1-benzhydrylpiperazine

Endpoint summary

Description of key information

Skin irritation:

In a K1 in vivo skin irritation study in New Zealand White rabbits according to OECD Guideline 404 and EU Method B.4, T000750 was observed to be non-irritating to the skin.

Eye irritation:

In a K2 Bovine Corneal Opacity and Permeability test, performed according to the guideline INVITTOX Protocol no. 98 and BCO-P SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997, T000750 induced serious eye damage and based on these results, it should be classified for eye damage category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the criteria of the CLP regulation (EC) No 1272/2008

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:

2006-10-19 to 2006-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented GLP compliant study, performed according to OECD test guideline 404 and EU Method B.4. No deviations were recorded that influenced the result of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

test item was put on a surgical gauze patch of approx 4x4 cm (instead of 6 cm²) to guarantee good contact

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

test item was put on a surgical gauze patch of approx 4x4 cm (instead of 6 cm²) to guarantee good contact

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV, Kreuzelweg 53, NL-5961 NM Horst/The Netherlands, Postbus 6174, NL-5960 AD Horst/The Netherlands
- Age at treatment: 14 weeks (male) and 11 weeks (females)
- Weight at study initiation: male: 2730 g and females 2511 and 2527 g
- Housing: standard laboratory conditions; individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks 4642 (batch no 77/05, Provimi Kliba AG) were provided for gnawing.
- Diet (e.g. ad libitum): pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no 51/06) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.
- Water (e.g. ad libitum): community tap water from Füllinsdorf, ad libitum
- Acclimatization period: 2006-10-19 to 2006-10-23 (one female); 2006-10-19 to 2006-10-24 (one male and one female); under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70%
- Air changes (per hr): approximately 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light/ 12 h dark. Music was played during the daytime light period.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Remarks:

purified water to moisten

Controls:

not required

Amount / concentration applied:

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

VEHICLE
- Concentration (if solution): 0.5 g was moistened with approximately 0.5 mL of purified water

Duration of treatment / exposure:

4 hours

Observation period:

7 days after treatment
Skin observation time points and termination:
The skin reaction was assessed at approximately 1, 24, 48 and 72 hours, as well as 7 days after exposure

Observations: viability/mortality and clinical signs: daily from acclimatization of the animals to the termination of test; body weights: at start of acclimatization, on the day of application and at termination of observation

Number of animals:

3 (1 male, 2 females)

Details on study design:

TEST SITE
- Area of exposure: left flank
- % coverage: no data
- Type of wrap if used: 0.5 g of T750 was placed on a surgical gauze patch (ca. 4cm x 4 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): flushed with lukewarm tap water
- Time after start of exposure: 4 hours

SCORING SYSTEM: grading of skin reaction according to Commission Directive 2004/73/EC, April 29, 2004. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately.

Irritation parameter:

erythema score

Basis:

animal: male

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal: female

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal: female

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks:

7 days

Irritation parameter:

edema score

Basis:

animal: all : 1 male and 2 females

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Irritant / corrosive response data:

A well-defined erythema was noted at the application site of two animals one hour after test item exposure whereas one animal expressed a very slight erythema at this reading. In addition, a very slight swelling was noted in two animals. A very slight erythema persisted up to the 48- or 72- hour reading in one and two animals, respectively. Scaling of the treated skin was observed in one animal 48 and 72 hours after the treatment.
No abnormal findings were observed on the treated skin of any animal 7 days after treatment, the end of the observation time.
Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.

Other effects:

Coloration: no staining produced by the test item of the treated skin was observed.
No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
The body weights of all rabbits were observed to be within the normal range of variability.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not induce significant or irreversible damage to the skin. T000750 is considered to be 'not irritating' to rabbit skin according to the referred classification criteria (Commission Directive 2001/59/EC of August 2001). Based on the criteria in the CLP Regulation, the test item should not be classified for skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-06-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well documented study performed according to following guideline: INVITTOX Protocol no. 98 and BCO-P SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997. Minor deviations to the protocol have been observed.

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol n. 98 "Bovine Corneal Opacity and Permeability Assay" (dated Feb 1994)

Deviations:

yes

Remarks:

Test substance concentration 10% i.s.o. 20% due to unabillity to create a homogenous solution/suspension.

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Deviations:

yes

Remarks:

Test substance concentration 10% i.s.o. 20% due to unabillity to create a homogenous solution/suspension.

GLP compliance:

not specified

Species:

other: bovine corneas

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Test system: freshly isolated bovine cornea
Source: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland

Collection of bovine eyes:
Freshly isolated bovine eyes were collected from the abattoir. After excess tissue was removed from the excised eyes, they were stored at room temperature in Hank's Balanced Salt Solution containing penicillin/streptomycin and then transported for further preparation. The eyes were used immediately after delivery in the laboraotry and within four hours after slaughtering.

Preparation of corneas:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity, and scratches were discarded. Each cornea was dissected from the eye using scalpel and rounded scissors. A rim of about 2-3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for the defects listed above.
Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium over night in a refrigerator at about 4°C. The preservation medium was composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Shortly before use, dextran was added.
Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching had to be avoided. After the anterior part of the holder was positioned on the top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. The posterior compartment had to be filled to return the cornea to its natural convex position. Care must be taken to assure no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated for about one hour at 32°C +/- 2°C in a water-bath.
At the end of the incubation period, the medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0min). For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged.

Vehicle:

physiological saline

Remarks:

natrium chloratum 0.9%

Controls:

other: negative control: saline; positive control: imidazole

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 10%
Until administration, the solution was stirred with a magnetic stirrer

VEHICLE
- Concentration (if solution): 0.9% sodium chloride in water

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

After the test item was rinsed off from the application side by changing cMEM several times until precipitates of the test item could be observed no longer, fresh cMEM was replaced in both compartments and opacity was measured (240min). To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step.

Number of animals or in vitro replicates:

9 bovine eyes in total (3 test, 3 negative controls, 3 positive controls).

Details on study design:

Preparation of the test item solution:
The test item was tested at a concentration of 10% in saline. Strong stirring with a magnetic stirrer resulted in a solution. Until administration, the solution was stirred with a magnetic stirrer.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): by rinsing with "complete minimum essential medium" (cMEM).
- Time after start of exposure: 240 minutes

OPACITY MEASUREMENT:
After recording the basal opacity of all corneas, the mean value of all corneas was calcualted. No cornea deviated from this by more than +/-3 units and no cornea was discarded. Sets of three corneas were used for treatment with the test item, the negative and positive controls, respectively.
Medium was completely removed from the anterior compartment and replaced by the test item, positive or negative control. The anterior compartment was plugged. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and will be incubated in a horizontal positioning a water-bath at 32°C +/- 2°C.

PERMEABILITY DETERMINATION:
Following the opacity readings after treatment, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32°C +/-2°C. Medium from the posterior compartment was removed with a 5 mL syringe, well mixed and transferred to a cuvette of 10 mm path length and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

In vitro score calculation:
The following formula was used to determine the in vitro score:
in vitro score = opacity value + (15 x OD490 value)
The in vitro score was calculated for each individual treatment and positive control cornea. The in vitro score value of each treated group was calculated from the individual in vitro score values
negative control:
in vitro score = opacity value + (15 x OD490 value)
Positive control and test item cornea:
in vitro score = corrected opacity value + (15 x corrected OD490 value)
Depending on the score obtained, the test item was classified into one of the following categories:
in vitro score 0 - 3: non eye irritant
in vitro score 3.1 - 25: mild eye irritant
in vitro score 25.1 - 55: moderate eye irritant
in vitro score 55.1 - 80: severe eye irritant
in vitro score > 80.1: very severe eye irritant

Irritation parameter:

in vitro irritation score

Value:

55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 6.2

Irritation parameter:

cornea opacity score

Value:

26.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 7

Irritation parameter:

other: permeability score

Value:

1.888

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 0451

Other effects / acceptance of results:

Treatment of the corneas with the test item resulted in a mean in vitro score of 55.0 +/- 6.2 after 240 minutes incubation, ranging from 49.1 to 61.4. The net value of the opacity score ranged from 19.3 to 33.3, the mean value was 26.7 +/- 7.0. The mean corrected permeability value of the corneas was 1.888 +/- 0.451, ranging from 1.448 to 2.348.
The in vitro score of saline , used as negative control was 2.1 +/- 1.4 (0.5 to 3.2) with the mean opacity value of 0.7 +/- 0.6 (0 to 1) and the mean permeability value of 0.096 +/- 0.057 (0.035 to 0.148).
The in vitro score of the positive control (imidazole, 20% dissolved in saline) was 82.8 +/- 13.4 (71.4 to 97.5) confirming the validity of the study. The corrected mean value of the opacity was 52.3 +/- 11.8, ranging from 39.3 to 62.3. The corrected mean value of the permeability was 2.031 +/-0.381, ranging from 1.608 to 2.347.

Before starting the permeability test, the dye solution sodium fluorescein was checked for its quality. The dye solution is valid for use, if a dilution of the stock solution containing 10 µg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.697.

According to the results obtained in this experiment, the test was considered acceptable.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The in vitro score of the test item was found to be 55.0 +/- 6.2. According to the in vitro irritation scale stated in the INVITTOX Protocol: Under the given test conditions the test item T750 is considered to be a moderate to severe eye irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

TalviOja (2007) investigated acute dermal irritation of T000750 in New Zealand White rabbits (3 rabbits after 4 hours of exposure to 0.5 g of test item). Skin reactions were recorded 1, 24, 48 and 72 hours after administration and scored according to the Draize scale.

No evidence of skin irritation was noted. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP Regulation (EC) No 1272/2008.

In addition, a Transcutaneous Electrical Resistance Test was performed by Meurer K (2006) to investigate the skin corrosion potential of T000750 in an in vitro test system using isolated skin discs from rats (24 hours of exposure to 149.2, 130.2, and 104.0 mg of test item). Based on the results described in the draft study report of the rat skin transcutaneous electrical resistance assay and the expert report, it is concluded that T000750 did not corrode rat skin under the experimental conditions reported. T000750 is considered to be non-corrosive in the TER test.

Eye irritation:

Deparade (2006) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 750 μl of T000750 (10% w/v suspension in physiological saline) was applied for 240 minutes. Both opacity and permeability were measured and the resulting objective values were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). A mean in vitro irritancy score of 55.0 (49.1 to 61.4) was calculated after 240 minutes of treatment. Since the test item induced an IVIS ≥ 55, it is concluded that according to the in vitro irritation scale stated in the INVITTOX Protocol, T000750 is considered to be a moderate to severe eye irritant and should be classified category 1 according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Justification for classification or non-classification

Skin irritation:

According to the in vivo acute dermal irritation study , no evidence for skin irritation was noted for T000750. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP Regulation (EC) No 1272/2008.

Eye irritation:

According to the in vitro eye irritation study (INVITTOX Protocol no. 98 and BCO-P SOP of Microbiological Associates Ltd., UK, Procedure Details April 1997), T000750 should be classifed for eye damage category 1.