Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 April 2019 to 03 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

11 April to ... 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley, RjHan:SD (CD®)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: on the first day of treatment, the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Weight at study initiation: on the first day of treatment, the males had a mean body weight of 424 g (range: 387 g to 453 g) and the females had a mean body weight of 268 g (range: 239 g to 292 g)
- Fasting period before study: no
- Housing: the animals were housed in a barriered rodent unit. F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (Le comptoir des sciures, Meyzieu, France).
- Diet (e.g. ad libitum): All animals had free access to SSNIFF rat/mouse pelleted maintenance diet, batch No. 94340868 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: Males were acclimated to the study conditions for a period of 7 days before treatment. Females were acclimated to the study conditions for a period of 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.


DETAILS OF FOOD AND WATER QUALITY:
The batches of diet, sawdust and wood shavings were analyzed by the suppliers for composition and contaminant levels.
Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 11 April 2019 To: 3 July 2019

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as an emulsion in the vehicle according to the procedure described below:
- weigh the required quantity of test item directly into a gauged flask,
- complete to final weight with vehicle.
Concentration of the dose formulatrion were the following: 10, 30 and 100 mg/mL.
Test item dose formulations were prepared on the days of treatment. A constant dosage volume of 10 mL/kg/day was used. Control animals (group 1) received the vehicle only.
The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
The dose formulations were maintained under delivery condition (at room temperature) throughout the administration procedure.
The control dose formulation was stirred just before administration and the test item dose formulations for at least 15 minutes before administration. The formulations were maintained under continuous magnetic stirring throughout the administration procedure.

VEHICLE : Water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dose formulations was performed by High Performance Liquid Chromatography with UV detection (HPLC/UV). A sample was taken from control and test item dose formulations and analyzed using the validated method on formulations used in Weeks 1, 3 and 7 (Acceptance criteria: Measured concentration = nominal concentration ± 15%).

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule:
- in the males: 2 weeks before mating, during the mating period (until evidence of mating), until euthanasia (at least 4 weeks in total)
- in the females:at least 2 weeks before mating, during the mating period (until evidence of mating), during gestation, during lactation until Day 13 p.p. inclusive, until euthanasia for any females with no delivery.

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor, on the basis of a 2 week preliminary study where males and females were dosed at 100, 300 or 1000 mg/kg/day for 14 days. In this study, there were no premature deaths, no clinical signs, no effects on food consumption or body weight, and no test item related organ weight differences or gross changes. Therefore, 1000 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment (if not random): Not applicable
- Fasting period before blood sampling for clinical biochemistry: Yes: Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

MORBIDITY AND MORTALITY: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia. The body weight of each female was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post coitum) (and on the day of euthanasia for any females which did not deliver), and on Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1 4, 4-8 and 8-13 p.p. During the mating period, food consumption was not measured for males or females.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY/COAGULATION: Yes
- Time schedule for collection of blood: on the day of euthanasia
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes (for at least 14 hours)
- How many animals: first five males and lactating females from each group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of euthanasia
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes (for at least 14 hours)
- How many animals: first five males and lactating females from each group
- Parameters checked in table [2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and Dose groups that were examined: The first five males and lactating females from each group euthanized as scheduled were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.
- Battery of functions tested: The following parameters were assessed and graded:
- in the cage: "touch escape" or ease of removal from the cage,
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
The following measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature.
Finally, motor activity was measured once by automated infra-red sensor equipment over a 60 minute period.

IMMUNOLOGY: No

OTHER: THYROIDE HORMONES
Blood samples were taken, in the first half of the morning (between 7.5 and 10 am), into tubes containing K3-EDTA as anticoagulant, as follows :
- at termination on Day 14 p.p. from all F0 females (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia),
- at termination from all F0 males (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia).
Blood was centrifuged within 2 hours after sampling (approximately 3000g for 10 minutes at +4°C). The plasma was transferred into 2 separate tubes (at least 125 μL in the first tube and the remaining plasma in the second tube) and frozen at -80°C. 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 sampled at termination. Plasma samples obtained on Day 14 p.p. from F0 females are kept at -80°C pending possible analysis.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)
A complete macroscopic post-mortem examination was performed on all F0 animals. This included examination of the external surfaces, all orifices, the cranial cavity, 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.
The tissues of F0 animals specified in the Tissue Procedure Table (Table 3)) were preserved in 10% buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's fixative).

HISTOPATHOLOGY: Yes (see table 3)
A microscopic examination was performed by the Principal Investigator on:
- all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and lactating females of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Preparation of histological slides (F0 animals) All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).

ORGAN WEIGHT: Yes (see Table 3)
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 14 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids (when applicable). The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

Statistics:

For body weight, food consumption and reproductive data, Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

For hematology, blood biochemistry, body weight (acclimation), hormones, anogenital distance, nipples/areolae, live birth index(es), sex-ratio and post-implantation loss, CITOX software was used to perform the statistical analysis.

PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism was noted at all doses in males and from 300 mg/kg/day in females (1/10 males at 100 mg/kg; 3/10 males and 2/10 females at 300 mg/kg and 10/10 males and 3/10 females at 1000 mg/kg during the pre-mating period; 4/10 females at 300 mg/kg and 9/10 females at 1000 mg/kg during gestation and 1/10 females at 1000 mg/kg during the lactation period). In females, ptyalism was observed during pre-mating and gestation periods and only once at 1000 mg/kg/day during lactation. The number of affected animals was mainly dose-related, as well as the duration of the effect (ranging from 1 day to half of whole dosing period in some males). This clinical sign was not noticed during detailed clinical observation as part of the Functional Observation Battery, demonstrating that ptyalism was transient after treatment administration. This finding was considered to be test item treatment-related but non-adverse.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no unscheduled deaths in males and females. One female was euthanized 26 days after the end of the mating period for no delivery. Macroscopic examination revealed that this female was not pregnant.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effect on mean body weight and mean body weight changes (see Table 4).
Statistically significant higher mean body weight gain was noticed between Days 8 and 13 p.p. in lactating females dosed at 100 and 1000 mg/kg/day when compared to controls. Taking into account the lack of dose-relationship, the fact that this was mainly due to 2 control females which lost weight on this interval and the absence of effect on mean body weight at the end of lactation period, this effect was not attributed to the test item treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on evaluated hematology and coagulation parameters (see Table 5).
In males, at 1000 mg/kg/day, a slight decrease in fibrinogen level (-12%) was observed. Despite a statistical significance (p<0.01), this was considered to be not toxicologically relevant based on absence of correlation with other parameters related to coagulation and inflammation (platelets, APTT, prothrombin time). In addition, no similar effect was observed in females.
At 300 mg/kg/day, a slight increase in lymphocyte counts (+33%) was observed. Despite a statistical significance (p<0.05), this was considered to be not toxicologically relevant since this was not dose-related mainly due to one animal (P24811) and, due to coagulation in tubes from 2 animals, impairing the analysis, results were obtained on 3 animals instead of 5. No similar effect was observed in females.
In females, at 1000 mg/kg/day, a slight decrease in hemoglobin level (-6%) was observed, correlating with a slightly lower packed cell volume (hematocrit, -8%) when compared to controls. Despite a statistical significance (p<0.05), this was considered to be not toxicologically relevant due to the low magnitude of variation. There was no similar effect in males.
A slight shorteningin APTT (-11%) was observed. Despite a statistical significance (p<0.05), this was considered to be not toxicologically relevant. There was no correlation with other parameters related to coagulation (platelets, fibrinogen, prothrombin time). In addition, no similar effect was observed in males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male, a dose dependent increase in total bilirubin level was observed (statistically significant from 300 mg/kg/day) when compared to controls. This effect was not considered test item treatment-related as all the values were of low magnitude and below the validated quantification range, i.e. in a physiological range and there were no concomitant effects on other hepatic function markers (ALAT, ASAT, ALP) or microscopic correlates. A similar effect was not noted in females.
A dose dependent minimal increase of albumin/globulin ratio was observed (statistically significant at 1000 mg/kg/day). This effect was considered test item treatment-related. However, changes were of low magnitude and poorly dose-related in females.
Total cholesterol concentration was slightly decreased at 1000 mg/kg/day when compared to controls ( 27%, p<0.01). This effect was considered test item treatment-related, although without correlation with variations in other markers (TRIG, ALAT, ASAT, ALP) or microscopic correlates. A similar effect was not observed in females.
In females, a slight dose dependent increase in glucose level was noticed when compared to controls (statistically significant at 1000 mg/kg/day, p<0.05). This effect was considered test item treatment-related, however there were no correlation with significant variations in other markers or microscopic correlates. A similar effect was not observed in males (see Table 6).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no effects of test item treatment on detailed clinical examination as part of the Functional Observation Battery.
There were no effects of test item treatment on reactivity to manipulation and different stimuli.
In female animals, the average number of horizontal movement was dose-dependently reduced when compared to controls (-50% at 1000 mg/kg/day, p<0.05). Since no hypoactivity was noticed neither at Functional Observation Battery nor at routine clinical examination, this effect was considered test item treatment related but non-adverse (see Table 7).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Spleen weights were generally increased in females at ≥ 100 mg/kg/day and reached statistical significance at ≥ 300 mg/kg/day. There were no correlating microscopic or hematology findings for these weight increases and therefore the organ weight changes were considered to have unlikely toxicological significance or relationship to test item administration.
In male animals, thymus absolute and relative to body weights were increased in males at 300 mg/kg/day only. Without a test item effect in high dose animals or a histopathological correlate this increase was not considered to have toxicological relevance. Additionally, absolute heart weight was statistically significantly decreased when compared to control animals in males at 1000 mg/kg/day, this very small magnitude change was not accompanied by microscopic or macroscopic effects and was considered to be due to individual variability and not a test item effect.
In female animals, decreased relative to body weight brain weight was observed at ≥ 300 mg/kg/day only and was not associated with a statistically significant increase in absolute brain weight. This very small magnitude decrease did not have a correlating microscopic finding or any clinical evidence, including Functional Observational Battery and motor activity results and was thus considered to have no toxicological relevance. Additionally, a statistically significant increase in absolute adrenal weights was observed for females at 300 mg/kg/day. Without a test item effect in high dose animals or a histopathological correlate, this was considered to have no toxicological relevance and was attributed to expected biological variability for rats of this strain and age (see table 8)

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test article-related gross findings were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test article-related microscopic findings were noted.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones (T4, TSH): There were no differences in treated F0 males from concurrent control means which could be considered to be test item-related (no dose relationship, no microscopic correlates, no statistical significance and/or slight variations from controls) (see table 9).

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Chemical analysis of the dose formulations:

Test item test concentrations in the administered dose formulations were analyzed in Weeks 01, 03 and 07. The test item concentrations remained within an acceptable range of variation (-7.8% to +3.4%) when compared to the nominal values (± 15% required). No test item was observed in the control dose formulations.

Table 4: Mean body weight (g) and mean body weight change (g)

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pre-mating (female) or whole study (males)
Day 15	511	499	504	493	278	277	284	283
% vs. controls		-2	-1	-4		0	+2	+2
Days 1 - end of period*	+130	+118	+126	+124	+12	+10	+15	+13
Gestation
Day 20p.c.	/	/	/	/	421	433	440	436
Days 0 - 20p.c.	/	/	/	/	+140	+148	+152	+149
Lactation
Day 13p.p.	/	/	/	/	362	373	377	377
Days 1 - 13p.p.	/	/	/	/	+36	+47	+40	+44

/: not applicable; In bold: body weight change.

*: Day 29 (male animals) or 15 (female animals).

No statistically significant differencesvs.controls.

Table 5: Hematology and coagulation (F0 animals)

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Lymphocytes (G/L)	13.60	16.07	18.12*	13.55	9.87	10.33	10.84	10.74
% vs. controls		+18	+33	0		+5	+10	+9
Hemoglobin (g/dL)	15.6	15.9	16.7	15.8	16.2	15.7	15.9	15.2*
% vs. controls		+2	+7	+1		-3	-2	-6
Packed Cell Volume (L/L)	0.48	0.48	0.50	0.48	0.51	0.48	0.49	0.47*
% vs. controls		0	+4	0		-6	-4	-8
MCHC (g/dL)	32.7	33.4	33.2	33.1	32.1	33.0*	32.7	32.3
% vs. controls		+2	+2	+1		+3	+2	+1
Fibrinogen (g/L)	3.17	3.23	3.14	2.80**	3.83	3.84	3.82	3.65
% vs. controls		2	-1	-12		0	0	-5
APTT (s)	15.5	15.1	14.6	15.5	14.5	14.3	14.3	12.9*
% vs. controls		-3	-6	0		-1	-1	-11

MCHC:mean cell hemoglobin concentration, PLT: platelet, APTT: activated partial thromboplastin time.

Statistical significance:*: p<0.05, **: p<0.01.

Table 6: Blood biochemistry (F0 animals)

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Total Bilirubin (µmol/L)	0.17	0.53	0.80*	0.84*	0.71	0.62	1.01	0.51
% vs. controls		+212	+371	+394		-13	+42	-28
Albumin (g/L)	36	36	37	35	36	35	36	35
% vs. controls		0	+3	-3		-3	0	-3
Albumin/globulin ratio	1.95	1.99	2.05	2.08*	1.94	2.11*	2.06	2.09
% vs. controls		+2	+5	+7		+9	+6	+8
Total cholesterol (mmol/L)	2.29	1.97	2.00	1.68**	2.68	2.62	2.46	2.38
% vs. controls		-14	-13	-27		-2	-8	-11
Glucose (mmol/L)	7.36	7.08	6.65	7.14	6.04	6.74	6.91	7.81*
% vs. controls		-4	-10	-3		+12	+14	+29
ALAT (U/L)	60	57	57	73	75	95	81	124**
% vs. controls		-5	-5	+22		+27	+8	+65

ALAT: alanine aminotransferase,Statistical significance:*: p<0.05, **: p<0.01.

In bold: test item treatment-related.

Table 7: Effects on motor activity

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Horizontal movements	592	499	611	577	503	409	326	251*
% vs. controls		-16	+3	-3		-19	-35	-50
Rearing	188	159	179	209	137	96	111	76
% vs. controls		-15	-5	+11		-30	-19	-45

Statistical significance:*: p<0.05.

Table 8: Summary of main organ weight data - terminal euthanasia (Day 30/Day 14p.p.)

 

	Female
Group	2	3	4
Dose (mg/kg/day)	100	300	1000
No. animals per group	10	10	10
Spleen (no. weighed)^a	5	5	5
Absolute value	18.7	29.6*	40.7*
% of body weight	14.1	22.1*	32.0*

^a: all values expressed as percent difference of control group means.

Based upon statistical analysis of group means,values highlighted in bold are significantly different from control group – P =<0.05; refer to data tables in the dedicated report for actual significance levels and tests used.

Table 9:  Mean thyroid hormone levels (±Standard Deviation) in F0 males at termination were the following:

 

Sex	Male
Dose level (mg/kg/day)	0	100	300	1000
T4 (ng/mL)	40.79 (± 7.12)	35.77 (± 7.16)	40.02 (± 6.39)	35.06 (± 3.57)
		-12	-2	-14
TSH (pg/mL)	2520 (± 1107)	2568 (± 1112)	1965 (± 1182)	1875 (± 1226)
		+2	-22	-26

in italic: differences from controls (%).

No statistically significant differencesvs.controls.

Conclusions:

The test item, SOPROMINE 1686, was administered daily by oral gavage to male and female Sprague Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 13 p.p., at dose levels of 100, 300 and 1000 mg/kg/day. Based on the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day based on the absence of adverse findings at this high-dose level.

Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item,SOPROMINE 1686,following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 13 post-partum (p.p.).

This study provides initial information:

-  on the possible health hazards likely to arise from repeated exposure over a relative limited period of time,

- on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The study was performed according to OECD guideline No. 422 and in compliance with GLP.

Methods

Three groups of ten male and ten female Sprague-Dawley rats (F0 animals) received the test item,SOPROMINE 1686(batch No.8230049),daily, by oral administration (gavage), 2 weeks before mating, during mating and, for the males until euthanasia, for the females throughout gestation until Day 13 post‑partum(p.p.) inclusive. The dose levels were 100, 300 and 1000 mg/kg/day. Another group of ten males and ten females received the vehicle (Drinking water treated by reverse osmosis) only and acted as a control group. A constant dosage volume of 10 mL/kg/day was used.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 3 and 7 were determined using a validatedHigh Performance Liquid Chromatography with UV detection (HPLC/UV) method.

The F0 animals were checked daily during the dosing period for mortality, morbidity and clinical signs. Detailed clinical observations were performed at least once a week. Body weight and food consumption were recorded once a week during premating, mating (food consumption not during mating), gestation (0, 7, 14 and 20p.c.)and lactation(Days 1, 4, 8 and 13p.p.)periods.

The F0 animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 13p.p.

A Functional Observation Battery (FOB) including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five F0 animals per sex and group at the end of the treatment period. Analysis of hematology and blood biochemistry parameters was performed from blood samples taken prior to euthanasia from at least five F0 animals per sex and group. Thyroid hormones (T4 and TSH) plasma levels were determined at termination from all F0 males.

The F0 males were euthanized after at least 4 weeks of treatment (Day 30) and the F0 females on Day 14p.p.Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, pituitary gland, prostate, seminal vesicles, spleen, testes and thymus) were recorded and a complete macroscopicpost‑mortemexamination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on long list of organs from five F0 animals per sex in the control and high-dose groups and on all macroscopic lesions.

Results

The test item concentrations were within an acceptable range of variation (± 15% of the nominal concentrations required). No test item was observed in the control dose formulations.

 In parental animals, there were no test item-related unscheduled deaths. Transient ptyalism after treatment was noted at all dose levels in males with dose-related incidence and in females from 300 mg/kg/day. Females were affected during pre-mating and gestation periods, but in a lower extent during lactation (1 out of 10 females at 1000 mg/kg/day). This clinical sign was considered to be test item-related and non‑adverse. There were no effects on mean body weight, mean body weight change and mean food consumption. There were no test item-related changes atreactivity to manipulation, to different stimuli and at motor activity evaluation (FOB) at any dose level, except a non-adverse dose-related decrease in horizontal movement in females.Hematology and coagulation parameter evaluation did not reveal differences from controls after test item administration. There were no differences between T4 and TSH levels from test item-treated and control male animals.

At pathology examination, there were no gross findings, microscopic findings or organ weight changes attributable to administration of SOPROMINE 1686.

Conclusion

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

EC Number:

942-252-2

IUPAC Name:

Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley, RjHan:SD (CD®)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
on the first day of treatment, the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Weight at study initiation:
on the first day of treatment, the males had a mean body weight of 424 g (range: 387 g to 453 g) and the females had a mean body weight of 268 g (range: 239 g to 292 g)
- Fasting period before study:
no
- Housing:
the animals were housed in a barriered rodent unit. F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (Le comptoir des sciures, Meyzieu, France).
- Diet (e.g. ad libitum):
All animals had free access to SSNIFF rat/mouse pelleted maintenance diet, batch No. 94340868 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum):
The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period:
Males were acclimated to the study conditions for a period of 7 days before treatment. Females were acclimated to the study conditions for a period of 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.


DETAILS OF FOOD AND WATER QUALITY:
The batches of diet, sawdust and wood shavings were analyzed by the suppliers for composition and contaminant levels.
Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 2°C
- Humidity (%):
50 ± 20%
- Air changes (per hr):
about 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light):
12 h/12 h

IN-LIFE DATES: From: 11 April 2019 To: 3 July 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was prepared as an emulsion in the vehicle according to the procedure described below:
- weigh the required quantity of test item directly into a gauged flask,
- complete to final weight with vehicle.
Concentration of the dose formulatrion were the following: 10, 30 and 100 mg/mL.
Test item dose formulations were prepared on the days of treatment. A constant dosage volume of 10 mL/kg/day was used. Control animals (group 1) received the vehicle only.
The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
The dose formulations were maintained under delivery condition (at room temperature) throughout the administration procedure.
The control dose formulation was stirred just before administration and the test item dose formulations for at least 15 minutes before administration. The formulations were maintained under continuous magnetic stirring throughout the administration procedure.

VEHICLE
: Water

The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.

Details on mating procedure:

Females were paired with males from the same dose level group. One female was placed with one male, in the latter's cage, during the night.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
The day of confirmed mating was designated Day 0 p.c.
Each female was placed with the same male until mating occurred.
The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dose formulations was performed by High Performance Liquid Chromatography with UV detection (HPLC/UV). A sample was taken from control and test item dose formulations and analyzed using the validated method on formulations used in Weeks 1, 3 and 7 (Acceptance criteria: Measured concentration = nominal concentration ± 15%).

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule:
- in the males: 2 weeks before mating, during the mating period (until evidence of mating), until euthanasia (at least 4 weeks in total)
- in the females:at least 2 weeks before mating, during the mating period (until evidence of mating), during gestation, during lactation until Day 13 p.p. inclusive, until euthanasia for any females with no delivery.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, on the basis of a 2 week preliminary study where males and females were dosed at 100, 300 or 1000 mg/kg/day for 14 days. In this study, there were no premature deaths, no clinical signs, no effects on food consumption or body weight, and no test item related organ weight differences or gross changes. Therefore, 1000 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment (if not random):
Not applicable
- Fasting period before blood sampling for clinical biochemistry:
Yes: Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- Rationale for selecting satellite groups:
Not applicable
- Post-exposure recovery period in satellite groups:
Not applicable
- Section schedule rationale (if not random):
Not applicable

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Not specified

MORBIDITY AND MORTALITY: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also evaluated.


BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of each male was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia. The body weight of each female was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post coitum) (and on the day of euthanasia for any females which did not deliver), and on Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1 4, 4-8 and 8-13 p.p. During the mating period, food consumption was not measured for males or females.

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-treatment period,
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on the day of euthanasia, to allow correlation with reproductive organs histopathology.

Sperm parameters (parental animals):

No sperm analysis was performed. At microscopic examination, special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (as follows)
- On Day 4 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible 4 males and 4 females per litter. Whenever necessary, partial adjustment (for example 5 males and 3 females) was permitted. No cross-fostering was performed.
Standardization of litter size was considered to reduce the litter size-induced variability in the growth and development of the pups and thus increase the sensitivity of statistical analysis. This also ensured that any adverse effects on pup growth and development are not masked by a test item treatment-related reduction in litter size


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Litter size:
The total litter size and sex of each pup were recorded on Day 1 p.p. Any gross external malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.

- Clinical signs:
The pups were observed daily for clinical signs and abnormal behavior.

- Body weight:
The body weight of each pup was recorded on Days 1, 4, 8 and 13 p.p.

- Pup development:
The following physical development measurements were performed in live pups of each litter:
- anogenital distance (AGD): on Day 1 p.p.,
- number of nipples and areolae in male pups: on Day 12 p.p.
The AGD was normalized to the cube root of body weight recorded on Day 1 p.p.

- Thyroid hormones:
Blood samples were taken, in the first half of the morning, into tubes containing K3-EDTA as anticoagulant, as follows:
- at termination on Day 4 p.p. from at least 2 culled pups/litter [chosen by manual randomization; approximately 0.25 mL per pup (to obtain approximately 0.5 mL of blood in total) was collected by decapitation under isoflurane anesthesia and then pooled per litter. A third pup was used in the case of total blood volume lower than 0.5 mL]. When they were insufficient pups in a litter to have 2 culled pups, only one culled pup was used for blood collection when available, otherwise there was no blood collection on Day 4 p.p.,
- at termination on Day 13 p.p. from at least 2 pups/litter [chosen by manual randomization; approximately 0.25 mL per pup (to obtain approximately 0.5 mL of blood in total. A third pup was used in the case of total blood volume lower than 0.5 mL) was collected from the vena cava immediately after euthanasia and then pooled per litter],
Blood was centrifuged within 2 hours after sampling (approximately 3000g for 10 minutes at +4°C). The plasma was transferred into 2 separate tubes (at least 125 μL in the first tube and the remaining plasma in the second tube) and frozen at -80°C. 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 Day 13 p.p.. Plasma samples obtained on Day 4 p.p. from pups were kept at -80°C pending possible analysis

GROSS EXAMINATION OF DEAD PUPS:
yes: Moribund pups and pups prematurely euthanized because of dying mother were euthanized by an intraperitoneal injection of sodium pentobarbital.
Found dead and prematurely euthanized pups were submitted to a detailed external examination (including orifices and buccal cavity), after euthanasia when applicable. Particular attention was paid to the external genital organs and to whether the pup has been fed (e.g. presence of milk in the stomach) when possible. Then, they were discarded without any further examination.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, after at least 14 hours fasting, all F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital and followed by exsanguination: males: after the end of the mating period (at least 4 weeks of treatment in total); females: on Day 14 p.p.

GROSS PATHOLOGY: Yes (see table 1)

A complete macroscopic post-mortem examination was performed on all F0 animals. This included examination of the external surfaces, all orifices, the cranial cavity, 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.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 14 p.p. and for one group 2 female euthanized on Day 26 p.c. due to no delivery. For any apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

The tissues of F0 animals specified in the Tissue Procedure Table (Table 3)) were preserved in 10% buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's fixative).

HISTOPATHOLOGY: Yes (see table 1)
A microscopic examination was performed by the Principal Investigator on:
- all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and lactating females of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Preparation of histological slides (F0 animals) All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).

ORGAN WEIGHT: Yes (see Table 1)
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 14 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids (when applicable). The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

Postmortem examinations (offspring):

SACRIFICE
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on Day 4 p.p. if blood sampled), followed by exsanguination when the thyroids were sampled:
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- surviving pups: on Day 13 p.p.

GROSS NECROPSY
Pups not selected on Day 4 p.p. were discarded without further examination.
Pups euthanized on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pups.


HISTOPATHOLOGY / ORGAN WEIGTHS
The body weight of 1 selected pup/sex/litter (euthanized on Day 13 p.p. was recorded before euthanasia. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
Preservation of tissues: Thyroids with parathyroids of the selected pup/sex/litter euthanized on Day 13 p.p. were preserved in 10% buffered formalin.

Statistics:

For body weight, food consumption and reproductive data, Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

For hematology, blood biochemistry, body weight (acclimation), hormones, anogenital distance, nipples/areolae, live birth index(es), sex-ratio and post-implantation loss, CITOX software was used to perform the statistical analysis.

PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01)

Reproductive indices:

The following parameters were calculated:

- pre-implantation loss:

Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea


- post-implantation loss (calculated with Excel):

Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantation sites


- mating index:

Number of mated animals
_____________________ x 100
Number of paired animals


- fertility index:

Number of pregnant female partners
_______________________________ x 100
Number of mated pairs


- gestation index:

Number of females with live born pups
________________________________ x 100
Number of pregnant females

Offspring viability indices:

- live birth index:

Number of live born pups on Day 1 p.p.
_________________________________ x 100
Number of delivered pups


- viability index on Day 4 p.p.:

Number of surviving pups on Day 4 p.p. (before culling)
_____________________________________________ x 100
Number of delivered pups


- lactation index on Day 13 p.p.:

Number of surviving pups on Day 13 p.p.
_____________________________________________ x 100
Number of surviving pups on Day 4 p.p. (after culling)


- AGD/cube root of body weight ratio (calculated with Excel):

AGD
______________
³√Body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism was noted at all doses in males and from 300 mg/kg/day in females (1/10 males at 100 mg/kg; 3/10 males and 2/10 females at 300 mg/kg and 10/10 males and 3/10 females at 1000 mg/kg during the pre-mating period; 4/10 females at 300 mg/kg and 9/10 females at 1000 mg/kg during gestation and 1/10 females at 1000 mg/kg during the lactation period). In females, ptyalism was observed during pre-mating and gestation periods and only once at 1000 mg/kg/day during lactation. The number of affected animals was mainly dose-related, as well as the duration of the effect (ranging from 1 day to half of whole dosing period in some males). This clinical sign was not noticed during detailed clinical observation as part of the Functional Observation Battery, demonstrating that ptyalism was transient after treatment administration. This finding was considered to be test item treatment-related but non-adverse.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no unscheduled deaths in males and females. One female was euthanized 26 days after the end of the mating period for no delivery. Macroscopic examination revealed that this female was not pregnant.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effect on mean body weight and mean body weight changes (see Table 4 of the Repeated dose toxicity study part in section 7.5.1).
Statistically significant higher mean body weight gain was noticed between Days 8 and 13 p.p. in lactating females dosed at 100 and 1000 mg/kg/day when compared to controls. Taking into account the lack of dose-relationship, the fact that this was mainly due to 2 control females which lost weight on this interval and the absence of effect on mean body weight at the end of lactation period, this effect was not attributed to the test item treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on evaluated hematology and coagulation parameters (see Table 5 in the Repeated dose toxicity study part in section 7.5.1).
In males, at 1000 mg/kg/day, a slight decrease in fibrinogen level (-12%) was observed. Despite a statistical significance (p<0.01), this was considered to be not toxicologically relevant based on absence of correlation with other parameters related to coagulation and inflammation (platelets, APTT, prothrombin time). In addition, no similar effect was observed in females.
At 300 mg/kg/day, a slight increase in lymphocyte counts (+33%) was observed. Despite a statistical significance (p<0.05), this was considered to be not toxicologically relevant since this was not dose-related mainly due to one animal (P24811) and, due to coagulation in tubes from 2 animals, impairing the analysis, results were obtained on 3 animals instead of 5. No similar effect was observed in females.
In females, at 1000 mg/kg/day, a slight decrease in hemoglobin level (-6%) was observed, correlating with a slightly lower packed cell volume (hematocrit, -8%) when compared to controls. Despite a statistical significance (p<0.05), this was considered to be not toxicologically relevant due to the low magnitude of variation. There was no similar effect in males.
A slight shorteningin APTT (-11%) was observed. Despite a statistical significance (p<0.05), this was considered to be not toxicologically relevant. There was no correlation with other parameters related to coagulation (platelets, fibrinogen, prothrombin time). In addition, no similar effect was observed in males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male, a dose dependent increase in total bilirubin level was observed (statistically significant from 300 mg/kg/day) when compared to controls. This effect was not considered test item treatment-related as all the values were of low magnitude and below the validated quantification range, i.e. in a physiological range and there were no concomitant effects on other hepatic function markers (ALAT, ASAT, ALP) or microscopic correlates. A similar effect was not noted in females.
A dose dependent minimal increase of albumin/globulin ratio was observed (statistically significant at 1000 mg/kg/day). This effect was considered test item treatment-related. However, changes were of low magnitude and poorly dose-related in females.
Total cholesterol concentration was slightly decreased at 1000 mg/kg/day when compared to controls ( 27%, p<0.01). This effect was considered test item treatment-related, although without correlation with variations in other markers (TRIG, ALAT, ASAT, ALP) or microscopic correlates. A similar effect was not observed in females.
In females, a slight dose dependent increase in glucose level was noticed when compared to controls (statistically significant at 1000 mg/kg/day, p<0.05). This effect was considered test item treatment-related, however there were no correlation with significant variations in other markers or microscopic correlates. A similar effect was not observed in males (see Table 6 in the Repeated dose toxicity study part in section 7.5.1).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no effects of test item treatment on detailed clinical examination as part of the Functional Observation Battery.
There were no effects of test item treatment on reactivity to manipulation and different stimuli.
In female animals, the average number of horizontal movement was dose-dependently reduced when compared to controls (-50% at 1000 mg/kg/day, p<0.05). Since no hypoactivity was noticed neither at Functional Observation Battery nor at routine clinical examination, this effect was considered test item treatment related but non-adverse (see Table 7 in the Repeated dose toxicity study part in section 7.5.1).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test article-related microscopic findings were noted.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones (T4, TSH): There were no differences in treated F0 males from concurrent control means which could be considered to be test item-related (no dose relationship, no microscopic correlates, no statistical significance and/or slight variations from controls) (see table 9 in the Repeated dose toxicity study part in section 7.5.1).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no effects on estrous cycle (number of cycles, cycle length) during the first 2 weeks of the treatment period.
At 1000 mg/kg/day, one female had one cycle shorter than 4 days. This had no incidence on mating abilities of this animal. This was therefore considered incidental and not test item treatment-related (see details in Table 2).
On Day 14 p.p., diestrus stage was noted in all females observed on Day 14 p.p. (with the exception of control female P25299 for which metestrus was noted), mainly correlating with the histopathology of the vagina of the control and high-dose females (with the exception of control female P25297 for which estrous stage was categorized as metestrus at histopathology, see § Microscopic examination).

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item-related effects on mean pairing, mating and fertility data nor on mean duration of gestation, mean pre-implantation loss rate and the number of pups delivered. (see details in Tables 3 and 4).

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related clinical signs at any dose level (see details in Table 5).

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on pup mortality. All pups found dead or cannibalized were found on Day 1 p.p., except one found cannibalized pup at 1000 mg/kg/day on Day 3 p.p. Most of them had no milk in the stomach at external examination. The global mean number of live pups per group was not affected and there was no dose-relationship in mean group data and in the number of litters affected. (see details in Tables 6 and 7)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The test item treatment had no effect on mean pup body weight and body weight gain (see details in Table 8).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no effects on mean pup sex ratio.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There were no effect of test item treatment on anogenital distance and normalized anogenital distance to the cube-root of mean body weight at Day 4 p.p.
Male pups from dam treated with test item at 300 mg/kg/day had a statistical significantly lower AGD when compared to controls (p<0.05). However, in absence of dose-level relationship and absence of development of nipples / areolae, this finding was considered to be incidental (see details in Table 9).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no test item-related effects on number of nipples and of areolae in male pups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Most of the pups found dead or cannibalized had no milk in the stomach at external examination.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones levels: There were no differences in T4 and TSH concentrations from control means considered to be test item-related (no dose-relationship, no statistical significance and/or slight variations from controls) (see details in Table 10).

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 2: Estrous cycle (First 2 weeks of the treatment period)

Mean data are summarized below:

 

Dose level (mg/kg/day)	0	100	300	1000
Number of cycles	3.0	3.0	3.0	3.1
Cycle length (days)	4.0	4.0	4.0	4.0
Number of rats cycling normally^a	10	10	10	9

^a: having a mean cycle of 4-5 days.

No statistically significant differencesvs.controls.

Table 3: Summary of the pairing, mating and fertility data

 

Dose level (mg/kg/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
Mating index (%)	100	100	100	100
Mean number of days taken to mate	2.3	4.0	2.8	2.6
Number of pregnant females	10	9	10	10
Fertility index (%)	100	90	100	100
Number of females with live born pup(s)	10	9	10	10
Gestation index (%)	100	100	100	100

M: males; F: females.

Table 4: The summary of delivery data

 

Dose level (mg/kg/day)	0	100	300	1000
Number of females which delivered	10	9	10	10
Mean duration of gestation (days)	22.0	21.8	21.7	21.8
Mean number ofcorpora lutea	14.7	15.0	15.4	16.2
Mean number of implantations	13.6	14.7	15.2	16.2
Mean pre-implantation loss (%)	10.1	2.5	2.0	0.0
Mean number of pups delivered	11.7	13.0	13.7	13.9
Mean number of live pups on Day 1p.p.	11.4	12.8	13.5	13.7
Mean post-implantation loss (%)	17.8	10.7	10.0	14.6
Live birth index on Day 1p.p.	98.0	98.6	98.7	98.8

No statistically significant differencesvs. controls.

Table 5: Pup clinical signs are summarized below:

Dose level (mg/kg/day)	0	100	300	1000
Absence of milk in stomach	2 (2)	1 (1)	2 (2)
Emaciated appearance	1 (1)
Hematoma (hindlimb/back/head)		5 (5)	3 (3)	5 (3)
Thinning of hair				9 (2)
Total of pups affected (litters affected/total of litters)	3 (3/10)	6 (5/9)	5 (4/10)	14 (5/10)

Table 6: Live birth, viability and lactation indexes:

 

Dose level (mg/kg/day)	0	100	300	1000
Live birth index on Day 1p.p. (%)	98	99	99	99
Mean number of live pups on Day 4p.p. (pre-culling)	11.4	12.8	13.5	13.6
Viability index on Day 4p.p.(%)	98	99	99	98
Mean number of live pups on Day 13p.p.	8.0	8.0	8.0	8.0
Lactation index on Day 13p.p.(%)	100	100	100	100

p.p.: post –partum.

No statistically significant differencesvs.controls.

 

 

Table 7: Distribution of prematurely dead pups:

 

Dose level (mg/kg/day)	0	100	300	1000
Number of found dead pups on Day 1p.p. (number of litters affected)	3 (2)	1* (1)	2 (2)	2 (1)
Number of cannibalized pups on Day 1p.p. (number of litters affected)	0	1 (1)	0	0
Number of cannibalized pups on Day 3p.p. (number of litters affected)	0	0	0	1 (1)

*: excluding cannibalized pups.

Table 8: Pup mean body weights (g) and pup mean body weight gains (g):

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300		1000
Body weight
Day 1p.p.	8.1	7.8	7.6	7.6	7.7	7.4	7.1		7.3
% vs. controls		-4	-6	-6		-4	-8		-5
Day 4p.p.(pre-culling)	11.5	10.7	10.5	10.5	10.8	10.2	9.8		10.2
% vs. controls		-7	-9	-9		-6	-9		-6
Day 4p.p.(post-culling)	11.5	10.7	10.5	10.5	10.8	10.3	9.9		10.2
% vs. controls		-7	-9	-9		-5	-8		-6
Day 8p.p.	20.2	19.0	18.3	18.4	18.5	18.3	17.5		17.7
% vs. controls		-6	-9	-9		-1	-5		-4
Day 13p.p.	33.3	32.0	30.8	30.7	30.3	30.9	29.9		29.7
% vs. controls		-4	-8	-8		+2	-1		-2
Body weight change
Days 1-4p.p.	3.4	2.9	2.8	2.9	3.1	2.9	2.7	2.9
Days 4-13p.p.	21.8	21.3	20.4	20.3	19.5	20.6	20.0	19.5

No statistically significant differencesvs.controls.

Table 9: Pup development: Mean anogenital distance (AGD, mm) data (±Standard Deviation) and normalized AGD at Day 4p.p.

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
AGD (mm)on Day 1p.p.	4.75 ± 0.73	4.51 ± 0.38	4.23* ± 0.38	4.33 ± 0.23	2.71 ± 0.61	2.61 ± 0.48	2.55 ± 0.33	2.56 ± 0.10
% vs. controls		-5	-11	-9		-4	-6	-6
Ratio AGD/^3√body weight (mm/g^1/3)	2.37 ± 0.38	2.28 ± 0.22	2.15 ± 0.19	2.20 ± 0.10	1.40 ± 0.37	1.35 ± 0.26	1.33 ± 0.18	1.32 ± 0.06
% vs. controls		-4	-9	-7		-4	-5	-6

Mean data based on litter data.

Statistical significance:*: p<0.05.

Table 10: Mean thyroid hormone levels (±Standard Deviation) in litter plasma on Day 13 p.p.:

 

Dose level (mg/kg/day)	0	100	300	1000
T4 (ng/mL)	38.84 (± 4.56)	39.68 (± 3.46)	36.03 (± 4.08)	35.30 (± 4.53)
		+2	-7	-9
TSH (pg/mL)	1268 (± 858)	1051 (± 228)	1097 (± 539)	1032 (± 344)
		-17	-13	-19

initalic: differences from controls (%).

No statistically significant differencesvs.controls.

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions and results of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day based on the absence of adverse findings at this high-dose level,
- the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 1000 mg/kg/day based on the absence of test item-related effects on mating and fertility at this dose level,
- the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day based on the absence of relevant findings on pups at this high-dose level.

Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item,SOPROMINE 1686,following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 13 post-partum (p.p.).The study was performed according to OECD guideline No. 422 and in compliance with GLP.

Methods

Three groups of ten male and ten female Sprague-Dawley rats (F0 animals) received the test item,SOPROMINE 1686,daily, by oral administration (gavage), 2 weeks before mating, during mating and, for the males until euthanasia, for the females throughout gestation until Day 13 post‑partum(p.p.) inclusive. The dose levels were 100, 300 and 1000 mg/kg/day. Another group of ten males and ten females received the vehicle (Drinking water treated by reverse osmosis) only and acted as a control group. A constant dosage volume of 10 mL/kg/day was used.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 3 and 7 were determined using a validatedHigh Performance Liquid Chromatography with UV detection (HPLC/UV) method.

The F0 animals were checked daily during the dosing period for mortality, morbidity and clinical signs. Detailed clinical observations were performed at least once a week. Body weight and food consumption were recorded once a week during premating, mating (food consumption not during mating), gestation (0, 7, 14 and 20p.c.)and lactation(Days 1, 4, 8 and 13p.p.)periods.

The F0 animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 13p.p.The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs and abnormal behaviour, and weighed on Days 1, 4, 8 and 13p.p.Anogenital distance on all pups on Day 1p.p.and the number of nipples/areolae in male pups on Day 12p.p.were observed.

A Functional Observation Battery (FOB) including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five F0 animals per sex and group at the end of the treatment period. Analysis of hematology and blood biochemistry parameters was performed from blood samples taken prior to euthanasia from at least five F0 animals per sex and group. Thyroid hormones (T4 and TSH) plasma levels were determined at termination from all F0 males and on Day 13p.p.from two pups/litter.

The F0 males were euthanized after at least 4 weeks of treatment (Day 30) and the F0 females on Day 14p.p.Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, pituitary gland, prostate, seminal vesicles, spleen, testes and thymus) were recorded and a complete macroscopicpost‑mortemexamination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on long list of organs from five F0 animals per sex in the control and high-dose groups and on all macroscopic lesions.

Pups not selected on Day 4p.p.were euthanized and discarded without further examination.

Selected pups were euthanized on Day 13p.p.andsubmitted to a detailed external examination with particular attention to the external genital organs.

 

Results

The test item concentrations were within an acceptable range of variation (± 15% of the nominal concentrations required). No test item was observed in the control dose formulations.

In parental animals, there were no test item-related unscheduled deaths. Transient ptyalism after treatment was noted at all dose levels in males with dose-related incidence and in females from 300 mg/kg/day. Females were affected during pre-mating and gestation periods, but in a lower extent during lactation (1 out of 10 females at 1000 mg/kg/day). This clinical sign was considered to be test item-related and non‑adverse. There were no effects on mean body weight, mean body weight change and mean food consumption. There were no test item-related changes atreactivity to manipulation, to different stimuli and at motor activity evaluation (FOB) at any dose level, except a non-adverse dose-related decrease in horizontal movement in females.Hematology and coagulation parameter evaluation did not reveal differences from controls after test item administration. There were no differences between T4 and TSH levels from test item-treated and control male animals.

There were no test item-related effects on estrous cycles during the first 2 weeks of treatment and at termina      tion, and on mean fertility, mating and delivery data.At pathology examination, there were no gross findings, microscopic findings or organ weight changes attributable to administration of SOPROMINE 1686.

In pups, there were notest item-related effects in terms ofpup mortality/viability, clinical signs, terminal external examination, development of nipples and areolae in male pups on Day 13 p.p.,percentage of male pups at birth, anogenital distance on Day 1p.p.ormean T4 and TSH levels on Day 13p.p.There were no significant effects on mean pup body weight.

In conclusion, based on the experimental conditions and results of this study:

.      the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day based on the absence of adverse findings at this high-dose level,

.      the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 1000 mg/kg/day based on the absence of test item-related effects on mating and fertility at this dose level,

.      the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day based on the absence of relevant findings on pups at this high-dose level.