Benzene, 4-bromo-1-chloro-2-[(4-ethoxyphenyl)(phenylmethoxy)methyl]-

Endpoint summary

Description of key information

Skin irritation/corrosion
Two studies are available:
1) An acute dermal irritation study (Latour, 2014) is available which is key study. This study showed that the test substance is not irritating.
2) An in vitro skin corrosion study (Buskens, 2014) is available which is supporting study. This study showed that the test substance is not corrosive.
Eye irritation
Two studies are available:
1) An acute eye irritation study (Latour, 2014) is available which is key study. This study showed that the test substance is slightly irritating.
2) An in vitro BCOP study (Verspeek-Rip, 2014) is available which is supporting study. This study showed that the test substance is not irritating.

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

Study period:

From 29 Oct to 08 Nov 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

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: between 12 and 24 weeks
- Weight at study initiation: at least 1.5 kg
- Housing: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet (e.g. ad libitum): Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days

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

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: watery ethanol (50% v/v)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.6 mL
- Concentration (if solution): 50% v/v

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: 2 cm*3 cm
- % coverage: The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage.
- Type of wrap if used:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The skin cleaned of residual test substance using tap water.
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to application) and after the final observation.
- Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: The exposure period is 4 hours.

Irritation parameter:

edema score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: The exposure period is 4 hours.

Irritant / corrosive response data:

No skin irritation was caused by 4 hours exposure to the test substance.
There was no evidence of a corrosive effect on the skin.
No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

Other effects:

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

Individual Skin Irritation Scores

Time after exposure (hours)	Animal
	798		796		799
	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
1	0	0	0	0	0	0
24	0	0	0	0	0	0
48	0	0	0	0	0	0
72	0	0	0	0	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

No skin irritation was caused by 4 hours exposure to the test substance.
Based on these results the test substance does not have to be classified and has no obligatory labelling requirement for skin irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments);
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 to 10 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Centre Lago, 01540 Vonnas, France
- Age at study initiation: between 22 and 25 weeks
- Weight at study initiation: at least 1.5 kg
- Housing: Animals were individually housed in labeled cages with perforated floors (dimensions 67 x 62 x 55 cm) and shelters (dimensions 40 x 32 x 23 cm).
- Diet (e.g. ad libitum): Pelleted diet for rabbits approximately 100 grams per day. Hay and wooden sticks were available during the study period
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days

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

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 58.3 mg (range 58.1 – 58.4 mg) (a volume of approximately 0.1 mL)

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 females

Details on study design:

Study design
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner one week later, after considering the degree of eye irritation observed in the first animal.

Preemptive pain management
One hour prior to instillation of the test substance, buprenorphine 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test substance, two drops of the topical anesthetic alcaine 0.5% were applied to both eyes.

Treatment
Immediately after the 1 hour observation, the treated eyes of two animals (806 and 808) were rinsed with approx. 50 mL tepid tap water, using a velocity of flow which did not affect the eye, to remove any visible residual test substance. For reference control the other eye was also rinsed.
Immediately after the 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.
Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg were administered by subcutaneous injection.
After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%.

Observations
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.
- Scoring system: Draize

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

ca. 0.56

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: One animal fully reversible within 48 hours, the other two fully reversible within 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: mean of 24, 48 and 72 hours

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Two animals appeared chemosis at 1 hour

Irritant / corrosive response data:

Instillation of approximately 58 mg of test substance (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in one animal and in 72 hours in the other two animals.
No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.
There was no evidence of ocular corrosion.
Remnants of the test substance were present in the eye on Day 1.

Other effects:

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

Individual eye irritation scores

Animal	Time after application	Cornea opacity	Iris	Conjunctivae
				Redness	Chemosis
806	1 hours 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	2 1 1 0	1 0 0 0
807	1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	2 1 1 0	0 0 0 0
808	1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	1 1 0 0	1 0 0 0

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results, the test substance does not have to be classified and has no obligatory labelling requirement for eye irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments),
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion

Two studies are available:

1) An acute dermal irritation study was conducted according to OECD 404 using rabbits (Latour, 2014). Key study.

This study showed that the test substance is not irritating.

2) An in vitro skin corrosion study was conducted according to OECD 431 using a human skin model (Buskens, 2014). Supporting study.

In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a human skin model test was performed. This study showed that the test substance is not corrosive. Based on this, in vivo skin irritation study in rabbit was performed to establish the possible skin irritating properties of the test substance.

Eye irritation

Two studies are available:

1) An acute eye irritation study was conducted according to OECD 405 using rabbits (Latour, 2014). Key study.

This study showed that the test substance is slightly irritating.

2) An in vitro BCOP study was conducted according to OECD 437 using bovine cornea (Verspeek-Rip, 2014). Supporting study.

In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, an in vitro BCOP test was performed. This study showed that the test substance is not irritating. Based on this, in vivo eye irritation study in rabbit was performed to establish the possible eye irritating properties of the test substance.

Justification for selection of skin irritation / corrosion endpoint:
This in-vivo study was conducted according to OECD 404 using rabbits.

Justification for selection of eye irritation endpoint:
This in-vivo study was conducted according to OECD 405 using rabbits.

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

Skin irritation/corrosion: Mean scores at 24, 48 & 72 hours for erythema were < 2.3 (actual value 0) for oedema were < 2.3 (actual value 0). In addition all reactions were fully reversible within the observation period.

Serious eye damage/eye irritation: Mean scores at 24, 48 & 72 hours for corneal opacity were < 1 (actual value 0), for iritis were < 1 (actual value 0), for conjunctival redness were < 2 (actual value 0.56) and for conjunctival oedema were < 1 (actual value 0).

Therefore in accordance with Regulation (EC) No. 1272/2008 Tables 3.2.2 & 3.3.2 the substance is not classified for the skin corrosion/irritation and serious eye damage/eye irritation endpoint.