 1,3-bis[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol and 1-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-3-[4-[[4-[3-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-2-hydroxy-propoxy]-3,5-dimethyl-phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol

Endpoint summary

Description of key information

In vitro skin irritation: The results obtained from an in vitro skin irritation test indicated that the test item revealed an inconclusive effect on skin irritation, most likely owing to physical effects of the test material. 
In vitro skin corrosion: The results obtained from an in vivo skin corrosion test indicated that the test item revealed no corrosive potential under the utilised test conditions. 
In vivo skin irritation: The results obtained from an in vivo skin irritation test indicated that the test item revealed no skin irritation potential under the utilised testing conditions.
In vitro eye irritation: The results obtained from an in vitro eye corrosion test indicated that the test item revealed no serious eye damaging potential under the utilised testing conditions 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle, Cedex, France
- Age at study initiation: 12 to 24 weeks old
- Weight at study initiation: At least 1.5 kg.
- Housing: Individually housed in labelled cages with perforated floors and shelters.
- Diet (e.g. ad libitum): Pelleted diets for rabbits at approximately 100g per day.
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark cycle.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):Each animal was treated by dermal application of 0.5 g of the test substance.

Duration of treatment / exposure:

Four hours

Observation period:

72 hours

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: The test substance was applied to the skin of one flank.
- Coverage: Approximately 2x3 cm
- Type of wrap if used: Applied to the skin on a metalline patch. This patch was mounted on micropore tape, which was wrapped around the abdomen and secured with a Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The skin was cleaned of residual test substance using tap water and watery ethanol (50% w/w).
- Time after start of exposure: 4 hours

SCORING SYSTEM: Scoring is based on a score of 1 to 4 for Erythema, eschar and oedema formation. Higher scores indicate a higher severity of effect.

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

924

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

929

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

931

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

924

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

931

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

931

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

There were no effects to 3 male rabbits of the test substance in terms of erythema and oedema formation.

Other effects:

No signs of systemic toxicity were observed in the test animals during the test period and no mortality occured.

Mean value irritation scores:

Animal No.	Mean score 24, 48 and 72 hours
	Erythema	Oedema
924	0	0
929	0	0
931	0	0

Interpretation of results:

not irritating

Conclusions:

The test substance elicited no skin irriation response after being applied dermally to rabbits for a period of 4 hours. As such, the test substance can be considered non irritating.

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

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Preparation of corneas:
All eyes were carefully examined for defects by holding the eyes to the light after submerging in physiological saline. Those exhibiting unacceptable
defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands)
containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)).
The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the
O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws.
The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

Test material applied in excess

Duration of treatment / exposure:

120 ± 10 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

Three eyes for the test substance, negative control and positive control substance.

Details on study design:

Details on treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 ul of the negative control and 10% (w/v) Benzalkonium chloride solution (positive control) were introduced onto the epithelium of the cornea. The test substance was applied directly on the corneas in such a way that the cornea was completely covered (in excess).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C, and then subsequently washed and possible pH effects noted. Subsequently the corneas were incubated for a further 240 ± 10 minutes at 32 ± 1°C. After the
incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). After the completion of the incubation period each cornea were inspected visually for dissimilaropacity patterns and the opacity determination was performed.

Opacity measurements:
The opacitometer determined the difference in the light transmission between each control or test substance treated cornea and an air filled chamber.The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative
control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive
control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the changein opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Permeability determination:
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled
according to holder number. 360 ul of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm
(OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was
1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the
experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using
cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before thedilution factor was applied to the readings.

Irritation parameter:

cornea opacity score

Value:

>= -2 - <= -1

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls were 0.0 for all three corneas. The individual positive control in vitro irritancy scores ranged from 132 to 165. The corneas treated with the positive control were turbid after 10 minutes of treatment.

The corneas treated with the test substance showed opacity values ranging from -2.0 to -1.0 and permeability values ranging from -0.002 to 0.006. The corneas were clear after the 10 minutes of treatment. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.0 to -0.9 after 10 minutes of treatment.

Treatment	Mean Opacity	Mean Permeability	Mean In vitro irritation Score
Negative control	0.0	0.0	0.0
Positive control (Benzalkonium Chloride)	103.0	3.287	152.3
Test substance	-1.3	0.002	-1.3

Interpretation of results:

not irritating

Conclusions:

Since the test substance induced an in vitro irritancy score (IVIS) ≤ 3, no classification is required for eye irritation or serious eye damage

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation: The results obtained from an in vitro skin irritation test indicated that the test item revealed an inconclusive effect on skin irritation, most likely owing to physical effects of the test material. This was further tested in vivo to confirm the result, with the in vivo test showing no evidence of skin irritation. An in vitro skin corrosion test also provided no indication of skin corrosion.

In vitro eye irritation/corrosion: The results obtained from an in vitro eye irritation/corrosion test (BCOP) indicated that the test item revealed no serious eye damaging potential under the utilised testing conditions.

Justification for selection of skin irritation / corrosion endpoint:
The conducted in vitro test using a constructed human epidermis skin model, provided an inconclusive result to skin irritation, most likely owing to physical properties of the test material. As such an in vivo test was conducted to confirm whether the test substance was considered a skin irritant; no irritation was observed during the study.

Justification for selection of eye irritation endpoint:
No indication of severe eye damage was observed.

Justification for classification or non-classification

Based on the available experimental data (no irritation observed in vivo) the test item is not classified with regards to skin irritation/corrosion according to Regulation (EC) 1272/2008 (CLP).

Based on the available experimental data the test item is not classified with regard to serious eye damage/irritation according to Regulation (EC) 1272/2008 (CLP).