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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12th June 2018 to 29th November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: The Guidelines for the Testing of Chemicals (the second edition), 421: Reproduction/Developmental Toxicity Screening Test, State Environmental Protection Administration )2013).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

600-273-1

Cas Number:

102089-74-7

Molecular formula:

C13H19NO3

IUPAC Name:

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

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Grade SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Technology Ltd.
- Age at study initiation: (P) - 56-62 days
- Weight at study initiation: (P) Males: 280 - 330g; Females: 190 - 230g;
- Housing: Suspension stainless steel cages on cage racks, 2 rats per cage at most, with corn cob bedding. During mating, the rats were housed in mating cages. After mating, females were housed in plastic cages, with a bedding of wood shaving.
- Diet: Sterilised growth and breeding feed from Beijing Keao Xiele Feed Co. ad libitum
- Water: Drinking water ad libitum
- Acclimation period: at least 10 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 40-70
- Photoperiod: (12 hrs dark / 12 hrs light)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed and placed in a jar, and corn oil added to give the required concentration. The jars were then mixed with the magnetic stirring apparatus for 1 minute. The test item was prepared daily.
VEHICLE
- Amount of vehicle: 5ml/kg bw

Details on mating procedure:

M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD0)
- After successful mating each pregnant female was caged in a plastic cage

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks of mating;
Up to day before scheduled kill, (except during childbirth)

Frequency of treatment:

Once daily in the morning, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Male and Female

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Male and Female

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Male and Female

No. of animals per sex per dose:

14

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily - morbidity and mortality
- Time schedule: Daily
- Cage side observations checked included: appearance, fur, activity, reaction, breathing, posture, excrement and urine.

DETAILED CLINICAL OBSERVATIONS: Yes, with handling
- Time schedule: Once per week in conjunction with weighing.
Any abnormality of head/neck including eyes, ears nose and mouth, back, abdomen, anus, skin colour around perineum, muscle tension, any trauma or tumour.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during pre-mating and mating period
Pregnant rats were weighed GD0, GD7, GD14 and GD20.
Prenatal rats and pups weighed PND0 and PND4.
Mother rats weighed once per week in order to adjust the dose.
All animals weighed on the day of the scheduled kill.

FOOD CONSUMPTION: The food ration was added weekly and food intake weighed the following day.

PARTURITION: Yes
- From GD21, observations made twice daily (morning and afternoon). Any difficulties were recorded.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1offspring (PND0):
stillbirths, live births, postnatal mortality, presence of grossly malformed pups, grossly puny pups.
PND4: signs of behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals at the end of the mating period.
- Maternal animals and non-mated females: All surviving animals - PND4 of the last of the parturition.

GROSS NECROPSY
- Gross necropsy consisted of external body orifices and internal examinations including the thoracic, cranial and abdominal cavities and their contents. The location, size, hardness and colour of abnormal findings were recorded. Special attention was paid to the reproductive organs. The number of implantation sites in the uteri were recorded for parous females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues - testes and epididymus were weighed.

Statistics:

Single factor analysis of variance and if there was significant difference, followed by Dunnett's test. The data from the clinical observations were validated by X2 test. The incidences of gross necroscopy and pathological findings were analysed by the unilateral Fischer's exact probability test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

6 males in the mid-dose group and 14 males in the high-dose group had soiled and damp perineal regions. The symptom incidence had a statistically significant increase compared to the control group (p ≤ 0.05 or p ≤ 0.01).

There were 5 females with soiled or damp perineal regions, 1 female with tumour and 2 females with dehairing in the high-dose group with later recovery. The symptom incidence had a statistically significant increase compared to the control group (p ≤ 0.01).

There was 1 mid-dose female with a soiled and damp perineal region, with later recovery. This was not statistically significant compared to the control group (p > 0.01).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the pre-mating and mating period, the mean body weight and body weight gain of all males in all dose groups had no statistically significant difference compared to the solvent control group (p > 0.01).
During the pre-mating period, there was no significant difference in females for body weight and body weight gain observed compared to the solvent control group (p > 0.01).
During the gestation period, the body weight gain of pregnant rats was higher than the pregnant rats in the control group. The body weight in the high-dose group at GD7, GD14 and GD20 was a statistically significant increase compared with the solvent control group (p ≤ 0.05 or p ≤ 0.01).

The body weight in the low-dose group in the second trimester of pregnancy (GD7 and GD14) had a statistically significant increase compared with the solvent control group (p ≤ 0.01) but the body weight gain during pregnancy was not significant compared to the solvent control (p>0.05). During the lactation period, there was no significant difference in mother rat body weight and body weight gain in all dose groups compared with the solvent control group (P>0.05)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the pre-mating period, the total food consumption of males in the high-dose group had a statistically significant increase compared to the control group (p ≤ 0.01). There was no significant difference in food consumption of males in the mid- and low-dose groups compared to the solvent control (p> 0.05).

During the pre-mating period, the mean food consumption of females in the first week in the high-dose group had a statistically significant increase compared to the control group (p ≤ 0.01) and the total food consumption of females in the high-dose group had a statistically significant increase compared with the solvent control group (p ≤ 0.01). No significant difference in food consumption of females was observed in the mid- and low-dose groups compared to the solvent control (p> 0.05). The food consumption of pregnant rats in all dose groups in the first 2 weeks had a statistically significant increase compared to the solvent control (p ≤ 0.05 or p ≤ 0.01) but there was no significant difference in the third week (p> 0.05). There was no significant difference in food consumption of the dams during the lactation period in any dose group compared to the solvent control (p> 0.05). According to these findings, the test item may activate an increase in food consumption but no dose dependent adverse effect was observed, so it was considered that the results had no correlation with test item toxicity.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were 14, 13, 14 and 14 mated rats in the solvent control and 100, 300 and 100 mg/kg.d respectively. There were 14, 13, 14 and 14 mated female rats and 14, 12, 14 and 13 pregnant female rats. All pregnant rats had live pups and 4, 1, 1 and 0 pregnant rats had at least one stillborn pup respectively. No pregnant rat died from dystopia and none had all stillbirths. There was no significant difference in the male mating index and fertility index compared to the solvent control (p > 0.05). The mean mating time of females in the low- and high-dose groups had a statistically significant increase compared to the control group (p ≤ 0.05) but not more than 4 days in total. This is in accordance with the normal physiological features of the Sprague-Dawley rat, so the result was not considered to be caused by the test item. There was no significant difference in gestation days in any dose group compared with the solvent control (p> 0.05). There was no significant difference in female mating index, fertility index and gestation index in all dose groups compared with the solvent control group (p > 0.05).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

2

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

3

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

4

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The stillbirth number on PND0 in the high-dose group had a statistically significant decrease compared with the control group (p≤0.05) but had no toxicological significance. The live birth index had a statistically significant increase compared with the control group (p≤0.05 or p≤0.01) but had no toxicological significance. All pups in 1, 1, 2 and 0 litters in the solvent control groups and 100, 300 and 1000 mg/kg.d respectively died during the periods from birth to PND4 and the pup viability index in the mid-dose group (p≤0.05) but no significant difference in the high-dose group was found and the result was absence of a dose relation among the dose group, so it was considered the decrease in the pup viability index in the mid-dose was caused by individual litter data and was not an adverse effect of the test item. There was no significant difference in average implantation number, live births number, live pup numbers on PND4, post implantation loss, sex ratio compared with solvent control group (P>0.05).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average litter weight in the high-dose group had a statistically significant increase compared with the control group in PND0 (p≤0.05) but had no toxicological significance. No significant difference of average litter weight in the high-dose group was found compared with the solvent control group in PND4 (p>0.05). There was no significant difference in average litter weight in low- and mid-dose groups compared with the solvent control group (p>0.05) on PND0 and PND4. All pups of animal 2212 were puny compared with the solvent control group and dead before PND4, no malformation or significant abnormalities in other live pups were observed from birth to study ending. This was considered accidental and had no correlation with the test item toxicology.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Exposure of rats to different doses of Boc-phenyl glycinol by oral gavage resulted in an incidence rate of soiled and damp perineal region for males in the 1000 and 300 mg/kg.d dose groups. This was a statistically significant increase compared with the control group and had dose correlation, so it was considered that the test item had some general toxicity to males at these dose levels. At the same time, the incidence rate of soiled and damp perineal region for females in the 1000 mg/kg.d group had a statistically significant increase compared with the control group, so it was considered that the test item had some general toxicity to females at that dose level.
For fertility and development, there was no adverse effect on the fertility of males and females in the 1000 mg/kg.d group. There was no adverse effect on the growth and development so it was considered that Boc-phenyl glycinol had no obvious fertility and developmental toxicity for pups at 1000 mg/kg.d dose level.
Based on the above results, it is considered that the No Observed Adverse Effect Level (NOAEL) for general toxicity to males for oral exposure to Boc-phenyl glycinol is 100 mg/kg.d; the Lowest Observed Adverse Effect Level (LOAEL) for general toxicity to males is 300 mg/kg.d; the NOAEL for general toxicity to females is 300 mg/kg.d.; the LOAEL is 1000 mg/kg.d.; the NOAEL for reproduction toxicity to parent rats for oral exposure to the test item is 1000 mg/kg.d.; the NOAEL for developmental toxicity to pups for oral exposure to the test item is 1000 mg/kg.d

Executive summary:

This study was conducted to produce initial information on the possible effects on reproduction/development toxicity of Boc-phenyl glycinol following oral exposure in rats. It is also to be used as a dose range finding study for other reproduction/development studies. The test method was designed to be compatible with The Guidelines for the Testing of Chemicals (the second edition), 421: Reproduction/Developmental Toxicity Screening Test, State Environmental Protection Administration (2013). 

The study was conducted in SD rats, all SPF grade. Based on the results of the preliminary test for the repeated dose oral toxicity, three doses of 1000, 300 and 100 mg/kg.d and a concurrent solvent control were used in the study. There were fourteen male and fourteen female rats in each group. All animals were dosed during the two weeks prior to mating, the two week mating period and up to the day before the scheduled kill. The test was terminated four days after the birth of the last litter of pups. Clinical observations were made daily, and body weight and food consumption were weighed weekly. The reproduction/development parameters were evaluated at the same tine. All parental animals were examined macroscopically for any abnormalities and pathological changes. A histopathological examination was made to the reproductive organs of rats in the control group and the high-dose group. 

During the study, 6 males in the mid-dose group and 14 males in the high-dose group had soiled and damp perineal region. The symptom incidence had a statistically significant increase compared to the control group (p≤0.05 or p≤0.01). There were 5 females found with soiled or damp perineal region, 1 female with tumour and 2 females with dehairing in the high-dose group. The symptom incidence had a statistically significant increase compared to the control group (p≤0.01). There was 1 female with soiled and damp perineal region in the mid-dose group.  No abnormalities were found in females in the low-dose group. No deaths were observed in any of the dose groups or the solvent control group.

No abnormalities were found in females in the low-dose group. No deaths were observed in any of the dose groups or the solvent control group. The body weight of parent rats in all dose groups had no significant difference compared with the solvent control groups (p>0.05). During the pre-mating period, there was no significant difference in body weight observed for females in any dose groups compared to the solvent control (p>0.05). During the gestation period, the body weight of pregnant rats was higher than that of pregnant rats in the solvent control group universally, but was of no toxicological significance. 

During the pre-mating period, the total food consumption of male and female rats in the high-dose group had a statistically significant increase compared with the control group (p≤0.01). At the same time, the food consumption of pregnant rats in all dose groups in the first 2 weeks had a statistically significant increase compared with the control group (p≤0.05 or p≤0.01). It was considered that the test item may activate the increase in food consumption, but no dose-dependent adverse effect was observed. Therefore, it was considered that the results had no correlation with test item toxicity. 

There was no significant difference in either the male mating index or fertility index in all dose groups compared to the solvent control (p>0.05). There was no significant difference in the female mating index, fertility index, gestation index or live birth index in any dose groups compared with the solvent control group (p>0.05). The mean mating time of females in the low- and high-dose groups had a statistically significant increase compared with the control group (p≤0.05) but not more than 4 days in total, which is in accordance with the normal physiological features of the SD rat, therefore it is considered that the result was not caused by a test item observed effect. There was no significant difference in gestation days in all dose groups compared with the solvent control group (P>0.05). No pregnant rat died from dystopia and no litter had all stillborn pups. 

There was no significant difference in average implantation number, live births number, live pups number on PND4, post-implantation loss and sex ratio compared with the solvent control (P>0.05). The live birth index had a statistically significant increase compared with the solvent control group (p≤0.05 or p≤0.01) but had no toxicological significance.

The average litter weight in the high-dose group had a statistically significant increase compared with the control group at PND0 (p≤0.05) but had no toxicological significance. All pups of animal 2212 were puny compared with the solvent control group and all died before PND4. No malformations or significant abnormalities were observed in any other live pups from birth to the end of the study. The gross necroscopy inspection and pathological examination on animals revealed no significant difference found to be related to test item treatment.  

Exposure of rats to different doses of Boc-phenyl glycinol by oral gavage resulted in an incidence rate of soiled and damp perineal region for males in the 1000 and 300 mg/kg.d dose groups. This was a statistically significant increase compared with the control group and had dose correlation, so it was considered that the test item had some general toxicity to males at these dose levels. At the same time, the incidence rate of soiled and damp perineal region for females in the 1000 mg/kg.d group had a statistically significant increase compared with the control group, so it was considered that the test item had some general toxicity to females at that dose level.
For fertility and development, there was no adverse effect on the fertility of males and females in the 1000 mg/kg.d group. There was no adverse effect on the growth and development so it was considered that Boc-phenyl glycinol had no obvious fertility and developmental toxicity for pups at 1000 mg/kg.d dose level.
Based on the above results, it is considered that the No Observed Adverse Effect Level (NOAEL) for general toxicity to males for oral exposure to Boc-phenyl glycinol is 100 mg/kg.d; the Lowest Observed Adverse Effect Level (LOAEL) for general toxicity to males is 300 mg/kg.d; the NOAEL for general toxicity to females is 300 mg/kg.d.; the LOAEL is 1000 mg/kg.d.; the NOAEL for reproduction toxicity to parent rats for oral exposure to the test item is 1000 mg/kg.d.; the NOAEL for developmental toxicity to pups for oral exposure to the test item is 1000 mg/kg.d