1,10-decanediyl diacrylate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined repeated oral dose toxicity study with Reproduction/Developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 04 June 2021 (study initiation) to 22 August 2021 (experimental completion date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No.422 without any deviation.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 04 June 2021 (study initiation) to 22 August 2021 (experimental completion date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 422 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The Sprague-Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study was designed such that it does not require an unnecessary number of animals to accomplish its objectives.
The Study plan was reviewed by the Test Facility Ethics Committee (CEC) in order to assess compliance with the corresponding authorized "project" as defined in Directive 2010/63/EU and in French decree No. 2013-118. During the study, the CEC was regularly informed of any amendments to the Study Plan that could had an impact on animal welfare.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at initiation of dosing: males were approximatively 10 weeks old, the females were approximatively 11 weeks old.
- Weight at initiation of dosing: males between 332 and 455 g, females between 214 and 311 g
- Housing: single housed in polycarbonate cages containing appropriate bedding. Individual housing of F0 animals was chosen as group housing for pregnant animals can adversely affect gestation and lactation, and to avoid aggressive behavior around mating. Toward the end of gestation and during lactation, autoclaved wood shavings were provided to females and their litter as nesting material. For psychological / environmental enrichment, animals were provided with rat hut and wooden log, except when interrupted by study procedures / activities.
- Diet: ad libitum, pellet of SSNIFF rat/mouse pelleted maintenance diet, batch No. 62874737 (SSNIFF Spezialdiäten GmbH, Soest, Germany), sterilized by irradiation.
- Water: ad libitum, municipal tap water freely available in water bottles.

It was considered that there were no known contaminants in the feed, water and enrichments that interfered with the objectives of the study.

ENVIRONMENTAL CONDITIONS (TARGETED CONDITIONS)
- Temperature: 20°C to 24°C
- Humidity: 30% to 70%
- Air changes: approximately 8 to 15 filtered, non-recycled air changes per hour
- Photoperiod: 12 hours light and 12 hours dark

IN-LIFE DATES: From: 29 June 2021 To: 22 August 2021

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a route of administration which is requested by the Regulatory Authorities for this type of test item.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: test item formulations were prepared on a weekly basis, based on test item dose formulation stability data and vehicle expiry. The stability of test item dose formulations was determined in Study No. 48659 VAS (Garapon, 2021) that demonstrate that the test item formulation is stable when prepared and stored under the same conditions as those used in the present study at a concentration between 2 and 200 mg/mL for 11 days. Control formulations (vehicle only) were prepared considering the expiry date of the used vehicle. All the formulations were stored at room temperature and protected from light.

Dosing formulations were prepared based on Study No. 48659 VAS (Garapon, 2021) describing the preparation procedure for a range of concentrations covering the lowest and the highest used in this study, at appropriate concentrations to meet dose level requirements. The control dose formulations were stirred continuously just before dosing. The test item dose formulations were stirred continuously at least 15 minutes before dosing and during dosing. All formulations were maintained under delivery conditions (at room temperature and protected from light).

VEHICLE
- Justification for use and choice of vehicle: the test item was found to be soluble and stable in the selected vehicle. The vehicle is also known to be non-toxic to animals.
- Concentration in vehicle: 20, 60 and 200 mg/mL for the low-, mid- and high-dose groups respectively.
- Amount of vehicle: 5 mL/kg bw (based on the most recent body weight)
- Lot/batch no.: MKCM3364 and MKCN9742

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLE COLLECTION AND ANALYSIS
Dose formulation samples were collected for analysis on weeks 1, 4 and 7 from all groups. Analyses were performed by GC/FID using a validated analytical procedure (48659 VAS.MET). Samples were analyzed to determine concentration. As acceptance criteria, individual samples concentration shall be of ±10%.

Stability analyses performed previously in conjunction with study No. 48659 VAS demonstrated that the test item is stable in the vehicle for 11 days when prepared and stored under the same conditions as in this study at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Males were treated 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed) and until euthanasia (at least 4 weeks in total).

Females were treated at least 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed), during gestation, during lactation until PND 13 inclusive or until euthanasia for females with no delivery (GD 24 or 26).

Frequency of treatment:

once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low-dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid-dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high-dose group

No. of animals per sex per dose:

10 in each dose group.

Refer also to table 1 in "Any other information on results incl. tables".

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a preliminary 2-week toxicity study performed in the same species (Study No. 48660 TSR (Privat, 2021)) in which the test item was administered to five males and five females per group at 250, 500 and 1000 mg/kg bw/day. The only test item clinical sign was hypersalivation at 500 but mainly 1000 mg/kg bw/day. The test item also induced minimally lower body weights and body weight gains in males at 1000 mg/kg bw/day. The increased incidence of accentuated lobular pattern noted in males and females at 1000 mg/kg bw/day was considered to be equivocal in the absence of microscopic examination.
Therefore, the high dose level selected for the present study was 1000 mg/kg bw/day. The low and intermediate dose levels were selected based on an approximately three-fold interval (i.e. 100 and 300 mg/kg bw/day).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: for all animals, at least once daily beginning upon arrival through termination, before dosing when applicable. Mortality was also checked for all animals at least twice daily a (at beginning and end of working day) beginning upon arrival through termination. In addition to the daily cage side observation, animals were observed between 1 to 3 hours post-dose on the days of dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a full clinical examination was performed at least once a week for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly for all animals (including all females prior mating), from at least Week -1, on Day 1 and throughout the study. For mated females the body weight was recorded on GD 0, 4, 7, 11, 14, 17 and 20, and PND 1, 4, 7, 10 and 13.

FOOD CONSUMPTION:
- Food consumption for each animal was quantitatively measured except during the mating period on weekly basis for all animals (including all females prior mating), from Day 1 until the start of the mating period. For mated females the periods in which food consumption was recorded were the following: GD 0-4, GD 4-7, GD 7-11, GD 11-14 and GD 14-20, PND 1-4, PND 4-7, PND 7-10 and PND 10-13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of euthanasia
- Animals fasted: Yes, min. of 14 hrs., max. of 18 hrs
- Method: from orbital sinus under light isoflurane anesthesia. Only for coagulation parameters, samples were processed to plasma before analysis.
- How many animals: 5 males and 5 lactating females
- Parameters checked in tables 2 and 3 under "Any other information on materials and methods incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of euthanasia
- Animals fasted: Yes, min. of 14 hrs., max. of 18 hrs
- Method: from orbital sinus under light isoflurane anesthesia. Samples were processed to plasma before analysis.
- How many animals: 5 males and 5 lactating females
- Parameters checked in table 4 under "Any other information on materials and methods incl. tables" were examined.

PLASMA HORMONES: Yes
- Time of blood sample collection: day of euthanasia
- Animals fasted: Yes, min. of 14 hrs., max. of 18 hrs
- How many animals: all F0 males (at termination) and females (PND 14)
- Method: from orbital sinus under light isoflurane anesthesia. Blood collection occurred in the first half of the morning. Samples were processed to plasma before analysis. The levels of the thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for F0 males and females sampled at termination.

OTHER:
ESTROUS CYCLE
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
- during the 2 weeks of the pre-dosing period,
- from the beginning of the dosing period during the pre-mating and mating periods, until the females were mated.

MATING
Animals were paired on the basis of one male and one female from the same group (not siblings) for a maximum of 14 days. Each female was placed with same male, in the latter's cage, during the night.
Mating was confirmed by the presence of sperm in the vaginal smear or a vaginal plug and was recorded and taken as Day 0 of gestation (GD0). Mated females were separated from the males and smearing ceased once mating had been confirmed. The pre-coital time was calculated for each female.

PREGNANCY AND PARTURITION
For each female, the following was recorded:
- Date of mating,
- Date of parturition (any sign of a difficult or prolonged parturition was recorded),
- Duration of gestation,
- Abnormalities of nesting or nursing behaviour,
- Number of implantation sites. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

Sacrifice and pathology:

On completion of the dosing period, after at least 14 hours fasting, animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination. Males were sacrificed after a minimum of 28 days of administration. Lactating females were sacrificed at PND 14 whilst females which did not delivered at GD 24 o GD 26 after body weight recording (without overnight fasting).

GROSS PATHOLOGY: Yes. A full macroscopic post-mortem examination was performed on all F0 animals. This included examination of the external surfaces and all orifices; the cranial cavity and the external surfaces of the brain and spinal cord; the thoracic, abdominal and pelvic cavities with their associated organs and tissues; and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. For F0 females, number of corpora lutea and implantation sites and the number of intrauterine implantations were recorded. The uterus of non-gravid females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.

For F0 animals, the organs detailed in table 5 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together.
In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair may be taken and entered as a tissue comment. Organ weight as a percent of body weight (using the terminal body weight recorded immediately before euthanasia) was calculated.

HISTOPATHOLOGY: Yes (see table 6). Representative samples of tissues were collected and preserved in 10% neutral buffered formalin (except for the eyes and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's fixative). Tissues identified in table 6 were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin, except for the testes and epididymides which were stained with hematoxylin/PAS.
A microscopic examination was performed on:
- all tissues listed in table 6 from the first five (and from all animals that did not conceive for Cowper’s glands, glans penis, levator ani plus bulbo cavernous muscle complex, testes, epididymides, prostate, seminal vesicles, mammary gland area, uterus, thyroid, vagina and ovaries) euthanized as scheduled males and lactating females of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups.
- reproductive organs from animals that did not conceive, to investigate possible causes.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Statistics:

See section "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hypersalivation (mild to moderate) was occasionally observed during the dosing period in 5/10 males and in 2/10 females at 1000 mg/kg bw/day. This sign was observed from Day 22 at the earliest until Day 43 at the latest. This non-adverse finding, commonly noted when a test item is administered by the oral route, was considered to be test item-related.
All the other clinical signs i.e. cutaneous observations on various parts of the body (areas of hair loss, scabs), were considered to be unrelated to the test item as they were also present in control animals and/or were reported sporadically in only a few animals.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred in males or females at any dose level. Due to absence of delivery, two females, of the 300 and 1000 mg/kg bw/day groups were euthanized on Day 24 or 26 p.c. (i.e. Day 43). These females were found to be non-pregnant at hysterectomy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For details refer also to Table 7. Few statistically significant differences were noted occasionally at 100 and 1000 mg/kg bw/day in females were considered to be incidental as they were isolated and of limited relevance (less than 10% vs. controls).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For details refer also to Table 8. No relevant effects on mean food consumption were observed at any dose level.
The statistically significant differences between controls and females at 100 or 1000 mg/kg bw/day was not attributed to the test item treatment as they were of low magnitude, rare and/or not dose related.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematology parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period, including the statistically significant lower mean hematocrit value noted in females at 100 mg/kg bw/day.
The coagulation parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The main differences observed in the blood biochemistry parameters between control and test item-treated animals are presented in Table 9. At 1000 mg/kg bw/day, when compared to controls, mean phosphorus level was lower in females (p<0.05). The change was of low magnitude and observed in one sex only, and there was an absence of effects on mean calcium and creatinine levels and of histopathological correlates. It was therefore considered to be of no toxicological significance.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Mean thyroid hormone levels (T4 and TSH plasma levels) in F0 males and females are presented in Table 10. At 1000 mg/kg bw/day, when compared to controls, there was a statistically significant increase in mean T4 plasma level in F0 males (p<0.001), together with a concurrent but not statistically significant decrease in mean TSH level. Eight of ten individual T4 values were above the HCD (only two in controls). This finding was considered test item-related but non adverse in view of the low magnitude of change and the absence of histopathological correlates.
In females and when compared to controls, there were higher mean T4 plasma levels from 300 mg/kg bw/day and lower mean TSH level from 100 mg/kg bw/day. These differences were of low magnitude and not statistically significant. Individual TSH values remained within the range of the control values (with the exception of one value at 100 mg/kg bw/day) and were not necessarily consistent with individual T4 values. Therefore, a test item relationship was considered to be unlikely.

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For details refer also to Table 11.
There were no test item-related changes in organ weights.
The few organ weight changes were not considered to be related to the test item as they were small in magnitude, not dose-related, had no gross or microscopic correlates and/or were not consistent between the sexes.
They included increased mean absolute and relative-to-body kidney weights in high-dose test item-treated females when compared to controls, achieving statistical significance (+11 and +8%; p<0.05). However, in view of the absence of microscopic correlates and of the high inter-individual variability, these differences were not considered to be test item-related.
There were also increased mean absolute thymus weights in high-dose test item-treated females when compared to controls, achieving statistical significance (+51%; p<0.05). However, in view of the absence of microscopic correlates and of the high inter-individual variability, this difference was not considered to be test item-related.
The increased mean absolute and relative-to-body uterus weights in mid-dose test item-treated females when compared to controls, achieving statistical significance (+27 and +26%; p<0.05) was also considered to be unrelated to the test item administration in view of the absence of test item-related microscopic findings in the high-dose group and of the high inter-individual variability.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related macroscopic findings.
All macroscopic findings either had no histologic correlates or correlated with common histologic findings in control rats and were considered to be incidental. This included the accentuated lobular pattern in the liver from 3/10 males treated at 1000 mg/kg bw/day that had no microscopic correlates.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, or are known common background findings in the rat kept under laboratory conditions (Greaves, 2012), including the slight accumulation of hyaline droplets in 1/5 males treated at 1000 mg/kg bw/day, the increased incidence and severity of mononuclear cell infiltrate in the prostate of males treated at 1000 mg/kg bw/day when compared to controls, the unilateral ocular malformation (retinal disorganization) in 1/5 females treated at 1000 mg/kg bw/day, and the focal hyaline cast in the kidney from 1/5 females treated at 1000 mg/kg bw/day.
The vaginal smears correlated with the histopathological examination of female reproductive tract.
The examination of reproductive organs of two males and two females, evenly distributed in the mid- and high-dose groups, that did not conceive revealed no noteworthy microscopic lesions except for one female receiving 300 mg/kg bw/day that had a decidual reaction in the uterus and was in proestrus stage. The decidual reaction is a proliferative response that mimics normal implantation site and may occur occasionally in young and old, nonpregnant or pseudopregnant rats (NTP Nonneoplastic Lesion Atlas – Uterus – decidual reaction). The other female (high-dose group) was also in proestrus stage.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

For detail refer also to Table 12. There were no test item-related effects on the mean estrous cycle parameters.
No effect on spermatogenesis was reported.
Details on reproductive data are reported in Table 13 and Table 14. No test item-related effects were observed on the precoital time (time taken to mate; all pairs mated within 4 days) or on mating/fertility indexes.
All females were pregnant, except two females at 300 or 1000 mg/kg bw/day, which accounts for the slightly lower mean fertility index noted at these dose levels. These isolated cases were considered as incidental.
No test item-related effects were observed on the number of corpora lutea, pre- and post implantation losses/pups delivered or on the duration of gestation at any dose level.

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Absence of adverse effects up to and including the highest dose-level tested.

Key result

Critical effects observed:

no

Chemical Analysis of the Dosage Forms: Concentration

No test item was found in the control dose formulations analysed in Weeks 1, 4 and 7.

For dose formulations, the measured concentrations of groups 2, 3 and 4 (low-, mid- and high-dose, respectively) analyzed in Weeks 1, 4 and 7 were found to be within an acceptable range of variations when compared with the nominal values (± 10% of their theoretical concentrations), except in Week 1 for group 4 (-13.0%). This was not considered to have a major impact on the study results or conclusion as the magnitude of the deviation was minor compared to the acceptable range (± 10%).

 

Table 7 - Mean Body Weights/Mean Body Weight Changes (g)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Pre-mating (males and females) or whole study
Day 1 (% vs. controls)	415	387 (-7)	413 (0)	393 (-5)	253	237 (-6)	263 (+4)	266 (+5)
Day 8	452	422	454	428	261	245	268	276
Day 15 (% vs. controls)	481	448 (-7)	491 (+2)	462 (-4)	269	254 (-6)	279 (+4)	284 (+6)
Days 1-8	+37	+34	+41	+35	+7	+8	+5	+10
Days 8-15	+29	+26	+38	+34	+8	+9	+11	+8
Days 1-15	+66	+60	+78	+69	+16	+17	+16	+18
Day 22	490	464	506	477	/	/	/	/
Day 29 (% vs. controls)	505	484 (-4)	530 (+5)	496 (-2)	/	/	/	/
Days 1-29	+90	+97	+116	+103	/	/	/	/
Gestation
GD 0	/	/	/	/	275	260	283	297*
(% from controls)						(-5)	(+3)	(+8)
GD 4	/	/	/	/	298	278	306	311
GD 7	/	/	/	/	305	287	315	323
GD 11	/	/	/	/	329	307*	335	343
(% from controls)						(-7)	(+2)	(+4)
GD 14	/	/	/	/	339	320	348	355
GD 17	/	/	/	/	371	353	388	389
GD 20 (% from controls)	/	/	/	/	426	410 (-4)	449 (+5)	442 (+4)
GD 0-4	/	/	/	/	+23	+18	+22	+14
GD 4-7	/	/	/	/	+8	+9	+10	+12
GD 7-11	/	/	/	/	+24	+21	+19	+20
GD 11-14	/	/	/	/	+11	+13	+14	+13
GD 14-17	/	/	/	/	+32	+33	+39	+34
GD -20	/	/	/	/	+55	+58	+61	+52
GD 0-20	/	/	/	/	+152	+150	+166	+145
Lactation
PND 1 (% from controls)	/	/	/	/	324	304 (-6)	334 (+3)	343 (+6)
PND 4	/	/	/	/	338	313*	348	359
PND 7	/	/	/	/	347	324*	354	369*
(% from controls)						(-7)	(+2)	(+6)
PND 10	/	/	/	/	357	341	370	384*
(% from controls)						(-4)	(+4)	(+8)
PND 13 (% from controls)	/	/	/	/	368	348 (-5)	376 (+2)	391 (+6)
PND 1-4	/	/	/	/	+14	+9	+14	+15
PND 4-7	/	/	/	/	+9	+11	+7	+10
PND 7-10	/	/	/	/	+10	+17*	+16	+15
PND 10-13	/	/	/	/	+11	+7	+6	+7
PND 1-13	/	/	/	/	+44	+44	+42	+47

Statistically significance: *: p<0.05

/: not applicable.

 

Table 8 - Mean Food Consumption (g/animal/day)

Sex		Male				Female
Dose level (mg/kg bw/day)		0	100	300	1000	0	100	300	1000
Pre-mating
Days 1-8		31	30	32	30	18	16	17	20*
(% from controls)			(-3)	(+3)	(-3)		(-11)	(-6)	(+11)
Days 8-15		29	26	31	29	17	16	17	18
Gestation
GD 0-4		/	/	/	/	21	19	22	21
GD 4-7		/	/	/	/	23	21	23	25
GD 7-11		/	/	/	/	24	21	22	23
GD -14		/	/	/	/	23	20*	23	24
GD 14-20		/	/	/	/	25	24	26	28*
Lactation
PND 1-4	/		/	/	/	40	36	34	37
PND 4-7	/		/	/	/	50	49	44	49
PND 7-10	/		/	/	/	63	61	52	61
PND 10-13	/		/	/	/	73	68	64	68

/: not applicable;

Statistical significance: *: p<0.05

 

Table 9 - Most relevant Blood Biochemistry Parameter Changes

Sex	Male				Female
Dose level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Phosphorus (mmol/L)	2.27	2.14	2.22	2.21	2.66	2.75	2.86	2.08*
(% from controls)		(-6)	(-2)	(-3)		(+4)	(+8)	(-22)

Statistical significance: *: p<0.05

 

Table 10 - Thyroid Hormones

	Thyroid hormones
	F0 males					F0 females
Dose level (mg/kg bw/day)	0	100	300	1000	HCD	0	100	300	1000	HCD
T4 (ng/mL) ± SD	46.94 ± 6.856	47.86 ± 6.175	53.84 ± 6.150	59.28# ± 7.782	[25.60; 54.17]	28.17 ± 6.554	28.33 ± 6.639	34.81 ± 7.668	35.75 ± 9.596	[20.1; 49.9]
(% from controls)		(+2.0)	(+14.7)	(+26.3)			(+0.6)	(+23.6)	(+26.9)
TSH (pg/mL) ± SD	1642 ± 1317.1	1604 ± 889.2	1274 ± 668.5	921 ±510.5	[627; 4455]	1323 ± 605.7	922 ± 406.9	1035 ± 472.4	909 ± 458.6	[421; 4038]
(% from controls)		(-2.3)	(-22.4)	(-43.9)			(-30.3)	(-21.8)	(-31.3)

HCD: Historical Control Data (OECD 421/422, 2016-2021), [min of individual values; max of individual values].

Statistical significance: #: p<0.001

 

Table 11 - Organ weight variations

Sex	Male			Female
Dose level (mg/kg bw/day)	100	300	1000	100	300	1000
Exam. animals / Num. of animals	10/10	10/10	10/10	10/10	10/10	10/10
- Final body weight	-5	+3	-3	-5	+2	+4
- Kidneys
.absolute	0	+6	+8	0	+2	+11*
.relative	+5	+3	+12	+6	-1	+8*
- Thymus
.absolute	-19	+12	-23	+3	+40	+51*
.relative	-14	+9	-17	+9	+33	+40
- Uterus
.absolute				+6	+27*	+19
.relative				+12	+26*	+15

Statistically significant from controls: *: p<0.05

The significance concerned the organ weights values and not the percentages.

 

Table 12 - Mean Estrous Cycle Parameters

Dose level (mg/kg bw/day)	0	100	300	1000	HCD
Number of females examined	10	10	10	10	110
Mean number of days of metestrus ± SD	3.2 ± 0.8	3.2 ± 0.9	3.4 ± 0.8	2.5 ± 0.8	[3.2;4.7]
Mean number of days of diestrus ± SD	5.3 ± 1.7	5.6 ± 1.7	5.7 ± 1.3	6.1 ± 0.7	[3.0;5.0]
Mean number of days of proestrus ± SD	3.1 ± 1.0	2.2 ± 1.4	2.3 ± 1.1	2.5 ± 1.1	[2.3;3.5]
Mean number of days of estrus ± SD	3.4 ± 0.7	4.0 ± 0.8	3.6 ± 0.7	3.9 ± 0.6	[3.5;4.7]
Mean number of cycles ± SD	2.9 ± 0.3	3.0 ± 0.0	2.9 ± 0.3	3.0 ± 0.0	[2.5;3.2]
Mean duration of cycles (days) ± SD	4.1 ± 0.3	4.0 ± 0.1	4.1 ± 0.2	4.1 ± 0.3	[3.8;5.2]
Mean percent of females cycling normally (%)	100	100	90.0	80.0	[80;100]
Mean percent of females with all stages (%)	100	90.0	100	100	[90;100]

No statistically significant differences vs. controls. HCD: Historical Control Data (OECD 421/422, 2017 to 2021, n =12 studies); /: not in HCD; [min study mean;max study mean]; 

 

Table 13 - Summary of Mating and Fertility Data

Dose level (mg/kg bw/day)	0	100	300	1000
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	10
Number of females mated	10	10	10	10
Mean number of days taken to mate	2.8	2.6	2.8	2.3
Number of pregnant females	10	10	9	9
Number of females with live born pup	10	10	9	9
Mating index (female, %)	100	100	100	100
Fertility index (%)	100	100	90	90

M: Males; F: Females.

No statistically significant differences vs. controls

 

Table 14 - Summary of Delivery and Litter Data

Dose level (mg/kg bw/day)	0	100	300	1000
Number of pregnant females	10	10	9	9
Number of females which delivered	10	10	9	9
Number of females with live concepti	10	10	9	9
Gestation index (%)	100	100	100	100
Mean duration of gestation (days)	21.4	21.3	21.2	21.4
Mean number of corpora lutea	16.4	16.5	16.7	15.7
Mean number of implantation sites	15.9	16.3	16.4	14.9
Mean pre-implantation loss (%)	3.1	1.2	1.6	3.4
Mean number of pups delivered	15.1	15.6	16.0	14.0
Mean post-implantation loss (%)	5.2	4.2	2.4	6.4

No statistically significant differences vs. controls

Conclusions:

Based on the experimental conditions of this study, the No Observed Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg bw/day based on the absence of adverse effects at this dose level.

Executive summary:

This combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD Guideline No. 422 and in compliance with GLP. 

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg bw/day, at a constant dosage volume of 5 mL/kg bw/day. A control group of ten male and ten female rats received the vehicle only (corn oil) under the same experimental conditions.

Males were treated for an overall period of approximately 4 weeks: 2 weeks before mating and then during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 7 to 8 weeks: 2 weeks before mating, through mating (up to 2 weeks) and gestation (3 weeks), until PND 13 inclusive.

The actual test item concentrations in the dose formulations were determined in Weeks 01, 04 and 07, using a validated GC/FID analytical method. Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before mating until the females had mated.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until PND 13. Hematology, coagulation, and blood biochemistry investigations were performed on the first five males and females in each group at scheduled euthanasia. Thyroid hormones (TSH and T4) were determined in males and females at termination. Males were euthanized after completion of the mating period. Dams were euthanized on PND 14. A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs (including animals that did not conceive). Designated organs were weighed, and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five control and high-dose males and lactating females, on all macroscopic lesions, and on reproductive organs from animals that did not conceive.

 

Actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variations (-10.4% to +0.6%) when compared to the nominal concentrations, except in Week 1 when the group 4 dose formulation was found to be -13.0% versus the nominal concentration.

 

There were no unscheduled deaths. Hypersalivation was sometimes observed in up to 7/20 animals at 1000 mg/kg bw/day. No test item-related effects were noted on mean body weight, mean body weight change or mean food consumption. There were no test item-related effects on estrous cycle. Mating, fertility and delivery data were unaffected by the test item treatment. At laboratory investigations (hematology, coagulation, blood biochemistry), there were no differences that could be related to test item treatment. When compared with controls, there was a statistically significant increase in mean T4 level (+26.3%, p<0.001) in males at 1000 mg/kg bw/day together with a concurrent but not statistically significant decrease in mean TSH level (-43.9%). This finding was considered test item-related but non adverse in view of the low magnitude of change and the absence of histopathological correlates. There were no test item-related effects on thyroid hormones levels in females. At pathology, there were no organ weight differences or macroscopic/microscopic findings related to the test item.

 

Based on the experimental conditions of this study:

• the No Observed Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg bw/day based on the absence of adverse effects at this dose level.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,10-decanediyl diacrylate

EC Number:

235-922-4

EC Name:

1,10-decanediyl diacrylate

Cas Number:

13048-34-5

Molecular formula:

C16H26O4

IUPAC Name:

10-(prop-2-enoyloxy)decyl prop-2-enoate

Test material form:

liquid: viscous

Remarks:

Colorless

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The Sprague-Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study was designed such that it does not require an unnecessary number of animals to accomplish its objectives.
The Study plan was reviewed by the Test Facility Ethics Committee (CEC) in order to assess compliance with the corresponding authorized "project" as defined in Directive 2010/63/EU and in French decree No. 2013-118. During the study, the CEC was regularly informed of any amendments to the Study Plan that could had an impact on animal welfare.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at initiation of dosing: males were approximatively 10 weeks old, the females were approximatively 11 weeks old.
- Weight at initiation of dosing: males between 332 and 455 g, females between 214 and 311 g
- Housing: single housed in polycarbonate cages containing appropriate bedding. Individual housing of F0 animals was chosen as group housing for pregnant animals can adversely affect gestation and lactation, and to avoid aggressive behavior around mating. Toward the end of gestation and during lactation, autoclaved wood shavings were provided to females and their litter as nesting material. For psychological / environmental enrichment, animals were provided with rat hut and wooden log, except when interrupted by study procedures / activities.
- Diet: ad libitum, pellet of SSNIFF rat/mouse pelleted maintenance diet, batch No. 62874737 (SSNIFF Spezialdiäten GmbH, Soest, Germany), sterilized by irradiation.
- Water: ad libitum, municipal tap water freely available in water bottles.

It was considered that there were no known contaminants in the feed, water and enrichments that interfered with the objectives of the study.

ENVIRONMENTAL CONDITIONS (TARGETED CONDITIONS)
- Temperature: 20°C to 24°C
- Humidity: 30% to 70%
- Air changes: approximately 8 to 15 filtered, non-recycled air changes per hour
- Photoperiod: 12 hours light and 12 hours dark

IN-LIFE DATES: From: 29 June 2021 To: 22 August 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

JUSTIFICATION FOR ROUTE OF EXPOSURE: the oral route of exposure was selected because this is a route of administration which is requested by the Regulatory Authorities for this type of test item.
The dose levels were selected based on a preliminary 2-week toxicity study performed in the same species (Study No. 48660 TSR (Privat, 2021)) in which the test item was administered to five males and five females per group at 250, 500 and 1000 mg/kg bw/day. The only test item clinical sign was hypersalivation at 500 but mainly 1000 mg/kg bw/day. The test item also induced minimally lower body weights and body weight gains in males at 1000 mg/kg bw/day. The increased incidence of accentuated lobular pattern noted in males and females at 1000 mg/kg bw/day was considered to be equivocal in the absence of microscopic examination.
Therefore, the high dose level selected for the present study was 1000 mg/kg bw/day. The low and intermediate dose levels were selected based on an approximately three-fold interval (i.e. 100 and 300 mg/kg bw/day).

PREPARATION OF DOSING SOLUTIONS: test item formulations were prepared on a weekly basis, based on test item dose formulation stability data and vehicle expiry. The stability of test item dose formulations was determined in Study No. 48659 VAS (Garapon, 2021) that demonstrate that the test item formulation is stable when prepared and stored under the same conditions as those used in the present study at a concentration between 2 and 200 mg/mL for 11 days. Control formulations (vehicle only) were prepared considering the expiry date of the used vehicle. All the formulations were stored at room temperature and protected from light.

Dosing formulations were prepared based on Study No. 48659 VAS (Garapon, 2021) describing the preparation procedure for a range of concentrations covering the lowest and the highest used in this study, at appropriate concentrations to meet dose level requirements. The control dose formulations were stirred continuously just before dosing. The test item dose formulations were stirred continuously at least 15 minutes before dosing and during dosing. All formulations were maintained under delivery conditions (at room temperature and protected from light).

VEHICLE
- Justification for use and choice of vehicle: the test item was found to be soluble and stable in the selected vehicle. The vehicle is also known to be non-toxic to animals.
- Concentration in vehicle: 20, 60 and 200 mg/mL for the low-, mid- and high-dose groups respectively.
- Amount of vehicle: 5 mL/kg bw (based on the most recent body weight)
- Lot/batch no.: MKCM3364 and MKCN9742

Details on mating procedure:

- M/F ratio per cage: 1:1 (not siblings)
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of gestation (GD0).
- Mated females were separated from the males and smearing ceased once mating had been confirmed.
- The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLE COLLECTION AND ANALYSIS
Dose formulation samples were collected for analysis on weeks 1, 4 and 7 from all groups. Analyses were performed by GC/FID using a validated analytical procedure (48659 VAS.MET). Samples were analyzed to determine concentration. As acceptance criteria, individual samples concentration shall be of ±10%.

Stability analyses performed previously in conjunction with study No. 48659 VAS demonstrated that the test item is stable in the vehicle for 11 days when prepared and stored under the same conditions as in this study at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Males were treated 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed) and until euthanasia (at least 4 weeks in total).

Females were treated at least 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed), during gestation, during lactation until PND 13 inclusive or until euthanasia for females with no delivery (GD 24 or 26).

Pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low-dose group

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid-dose group

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high-dose group

No. of animals per sex per dose:

10 in each dose group.

Refer also to table 1 in "Any other information on results incl. tables".

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the oral route of exposure was selected because this is a route of administration which is requested by the Regulatory Authorities for this type of test item. The dose levels were selected based on a preliminary 2-week toxicity study performed in the same species (Study No. 48660 TSR (Privat, 2021)) in which the test item was administered to five males and five females per group at 250, 500 and 1000 mg/kg bw/day. The only test item clinical sign was hypersalivation at 500 but mainly 1000 mg/kg bw/day. The test item also induced minimally lower body weights and body weight gains in males at 1000 mg/kg bw/day. The increased incidence of accentuated lobular pattern noted in males and females at 1000 mg/kg bw/day was considered to be equivocal in the absence of microscopic examination.
Therefore, the high dose level selected for the present study was 1000 mg/kg bw/day. The low and intermediate dose levels were selected based on an approximately three-fold interval (i.e. 100 and 300 mg/kg bw/day).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: for all animals, at least once daily beginning upon arrival through termination, before dosing when applicable. Mortality was also checked for all animals at least twice daily a (at beginning and end of working day) beginning upon arrival through termination. In addition to the daily cage side observation, animals were observed between 1 to 3 hours post-dose on the days of dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a full clinical examination was performed at least once a week for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly for all animals (including all females prior mating), from at least Week -1, on Day 1 and throughout the study. For mated females the body weight was recorded on GD 0, 4, 7, 11, 14, 17 and 20, and PND 1, 4, 7, 10 and 13.

FOOD CONSUMPTION:
- Food consumption for each animal was quantitatively measured except during the mating period on weekly basis for all animals (including all females prior mating), from Day 1 until the start of the mating period. For mated females the periods in which food consumption was recorded were the following: GD 0-4, GD 4-7, GD 7-11, GD 11-14 and GD 14-20, PND 1-4, PND 4-7, PND 7-10 and PND 10-13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of euthanasia
- Animals fasted: Yes, min. of 14 hrs., max. of 18 hrs
- Method: from orbital sinus under light isoflurane anesthesia. Only for coagulation parameters, samples were processed to plasma before analysis.
- How many animals: 5 males and 5 lactating females
- Parameters checked in tables 2 and 3 under "Any other information on materials and methods incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of euthanasia
- Animals fasted: Yes, min. of 14 hrs., max. of 18 hrs
- Method: from orbital sinus under light isoflurane anesthesia. Samples were processed to plasma before analysis.
- How many animals: 5 males and 5 lactating females
- Parameters checked in table 4 under "Any other information on materials and methods incl. tables" were examined.

PLASMA HORMONES: Yes
- Time of blood sample collection: day of euthanasia
- Animals fasted: Yes, min. of 14 hrs., max. of 18 hrs
- How many animals: all F0 males (at termination) and females (PND 14)
- Method: from orbital sinus under light isoflurane anesthesia. Blood collection occurred in the first half of the morning. Samples were processed to plasma before analysis. The levels of the thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for F0 males and females sampled at termination.

PREGNANCY AND PARTURITION
For each female, the following was recorded:
- Date of mating,
- Date of parturition (any sign of a difficult or prolonged parturition was recorded),
- Duration of gestation,
- Abnormalities of nesting or nursing behaviour,
- Number of implantation sites. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
- during the 2 weeks of the pre-dosing period,
- from the beginning of the dosing period during the pre-mating and mating periods, until the females were mated.

Sperm parameters (parental animals):

The stages of spermatogenesis in the male gonads were determined and the histopathology of interstitial testicular cell structure was performed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes by random selection
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible). Whenever necessary, partial adjustment (for example 5 males and 3 females) was allowed; extra pups were euthanized by an intraperitoneal injection of sodium pentobarbital. No cross-fostering was performed.

PARAMETERS EXAMINED
The following parameters were examined for each litter:
- number of pups born (live, dead and cannibalized) on PND 1
- external abnormalities of the pups
- number and sex of pups alive on PND 1, 4, 7, 10 and 13 at least
- clinical signs and abnormal behavior on a daily basis; the first clinical examination (on PND 1) included: gross external examination (e.g. external visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture; presence of umbilical cord, lack of milk in the stomach, presence of dried secretion)
- qualitative assessment of body temperature, activity and reaction to handling
- individual Body Weight of Live pups on PND 1, 4, 7, 10 and 13
- anogenital distance (AGD) on PND 1 (normalized to the cube root of PND 1 body weight) (AGD normalized = AGD / body weight^¿)
- number of nipples and of areolae in male pups on PND 12
- thyroid hormones: from 2 culled pups/litter (PND 4) and 1 pup/sex/litter (PND 13) for all groups blood was collected and processed to plasma. The levels of the thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for pups sampled on PND 13. Plasma samples obtained on PND 4 from pups were kept at 80°C pending possible analysis but finally not analysed.

GROSS EXAMINATION OF DEAD PUPS:
yes, found dead or moribund animals (euthanized by intraperitoneal injection of sodium pentobarbital) were necropsied and macroscopic post-mortem examination of the principal thoracic and abdominal organs, comprising also a detailed external examination (including orifices and buccal cavity), was performed on all found dead and prematurely euthanized pups. Particular attention was paid to the external genital organs, the internal reproductive organs and to whether the pup had been fed (e.g. presence of milk in the stomach) when possible. Macroscopic lesions were preserved.

Postmortem examinations (parental animals):

On completion of the dosing period, after at least 14 hours fasting, animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination. Males were sacrificed after a minimum of 28 days of administration. Lactating females were sacrificed at PND 14 whilst females which did not delivered at GD 24 o GD 26 after body weight recording (without overnight fasting).

GROSS PATHOLOGY: Yes. A full macroscopic post-mortem examination was performed on all F0 animals. This included examination of the external surfaces and all orifices; the cranial cavity and the external surfaces of the brain and spinal cord; the thoracic, abdominal and pelvic cavities with their associated organs and tissues; and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. For F0 females, number of corpora lutea and implantation sites and the number of intrauterine implantations were recorded. The uterus of non-gravid females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.

For F0 animals, the organs detailed in table 5 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together.
In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair may be taken and entered as a tissue comment. Organ weight as a percent of body weight (using the terminal body weight recorded immediately before euthanasia) was calculated.

HISTOPATHOLOGY: Yes (see table 6). Representative samples of tissues were collected and preserved in 10% neutral buffered formalin (except for the eyes and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's fixative). Tissues identified in table 6 were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin, except for the testes and epididymides which were stained with hematoxylin/PAS.
A microscopic examination was performed on:
- all tissues listed in table 6 from the first five (and from all animals that did not conceive for Cowper’s glands, glans penis, levator ani plus bulbo cavernous muscle complex, testes, epididymides, prostate, seminal vesicles, mammary gland area, uterus, thyroid, vagina and ovaries) euthanized as scheduled males and lactating females of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups.
- reproductive organs from animals that did not conceive, to investigate possible causes.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

SACRIFICE
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on PND 4 when blood sampled), followed by exsanguination at necropsy:
- unselected pups: PND 4
- surviving pups: PND 13

The animals (unselected on PND 4 and PND 13) were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs, comprising also a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs and the internal reproductive organs. Any macroscopic lesions (all pups) and thyroid with parathyroids (1 PND 13 pup/sex/litter) were preserved.

HISTOPATHOLOGY / ORGAN WEIGTHS (see table 6)
The body weight of one selected pup/sex/litter (or of two pups from the same sex when there was only one sex in the litter) euthanized on PND 13 was recorded before euthanasia. Thyroids with parathyroids of the selected pup/sex/litter euthanized on PND 13 were preserved in 10% neutral buffered formalin.

Statistics:

See section "Any other information on materials and methods incl. tables".

Reproductive indices:

Pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] x 100

Post-implantation loss: [(Number of implantation sites - Number of live pups) / Number of implantation sites] x 100

Mating index (%): (Number of mated animals) / (Number of paired animals) x 100

Fertility index (%): (Number of pregnant females partners) / (Number of mated pairs) x 100

Gestation index (%): (Number of females with live born pups) / (Number of pregnant females) x 100

Offspring viability indices:

Live birth index: (Number of live born pups on PND 1 / Number of delivered pups) x 100

Viability index on PND 4: (Number of surviving pups on PND 4 (before culling) / Number of delivered pups) x 100

Lactation index on PND 13: (Number of surviving pups on PND 13 / Number of surviving pups on PND 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hypersalivation (mild to moderate) was occasionally observed during the dosing period in 5/10 males and in 2/10 females at 1000 mg/kg bw/day. This sign was observed from Day 22 at the earliest until Day 43 at the latest. This non-adverse finding, commonly noted when a test item is administered by the oral route, was considered to be test item-related.
All the other clinical signs i.e. cutaneous observations on various parts of the body (areas of hair loss, scabs), were considered to be unrelated to the test item as they were also present in control animals and/or were reported sporadically in only a few animals.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred in males or females at any dose level. Due to absence of delivery, two females, of the 300 and 1000 mg/kg bw/day groups were euthanized on Day 24 or 26 p.c. (i.e. Day 43). These females were found to be non-pregnant at hysterectomy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For details refer also to Table 7. Few statistically significant differences were noted occasionally at 100 and 1000 mg/kg bw/day in females were considered to be incidental as they were isolated and of limited relevance (less than 10% vs. controls).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For details refer also to Table 8. No relevant effects on mean food consumption were observed at any dose level.
The statistically significant differences between controls and females at 100 or 1000 mg/kg bw/day was not attributed to the test item treatment as they were of low magnitude, rare and/or not dose related.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematology parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period, including the statistically significant lower mean hematocrit value noted in females at 100 mg/kg bw/day.
The coagulation parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The main differences observed in the blood biochemistry parameters between control and test item-treated animals are presented in Table 9. At 1000 mg/kg bw/day, when compared to controls, mean phosphorus level was lower in females (p<0.05). The change was of low magnitude and observed in one sex only, and there was an absence of effects on mean calcium and creatinine levels and of histopathological correlates. It was therefore considered to be of no toxicological significance.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Mean thyroid hormone levels (T4 and TSH plasma levels) in F0 males and females are presented in Table 10. At 1000 mg/kg bw/day, when compared to controls, there was a statistically significant increase in mean T4 plasma level in F0 males (p<0.001), together with a concurrent but not statistically significant decrease in mean TSH level. Eight of ten individual T4 values were above the HCD (only two in controls). This finding was considered test item-related but non adverse in view of the low magnitude of change and the absence of histopathological correlates.
In females and when compared to controls, there were higher mean T4 plasma levels from 300 mg/kg bw/day and lower mean TSH level from 100 mg/kg bw/day. These differences were of low magnitude and not statistically significant. Individual TSH values remained within the range of the control values (with the exception of one value at 100 mg/kg bw/day) and were not necessarily consistent with individual T4 values. Therefore, a test item relationship was considered to be unlikely.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, or are known common background findings in the rat kept under laboratory conditions (Greaves, 2012), including the slight accumulation of hyaline droplets in 1/5 males treated at 1000 mg/kg bw/day, the increased incidence and severity of mononuclear cell infiltrate in the prostate of males treated at 1000 mg/kg bw/day when compared to controls, the unilateral ocular malformation (retinal disorganization) in 1/5 females treated at 1000 mg/kg bw/day, and the focal hyaline cast in the kidney from 1/5 females treated at 1000 mg/kg bw/day.
The vaginal smears correlated with the histopathological examination of female reproductive tract.
The examination of reproductive organs of two males and two females, evenly distributed in the mid- and high-dose groups, that did not conceive revealed no noteworthy microscopic lesions except for one female receiving 300 mg/kg bw/day that had a decidual reaction in the uterus and was in proestrus stage. The decidual reaction is a proliferative response that mimics normal implantation site and may occur occasionally in young and old, nonpregnant or pseudopregnant rats (NTP Nonneoplastic Lesion Atlas – Uterus – decidual reaction). The other female (high-dose group) was also in proestrus stage.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

For detail refer also to Table 12. There were no test item-related effects on the mean estrous cycle parameters.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effect on spermatogenesis was reported.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Details on reproductive data are reported in Table 13 and Table 14. No test item-related effects were observed on the precoital time (time taken to mate; all pairs mated within 4 days) or on mating/fertility indexes.
All females were pregnant, except two females at 300 or 1000 mg/kg bw/day, which accounts for the slightly lower mean fertility index noted at these dose levels. These isolated cases were considered as incidental.
No test item-related effects were observed on the number of corpora lutea, pre- and post implantation losses/pups delivered or on the duration of gestation at any dose level.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Absence of adverse effects up to and including the highest dose-level tested.

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Absence of adverse effects up to and including the highest dose-level tested.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Table 15 presents the numbers of pups with findings at gross examination on PND 1 (external examination, qualitative assessment of body temperature and/or activity/reaction to handling). Table 16 summarizes the recorded clinical signs in lactating pups.
No test item-related clinical signs were observed at any dose level.
The few findings recorded during the lactation period were not attributed to the test item treatment as they were reported in isolated litters and/or were not dose-related and/or are routinely observed in rat pups of this strain and age.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effects were observed on the incidence of pups found dead or cannibalized at any dose level as shown in Table 17 and Table 18. There were no test item-related effects on pup mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Refer to Table 19 for details on pups body weight. There were no test item-related effects on mean body weight or mean body weight gain in pups at any dose-level.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Refer to Table 20 and Table 21 for details on pups thyroid hormones. The mean levels of thyroid hormones were unaffected by the test item treatment on PND 13 pups from both sexes.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to Table 22 for details. When compared to controls, there was a trend towards a higher mean percentage of males in test item-treated groups. As this difference was not statistically significant, was close to the HCD range, and was not accompanied by correlating changes (e.g. external genital abnormalities, anogenital distance alteration), it was considered to be incidental and unrelated to the test item treatment. Moreover, the control data was slightly low because of one litter with 18.8% of males (control mean at 52% when excluding this litter).

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

For details refer to Table 23. No test item-related effects on the anogenital distance were observed in males or females at any dose level (no statistical significance, no dose-relationship and/or only small difference from controls).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples or areolae were noted in any male pups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related macroscopic findings.
The scab seen in 1/9 females treated at 1000 mg/kg bw/day was considered to be incidental.

Histopathological findings:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Absence of adverse effects up to and including the highest dose-level tested.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Chemical Analysis of the Dosage Forms: Concentration

No test item was found in the control dose formulations analysed in Weeks 1, 4 and 7.

For dose formulations, the measured concentrations of groups 2, 3 and 4 (low-, mid- and high-dose, respectively) analyzed in Weeks 1, 4 and 7 were found to be within an acceptable range of variations when compared with the nominal values (± 10% of their theoretical concentrations), except in Week 1 for group 4 (-13.0%). This was not considered to have a major impact on the study results or conclusion as the magnitude of the deviation was minor compared to the acceptable range (± 10%).

 

Table 7 - Mean Body Weights/Mean Body Weight Changes (g)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Pre-mating (males and females) or whole study
Day 1 (% vs. controls)	415	387 (-7)	413 (0)	393 (-5)	253	237 (-6)	263 (+4)	266 (+5)
Day 8	452	422	454	428	261	245	268	276
Day 15 (% vs. controls)	481	448 (-7)	491 (+2)	462 (-4)	269	254 (-6)	279 (+4)	284 (+6)
Days 1-8	+37	+34	+41	+35	+7	+8	+5	+10
Days 8-15	+29	+26	+38	+34	+8	+9	+11	+8
Days 1-15	+66	+60	+78	+69	+16	+17	+16	+18
Day 22	490	464	506	477	/	/	/	/
Day 29 (% vs. controls)	505	484 (-4)	530 (+5)	496 (-2)	/	/	/	/
Days 1-29	+90	+97	+116	+103	/	/	/	/
Gestation
GD 0	/	/	/	/	275	260	283	297*
(% from controls)						(-5)	(+3)	(+8)
GD 4	/	/	/	/	298	278	306	311
GD 7	/	/	/	/	305	287	315	323
GD 11	/	/	/	/	329	307*	335	343
(% from controls)						(-7)	(+2)	(+4)
GD 14	/	/	/	/	339	320	348	355
GD 17	/	/	/	/	371	353	388	389
GD 20 (% from controls)	/	/	/	/	426	410 (-4)	449 (+5)	442 (+4)
GD 0-4	/	/	/	/	+23	+18	+22	+14
GD 4-7	/	/	/	/	+8	+9	+10	+12
GD 7-11	/	/	/	/	+24	+21	+19	+20
GD 11-14	/	/	/	/	+11	+13	+14	+13
GD 14-17	/	/	/	/	+32	+33	+39	+34
GD -20	/	/	/	/	+55	+58	+61	+52
GD 0-20	/	/	/	/	+152	+150	+166	+145
Lactation
PND 1 (% from controls)	/	/	/	/	324	304 (-6)	334 (+3)	343 (+6)
PND 4	/	/	/	/	338	313*	348	359
PND 7	/	/	/	/	347	324*	354	369*
(% from controls)						(-7)	(+2)	(+6)
PND 10	/	/	/	/	357	341	370	384*
(% from controls)						(-4)	(+4)	(+8)
PND 13 (% from controls)	/	/	/	/	368	348 (-5)	376 (+2)	391 (+6)
PND 1-4	/	/	/	/	+14	+9	+14	+15
PND 4-7	/	/	/	/	+9	+11	+7	+10
PND 7-10	/	/	/	/	+10	+17*	+16	+15
PND 10-13	/	/	/	/	+11	+7	+6	+7
PND 1-13	/	/	/	/	+44	+44	+42	+47

Statistically significance: *: p<0.05

/: not applicable.

 

Table 8 - Mean Food Consumption (g/animal/day)

Sex		Male				Female
Dose level (mg/kg bw/day)		0	100	300	1000	0	100	300	1000
Pre-mating
Days 1-8		31	30	32	30	18	16	17	20*
(% from controls)			(-3)	(+3)	(-3)		(-11)	(-6)	(+11)
Days 8-15		29	26	31	29	17	16	17	18
Gestation
GD 0-4		/	/	/	/	21	19	22	21
GD 4-7		/	/	/	/	23	21	23	25
GD 7-11		/	/	/	/	24	21	22	23
GD -14		/	/	/	/	23	20*	23	24
GD 14-20		/	/	/	/	25	24	26	28*
Lactation
PND 1-4	/		/	/	/	40	36	34	37
PND 4-7	/		/	/	/	50	49	44	49
PND 7-10	/		/	/	/	63	61	52	61
PND 10-13	/		/	/	/	73	68	64	68

/: not applicable;

Statistical significance: *: p<0.05

 

Table 9 - Most relevant Blood Biochemistry Parameter Changes

Sex	Male				Female
Dose level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Phosphorus (mmol/L)	2.27	2.14	2.22	2.21	2.66	2.75	2.86	2.08*
(% from controls)		(-6)	(-2)	(-3)		(+4)	(+8)	(-22)

Statistical significance: *: p<0.05

 

Table 10 - Thyroid Hormones

	Thyroid hormones
	F0 males					F0 females
Dose level (mg/kg bw/day)	0	100	300	1000	HCD	0	100	300	1000	HCD
T4 (ng/mL) ± SD	46.94 ± 6.856	47.86 ± 6.175	53.84 ± 6.150	59.28# ± 7.782	[25.60; 54.17]	28.17 ± 6.554	28.33 ± 6.639	34.81 ± 7.668	35.75 ± 9.596	[20.1; 49.9]
(% from controls)		(+2.0)	(+14.7)	(+26.3)			(+0.6)	(+23.6)	(+26.9)
TSH (pg/mL) ± SD	1642 ± 1317.1	1604 ± 889.2	1274 ± 668.5	921 ±510.5	[627; 4455]	1323 ± 605.7	922 ± 406.9	1035 ± 472.4	909 ± 458.6	[421; 4038]
(% from controls)		(-2.3)	(-22.4)	(-43.9)			(-30.3)	(-21.8)	(-31.3)

HCD: Historical Control Data (OECD 421/422, 2016-2021), [min of individual values; max of individual values].

Statistical significance: #: p<0.001

 

Table 11 - Organ weight variations

Sex	Male			Female
Dose level (mg/kg bw/day)	100	300	1000	100	300	1000
Exam. animals / Num. of animals	10/10	10/10	10/10	10/10	10/10	10/10
- Final body weight	-5	+3	-3	-5	+2	+4
- Kidneys
.absolute	0	+6	+8	0	+2	+11*
.relative	+5	+3	+12	+6	-1	+8*
- Thymus
.absolute	-19	+12	-23	+3	+40	+51*
.relative	-14	+9	-17	+9	+33	+40
- Uterus
.absolute				+6	+27*	+19
.relative				+12	+26*	+15

Statistically significant from controls: *: p<0.05

The significance concerned the organ weights values and not the percentages.

 

Table 12 - Mean Estrous Cycle Parameters

Dose level (mg/kg bw/day)	0	100	300	1000	HCD
Number of females examined	10	10	10	10	110
Mean number of days of metestrus ± SD	3.2 ± 0.8	3.2 ± 0.9	3.4 ± 0.8	2.5 ± 0.8	[3.2;4.7]
Mean number of days of diestrus ± SD	5.3 ± 1.7	5.6 ± 1.7	5.7 ± 1.3	6.1 ± 0.7	[3.0;5.0]
Mean number of days of proestrus ± SD	3.1 ± 1.0	2.2 ± 1.4	2.3 ± 1.1	2.5 ± 1.1	[2.3;3.5]
Mean number of days of estrus ± SD	3.4 ± 0.7	4.0 ± 0.8	3.6 ± 0.7	3.9 ± 0.6	[3.5;4.7]
Mean number of cycles ± SD	2.9 ± 0.3	3.0 ± 0.0	2.9 ± 0.3	3.0 ± 0.0	[2.5;3.2]
Mean duration of cycles (days) ± SD	4.1 ± 0.3	4.0 ± 0.1	4.1 ± 0.2	4.1 ± 0.3	[3.8;5.2]
Mean percent of females cycling normally (%)	100	100	90.0	80.0	[80;100]
Mean percent of females with all stages (%)	100	90.0	100	100	[90;100]

No statistically significant differences vs. controls. HCD: Historical Control Data (OECD 421/422, 2017 to 2021, n =12 studies); /: not in HCD; [min study mean;max study mean]; 

 

Table 13 - Summary of Mating and Fertility Data

Dose level (mg/kg bw/day)	0	100	300	1000
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	10
Number of females mated	10	10	10	10
Mean number of days taken to mate	2.8	2.6	2.8	2.3
Number of pregnant females	10	10	9	9
Number of females with live born pup	10	10	9	9
Mating index (female, %)	100	100	100	100
Fertility index (%)	100	100	90	90

M: Males; F: Females.

No statistically significant differences vs. controls

 

Table 14 - Summary of Delivery and Litter Data

Dose level (mg/kg bw/day)	0	100	300	1000
Number of pregnant females	10	10	9	9
Number of females which delivered	10	10	9	9
Number of females with live concepti	10	10	9	9
Gestation index (%)	100	100	100	100
Mean duration of gestation (days)	21.4	21.3	21.2	21.4
Mean number of corpora lutea	16.4	16.5	16.7	15.7
Mean number of implantation sites	15.9	16.3	16.4	14.9
Mean pre-implantation loss (%)	3.1	1.2	1.6	3.4
Mean number of pups delivered	15.1	15.6	16.0	14.0
Mean post-implantation loss (%)	5.2	4.2	2.4	6.4

No statistically significant differences vs. controls

 

Table 15 - Findings in F1 Pups on PND 1 Including the Prematurely Dead Pups

Dose level (mg/kg bw/day)	0	100	300	1000
Nb. of litters examined	10	10	9	9
Nb. of pups examined	150	154	143	126
- gross external examination, n (L)	5(3)	1(1)	10(4)	4(3)
- qualitative assessment of body temperature, n (L)		1(1)	15(1)
- activity/reaction to handling, n (L)		1(1)
Litters affected, Nb. (%)	3 (30.0)	1 (10.0)	4 (44.4)	3 (33.3)
Pups affected, Nb. (%)	5 (3.3)	1 (0.6)	20 (14)	4 (3.2)

n: number of pups, L: number of litters affected.

Table 16 - Incidence of Clinical Signs in Lactating Pups Including the Prematurely Dead Pups

Dose level (mg/kg bw/day)	0	100	300	1000
Hematoma on head, n (L)	2 (2)	1 (1)	4 (2)	4 (3)
Hematoma on neck, n (L)	3 (2)
Hematoma on body, n (L)			1 (1)
Cold to the touch, n (L)		1 (1)	15 (1)
Hypoactivity, n (L)		1 (1)
Stiffness, n (L)			4 (1)
Emaciated appearance, n (L)		1 (1)
Absence of milk in stomach, n (L)		1 (1)	3 (1)
Desquamation on back, n (L)				1 (1)
Thinning of hair, n (L)	8 (1)		3 (2)
Wound on muzzle, n (L)			2 (1)
Generalized pallor			1 (1)
Necrosed tail, n (L)	1 (1)		1 (1)
Scab on tail, muzzle and/or mouth, n (L)	1 (1)		1 (1)	1 (1)

n: number of pups, L: number of litters affected.

-: no clinical signs.

 

Table 17 - Incidence of Mortality - Summary

Dose level (mg/kg bw/day)	0	100	300	1000
Number of pups delivered (number of litters)	151 (10)	156 (10)	144 (9)	126 (9)
Number of pups found dead and/or cannibalized during the lactation period (number of litters affected)	2 (2)	7 (4)	25# (4)	2 (1)

Statistical significance: #: p<0.001

Table 18 - Incidence of Mortality - Details

Dose level (mg/kg bw/day)	0	100	300	1000
Number of sacrificed moribund pups during lactation period between PND 1 to 4	0 (0)	0 (0)	2 (1)	0 (0)
Number of pups found dead during lactation period between PND 1 to 4	2 (2)	5 (3)	12 (2)	2 (1)
Number of pups cannibalized during lactation period between PND 1 to 4	0 (0)	1(1)	11 (3)	0 (0)

 

Table 19 - Pup Body Weight and Body Weight Change

Sex	Males				Females
Dose level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Mean body weight (g)
Day 1	7.2	6.8	7.2	7.5	6.9	6.5	6.7	7.0
PND 4 preculling	10.2	10.0	10.2	10.9	9.9	9.4	9.5	10.0
PND 7	18.0	17.4	18.2	18.6	17.3	16.4	17.3	17.4
PND 10	26.5	26.3	27.6	27.5	25.6	24.7	26.3	26.1
PND 13	35.7	35.4	37.5	36.5	34.6	33.6	36.0	35.1
Mean body weight gain (g)
PND 4-13	25.5	25.3	27.3	25.6	24.7	24.2	26.4	25.0

No statistically significant differences vs. controls

 

Table 20 - Thyroid Hormones in Male Pups on PND13

	Thyroid hormones
Dose level (mg/kg bw/day)	0	100	300	1000
T4 (ng/mL) ± SD	42.77± 7.011	40.90± 8.135	40.49 ±7.651	42.39± 5.513
(% from controls)		(-4.4)	(-5.3)	(-0.9)
TSH (pg/mL) ± SD	751± 400.4	649 ±241.9	722 ±391.1	608 ±153.0
(% from controls)		(-13.7)	(-3.9)	(-19.1)

No statistically significant differences vs. controls.

 

Table 21 - Thyroid Hormones in Female Pups on PND13

	Thyroid hormones
Dose level (mg/kg bw/day)	0	100	300	1000
T4 (ng/mL) ± SD	40.40± 6.986	40.58± 7.157	39.63± 8.355	41.02 ±5.686
(% from controls)		(+0.4)	(-1.9)	(+1.5)
TSH (pg/mL) ± SD	724± 164.7	886 ±323.8	821 ±520.6	825± 420.6
(% from controls)		(+22.3)	(+13.4)	(+13.9)

No statistically significant differences vs. controls.

 

Table 22 - Sex Ratio (mean % of Males ± SD)

Dose level (mg/kg bw/day)	0	100	300	1000	HCD
Sex ratio on PND 1 (% of males) ± SD	49 ± 13.0	54 ± 14.0	55 ± 13.3	58 ± 12.0	[38.4;57.0]
(% from controls)		(+9)	(+11)	(+18)
Sex ratio on PND 13 (% of males) ± SD	49 ± 3.8	54 ± 16.2	56 ± 16.7	50 ± 4.3	[41.5;52.6]
(% from controls)		(+11)	(+14)	(+2)

HCD: Historical Control Data (OECD 421/422, 2017 to 2021, n =12 studies); [min study mean;max study mean]

No statistically significant differences vs. controls.

 

Table 23 - Anogenital Distance Corrected for Body Weight

Sex	Male				Female
Dose level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Number of litters	10	10	9	9	10	10	9	9
Mean AGD ± SD	3.7 ± 0.42	4.0 ± 0.37	3.9 ± 0.31	4.2 ± 0.51	2.1 ± 0.28	2.1 ± 0.25	2.1 ± 0.11	2.0 ± 0.23
AGD/BW^1/3± SD	1.9 ± 0.24	2.1 ± 0.18	2.0 ± 0.15	2.2 ± 0.18	1.1 ± 0.15	1.1 ± 0.11	1.1 ± 0.09	1.0 ± 0.12

No statistically significant differences vs. controls.

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study:
- the No Observed Effect Level (NOAEL) for parental toxicity and reproductive performance (mating and fertility) was considered to be 1000 mg/kg bw/day based on the absence of adverse effects at this dose level,
- the NOAEL for toxic effects on progeny was considered to be 1000 mg/kg bw/day based on the absence of adverse effects on pups in the study.

Executive summary:

This combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD Guideline No. 422 and in compliance with GLP. 

In this study, three groups of ten male and ten female Sprague-Dawley rats received the test item daily by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg bw/day, at a constant dosage volume of 5 mL/kg bw/day. A control group of ten male and ten female rats received the vehicle only (corn oil) under the same experimental conditions.

Males were treated for an overall period of approximately 4 weeks: 2 weeks before mating and then during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 7 to 8 weeks: 2 weeks before mating, through mating (up to 2 weeks) and gestation (3 weeks), until PND 13 inclusive.

The actual test item concentrations in the dose formulations were determined in Weeks 01, 04 and 07, using a validated GC/FID analytical method.

Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before mating until the females had mated.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until PND 13. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on PND 4 by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on PND 4 were sampled, euthanized and discarded without further examination. The pups were observed daily for clinical signs or abnormal behavior and were weighed on PNDs 1, 4, 7, 10 and 13. Their anogenital distances were measured on PND 1 and the number of nipples and areolae in male pups were counted on PND 12.

Hematology, coagulation, and blood biochemistry investigations were performed on the first five males and females in each group at scheduled euthanasia. Thyroid hormones (TSH and T4) were determined in males and females at termination, and in at least two pups/litter euthanized on PND 13. Males were euthanized after completion of the mating period. Dams were euthanized on PND 14. A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs (including animals that did not conceive). Designated organs were weighed, and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five control and high-dose males and lactating females, on all macroscopic lesions, and on reproductive organs from animals that did not conceive.

Pups were euthanized on PND 13 and submitted to a detailed external examination with particular attention to the external genital organs.

 

Actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variations (-10.4% to +0.6%) when compared to the nominal concentrations, except in Week 1 when the group 4 dose formulation was found to be -13.0% versus the nominal concentration.

 

There were no unscheduled deaths. Hypersalivation was sometimes observed in up to 7/20 animals at 1000 mg/kg bw/day. No test item-related effects were noted on mean body weight, mean body weight change or mean food consumption. There were no test item-related effects on estrous cycle. Mating, fertility and delivery data were unaffected by the test item treatment. At laboratory investigations (hematology, coagulation, blood biochemistry), there were no differences that could be related to test item treatment. When compared with controls, there was a statistically significant increase in mean T4 level (+26.3%, p<0.001) in males at 1000 mg/kg bw/day together with a concurrent but not statistically significant decrease in mean TSH level (-43.9%). This finding was considered test item-related but non adverse in view of the low magnitude of change and the absence of histopathological correlates. There were no test item-related effects on thyroid hormones levels in females. At pathology, there were no organ weight differences or macroscopic/microscopic findings related to the test item.

 

Observations in the pups from birth until PND 13 did not show any test item-related effects on mortality, viability, clinical signs, mean body weight, mean body weight change, mean sex ratio, mean anogenital distance or numbers of nipples and areolae. Thyroid hormone analysis did not reveal any disturbances in pups on PND 13. At necropsy, there were no test item-related findings.

 

Based on the experimental conditions of this study:

• the No Observed Effect Level (NOAEL) for parental toxicity and reproductive performance (mating and fertility) was considered to be 1000 mg/kg bw/day based on the absence of adverse effects at this dose level,


• the NOAEL for toxic effects on progeny was considered to be 1000 mg/kg bw/day based on the absence of adverse effects on pups in the study.