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

Endpoint summary

Description of key information

The results from the acute oral and acute dermal toxicity studies indicated that there was no indication of toxicity. The results from the acute inhalation toxicity study indicated that there was a positive indication of toxicity.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 October 2015 to 23 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17th Dec 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA 712-C-98-190 (1998)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Age of animals at dosing: Young healthy adult rats, 8 weeks old
Body weight at treatment: 190 – 203 g
Acclimatization period: 5 or 6 days

Husbandry
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 522/6
Housing: 3 animals / cage
Cage type: Type II polypropylene/polycarbonate
Bedding: Lignocel 3/4-S Hygienic Animal Bedding” produced by J. Rettenmaier & Söhne GmbH & Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study.
A copy of the Certificate of Analysis is retained in the archive at CiToxLAB Hungary Ltd.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.5 – 24.8 °C
Relative humidity: 30 – 68 %
Ventilation: 15 – 20 air exchanges/hour
Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and othernormal rodent activities.
The temperature and relative humidity were recorded twice daily during the study.

Food and Water Supply
Animals received ssniff SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (Batch No.: 930 3907, expiry date: December 2015), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 ml bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water 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 at CiToxLAB Hungary Ltd.

Animal Identification
Animals were individually identified using numbers written on the tail with an indelible marker pen. The numbers were given on the basis of CiToxLAB Hungary Ltd.' s Master File, for each animal allocated to the treatment groups. The cages were identified by cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1 % (w/v) methylcellulose solution

Details on oral exposure:

A single oral gavage administration was followed by a fourteen-day observation period. On the night before treatment, the animals were fasted. The food but not water was withheld during an overnight period. Animals were weighed just before treatment. The test item was administered by oral gavage in the morning. The food was returned 3 hours after the treatment.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

2 groups of 3 females

Control animals:

no

Details on study design:

Clinical Observations
Clinical observations were performed on all surviving animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body Weight Measurement
The body weights of the animals were recorded on the day before treatment (Day -1), on the day of the treatment (Day 0) and weekly thereafter, where possible. Moreover, the body weight of found dead animals was recorded at necropsy.

NECROPSY
Macroscopic examination was performed on all animals. The animals were sacrificed by exsanguination under pentobarbital anaesthesia (Euthanimal 40%; Lot No.: 1409236-06, Expiry Date: September 2017, Produced by: AlfasanNederland BV, Woerden, Netherlands). After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed. Macroscopic abnormalities were recorded.

EVALUATION OF THE RESULTS
The method used was not intended to allow the calculation of a precise LD50 value.
Clinical signs, body weight, body weight gain and gross macroscopic data were tabulated.

DEVIATION FROM THE STUDY PLAN
At the request of the Sponsor the test item was not ranked into classes of Globally Harmonized Classification System. This deviation has no impact on the results or integrity of the study.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

N-Boc-D-phenylglycinol caused mortality in one of six (1/6) animals at a dose level of 2000 mg/kg bw on Day 1.

Clinical signs:

other: Treatment with N-Boc-D-phenylglycinol at the dose level of 2000 mg/kg bw caused decreased activity, prone position, piloerection, cold body temperature (whole body is cold to touch) and decreased respiratory rate in all animals (6/6) on Day 0. In addition

Gross pathology:

In the dead animal dark/red discoloration of the non-collapsed lungs was visible. It is considered to be typical agonal or post mortem change. In the surviving animals no macroscopic observations were present after termination (Day 14).

Interpretation of results:

study cannot be used for classification

Conclusions:

At the request of the Sponsor the test item was not ranked into classes of Globally Harmonized Classification System. This deviation has no impact on the results or integrity of the study.

Under the conditions of this study, the acute oral LD50 value of the test item N-Boc-D-phenylglycinol was found to be above 2000 mg/kg bw in female CRL:(WI) rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 05 2018 to March 14 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

The Guidelines for the testing of chemicals "Acute Inhalation Toxicity-acute class method" (436) published by the Ministry of Enviornmental Protection of People's Republic of China in the year of 2013/
The classification is according to the Globally Harmonized System of Classification and Labeling of Chemicals (seventh edtion, 2017).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species: Rat
Strain: Sprague-Dawley
Grade: SPF
Supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal Production License: SCXK. (Jing) 2016-0006
Animal Certificate No.: 11400700307125
Justification of test system: According to Acute Inhalation Toxicity of the MEP Guideline for Testing of Chemicals rat is the preferred strain for the heredity characters, stability, and available background data.
Number of animals: 6 animals (3 male and 3 female) were ordered and used. The females were nulliparous and non-pregnant.
Age and body weight: When exposure the age was between 63~69days and the body weight was between282~294g for male rats and 247~26lg for female rats. The body weights were within ±20% of the mean weight for each sex when exposed.

Physical examination and acclimatization
A physical examination, weighing and marking on the hair and cage card identifying was made in 24 hours after animals' arrival. After the physical examination, 12 days acclimatization period started. Animals were acclimated to the restraining tubes twice prior to dosing in order to minimize stress and uncomfortableness for restraining tubes. The first pre-adaption was about lh. The second pre-adaption was about 2 hours. No abnormalities were found during both restrainings. One or two animals were paired housed per cage during the acclimatization period.

Test conditions
Husbandry: Animals were housed in Room A120-l of the facility. Animals were raised in suspended, stainless steel cages (L32.0cm xW28.0cmxH20.0cm) on cage racks (L167.0cmxW70.0cmxH17l.0cm). There were 10 cages per layer, and 4 layers per rack. Animals were housed individually after exposure.
Environmental controls: The temperature and humidity were automatically controlled and recorded. The target value of animal room temperature was 19 C - 25 C, of the relative humidity was 40% ~ 70% and the light cycle was 12 hour light and
12 hour dark.
Food and water: Animals were provided with rodent complete nutrition pellet diet supplied by Beijing keaoxieli Feed CO., LTD. Analysis report of diet was provided by the supplier. Water was purified using the HT-RO 1000 purity system. Drinking water was routinely analyzed. Diet and drinking water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. During the test, diet and water were available to the animals ad libitum except for exposure.
Animal welfare: The animal use for this study complies with the national animal welfare laws and regulations (instructive notions with respect to caring for laboratory animals) (2006, PRC Ministry of Science).The animal care and use activities required for the conduct of this study were reviewed and approved by the testing facility Animal Care and Use Committee (lACUC).

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

not specified

Mass median aerodynamic diameter (MMAD):

1.97 µm

Geometric standard deviation (GSD):

1.59

Remark on MMAD/GSD:

Aerosol Instrument for Aerodynamic Particle Sizer (APS) 3321 was used to asses the particle size distribution of the test atmosphere.

Details on inhalation exposure:

Equipment: HOPE-MED 8052H dynamic snout only dust inhalation instrument was used.

Atmosphere Generation System: The test item was aerosolized using a stainless steel aerosol generation system. The test item was infused into generation system through peristaltic pump and mixed with compressed air. Target concentration was achieved by adjusting air flow rate and pump infusion velocity.

Exposure Method
Test item preparation: Test item had been grinded with 10% carbon-white. Before exposure, each rat was restrained in a confined transparent polycrylic tube. The exposure tubes were installed in the portholes of the inhalation chamber and the chamber was sealed up. Filtered and compressed air was mixed with quantitative test item and aerosol was sent to exposure chamber(0.04m3). The test item moving speed and exposure airflow rate had been adjusted. The aerosol had been continuously generated from generation system on the top of the chamber with an aerosol producer. A slight negative pressure was maintained in outer plenum of chamber to prevent leakage of the test substance into the surrounding area. The exhausted air was removed from the outlet at the bottom of the chamber to absorption unit. Equipment diagram is shown in appendix Figure 2.
Concentration trial: Before commencement of the exposure, technical trial had been conducted (without animals) using the inhalation system. The two concentrations' error were fallen within +20%, so the exposure had been done.

Exposure Duration: 4 hours in rats.

Monitoring of Exposure Conditions
The actual concentrations, particle size distribution at the animals' breathing zone, chamber airflow, chamber temperature, relative humidity and oxygen concentration were determined during exposure period. Detection frequency and methods see below table 2.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

2970 mg/m^3

No. of animals per sex per dose:

3 males and 3 females

Control animals:

no

Details on study design:

Clinical Observation
Clinical observations were recorded once during the exposure and twice with more than 30 minutes interval after exposure on the exposure day and then once daily for up to end observation.
Observation and record was conducted including animal fur changes, eyes, and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behavior patterns. Attention was directed to tremors, convulsions, salivation, diarrhoea, lethargy, sleep, and coma.

Body Weights
The animals were weighed in the first 24 hours after arrival, on the day of exposure prior to exposure (day 0), and on day 1, day 3, day 7 and day 14.

Necropsy and Histopathology
All surviving animals were dissected at the end of the study after anesthetizing with ether inhalation and killed by bloodletting. Nose, pharynx, larynx, trachea, and lung were examined. The necropsy included following examinations such as the external features of the carcass, external body orifices, the abdominal, thoracic, and their contents of all animals, and the location, size, hardness, and the color. No abnormalities were found in female and male animals at the gross necropsy, so histopathological examination was not performed.

Evaluation of Data
The number of animals in each group, the symptoms, the gross anatomy of the surviving animals and the frequency of various types of lesions were counted separately. The incidence of the above items in different groups for different sex and the mean and standard deviation of body weight at different times were calculated .
The inhalation toxicity LC50 range was found. According to GHS criteria for the acute inhalation toxicity (as shown in table 3 below) the test item category was given. Because unit of LC50 was mg/m3, the LC5o divided by the 1000 was converted to mg/L units when test item classification was conducted.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2 970 mg/m³ air (nominal)

Based on:

test mat.

Remarks on result:

other: 2970+/-67 mg/m³

Mortality:

No animals were found dead during the test period.

Clinical signs:

other: none observed

Body weight:

The body weight of male and female animals showed increased trend during the observation period.

Gross pathology:

No abnormalities were found in female and male animals at the gross necropsy.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Based on the results, the LD50 in SD rats for Boc-Phenyl-Glycinol is more than 2970+/-67 mg/m³ and it is classified to 4 or more than 4 according to GHS classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

2 970 mg/m³ air

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2015 to 23 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

OECD Guidelines for Testing of Chemicals (No.: 402, 24th Feb 1987)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

Commission Regulation (EC) No 440/2008, B.3 (L142, 30 May 2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Version / remarks:

OPPTS 870.1200 (EPA 712-C-98-192, August, 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: CRL:(WI) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
Hygienic level at arrival: SPF
Hygienic level during
the study: Standard housing conditions
Justification of strain: The Wistar rat is one of the standard rodent species used in acute toxicity studies
Number of animals: 5 animals/sex
Sex: Male and female, female rats were nulliparous and non-pregnant.
Age of animals at study start: Young adult rats
Body weight range at dosing: Between 220 g and 252 g
Acclimatization time: 6 days

Husbandry
Animal health: Only healthy animals were used for the study. The veterinarian certified the health status.
Room-Box: 242/2
Housing: Individual caging
Cage type: Type II. polypropylene/polycarbonate
Bedding: Lignocel ¾S Hygienic Animal Bedding (produced by J. Rettenmaier & Söhne GmbH & Co.KG, Germany) was available to animals during the study.
A copy of the Certificate of Analysis is retained in the archives at CiToxLAB Hungary Ltd.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.3 – 24.8 °C
Relative humidity: 32 – 64%
Ventilation: 15-20 air exchanges/hour
Enrichment: Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.
Temperature and relative humidity were recorded twice daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum, and tap water from the municipal supply, as for human consumption from a 500 ml bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study and the water was considered fit for human consumption.
The batch of feed employed in the study was as follows:
 930 3907, expiry date: December 2015
The supplier provided an analytical certificate for the batch used. Copy of the certificates will be archived with the raw data.
Water 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 at CiToxLAB Hungary Ltd.

Animal Identification
The individual identification was performed using numbers written on the tail with a marker pen. The numbers were given on the basis of CiToxLAB Hungary Ltd.' s Master File for each animal allocated to the treatment groups. The cages were identified by cards containing information about study code, sex, dose group, cage number and individual animal number.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

A single dermal application was made and was followed by a fourteen-day observation period. Sufficient water was used to dampen the test material to ensure good contact with the skin.

Procedure
The back of each animal was shaved (approximately 10 % area of the total body surface) approximately 24 hours prior to treatment. The test item was applied as a single dose, moistened with water to the shaved skin and remained in contact with the skin for the 24-hour exposure period. Sterile gauze pads were placed on the skin of rats to cover the test item. These gauze pads were kept in contact with the skin using a patch with adhesive hypoallergenic plaster. The entire trunk of the animal was then wrapped with semi occlusive plastic wrap for 24 hours.
At the end of the exposure period, the area of skin treated with the test item was washed with water of body temperature.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw of the test item

No. of animals per sex per dose:

2 groups, 1 with 5 males, 1 with 5 females

Control animals:

no

Details on study design:

Clinical Observations
Clinical observations were performed on the day of treatment at 1 and 5 hours after application of the test item and once each day for 14 days thereafter. Observations included the skin and fur, eyes and mucous membranes, the respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Skin Irritation
Adverse skin reactions at the site of application were recorded daily following the removal of the dressing.

Measurement of Body Weight
The body weights were recorded on Day 0 (before test item administration) and on Days 7 and 14 just before necropsy.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

Test item did not cause mortality at the dose level of 2000 mg/kg bw.

Clinical signs:

other: There were no systemic clinical signs noted in any animal throughout the study.

Gross pathology:

No macroscopic observations were noted at a dose level of 2000 mg/kg bw.

Other findings:

No local dermal signs were observed after treatment with the test item during the 14 days observation period.

Interpretation of results:

study cannot be used for classification

Conclusions:

At the request of the Sponsor the test item was not ranked into classes of Globally Harmonized Classification System. This deviation has no impact on the results or integrity of the study.

The acute dermal median lethal dose (LD50) of the test item N-Boc-D-phenylglycinol was found to be greater than 2000 mg/kg body weight in male and female CRL:(WI) rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw