Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

Endpoint summary

Description of key information

Based on the results from both the skin irritation/corrosion and eye irritation studies, there was no indication of irritation or corrosion.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2015 to 07 January 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Justification for test system used:

The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Details on test system:

Human Skin
EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.: 15-EKIN-040, Expiry Date: 12 October 2015) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin-SM Test Kits used in the present study) and are documented in Appendix 2.

Kit Contents
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 15 MAIN3 041; Exp. Date: 14 October 2015)
A flask of sterile “Assay Medium”
(Batch No.: 15 ESSC 041; Exp. Date: 14 October 2015)

Number of Replicate Wells
In this assay, two replicates per test item per time point were used. Two negative controls and two positive controls were also run in the assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.

Kit Reception
In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKIN-SM kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- 20 mg of test item was applied evenly to the epidermal surface of each of two test units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.6-23.9°C) covered with the plate lids.

Number of replicates:

two test units

Irritation / corrosion parameter:

other: optical density

Run / experiment:

Mean

Value:

0.831

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

93.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.889).
The positive control treated tissues showed 0.9% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 15.9%.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro EPISKIN model test with N-Boc-D-phenylglycinol, the results indicate that the test item is not corrosive to the skin.

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:

02 October 2015 to 07 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EC) No 1152/2010 ammending Regulation (EC) No 440/2008 (08 December 2010, B.48)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

Strain of chicken: COBB 500
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Dose: 30 mg bw
Positive Control: 30mg powdered Imidazole
Negative Control: 30 μL of physiological saline

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Number of animals or in vitro replicates:

4-5 heads

Details on study design:

Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

RETENTION OF CHICKEN’S EYES
Retention of chicken’s eyes
At the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin, Manufacturer: Reanal, Batch number: KTM10531, Expiry date: February 2017) was used for potential histopathology and stored at room temperature.

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean at up to 75 min

Value:

-0.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean at up to 240 min

Value:

-1.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum corneal opacity

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in each experiment. These experiments were considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these in vitro eye irritation assays in isolated chicken eyes with N-Boc-D-phenylglycinol (Batch number: LS-53-20150815), the test item is a non-irritant, UN GHS Classification: Non-classified

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Novermber 2015 to 26 January 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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

EXPERIMENTAL ANIMALS
Species and strain: New Zealand White rabbits
Source: S&K-LAP Kft.
2173 Kartal, Császár út 135, Hungary
Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined prior to starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
Number of animals: 3 animals
Age of animals at treatment: 16-17 weeks old (young adult)
Sex: Male
Body weight range on the day of treatment: 3658 g – 3988 g
Body weight range
before euthanasia: 3692 g – 4178 g
Date of receipt: 21 September 2015
Acclimatization time: at least 43 days
Animal identification: The individual identification was by engraved ear tag. The cages were marked with individual identity cards with information about study code, sex, dose, cage number and individual animal number.

HUSBANDRY
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 618
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature
during the study: 19.6 – 23.0 °C
Relative humidity
during the study: 31 – 79 %
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity values were measured continuously. The measured range was checked at least daily during the acclimatisation and experimental phases.

FOOD AND FEEDING
Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út 0399, Hungary, ad libitum. Animals were provided with the following batches:
• 0740 08 15, expiry date: 16 November 2015
• 0002796034, expiry date: 21 December 2015
The details of the diet used will be archived with the raw data and are not reported.

WATER SUPPLY AND QUALITY CONTROL OF WATER
The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system. The quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u. 36., Hungary). The quality control results are retained in the archives of CiToxLAB Hungary Ltd.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1g

Duration of treatment / exposure:

one hour

Duration of post- treatment incubation (in vitro):

none

Number of animals or in vitro replicates:

3 males

Details on study design:

Clinical Observations and Evaluation of Ocular Irritation
The eyes were examined at 1, 24, 48, 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded. At the end of the observation period, the animal was sacrificed by intramuscular injections of Ketamin 10% (Ketamidor) and Xylazin 2% (Primazin 2%) followed by i.v. pentobarbital sodium. Death was verified by checking pupil and corneal reflex and the absence of respiration.
All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.56

Interpretation of results:

GHS criteria not met

Conclusions:

The test item N-Boc-D-phenylglycinol, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 1 week.
According to Regulation (EC) No 1272/2008, N-Boc-D-phenylglycinol does not require classification as an eye irritant.
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, N-Boc-D-phenylglycinol does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available