N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jan - 12 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

EC Number:

701-177-3

Molecular formula:

C21H39NO3

IUPAC Name:

N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 62 - 69 days
- Weight at study initiation: 215.8 - 273.8 g
- Housing: individually in MAKROLON cages (type III plus) with granulated textured wood bedding (Granulat A2, J. Brandenburg, Goldenstedt/Arkeburg, Germany)
- Diet: Ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light):12 / 12

IN-LIFE DATES: From: 13 Jan To: 11 Feb 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous hydroxypropylmethylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared daily by dissolving appropriate amounts of the test material in 0.5% aqueous hydroxypropylmethylcellulose gel. Dosing solutions were administered within 4 h after preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is poorly water soluble. Therefore, an aqueous suspension of 0.5% hydroxypropylmethylcellulose gel was used as vehicle.
- Amount of vehicle: 10 mL/kg bw/day
- Lot/batch no.: 18D04-B03
- Supplier: Fagron Services B.V, Uitgeest, The Netherlands

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC with UV detection was employed to quantify the test item in the dose formulations. The analytical method was validated by LPT and reported in LPT Study No. 37647.
The homogeneity of the dose formulations was assessed on 21 and 24 January 2020 at the start of treatment, during (middle) administration and before administration to the last animal of the test item group. The concentration was analysed on 4 February 2020 during treatment before administration to the last animal of the group. The range of % nominal concentration was 93.8 - 105.9% for the homogeneity and 95.0 - 103.5% for concetration analysis.
In the method validation, also a stability assessment of the dose formulations was conducted. The samples of all storage conditions (24 h at room temperature and 7 or 64 days at -20 °C) met the acceptance criteria.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight, every night with the same male and female until proof of pregnancy was observed
- Verification of same strain and source of both sexes: yes, only sexually mature ('proved') male rats of the same breed served as partners.
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 6 to 20 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

15 days, until Day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Dose reduction to 750 mg/kg bw/day due to one mortality, poor health and pronounced reduction of body weight for several animals.

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

Dose reduction from 1000 mg/kg bw/day due to one mortality, poor health and pronounced reduction of body weight for several animals.

No. of animals per sex per dose:

25 P females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a foregoing range finding study, in which the test substance was administered orally to female rats at dose levels of 100, 300, 1000 mg/kg bw/day from Day 6 to Day 20 of pregnancy (LPT Study Report No. 37716). Adverse effects of reduced body weight, body weight gain and food consumption of the dams and reduced fetal weight and placental weight as secondary effects due to maternal toxicity were observed at 1000 mg/kg bw/day. Therefore, 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the main study.
- Fasting period before blood sampling for dam thyroid hormones: overnight
- Time of day for dam blood sampling: in the morning of day of sacrifice
- Blood samples collected from: retrobulbar venous plexus under isoflurane anesthesia
- Anesthesia: yes, isoflurane

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: viability (check for dead or moribund animals), behavioral changes, reaction to treatment, illness and the signs of abortion or premature delivery, appearance, change and disappearance of clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT AND BODY WEIGHT GAIN: Yes
- Time schedule for body weight examinations: first measurement on Day 0 of pregnancy, daily afterwards
- Time schedule for body weight gain examination: calculated in intervals (gestation day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily inspection of drinking water bottles

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: thyroid and gravid uterus including cervix were weighed, macroscopic examination of the internal organs of the dams were performed

OTHER: A macroscopic examination of the internal organs of the dams was performed

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes, incl. distributions in the uterine horns
- Number of implantations: Yes, incl. distributions in the uterine horns
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- No. of animals: 20 for the control, 100, and 300 mg/kg bw/day groups, 19 for the 1000/750 mg/kg bw/day group
- Plasma: No
- Serum: Yes
- Volume collected
: 900 µL (300 µL each for T3, T4 and TSH analysis)

Fetal examinations:

- No. of fetuses examined: 20 litters/group examined
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes
- Parameters examined:
- Macroscopic inspection (gross evaluation)
- Number of fetuses (alive and dead) and placentae (location in the uterus and assignment of fetuses)
- Sex and viability
- Number and size of resorptions
- Corpora lutea in the ovaries
- Implantations and location of fetuses in the uterus
- Weights of fetuses and weights of the placentae
- Ano-genital distance (AGD) of live fetuses
- External inspection for damages (especially malformations)
- Determination of number and kind of retardations, variations or malformations
- Comparison of external sex with internal (gonadal) sex
- Examination of incomplete testicular descent/cryptorchism in male fetuses

Statistics:

Parametrical data:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normalityof distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).

Indices:

Malformed fetuses:
Total malformation rate [%] = (malformed fetuses per group / fetuses per group) x 100

Fetuses with variations:
Total variation rate [%] = (fetuses per group with variations / fetuses per group) x 100

Fetuses with retardations:
Total retardation rate [%] = (fetuses per group with retardations / fetuses per group) x 100

Group indices:
Pre-implantation loss [%] = [(corpora lutea per group - implantations per group) / corpora lutea per group] x 100

Post-implantation loss [%] = [(implantations per group - living fetuses per group) / implantations per group] x 100

Indices per litter:
Pre-implantation loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group

Post-implantation loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Salivation (21/21), decreased water consumption at the beginning of dosing (4/21), increased water consumption during second half of dosing period (20/21), breathing sounds (6/21), clear discharge from vagina and anus (5/21), reduced motility (4/21), hemorrhagic nose/snout (4/21), soft feces (4/21) and piloerection (3/21); adverse.
100 and 300 mg/kg bw/day: Salivation (3/20 at 100 mg/kg bw/day, 11/20 at 300 mg/kg bw/day) only for short period; non-adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

1000/750 mg/kg bw/day: One animal prematurely sacrificed on GD 8 due to severe clinical signs (gasping, severe salivation and a hemorrhagic nose/snout).
100 mg/kg bw/day: One animal found dead on GD 11 due to misgavage.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Decreased body weight from GD 8 - GD 21 (at maximum 12.3% below the value of controls), reduced body weight gain (GD 0 - GD 20 and GD 6 - GD 20); adverse.
300 mg/kg bw/day: Decreased body weight gain (GD 6 - GD 20); non-adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Decreased food consumption from GD 6 - GD 16 (at maximum 40.9% below the value of the controls); adverse.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Statistically significant decreased gravid uterus weight (22.0% below the value of the controls)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000/750 mg/kg bw/day: Thickened and yellow discoloured cardia region of the stomach (3/20 at 300 mg/kg bw/day, 9/21 at 1000/750 mg/kg bw/day), hemorrhagic foci (2/20 at 300 mg/kg bw/day).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In all groups thyroids revealed single or multiple keratinized cysts; incidental.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone levels
300 and 1000/750 mg/kg bw/day: Statistically significant, dose-response related decrease of T3 and T4 serum levels (T3: 30.2% or 29.8% below controls; T4: 19.8% or 28.7% below controls) and dose-dependent increase of TSH serum levels (not statistically significant); hormone levels were within the range of historical control data of the test facility and no correlation with thyroid weights and histopathology noted.

Details on results:

MORTALITY
In the high dose group (1000 mg/kg bw/day) one dam was prematurely sacrificed on GD 8 due to signs of severe toxicity (gasping, severe salivation and a hemorrhagic nose/snout). In addition at necropsy, this dam had an emaciated body and highly dilated and inflated intestines without content. Evaluation of the pregnancy status by staining of the uterus according to SALEWSKI revealed that the animal was not pregnant. As a consequence and due to further signs of toxicity (clinical signs, reduced body weight, reduced food consumption), the high dose level was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day thereafter. In the low dose group (100 mg/kg bw/day), one animal was found dead on GD 11. Necropsy revealed a thorax filled with liquid and dark-red discolored lungs. Therefore, the premature death of this animal was considered to be due to a misgavage and the dam was excluded from the study. Therefore, no premature test item-related deaths occurred in the control group and the low- and mid-dose group (100 or 300 mg/kg bw/day).

CLINICAL SIGNS
In the high-dose group (1000/750 mg/kg bw/day), several clinical signs and changes of the external appearance and the feces were noted. Salivation and increased water consumption were noted for almost all animals of the high-dose group (1000/750 mg/kg bw/day) and therefore, were considered to be test item-related. Breathing sounds, clear discharge from vagina and anus, decreased water consumption, reduced motility, hemorrhagic nose/snout, soft feces and piloerection were considered to be test item-related and adverse.
No test item-related clinical signs or changes in the external appearance that were considered to be of toxicological relevance were noted in the control group and the low- and mid-dose groups (100 or 300 mg/kg bw/day). Salivation was noted for 3/20 dams of the low dose group (100 mg/kg bw/day) and 11/20 animals of the intermediate dose group (300 mg/kg bw/day). However, as salivation was noted for only up to 5 days per animal, salivation was considered to be due to high amounts of test item administered and/or local adverse effects in the stomach and therefore, not toxicologically relevant. Furthermore, breathing sounds were noted for one mid-dose dam on GD 17.

BODY WEIGHT DEVELOPMENT
In the high-dose group (1000/750 mg/kg bw/day), a decreased body weight was noted from GD 8 until study termination on GD 21 (at maximum 12.3% below the value of the control group on GD 20, statistically significant at p ≤ 0.01). This distinct decrease of the body weight that lasted for almost the complete dosing period, was considered to be toxicologically relevant. The decreased body weight in the high-dose group (1000/750 mg/kg bw/day) also led to a toxicologically relevant reduction for the body weight gain from GD 0 to GD 20 and from GD 6 to GD 20.
No test item-related differences in body weight were noted for the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively). In accordance with the body weight, also no test item-related difference between the control group and the animals treated with 100 or 300 mg/kg bw/day was noted for the body weight gain from GD 0 to 20 and in the dosing period from GD 6 to GD 20. In the mid-dose group (300 mg/kg bw/day), a statistically significantly lower body weight gain was noted between GD 6 and GD 20 (7.8% below the value of the control group). However, this small difference in body weight gain was considered to be not toxicologically relevant as it was < 10%.

FOOD AND DRINKING WATER CONSUMPTION
In the high-dose group (1000/750 mg/kg bw/day), a reduction of the food consumption was noted from GD 6 to GD 16 (at maximum 40.9% below the value of the control group between gestation days 7 to 8, with the exception of the period from GD 13 to GD 14). The long-lasting and distinct reduction in food consumption was considered to be test item-related. Almost all high dose females (1000/750 mg/kg bw/day) displayed increased water consumption and 4 females had a decreased water consumption. The females nos. 76 and 77 had a decreased water consumption on GD 18 and 19 or on GD 19 and increased water consumption from GD 20 until study termination on GD 21. Female no. 95 displayed an increased water consumption on GD 13 and a decreased water consumption from GD 14 to GD 21. Animal no. 78 was noted with decreased water consumption only from GD 18 to GD 21. The other animals displayed increased water consumption only.
At the start of the dosing period between GD 6 and GD 9, a reduced food consumption was noted for the low- (100 mg/kg bw/day) and mid- (300 mg/kg bw/day) dose groups (at maximum 9.9% below the value of the control group between GD 8 and GD 9 for the mid-dose group). However, these reductions in the food consumption for the low- and mid-dose groups were considered to be spontaneous. In addition, single occurrences of a statistically significantly reduced food consumption from GD 10 to GD 11 and from GD 15 to GD 16 for the mid-dose group (7.7% or 6.9% below the value of the control group) were considered to be spontaneous.

THYROID HORMONE LEVELS
A statistically significant dose-response related decrease was noted for the serum levels of T3 and T4 for the animals dosed with 300 or 1000/750 mg/kg bw/day (T3: 30.2% or 29.8% below the value of the control group; T4: 19.8% or 28.7% below the value of the control group). The serum levels of TSH were in tendency dose-dependently increased (not statistically significant). However, the levels of the mid- (300 mg/kg bw/day) and high-dose group (1000/750 mg/kg bw/day) for T3, T4 and TSH were within the range of the LPT historical control data and no correlation to thyroid weights and histopathology was noted in this study and in the 90-day sub-chronic toxicity study (LPT Study No. 37647; LPT, 2020a in section 7.5 of the IUCLID dossier). Therefore, the decreased levels of T3 and T4 and the increased levels of TSH observed in the mid- and high-dose groups are considered to be not toxicologically relevant.

NECROPSY
No test item-related observations were noted for the dams of the low-dose group (100 mg/kg bw/day) during the macroscopic inspection of the organs and tissues. In the mid-dose group (300 mg/kg bw/day), in 3/20 dams the cardia region of the stomach was thickened, yellow discolored and in two cases also with hemorrhagic foci. In the high-dose group (1000/750 mg/kg bw/day), thickened and discolored cardia region of the stomach was noted for 9/21 animals (20 dams and animal no. 86 with a total loss of implantations). Due to the dose-dependence relationship, the local changes in form of the thickened cardia region were considered to be test item-related and are believed to be caused by the chemical (irritating/corrosive) properties of the test item.

HISTOPATHOLOGY AND THYROID WEIGHTS
In all groups, histopathological examination of the thyroids revealed single or multiple keratinised cysts. However, these findings were considered to be coincidental and not test item-related. No test item-related differences to the control group were noted for the absolute thyroid weights of the animals in the dose groups (100, 300 or 1000/750 mg/kg bw/day).

GRAVID UTERUS WEIGHT, CARCASS WEIGHT AND BODY WEIGHT GAIN FROM GD 6
In the high-dose group (1000/750 mg/kg bw/day), statistically significant reductions were noted for the gravid uterus weight (22.0% below the value of the control group) and for the carcass weight (8.2% below the value of the control group). No test item-related differences were noted between the gravid uterus and carcass weight of the control dams and the dams of the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively).
In the high-dose group (1000/750 mg/kg bw/day), a decrease was noted also for the net body weight gain between GD 6 and GD 21 (-18.3 g in the high dose group compared to +3.2 g in the control group). The distinctly reduced net body weight gain was considered to be test item-related. For the net body weight from GD 6 to 21, no test item-related differences were noted between the dams of the control group and the dams of the low- and mid-dose groups (100 an 300 mg/kg bw/day).

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Due to an increased number of resorptions, the index of post-implantation loss is also increased.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Increased number of resorptions (33 resorptions vs. 13 resorptions in the control group).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

In the high-dose group (1000/750 mg/kg bw/day), an increase was noted for the number of resorptions. In detail, in the control group 13 resorptions were noted and 14 resorptions each occurred in the low- and mid-dose groups. In the high-dose group 33 resorptions were noted, including animal no. 86 that lost all of its 15 implantations. Furthermore, a statistically significantly increased number of late resorptions was noted for the high-dose group (1000/750 mg/kg bw/day; 4 dams with one late resorption each in the high-dose group compared to no late resorptions in the control group). Since also the number of late resorption was above the upper limit of the LPT historical control data range, the increased number of resorptions noted for the high dose group was considered to be test item-related.
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively).

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

early or late resorptions

2

Key result

Dose descriptor:

NOAEL

Remarks:

local toxicity

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

gross pathology

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: local changes in cardia region of stomach

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000/750 mg/kg bw/day: Decreased weights of male and female fetuses, alone and for male and female fetuses combined (12.8%, 13.5% and 12.9% below the value of the controls), decreased fetal weights were considered secondary to reduced body weight (gain) and food consumption of dams.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: One fetus with open cranial roof (cranioschisis) and one fetus with a short tail.

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: One open skullcap and one case of brachycaudia (refer also to cranioschisis and short tail reported under 'External malformations').

Other effects:

no effects observed

Description (incidence and severity):

Testicular development
No cryptorchism and no testicular malposition noted.

Details on embryotoxic / teratogenic effects:

No dead fetus was noted and no test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000/750 mg/kg bw/day).
The placental weights showed no test item-related differences between the control group and the dose groups and no test item-related differences were noted for the fetal weights of the low and intermediate dose group (100 and 300 mg/kg bw/day) compared to the control group. For the fetal weights of the high dose group (1000/750 mg/kg bw/day), however, a decrease was noted for the male and female fetuses alone and also for the male and female fetuses combined (12.8%, 13.5% and 12.9% below the value of the control group, statistically significant). The decreased fetal weights were considered to be secondary to the reduced body weight (gain) and the reduced food consumption of the dams as the low fetus weights of the individual animals correspond to a very low food consumption and reduced net body weight gain of the dams.
No test item-related difference in the number of runts were noted for dose groups (100, 300 and 1000/750 mg/kg bw/day) in comparison to the control group. One runt each was noted in the control group and the mid-dose group (300 mg/kg bw/day), three runts in the low-dose group (100 mg/kg bw/day) and two runts were noted in the high-dose group (1000/750 mg/kg bw/day).
No test item-related differences to the control group were noted for the fetal anogenital distance of the dose groups (100, 300 and 1000/750 mg/kg bw/day).

MACROSCOPIC INSPECTION OF FETUSES
No macroscopically visible external observations were noted for the fetuses of the control group and the fetuses of the mid- and high-dose groups (300 and 1000/750 mg/kg bw/day) during the external inspection at laparotomy. In the low-dose group (100 mg/kg bw/day), two fetuses with a malformation were noted: One fetus was noted with an open cranial roof (cranioschisis) and one fetus had a short tail. The observations were also noted during the soft tissue examination of these two fetuses. However, as no malformations were noted in the mid- (300 mg/kg bw/day) and high-dose groups (1000/750 mg/kg bw/day), the occurrence of two fetuses with malformations in the low-dose group was considered to be spontaneous and not test item-related.
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the dose groups (100, 300 and 1000/750 mg/kg bw/day).
No cryptorchism and no testicular malposition were noted during assessment of the testicular development of the male fetuses of the control group and the dose groups (100, 300 and 1000/750 mg/kg bw/day).

SKELETAL EXAMINATION
Malformations:
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 and 1000/750 mg/kg bw/day) during the skeletal examination according to DAWSON.

Variations:
Skeletal variations were noted for the ribs (less than 13 ribs ossified or ribs wavy) and the sternum (bipartite, dumbbell-shaped or misaligned to a slight degree). No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 and 1000/750 mg/kg bw/day).

Retardations:
Retardations (i.e. delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (only one body ossified or all bodies unossified), the sacral vertebral bodies (unossified), the os pubis (incompletely ossified) and the metacarpalia/metatarsalia (absence of ossification in metacarpalia/metatarsalia 2 to 5). No test item-related difference in the incidence of skeletal retardations at 100, 300 and 1000/750 mg/kg bw/day was noted during skeletal examination according to DAWSON.

SOFT TISSUE EXAMINATION
Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the mid- and high-dose groups (300 and 1000/750 mg/kg bw/day) during the soft tissue examination according to WILSON. In the low-dose group (100 mg/kg bw/day), two malformations were noted in form of an open skullcap and in form of a brachycaudia (see also the observations of cranioschisis and short tail). However, as no malformations were noted in the mid- and high-dose groups, the occurrence of two fetuses with malformations in the low-dose group was considered to be spontaneous and not test item-related.

Variations
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle or distinct cystic area in the cerebrum), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (hemorrhagic focus/foci). No test item-related and statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 and 1000/750 mg/kg bw/day).

Unclassified observations
No unclassified observations were noted for the control group. An unclassified observation in form of a thoracic cavity filled with blood was noted for one fetus each of the low- and mid-dose groups (100 and 300 mg/kg bw/day, respectively) and for three fetuses of the high-dose group (1000/750 mg/kg bw/day). This observation was considered to be a preparation-induced artefact.

ASSESSMENT OF FETAL ALTERATIONS
No test item-related malformations or variations were noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON). Furthermore, no test item-related retardations (delay in ossification) were noted in any of the dose groups (100, 300 and 1000/750 mg/kg bw/day).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

development

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

external: tail

Description (incidence and severity):

100 mg/kg bw/day: one fetus with open cranial roof (cranioschisis) and one fetus had a short tail, observations were considered spontaneous and not treatment-related.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Detailed tables summarising study data are provided under 'Attached background material'. Summary tables demonstrating key results are provided below.

 

Table 1: Summary of animals examined

	Group 1 Control	Group 2 100 mg/kg bw/day	Group 3 300 mg/kg bw/day	Group 4 1000/750 mg/kg bw/day
Treated dams	25	25	25	25
Not pregnant dams	2	0	1	0
Dams with non-test item-related premature death	0	1*	0	0
Prematurely sacrificed dams	0	0	0	1**
Dams without viable fetuses	0	0	0	1***
Not evaluated dams (spare animals)	3	4	4	3
Evaluated litters	20	20	20	20

*: The death of dam no. 26 that was found dead on GD 11 was considered to be due to a misgavage. Therefore, dam no. 26 was excluded from the study.

**: Dam no. 82 was prematurely sacrificed due to severe signs of toxicity.

***: Dam no. 86 was noted with resorptions of all implants.

 

Table 2: Summary of animals evaluated

	Group 1 Control	Group 2 100 mg/kg bw/day	Group 3 300 mg/kg bw/day	Group 4 1000/750 mg/kg bw/day
Animal nos. of mated rats	1 - 25	26 - 50	51- 75	76 - 100
Animal nos. with evaluable litters at laparotomy	1, 4 - 22	27 - 46	51, 53 - 71	76 - 81, 83 - 85, 87 - 97
Dams not pregnant (animal nos.)	2, 3	None	52	None
Dams with non-test itemrelated premature death (animals nos.)	None	26*	None	None
Prematurely sacrificed dams (animal nos.)	None	None	None	82**
Dams without viable fetuses (animal nos.)	None	None	None	86***
Spare animals (animal nos.)	23 - 25	47 - 50	72 - 75	98 - 100

*: The death of dam no. 26 that was found dead on GD 11 was considered to be due to a misgavage. Therefore, dam no. 26 was excluded from the study.

**: Dam no. 82 was prematurely sacrificed due to severe signs of toxicity.

***: Dam no. 86 was noted with resorption of all implants.

 

Table 3: Clinical signs, changes of external appearance and the feces noted for the high dose group

	Observations in group 4 (1000/750 mg test item/kg bw/day)*
Observation	Affected dams	First to last day seen	Number of days observed (min - max) in at least 1 animal
Salivation (slight to severe)	21/21	GD 6 - GD 21	1 - 11
Increased water consumption	20/21	GD 11 - GD 21	1 - 9
Breathing sounds	6/21	GD 7 - GD 21	2 - 5
Discharge from vagina and anus (clear)	5/21	GD 7 - GD 16	1 - 6
Decreased water consumption	4/21	GD 14 - GD 21	1 - 8
Motility reduced	4/21	GD 9 - GD 19	1 - 4
Hemorrhagic nose/snout	4/21	GD 8 - GD 18	1
Soft feces	4/21	GD 11 - GD 16	2 - 5
Piloerection	3/21	GD 10 - GD 21	1 - 12

*: The prematurely sacrificed animal no. 82 was excluded in this table.

 

Table 4: Body weight gain from GD0 to GD20 and from GD6 to GD20

Group	Time interval Gestation day 0 - 20 (whole study period)			Time interval Gestation day 6 - 20 (whole treatment period)
	Gain [g]	Gain [%]	Difference to control [%]	Gain [g]	Gain [%]	Difference to control [%]
Control	+171.8	+71.0	n.a.	+131.6	+46.6	n.a.
Group 2 100 mg/kg bw/day	+163.3	+67.9	-5.0	+124.5	+44.5	-5.4
Group 3 300 mg/kg bw/day	+164.8	+68.6	-4.1	+121.3*	+42.7	-7.8
Group 4 1000/750 mg/kg bw/day	+120.6**	+49.8	-29.8	+82.7**	+29.4	-37.1

n.a.: Not applicable

*/**: Statistically significant at p ≤ 0.05/0.01 (DUNNETT test).

 

Table 5: Thyroid hormone levels determined in this study and LPT background data on T3 and T4 levels

Thyroid hormone levels	Values observed in this study [mean per dam]	LPT Background Data*** Mean value ± SD (range) of the individual control groups (n = 5 control groups; data taken from 2019 to June 2020)
T3 [ng/mL]	Control: 2.46 Group 2: 2.21 Group 3: 1.72 **/**** Group 4: 1.72 **/****	2.17 ± 0.86 1.22 - 8.64	control groups
T4 [nmol/L]	Control: 19.14 Group 2: 18.47 Group 3: 15.35 */**** Group 4: 13.66 **/****	20.27 ± 4.71 11.82 - 37.82	control groups
TSH (ng/mL)	Control: 1.03 Group 2: 1.07 Group 3: 1.49 Group 4: 2.28	0.85 ± 0.87 0.08 - 4.75	control groups

*/**: Statistically significant at p ≤ 0.05/0.01 (DUNNETT test).

***: Not audited by QAU.

****: Considered to be spontaneous as incidences were within LPT background data range

 

Table 6: Macroscopic changes noted during necropsy

Group	Animal no.	Observation
Group 1 Control	-	No observation noted
Group 2 (100 mg/kg bw/day)	No. 26 (PD)	Thorax: filled with liquid Lungs: dark-red discolored
	No. 35	Uterus: amniotic fluid red discolored
Group 3 (300 mg/kg bw/day)	No. 54	Stomach: cardia region thickened, yellow discolored
	No. 55	Stomach: cardia thickened, yellow discoloured, 2 hemorrhagic foci (approx. 1 mm in diameter)
	No. 59	Stomach: cardia region thickened, yellow discoloured, 2 hemorrhagic foci (approx. 1-2 mm in diameter)
Group 4 (1000/750 mg/kg bw/day)	No. 78	Intestines: inflated Stomach: cardia region thickened, white discolored
	No. 80	Stomach: cardia region thickened, yellow discolored
	No. 82 (PS, NP)	Body: emaciated Intestines: without content, strongly dilated, inflated
	No. 85	Stomach: cardia region thickened, yellowish discolored
	No. 87	Stomach: mucosa thickened, cardia region yellowish discolored
	No. 88	Stomach: mucosa thickened in cardia region
	No. 89	Stomach: cardia region thickened, yellowish discolored
	No. 90	Stomach: cardia region thickened, yellowish discolored
	No. 91	Stomach: cardia region thickened, white discolored
	No. 92	Stomach: cardia region thickened, yellowish discolored

PD: Premature death

PS: Premature sacrifice

NP: Not pregnant

 

Table 7: Overview of reproduction parameters

Parameter		Group 1 Control (n = 20)	Group 2 (100 mg/kg bw/day) (n = 20)	Group 3 (300 mg/kg bw/day) (n = 20)	Group 4 (1000/750 mg/kg bw/day) (n = 21)
Corpora lutea	total mean per dam	336 16.8	316 15.8	321 16.1	337 16.0
Implantation sites	total mean per dam	330 16.5	316 15.8	319 16.0	330 15.7
Resorptions	total mean per dam	13 0.7	14 0.7	14 0.7	33 1.6
Early resorptions	total mean per dam	13 0.7	14 0.7	14 0.7	29 1.4
Late resorptions	total mean per dam	0 0.0	0 0.0	0 0.0	4 0.2*
Live fetuses	total mean per dam	317 15.9	303 15.2	305 15.3	297 14.9
Dead fetuses	total	0	0	0	0
Pre-implantation loss [%]	per group mean per dam	1.8 1.7	0.0 0.0	0.6 0.6	2.1 2.0
Post-implantation loss [%]	per group mean per dam	3.9 3.8	4.4 4.5	4.4 4.2	10.0 10.2

*: Statistically significant at p ≤ 0.05 (DUNNETT test).

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

 

Table 8: LPT background data on number of late resorptions

Reproduction parameters	Values observed in this study [mean per dam]	LPT Background DataJ** Mean value ± SD (range) of the individual control or test groups (n = 12 control or data taken from 2016 to July 2017)
Late resorptions	Control: 0.0 Group 2: 0.0 Group 3: 0.0 Group 4: 0.2*	0.03 ± 0.05 0.0 - 0.1	control groups

*: Statistically significant at p ≤ 0.05 (DUNNETT test).

**: Not audited by QAU

 

Table 9: Male to female ratio for the fetuses of the test groups

	Ratio [male/female fetuses]
Control	1.10
100 mg/kg bw/day	1.06
300 mg/kg bw/day	0.97
1000/750 mg/kg bw/day	1.14

 

Table 10: Statistically significant, considered to be not test item-related differences in the incidence of fetal skeletal retardations

Skeletal retardations	Values observed in this study [fetal incidence in % per group]	LPT Background Data*** Mean value ± SD (range) of the individual control or test groups [fetal incidence in % per group] (n = 15 control or n = 45 test item groups, data taken from 2016 to July 2017)
Absence of ossification in metacarpalia 2 to 5	Control: 0.6 Group 2: 0.7 Group 3: 0.7 Group 4: 7.4 **/****	3.6 ± 4.0 0.0 - 15.0	control groups
Absence of ossification in metatarsalia 2 to 5	Group 2: 0.0 Group 3: 0.0 Group 4: 2.7 */****	1.2 ± 1.8 0.0 - 4.6	control groups
Caudal vertebral bodies only one body ossified	Control: 0.0 Group 2: 0.0 Group 3: 0.0 Group 4: 2.7 */****	1.0 ± 2.1 0.0 - 7.9	control groups
Hyoid unossified	Control: 26.4 Group 2: 30.9 Group 3: 25.5 Group 4: 12.2 **/*****	54.2 ± 16.3 24.3 - 77.6	control groups
Sternebra(e) incompletely ossified	Control: 0.0 Group 2: 0.0 Group 3: 2.0 Group 4: 2.7 */****	10.3 ± 6.1 3.4 - 26.7	control groups
Thoracic vertebral body/bodies bipartite	Control: 5.0 Group 2: 0.7 */***** Group 3: 0.7 */***** Group 4: 1.4 */*****	2.6 ± 1.5 0.7 - 5.1	control groups

*/**: (p ≤ 0.05 / p ≤ 0.01) Fisher or Chi² - test

***: Not audited by QAU.

****: Considered to be spontaneous as incidences were within LPT background data range.

*****: A decreased incidence was considered to be spontaneous.

 

Table 11: Results of the test item formulation analysis

Parameter	Sampling	Range of % nominal concentration
Homogeneity	at the start of administration, during administration and before administration to the last animal of each dose group	93.8% - 105.9%*
Concentration	before administration of the last animal of each dose group at a time when the majority of animals was dosed	95.0% - 103.5%

*: Contains results before and after high dose level adjustment from 1000 mg/kg to 750 mg/kg.

 

Applicant's summary and conclusion

Conclusions:

The pre-natal developmental toxicity in rats after oral administration by gavage was investigated according to OECD guideline 414 under GLP conditions. N-methyl-N-[C18-(unsaturated)alkanoyl]glycine exhibited distinct maternal toxicity at doses > 300 mg/kg bw/day. Reduced pup weights observed were a secondary reaction to maternal toxicity. The No-Observed-Adverse-Effect-Levels (NOAEL) for maternal systemic, maternal developmental and fetal developmental toxicity were, therefore, established to be 300 mg/kg bw/day.