Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2017 - 06 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate

EC Number:

947-903-4

Cas Number:

1917323-93-3

Molecular formula:

C28H42Ca3N4O16 and C10H16CaN2O5

IUPAC Name:

Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate

Test material form:

other: highly viscous, semi-solid mass

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hsd.Han:Wist rat

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90.H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: maless/females 13-14 weeks
- Weight at study initiation: (P) Males: 360-448 g; Females: 218-255 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water : ad libitum, tap water from municipal supply
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua purificata (Distilled water) Quality: Ph.Hg.VIII.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility not longer than for 24 hours before the use. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week, animals received volumes based on the actual body weight on Day 0.

VEHICLE
- Concentration in vehicle: 200 mg/mL, 60 mg/mL and 20 mg/mL
- Dose volume: A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups.
- Lot/batch no.: 1702-5502

Details on mating procedure:

- M/F ratio per cage: 1:1 mating
- Length of cohabitation: until copulation or 14 days have elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions (control of concentration and homogeneity) was performed once during the dose range finding study (Section 7.5.1). Samples were taken on study Day 0 and measurement was conducted one day thereafter. Five samples were taken from each test concentration and five samples were taken from the control sample. The samples were stored at 5 ± 3°C before the analysis. Formulation samples were diluted with acetonitrile : water = 1 : 9 (v/v) mixture and analysed by an HPLC method with UV detection.

Duration of treatment / exposure:

Male animals were dosed for 56 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period of 28-41 days) and then they were subjected to necropsy one day after the last treatment. Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 13-19 (altogether for 55-56 days depending on day of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum.

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose setting is based on findings obtained in an OECD 407 dose range finding study performed with Retardan 200 P (study no. 666-400-1746, non-GLP) and in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table No.1 were included.

BODY WEIGHT: Yes
Parental males were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum day 0 (within 24 hours after parturition), 4 and 13. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight was measured on day of necropsy for animals subjected to organ weighing.

FOOD CONSUMPTION
The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period except mating phase: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals.

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.
Vaginal smear were also prepared on the day of the necropsy.

Sperm parameters (parental animals):

Parameters examined in male parental generation [P]:
- testis weight, epididymis weight, prostate weight and seminal vesicles with coagulating glands as a whole

Litter observations:

On post-partal day 4, the size of each litter was adjusted to four pups per sex per litter.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) presence of gross anomalies
Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete, post-natal day 0) and on postnatal day 4. The anogenital distance of each pup was determined on postnatal day 4.
Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum T4 levels.
The number of nipples/areolae in male pups was counted on postnatal day 13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

CLINICAL PATHOLOGY
Clinical pathology examinations including hematology, blood coagulation and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment. Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran CP® anesthesia. Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry.In addition, blood samples were collected for possible determination of serum levels of thyroid hormones.

HEMATOLOGY
WBC (White Blood Cell leukocyte count), RBC (Red Blood Cell erythrocyte count), HGB (Hemoglobin concentration), HCT (Hematocrit, relative volume of erythrocytes), MCV (Mean Corpuscular erythrocyte Volume), MCH (Mean Corpuscular erythrocyte Hemoglobin), MCHC (Mean Corpuscular erythrocyte Hemoglobin Concentration), PLT (Platelet thrombocyte count), RET (Reticulocytes),Differential white blood cell count

CLINICAL CHEMISTRY
ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration),
UREA (Urea concentration), GLUC (Glucose concentration),CHOL (Cholesterol concentration), Na+ (Sodium concentration),K+ (Potassium concentration), ALB (Albumin concentration), TPROT (Total Protein concentration)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (T4) as follows:
-from all dams and at least two pups per litter on day 13
-from all parent male animals at termination

SACRIFICE
-Parental male animals: after the optionally extended post-mating period on Day 56.
-Not mated female animal: on Day 55.
-Non-pregnant or not-delivered (but mated) female animals: on Day 55.
-Dams not selected for toxicology examinations: on post-partum day 13 or shortly thereafter (Day 55 or 56).
-Dams selected for toxicology examinations: shortly after post-partum day 13 (Day 55).
-Offspring: on postnatal day 13 or shortly thereafter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examinations were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma.
The following organs were preserved: Adrenal glands, Aorta, Bone with marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with vagina), Gross lesions, Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, Small intestines(representative regions: duodenum, ileum, jejunum), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high dose groups.

Postmortem examinations (offspring):

On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test). Dead pups found were subjected to necropsy by a macroscopic examination. Any observed abnormalities were recorded.

Statistics:

The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations.
The behavior and physical condition of animals in 100, 300, and 1000 mg/kg bw/day and in the control group were considered to be not impaired during the entire observation period. Slight alopecia was observed on the forelimbs of one male animal in the control group (1/12) from Day 7 until Day 47. This finding was considered to be individual ones occurring commonly in experimental rats and not related to the test item.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not adversely affected by the test item in male or female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period. Lower mean body weight gain was observed in male animals at 100, 300 and 1000 mg/kg bw/day comparing to their control during the second and fifth week of the treatment period and higher mean body weight gain was detected in males at 300 and 1000 mg/kg bw/day comparing to control on the third week of treatment period. At the end of the whole treatment period the summarized body weight and body weight gain were similar to that of the control group in male animals at 100, 300 or 1000 mg/kg bw/day.
Lower mean body weight gain was observed in female animals at 1000 mg/kg bw/day comparing to their control during the first week of the gestation period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item or treatment related changes in the food consumption of male and female animals at any dose level (100, 300 and 1000 mg/kg bw/day). Statistical significance was noted for the slightly higher mean daily food consumption in male animals at the 1000 mg/kg bw/day group in two periods: between Days 20 and 27 and between Days 48 and 54, which was judged to be not treatment related.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day. Compared to their control group, the mean white blood cell count (WBC) and the mean percentage of basophil granulocytes (BASO) were lower in male animals at 100 mg/kg bw/day. Similarly, the WBC was below the control value in male animals at 300 mg/kg bw/day. All examined hematological parameters were comparable in female animals in the control and in all test item treated groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
In the male animals at 1000 mg/kg bw/day, the mean potassium (K+) concentration was statistically significantly higher than in the control group.
In female animals, statistical significances were noted for the slightly lower mean sodium (Na+) concentration at 300 and 1000 mg/kg bw/day and for the lower mean concentration of glucose (GLUC) at 1000 mg/kg bw/day. These changes were with minor degree and these were considered to have no toxicological relevance.
T4 levels were lower than in the control group in parental male animals at 100, 300 and 1000 mg/kg bw/day in a dose related manner.
Lower mean serum T4 levels were detected with respect to their control in female animals at 100, 3000 and 1000 mg/kg bw/day groups however independently from the doses. Moreover, the inter-individual variation was high in the control and 100, 300 and 1000 mg/kg bw/day groups.
There were no related microscopic lesions in the organs of the hypothalamicpituitary-thyroid axis in the examined animals. Therefore this finding was not considered to be adverse under the conditions of this study.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations did not demonstrate any test item related changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (100, 300 and 1000 mg/kg bw/day, control).

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):

Histopathological examinations did not reveal any test item related alterations in the organs or tissues of male or female animals at 1000 mg/kg/bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals; full histopathology in selected animals).
In some female animals (3/12 control including one non-pregnant female; 1/4 at 100 mg/kg bw/day; 1/4 (non-pregnant) at 300 mg/kg bw/day) dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
Alveolar emphysema in minimal or mild degree was observed in the lungs of male (1/5 control and 1/5 at 1000 mg/kg bw/day) and female (1/5 control) animals. This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted for male animals (3/5 control and 1/5 at 1000 mg/kg bw/day) and female animals (1/5 control and 1/5 at 1000 mg/kg bw/day). Hyperplasia of BALT is a physiological immune-morphological phenomenon, without toxicological significance.
Acute hemorrhage in the thymus was observed in one male at 100 mg/kg bw/day (1/1; thymus was examined only in one animal at 100 mg/kg bw/day due to its macroscopic observation), in two males at 1000 mg/kg bw/day (2/5), in one female at 100 mg/kg bw/day (1/1) and in one female at 300 mg/kg bw/day. This finding was considered as consequence of hypoxia, dyspnoe and circulatory disturbance developed during exsanguinations.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the animals.
The cytomorphology of the endocrine glands were the same in the control and the treated animals.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A test item influence on the estrous cycle was not detected at any dose levels (100, 300 or 1000 mg/kg bw/day).
There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in estrous or diestrous during the pre-treatment and pre-mating period.
In the pre-mating period statistically significant difference was observed when compared to their control in the lower mean number of days in proestrous at 100 mg/kg bw/day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In all male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly lower fertility indices were observed compared to the respective control in 1000 mg/kg bw/day group (male and female). However, the values were comparable with the historical control. Therefore, this slight change was not considered to be adverse. There was statistically significant difference between the control and test item treated groups of male and female animals at 100, 300 and 1000 mg/kg bw/day in copulatory indices. One pair of control group failed to mate during the 14-day period resulting in lower copulatory indices. Thus test item treated groups showed statistically significantly higher copulatory indices. This finding was not considered as biologically relevant.Statistical significance was detected at the slightly lower fertility index in male and female animals at 1000 mg/kg bw/day comparing to their control. In this group 4 out 12 females were non-pregnant but mated (sperm positive). However, the fertility indices were relatively low in all groups in this study – control, 100, 300 and 1000 mg/kg bw/day; 82%, 75%, 75% and 67%, respectively; male and female – relative to the historical control: males: 88 %, n=162; females: 89%, n=162, individuals; SD=12, n=13 groups, min=68.8 %, max=100 %).
In the female animals, statistical significance was also noted for the lower gestation index at 300 mg/kg bw/day because one pregnant female in this group did not deliver. This slight, only statistically significant difference was considered to be indicative of biological variation.
There were no differences between the control and test item administered groups in the mean pre-coital interval or in the mean conceiving days.

Details on results (P0)

In the female animals the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose 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):

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring with signs (cold, not suckled) at 100, 300 and 1000 mg/kg bw/day was slightly higher than in the control group on postnatal day 0. However, this finding was not considered to be toxicologically relevant as no dose dependence was observed and it was transient and detected shortly after the delivery. Moreover, this observation was not associated with any influence on the development of the offspring.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices. The mean number of dead offspring per litter was similar in all groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean pup weights as well as the mean pup weight gain were similar in the control and in all test item treated groups (100, 300 and 1000 mg/kg bw/day) on postnatal days 0, 4 and 13.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In PN13 pups, there were no significant differences in the T4 levels of control and test item treated groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination. There were no macroscopic changes in the organs or tissues of stillborn offsprings (2/2) at 100 mg/kg bw/day subjected to necropsy on postnatal day 0. Autolyzed visceral organs were noted on post-natal day 0 for three pups (3/3) at 100 mg/kg bw/day and for one pup (1/1) at 300 mg/kg bw/day.

Other effects:

no effects observed

Description (incidence and severity):

There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0, 4 or 13
The anogenital distances (AGD; absolute and normalized) were not affected by the test item at 100, 300 and 1000 mg/kg bw/day.
Statistical significances were observed at the shorter anogenital distances (absolute and normalized as well) in female pups of the 1000 mg/kg bw/day treated dams. These minor changes were considered to be indicative of biological variations and not related to the test item.
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on postnatal day 13.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

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

Table 1: SUMMARY OF THYROID HORMONE (T4) LEVEL

		Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Parent Male	Mean	228.33	140.54 **	104.04 **	90.82 **	DN
	SD	52.59	58.42	37.38	28.33
	n	12	12	12	10
	±%		-38	-54	-60
Parent Female	Mean	200.33	177.80	195.51	186.89	NS
	SD n	122.19         8	100.42         9	113.72          8	97.76       8
	±%		-22	-14	-18
Offspring PN4	Mean	141.90
	SD	-	-	-	-
	n	1	-	-	-
	±%
Offspring PN13	Mean	70.95	64.78	99.97	75.33	NS
	SD	12.55	12.71	97.88	24.55
	n	8	8	7	4
	±%		-9	41	6

REMARKS : ±%= Percent Deviation Versus Control
NS = Not Significant
*= p<0.05
** = p< 0.01

U = Mann-Whitney U - test Versus Control
DN = Duncan's multiple range test
PN = Postnatal day

T4 = Free thyroid - T4 - hormone concentration

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, the test item did not cause signs of systemic toxicity in parental Wistar rats at 100, 300 or 1000 mg/kg bw/day doses administered by oral gavage. The test item did not adversely influence the reproductive performance or fertility in parental male and female rats at all doses. The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.
The NOAEL for systemic toxicity of male/female rats is 1000 mg/kg bw/day, the NOAEL for reproductive performance of male/female rats is 1000 mg/kg bw/day and the NOAEL for F1 Offspring is 1000 mg/kg bw/day.

Executive summary:

The purpose of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according OECD TG 422 was to obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum.

Four groups of Hsd.Han:Wistar rats (n=12/sex/group) were administered with the test item by gavage once a day at 0 (vehicle), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. The concentrations of the test item in the dosing formulations varied in the acceptable range between 92 % and 100 % of the nominal values confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 56 days). Females were additionally exposed through the gestation period and up to lactation days 12-18, up to the day before necropsy (altogether for 55-56 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter.

Blood samples were collected for determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. T4 serum levels were determined in parental male animals and in pups sampled on post-natal day 13.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) and pituitary in the control and high dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (distilled water) only. Historical control data were also considered.

Parental generation

Mortality

There was no mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study.

Clinical observation

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations. At the same intervals, the behavior and physical condition of the animals was not impaired at each dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period.

Body weight and body weight gain

Test item related changes in the body weight or body weight gain were not detected. The body weight development was not disturbed and it was comparable in the control and test item treated groups.

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at 100, 300 and 1000 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

Reproductive performance

Slightly lower fertility indices were observed compared to the respective control in 1000 mg/kg bw/day group (male and female). However, the values were comparable with the historical control. Therefore, this slight change was not considered to be adverse.

Clinical pathology

Clinical pathology examinations (hematology, blood coagulation or clinical chemistry) did not reveal alterations related to the test item.

Clinical biochemistry

T4 levels were lower than in the control group in parental male animals at 100, 300 and 1000 mg/kg bw/day in a dose related manner.

Lower mean serum T4 levels were detected with respect to their control in female animals at 100, 3000 and 1000 mg/kg bw/day groups however independently from the doses. Moreover, the inter-individual variation was high in the control and 100, 300 and 1000 mg/kg bw/day groups.There were no related microscopic lesions in the organs of the hypothalamicpituitary-thyroid axis in the examined animals. Therefore this finding was not considered to be adverse under the conditions of this study.

Necropsy

Specific macroscopic alterations related to the test item were not found at necropsy.

Organ weight

There were no test item related changes in the weights (absolute and relative to body or brain weights) of selected organs in the animals at any dose level.

Histopathology

Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day.

There were no changes related to the test item in the organs or tissues of selected animals (male or female) at 1000 mg/kg bw/day.

 

Offspring

No adverse findings on the offspring’s development were detected (mortality, clinical signs and body weight, anogenital distance and nipple retention in male pups, necropsy). In PN13 pups, there were no significant differences in the T4 levels of control and test item treated groups.

Conclusion

Under the conditions of the present study, the test item did not cause signs of systemic toxicity in parental male and female Hsd.Han: Wistar rats at 100, 300 or 1000 mg/kg bw/day doses administered by oral gavage.

The test item did not adversely influence the reproductive performance or fertility (gonad function, mating behavior, conception, parturition) in parental male and female rats at the doses of 100, 300 and 1000 mg/kg bw/day. The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows: NOAEL for systemic toxicity of male/female rats = 1000 mg/kg bw/day, NOAEL for reproductive performance of male/female rats = 1000 mg/kg bw/day, NOAEL for F1 Offspring =1000 mg/kg bw/day.