Reaction mass of (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl palmitate and (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl stearate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl palmitate

Molecular formula:

C36 H67 N O6

IUPAC Name:

(9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl palmitate

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 200 to 225 g for males and 175 to 200 g for females
- Housing:
The animals were housed in a limited access rodent facility. From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate,Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.

- Diet (e.g. ad libitum): ad libitum (laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4,
20019, SettimoMilanese (MI), Italy)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01.03.2021 To: 14.04.2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test item was suspended in the vehicle. The preparations were made daily at concentration of 25, 75 and 250 mg/mL, unless specified otherwise based on stability data stated in the validation study (section 3.4). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations (ERBC Study no. A4158). The analysis confirmed a 11 days stability at room temperature in the range from 25 to 250 mg/mL.
Samples of the preparations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the homogeneity and concentration.
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC. The validated software used for this activity was Analyst 1.6.2. Results of the analyseswere within the acceptability limits stated in ERBC SOPs for concentration of suspension (85-115%),

Details on mating procedure:

- M/F ratio per cage: 1:1 (one exception since one low dose male was found dead before the start of the mating phase)
- Length of cohabitation: until positive identification occured
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.

Duration of treatment / exposure:

- Males: at least 28 days. The duration of treatment covered a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.

- Females: up to 60 days. The duration of treatment covered a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Test group 1 (Control)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Test group 2

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Test group 3

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Test group 4

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels have been selected in consultation with the Sponsor based on information from previous studies.
In a previous repeated-dose toxicity study (OECD 407) with a structural similar substance the NOAEL was considered to be 1000 mg/kg/day

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Mortality: Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
- Clinical observations: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once a week
- Males: weekly from allocation to termination.
- Females: at weekly interval from allocation, where possible, to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
- Dams were weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: weekly during the pre-mating period, starting Day 1 of dosing up to mating.
- Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum

WATER CONSUMPTION (if drinking water study): No

CLINICAL BIOCHEMISTRY: Yes
- Anaesthetic used for blood collection (for hormone determination) : Yes (Isoflurane)
- How many animals: all parental males and females/ Pups: from each litter (1 sample for males and 1 sample for females)
- Parameter checked: T4, TSH (serum)

REPRODUCTIVE PERFORMANCE
- Mating: 1:1 (with one exception since one low dose male was found dead before the start of the mating phase)
- Parturition and gestation length: A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition was complete) was defined as Day 0 post partum. On gestation Day 22, one female (Group 3) gave birth to 8 dead pups. The delivery was not completed since the day after (Day 23 post coitum) a total of 11 pups, 8 dead and 3 alive, were counted in the cage and the dam was found dead. At necrospy, additional 3 dead foetuses were recorded in the uterus.

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
- Number of implantations: Yes
- Number of total resorptions: Yes
- Number of early/late resorptions: No

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: approximately 0.8 mL

Fetal examinations:

- External examinations: Yes, all pups
- Soft tissue examinations: necropsy observations macroscopically
- Skeletal examinations: No
- Head examinations: No
- Sex ratio: Yes
- Pup body weight data: Yes
- Pup clinical observations. Yes

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Indices:

Prenatal loss (%) = (no.of visible implantations − live litter size at birth) / no.of visible implantations x 100

Postnatal loss at Day 4 post partum (%) = (Live litter size at birth - live litter size at Day 4 (before culling)/ Live litter size at birth x 100

Postnatal loss at Day 13 post partum (%) = (Live litter size on Day 4 (after culling) - Live litter size on Day 13)/ Live litter size on Day 4 (after culling) x 100

Pup loss at Day 0 post partum (%) = (Total litter size - Live litter size)/ Total litter size x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were recorded in females before pairing. During gestation, signs of difficulty to delivery were recorded in one female at 100 mg/kg/day.
No treatment-related clinical signs were recorded during post partum phase.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On gestation day 22, one female at 100 mg/kg/day was sacrificed following the occurrence of important clinical signs like prolapse of the uterus, pallor, piloerection and coldness to touch. The presence of these signs was related to difficulty in delivery.
One female at 300 mg/kg/day was found dead on gestation day 23. This female gave birth 8 pups on gestation day 22 (found dead) and additional 3 live pups were found in the cage on Day 23 post coitum. Both females had a prolonged time of gestation without completing the parturition and the impaired delivery was the reason of the humane sacrifice for the low dose female (100 mg/kg/day) and the occurrence of premature death for the mid-dose female (300 mg/kg/day). The gross and microscopic evaluation did not allow to establish the cause of impaired parturition. However, since these findings occurred in single animals, they could be considered unrelated to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry 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):

There were no treatment-related changes in terminal body weight when compared to controls.

Statistically significant increase in absolute and relative mean liver weight for high dose females (23% of mean relative weight) was noted and correlated microscopically with centrilobular hepatocellular hypertrophy. This was considered treatment-relatd.
Any organ weight changes were within the range of occasionally
observed and expected spontaneous changes in rats of the same age and considered unrelated to treatment.
Table included under "Any other information on results incl. tables".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic observations associated with the oral administration of the test item were present in the liver of high dose females.
- In the liver, treatment-related microscopic observations consisted in centrilobular hepatocellular hypertrophy of
minimal to mild severity was present in most females (7/10) at the high dose. Hepatocyte hypertrophy
consisted of enlarged hepatocytes containing variable amounts of fine granular pale eosinophilic (ground glass) cytoplasm.

- Any other microscopic observation had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of implantation sites did not show significant differences between groups as well as the pre-natal loss.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Two females, one control and one at 100 mg/kg/day lost the litter on Day 0 post partum

Early or late resorptions:

not examined

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

On gestation Day 22, one female (no. 51 of Group 3) gave birth to 8 dead pups. The delivery was not completed since the day after (Day 23 post coitum) a total of 11 pups, 8 dead and 3 alive, were counted in the cage and the dam was found dead. At necrospy, additional 3 dead foetuses were recorded in the uterus.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The gestation length was similar in all groups.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

– 8/10 in the control group (0 mg/kg/day)
– 8/9 in the low dose group (100 mg/kg/day)
– 8/9 in the mid-dose group (300 mg/kg/day)
– 9/10 in the high dose group (1000 mg/kg/day)

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: effect level based on females.

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distance (normalized to the cube root of the body weigh on Day 1 post partum) did not differ in male pups of treated groups when compared to controls whereas it was significantly increased in females of all treated groups (up to 28%, respect to the control group).
Although a dose relation was evident, the statistical differences were not considered of toxicological significance but rather due to unexpected lower control, being all values in the range of the historical control data (HCD). Table included under "Any other information on results incl. tables".

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs:
The observation of pups did not reveal treatment-related findings. Pale or small appearance, the presence of wound in the muzzle, not opened eye, or tip of tail missing were seen as single occurrences regardless the treatment groups. No nipple/aerola was recorded in any male pup.

Necropsy:
Findings at necropsy included the absence of milk in the stomach from pups sacrificed for humane kill. All autolysed organs were frequently found in decedent pups. The necropsy of culled pups (Day 4 post partum) and those sacrificed at termination (Day 14 post partum) did not show treatment-related changes.

Organ weight:
No significant changes were seen in thyroid weight from male and female pups of treated groups compared to controls.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Parental liver weights

		Males				HCD	Females				HCD
Dose level (mg/kg/day)		Control	100	300	1000		Control	100	300	1000
Liver	Abs. weight (g)	12.282	14.248*	14.946*	16.092*	14.52	12.234	12.608	13.924	15.227*	11.29
	Rel. weight (g)	2.798	3.210*	3.350*	3.636*	3.65	3.955	4.221	4.534	4.854*	3.83
* = Statistical significant from control at p<0.05

 

Table 2: Thyroid hormone measurements in male and female pups

Pups Day14 Post Partum_Males (Group mean data)
		Group
Parameter/units		1	2	3	4
Thyroxine nmol/L	Mean SD N	54.9 10.1 7	52.3 6.5 7	52.5 6.9 8	53.3 2.8 9
Thyroid Stimulating Hormone ng/mL	Mean SD N	3.80 0.65 7	3.60 0.68 7	3.83 0.72 8	3.67 0.85 9
Controls from group(s): 1                           Subgroup(s): 1 * = mean value of group is significantly different from control at p < 0.05 + = mean value of group is significantly different from control at p < 0.01
Pups Day14 Post Partum_Females (Group mean data)
		Group
Parameter/units		1	2	3	4
Thyroxine nmol/L	Mean SD N	56.0 7.6 7	55.3 10.5 7	55.6 8.9 8	54.8 8.0 9
Thyroid Stimulating Hormone ng/mL	Mean SD N	3.54 0.53 7	3.71 0.53 7	4.33 0.89 8	3.67 0.76 9
Controls from group(s): 1                           Subgroup(s): 1 * = mean value of group is significantly different from control at p < 0.05 + = mean value of group is significantly different from control at p < 0.01

 

Table 3 Anogenital distance

Females_ANOGENITAL DISTANCE ON DAY 1 POST PARTUM – GROUP MEAN DATA
Anogenital Distance (normalized) mmg/g^1/3)^
	Group
	1	2	3	4	HCD
Mean SD N	1.07 0.12 7	1.28* 0.17 7	1.33* 0.12 8	1.37* 0.05 9	1.39
^ = normalised to the cube root of the body weight collected on Day 1 post partum (mm/g) Statistical analysis: Kruskall Wallis test T test if group mean differences are different from control at p < 0.05 * = mean value of group is significantly different from control
Males_ANOGENITAL DISTANCE ON DAY 1 POST PARTUM – GROUP MEAN DATA
Anogenital Distance (normalized) mmg/g^1/3)^
	Group
	1	2	3	4	HCD
Mean SD N	2.52 0.16 7	2.50 0.10 7	2.62 0.18 8	2.47 0.17 9	2.06
^ = normalised to the cube root of the body weight collected on Day 1 post partum (mm/g) Statistical analysis: Kruskall Wallis test T test if group mean differences are different from control at p < 0.05 * = mean value of group is significantly different from control

 

Table 4 Historical Control Data of the Anogenital distance

Anogenital Distance- Historical Control Data
	Mean mmg/g^1/3	Max mmg/g^1/3	Min mmg/g^1/3	N
AGD (mmg/g^1/3) Male Pups	2.06	2.96	1.05	183	Pups
AGD (mmg/g^1/3) Female Pups	1.39	8.19	0.55	155	Pups
Average study
AVG_study AGD (mmg/g^1/3) Male Pups	2.05	2.26	1.94	3	studies
AVG_study AGD (mmg/g^1/3) Male Pups	1.35	1.85	0.93	3	studies