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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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 species / strain selection:

The Wistar Hannover rat is the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.

Sex:

male/female

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 oral 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%),

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

Positive control:

no

Examinations

Observations and examinations performed and frequency:

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)

Sacrifice and pathology:

SACRIFICE
Parental animals that had completed the scheduled test period were killed by exsanguinations under isoflurane anaesthesia. One female of the low dose group was sacrificed for humane reasons, before the scheduled test period, under carbon dioxide asphyxiation.
The males were killed at completion of mating of all females (after 35 days of treatment). The females with live pups were killed on Day 14 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. One control female did not show evidence of copulation, however it gave birth. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

GROSS NECROPSY
- Females: All females were also examined for the number of visible implantation sites (pregnant
animals). Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS
- Parental animals: From all animals completing the scheduled test period, the following organs/tissues were dissected free of fat and weighed: Abnormalities, epididymides, liver, ovaries with oviducts, prostate gland (dorsolateral and ventral), seminal vesicles with coagulating glands, testes, thyroid gland, uterus – cervix, vagina.
- The ratios of organ weight to body weight was calculated for each animal.

TISSUE FIXATION
Samples from all the tissues/organs mentioned under “organ weights” were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, Harderian glands, ovaries, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol)

HISTOPATHOLOGY
- The following tissues were required for histopathological examination: Abnormalities, epididymides, liver, ovaries with oviducts, prostate gland (dorsolateral and ventral), seminal vesicles with coagulating glands, testes, thyroid gland, uterus – cervix, vagina.
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
- The examination was restricted as described:
o Tissues mentioned in “organ weights” from all males and females in the control and high dose groups killed at term. The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also included.
o Tissues mentioned in “organ weights” from all animals killed or dying during the treatment period.
o All abnormalities in all groups.
- The examination of liver and thyroid was extended to animals of the low and mid-dose groups of both sexes based on treatment-related changes observed in the high dose group.

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs were recorded in males throughout the study.
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:

effects observed, treatment-related

Description (incidence and severity):

A dose-related increase in the TSH level was observed in parental males. The increase was 63% at 300 mg/kg/day and 113% at 1000 mg/kg/day, compared to controls. This finding could be related with the follicular cell hypertrophy of thyroid gland observed at histopathology and, due to the absence of T4 changes, could represent a subclinical hypothyroidism. Table included under "Any other information on results incl. tables".

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.
Treatment-related increases in the liver and thyroid weights, absolute or relative to body weight, were observed at all dose levels with a dose relationship.
The statistically significant increase in absolute and relative thyroid weight for mid and high dose males (21% of mean relative weight, respectively) correlated microscopically with follicular cell hypertrophy of thyroid gland. In addition, statistically significant increase in absolute and relative mean liver weight for low, mid- and high dose males (15%, 20% and 30% of mean relative weight, respectively) and high dose females (23% of mean relative weight) was noted and for mid and high dose males and high dose females correlated microscopically with centrilobular hepatocellular hypertrophy.
Any organ weight changes other than those listed above 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 thyroid and liver.
- In the thyroid gland, treatment-related microscopic observations consisted in follicular cell hypertrophy which was characterised by the presence of increased size and height of follicular cells with diffuse distribution. Follicular lumen was decreased in size and filled with normal staining to pale colloid and the cytoplasm of the follicular cells was enriched with small clear vacuoles. The above mentioned finding was mild to marked in all males (10/10) and moderate in one female (1/10) of high dose group and of mild severity in males (3/10) of mid-dose group. In males at the mid and high dose, this finding correlated with
changes in thyroid related hormone levels measured in parental males (increased values of TSH) and with the increased absolute and relative thyroid mean weights. Follicular cell hypertrophy of the thyroid gland was considered treatment-related.
- In the liver, treatment-related microscopic observations consisted in centrilobular hepatocellular hypertrophy of
minimal to mild severity was present in all males (10/10) and most females (7/10) at the high
dose and of minimal severity in few males (2/10) of the mid-dose group. Hepatocyte hypertrophy
consisted of enlarged hepatocytes containing variable amounts of fine granular pale
eosinophilic (ground glass) cytoplasm.
- There were no microscopic observations in the
testes as per specific stage aware evaluation using PAS stain.
- Any microscopic observations other than those listed above 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.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: the relationship between thyroid hypertrophy and TSH higher level is not necessarily indicative of thyroid dysfunction since the thyroid hormone changes did not include low serum of T4

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Liver and thyorid 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
Thyroid	Abs. weight (g)	0.0230	0.0254	0.0284*	0.0283*	0.026	0.0221	0.0216	0.0238	0.0230	0.023
	Rel. weight (g)	0.0053	0.0057	0.0064	0.0064	0.006	0.0074	0.0072	0.0078	0.0074	0.008
* = Statistical significant from control at p<0.05

 

Table 2: Thyroid hormone measurements in parental males and male and female pups

Parental Males (Group mean data)
		Group
Parameter/units		1	2	3	4
Thyroxine nmol/L	Mean SD N	41.4 3.9 10	41.4 3.6 9	44.8 5.3 10	38.1 4.1 10
Thyroid Stimulating Hormone ng/mL	Mean SD N	8.79 1.99 10	9.6 1 1.74 9	14.34+ 4.24 10	18.68+ 9.13 10
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_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

Applicant's summary and conclusion

Conclusions:

In conclusion after repeated dose, treatment-related effects were seen in parental animals at 300 and 1000 mg/kg/day characterized by morphological changes in thyroid and liver tissues (hypertrophy) as well as by TSH alteration in males (increase). Nevertheless, the relationship between thyroid hypertrophy and TSH higher level is not necessarily indicative of thyroid dysfunction since the thyroid hormone changes did not include low serum of T4. No adverse effects were observed regarding developmental and reproductive parameters.
Based on these results, the NOAEL (No Observed Adverse Effect Level) can be conservatively
set at 100mg/kg/day for general repeated dose toxicity. However, no clear indications of
adverse effects were seen at the higher dose levels.

Executive summary:

For more details please refere to sextion 7.8.1