Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-[[4-(diethylamino)-6-[(4-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:4)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

see Section: Any ather information on materials and methods

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[diethylamino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Cas Number:

42355-78-2

Molecular formula:

C40H40N12Na4O12S4

IUPAC Name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[diethylamino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CRL

Details on test animals or test system and environmental conditions:

Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species and because rat is recommended according to the test guideline
Strain: Wistar CRL (SPF quality - guaranteed)
Supplier: Charles River SPF breeding, supplied via VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
Sex: sexually adult females (males – only for mating)

Total number of animals: 25 probably pregnant females for each dose level (total number of animals before mating: 100 females and 25 males)
Acclimatization: 13 days
Age at start of the study: 12 weeks
Housing conditions: SPF conditions
Light cycle: 2 hour light/12 hour dark
Microclimate: 22±3 °C, relative humidity 30–70 %

Animal per cage: before mating 2 rats of the same sex in one cage,
during mating period one male and two females in one cage were housed.
pregnant females were then placed individually.
Bedding: sterilized clean shavings of soft wood or sterilized LIGNOCEL (raw material - spruce; producer: J.Rettenmaier&söhne, Germany).
Food: complete pelleted diet for rats and mice in SPF breeding (Altromin Spezialfutter) was used.
Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany. Diet was sterilized before using.
Water: free access to drinking water (water ad libitum).
Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Quality standard ČSN 757 111.
Selection of animals: pregnant females were randomly assigned to the experimental groups after determination of pregnancy.
Identification of animals: the animals were identified by the colour marks (colour for veterinary usage) on their fur, each cage was marked with the number of animals, sex, number of cage, name and dose level of the test item.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
Batch No.:1905170334
Expiration: 5/2021
Manufacturer: Ardeapharma Ševětín, Czech Republic

CONCENTRATIONS
- Concentration Level 10 mg/10 mL:
The test item was weighed into glass beaker calibrated to 100 mL and dissolved in vehicle in ultrasonic bath for 5 min. After this the solution was stirred by magnetic stirrer (300 rpm) for 10 minutes.
- Concentration Level 1000 mg/10 mL:
The test item was weighed into glass beaker calibrated to 100 mL and dissolved in vehicle (ca 80 % of total volume) in ultrasonic bath together with occasional mixing by glass rod for 25 minutes. After this the glass beaker was diluted to mark by vehicle and the application form was stirred by magnetic stirrer (350 rpm) for 35 min.

The beaker with test item was during dissolving, homogenisation and stability measuring covered by alluminium foil due to possible light unstability of test item. This measure is recommended for further test item application form preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the application forms was checked by determination of a peak area of the test item in three places of application form (at the bottom, in the middle and on the surface).
Stability of the application form 10 mg/10 mL was checked by analyses of the application form within 120 min (at the time 0, 30, 60, 90 and 120 min). Time interval 0 min represents for this concentration the time after 5 minutes dissolving in ultrasonic bath and 5 minutes of mixing by magnetic stirrer (300 rpm).
Stability of the application form 1000 mg/10 mL was checked by analyses of the application form within 180 min (at the time 0, 30, 60, 90, 120, 150 and 180 min). Time interval 0 min represents for this concentration the time after 25 minutes dissolving in ultrasonic bath and 35 minutes of mixing by magnetic stirrer (350 rpm).

Results of Analysis
It follows from the results of analyses (homogeneity and stability) that the application form 10 mg /10 mL) of the test item, at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes from the finalization of application form preparation.
While the application form 1000 mg /10 mL of the test item is homogenous and stable at least for 180 minutes from the finalization of application form preparation.

Details on mating procedure:

After acclimatization females were mated with males (1 male and 2 females). Vaginal smears were carried out daily in the morning to control fertilization (first time: 24 hours after
the first removing to male). Presence of sperms was examined. Day 0 of pregnancy was the day on which sperms in vaginal smears were observed. Pregnant females were randomly
distributed to experimental groups.

Frequency of treatment:

daily - 7 days per week at the same time (8.00 – 10.00 am)

Duration of test:

Exposition lasted from implantation (the 5th day after fertilization) to one day prior to the day of scheduled euthanasia (the 19th day after fertilization).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day

No. of animals per sex per dose:

25 probably pregnant females for each dose level
Male rats serve only for mating (they are not administered by the test item and examined)

Control animals:

yes, concurrent vehicle

Details on study design:

Females were without feed two hours before application and two hours after application of the test item.

Dose selection rationale:
Based on the result of the DRF study on the substance, the selected highest tested dose for the main study should have been 1000 mg/kg bw/day, since no significant toxicological effects have been observed at this dose and it could have been considered as limit dose. However, it should be noticed that this study has been performed not only to investigate the toxicological properties of the substance in itself, but also and mainly to compare the effects within the members of the category; therefore the highest tested dose has been selected as below reported, taking into account the existing following studies:

• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on kidney at 750 mg/kg bw/day
• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on testes at 750 mg/kg bw/day
• 3a-MSA – Subacute toxicity study – Effects to be confirmed on haematological parameters, liver and kidney starting from 200 mg/Kg bw/day
• 3a-MSA – Two generation toxicity study - Relevant toxicological effects mainly in terms of maternal toxicity at 1000 mg/kg bw/day and minor effects at 300 mg/kg bw/day

Indeed, in this respect all systemic studies concerning repeated dose, reproductive and developmental toxicity (OECD 422, 408 and 414) have been set on similar dose level in order to be able to compare all members of the category on the specific organ effects.
The lowest and the medium doses have been selected in order to have a coherent spacing of doses respect to the highest selected one, also taking into account all the available data on the repeated dose toxicity ( 3a-MSA, 1-DSA).

Examinations

Maternal examinations:

- Examination of vaginal smears of females
Each morning in the mating period vaginal smears were prepared from all mated females. These smears were stained and examined microscopically for presence of spermatozoa. Day 0 of pregnancy was the day when sperms were observed.

- Body weight of females
The body weight of pregnant females was recorded on automatic balances with group mean computing module. First weighing was performed on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy. Weight increments were computed as a mean per group (in grams).

- Food consumption of females
Food consumption was determined at three-day intervals; it coincided with the terms of body weight recording.

- Mortality of females
All rats were examined for vitality or mortality changes daily during the acclimatization, mating and pregnancy.

- Health condition control of females
The health condition was controlled daily during the acclimatization period, during the mating period and during pregnancy. Pre-experimental control of all females was performed to ensure that only the females exhibiting normal behavioral activity would be entered into the study. In administration period this observation was performed before application and immediately after application.

- Clinical observations of the females
During the administration period clinical observation was made in order to record possible clinical effect of the test item application and all changes in behavior of females. Females were observed in natural conditions in their cages after application - once a day at the similar time each day.

- Determination of thyroid hormones
The blood samples for this examination were taken from orbital plexus by glass micropipette under the light ether narcosis on the 20th day of pregnancy. The blood serum samples were prepared by spontaneous coagulation and they were centrifuged 10 minutes in centrifuge and then were serum samples frozen. Blood samples from the pregnant females were assessed for serum levels of thyroid hormones (T3, T4, TSH) by ELISA kit.

-Biometry and histopathological examination of thyroid gland
During macroscopic examination the thyroid glands were removed from all pregnant females and were preserved in fixation medium. The thyroid weights were determined after fixation. For histopathological processing the routine histological paraffin technique with synoptic haematoxylin-eosin staining was used. The histopathology was performed in the control and high dose group. Treatment-related changes were not observed at the high dose group therefore detailed histological examination of thyroid gland was performed only for all high dose and control females.

- Biometry of uterus and macroscopical examination of females
On the 20th day of pregnancy the females were narcotised and sacrificed by cutting the neck spine and medulla. The revision of the external surface of the body was performed.
During macroscopic all orifices, the cranial, thoracic and abdominal cavities were examined and uterus (incl. the cervix) was removed and weighed.
In gravid uterus number of viable foetuses, number of dead foetuses, number of early resorption (implantation without recognizable embryo/foetus) and number of late resorption (dead embryo or foetus with external degenerative changes) were recorded. The number of corpora lutea of both ovaries were found out.
Uteri of non-pregnant females were examined to confirm the non-pregnant status (by the help of ammonium sulphide staining)
The absolute weight of uterus was recorded. Afterwards the relative weight of uterus was computed according to the following formula:

Relative weight of uterus = (absolute weight of uterus ×100)/(body weight of female on 20th day)

Also the correction of body weight was performed according to the following formula:

Corrected body weight = body weight of female on 20th day - weight of uterus

Ovaries and uterine content:

In uterus number of viable foetuses, number of dead foetuses and number of resorptions (implantation without recognisable embryo/foetus or dead embryo or foetus with external degenerative changes) were recorded.
Number of corpora lutea on ovaries was also recorded.
Preimplantation and postimplantation losses were calculated from number of implantations (number of foetuses plus number of resorptions), corpora lutea and resorptions.

Preimplantation loss – IUDE (Intra Uterine Death Early):
Preimplantation loss = (corpora lutea - implantations)/(corpora lutea) ×100

Postimplantation loss – IUDL (Intra Uterine Death Late):
Postimplantation loss = resorptions/implantations ×100

Fetal examinations:

Sex, individual body weights and anogenital distance (AGD) of foetuses were recorded. A digital caliper was used for AGD measurements. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight.

Each foetus was examined for external alterations:
- symmetry of fore and hind limbs
- number of fingers
- closing or opening of eye fissures and external auditory canal
- symmetry of head
- integrity of superior palatum
- status of umbilicus
- genital papilla

One half of each litter (one half of female and male foetuses) was examined for soft tissue alterations using careful gross dissection.
Second half of each litter was processed and microscopically examined for skeletal and cartilage alterations.
Single staining displayed only ossified skeletal structures was used: the foetuses were fixed in ethanol, macerated in potassium hydroxide solution, stained with Alizarin red and placed in glycerine-based solution.
The skeletal examination was performed using a stereomicroscope and included examination of skull, clavicle, scapula, sternebra and sternum, ribs, vertebrae, pelvic girdle, forelimb/hindlimb.

Physiological appearance:
- Vertebrae - cervical vertebrae: 7, thoracic v.: 13, lumbar v.: 6, sacral v.: 4.
- Ribs:
“True ribs”:7 vertebrosternal ribs, upper thoracic ribs that are attached directly to the sternum
“False ribs”:3, long costal cartilage, but not articulated with sternum
“False floating ribs”:3, short costal cartilage, and not articulated with sternum
- Sternum: 6 ossification sites

Statistics:

For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups.

The parametric tests were used for statistical evaluation of:
•body weight of females (5th, 8th, 11th , 14th , 17th , 20th day of pregnancy)
•corrected body weight (subtraction weight of uterus from surgery body weight of females)
•food consumption (per interval)
•mean weight of foetuses (males, females, both sex)
•anogenital distance
•thyroid hormones
•biometry of thyroid gland (absolute and relative weight )
•biometry of uteri (absolute and relative weight )
•preimplantation (IUDE) and postimplantation (IUDL) losses

As the first step the test for normality (Shapiro-Wilk test) was performed. If the data were not normally distributed the transformation of data was performed (Box-Cox transformation). If the data were not normal distributed after transformation the non-parametric tests (Kruskal-Wallis Test and Mann-Whitney test) for comparison of the medians were performed.
If data were normally distributed after transformation, the Variance check (Levene’s test) to verify standard deviations within each group was used. One-Way ANOVA (probability level 0.05) was used to detect whether there were any significant differences amongst the means and then the post hoc statistical testing (Fisher's least significant difference - LSD test) for only statistical significant differences was performed.

The non-parametric tests were used for statistical evaluation of following parameters:
•number of corpora lutea, number of implantations, number of resorptions
•number of live foetuses (males, females, both sex)
•number of dead foetuses
The two-groups Mann-Whitney test (probability level 0.05) was applied.

The categorical data (skeletal foetal findings) were analyzed using the generalized linear mixed models with Poisson distribution.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical changes indicating the dysfunction of organism were found in the control and treated groups during the whole study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) were used for calculation of mean body weights. The statistical evaluation was performed from the 5th to 20th day of pregnancy.
The body weights of treated females at all dose levels were similar to the control group during whole study. No statistically significant changes were observed. The body weight increments at all dose levels were comparable with the body weight increment of the control females.
The lowest body weight increment was recorded at the middle dose level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Only females which were found to be pregnant 20th day of gestation (females with live foetuses) were used for calculation of mean food consumption, although is it is not a feeding study. The statistical evaluation was performed from the 5th to 20th day of pregnancy.
The average food consumption of all treated groups was comparable with the control group during whole study. Statistically significant differences were not detected.

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

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Corrected body weight of all treated females (the necropsy body weight of female minus weight of uterus) was not statistically significantly changed compared to control females. Values of corrected body weight of treated females were similar to the control females.
The uteri of all females were weighed, but only females which were found to be pregnant 20th day of gestation were used for calculation of mean weight of uterus. The absolute weight of uterus was recorded and the relative weight of uterus was computed. The statistical evaluation of absolute and relative weight of uterus was performed.
The mean absolute and relative weights of uterus at all dose levels were comparable with the control group. Statistically significant differences in uterus biometry were not detected in females of any dose level.
The thyroid glands of all females were weighed (after fixation), but only females which were found to be pregnant 20th day of gestation were used for calculation of mean weight of thyroid gland. The absolute weight of thyroid gland was recorded and the relative weight of thyroid gland was computed. The statistical evaluation of absolute and relative weight of thyroid glands was performed.
Statistically significant differences of thyroid weights were not detected in females of any dose level. Absolute and relative weights of the thyroid glands were similar in the treated and control females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination was performed in all females (including females without foetuses). No finding was noted at necropsy in treated and control females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histopathology of the thyroid glands was performed for the control females and high dose in first instance. Histological examination of thyroid glands revealed no pathological changes. Because the treatment related changes were not recorded, histopathology of thyroid gland at doses 100 and 300 mg/kg/day was not performed.

Other effects:

no effects observed

Description (incidence and severity):

Blood samples from the females which were found to be pregnant 20th day of gestation were assessed for serum levels of thyroid hormones (T3, T4, TSH).
No statistically significant differences were recorded in serum levels of thyroid hormones T3 and T4 in females from treated groups against control females. Serum level of TSH was slightly decreased in females at the middle dose level, but also without statistical significance.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Preimplantation losses (IUDE) and postimplantation losses (IUDL) in all treated groups were similar or decreased in comparison with control group. Statistically significant differences were not detected.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of resorptions were similar or lower at all dose levels in comparison with the control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

Only females which were found to be pregnant on the 20th day of gravidity were used for calculation of mean and standard deviation values. The statistical evaluation was performed for the number of live foetuses, the number of live male foetuses, the number of live female foetuses and number of dead foetuses per litter. Statistically significant differences of above mentioned parameters were not detected in any dose level.
One dead foetus was found out in the highest dose level (0 – 0 – 0 – 1). The highest number of foetuses was recorded in the lowest dose level. The average total number of foetuses in litter was well balanced in all groups.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

dead fetuses

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean body weight of foetuses was decreased at the middle dose level compared to the control group. Although the variation between individual body weight of foetuses at this dose level against control foetuses was observed, this difference was not statistically significant. Male foetuses were heavier than females in all groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The statistical evaluation was performed for the number of live foetuses, the number of live male foetuses, the number of live female foetuses and number of dead foetuses per litter. Statistically significant differences of above mentioned parameters were not detected in any dose level.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean body weight of foetuses was slightly decreased at the highest dose level compared to the control group, statistical significance was not detected. Male foetuses were heavier than females in all groups.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

The anogenital distance (AGD) of each foetus was measured by digital caliper on 20th of pregnancy of females and the corrected AGD was calculated. The statistical evaluation was performed for AGD and corrected AGD of male and female foetuses.
The AGD and corrected AGD of male foetuses at all dose levels were comparable with control.
The statistically significantly increased female AGD was recorded at the middle dose level, but the corrected AGDs of female foetuses at all dose levels were comparable with control.

Examination of symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla were performed.
One dead foetus was found out in the highest dose level (0 – 0 – 0 – 1). No external changes were recorded.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Examination of foetal skeleton indicated mainly delayed development of the skeleton at all dose levels and control group.
Incomplete ossification of foetal cranium was found out during examination in all groups including control group. With delayed development were affected mostly parietal bone, interparietal bone, supraoccipital bone, arcus zygomaticus, squamous part of temporal bone, less frequently frontal bone, basioccipital bone, basisphenoid and nasal bone. This delayed development was not related probably to the treatment, because the occurrence of these findings are seen commonly also in high percentage in control foetuses. The highest portions of litters with incomplete ossification of parietal bone, interparietal bone, supraoccipital bone, frontal bone, nasal bone and basisphenoid were recorded at the highest dose level, this may be associated with the lower body weight of the foetuses at this dose level.
Other findings on cranial bones which were observed in all test groups including control group were hole in the supraoccipital bone. The frequency of holes in the supraoccipital bone was similar or lower at dose levels compared to the control group. Other changes of foetal cranium were found only sporadically.

Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded in foetuses of all treated groups as well as control. It is normal variability in the schedule of ossification; ossification of sternum have not to be complete on the 20th day of pregnancy. These findings were not related to the treatment, because the occurence of these findings was comparable or lower to the control group.

Examination of vertebrae revealed high incidence of dumbbell ossification of vertebrae thoracic centrum in all test groups including control group. There was recorded increased dose-dependent incidence of litters with dumbbell ossification of vertebrae thoracic centrum in the treated groups compared to the control group (76.19 % – 79.17 % – 85.00 % – 100.00 %) but the incidence of affected foetuses with this finding at treated groups was quite comparable with control group (33.33 % – 33.16 % – 31.25 % – 40.63 %), highlighting no dose-dependency of the effect. The statistical significance was not detected. This delay development is not related to the lower body weight of foetuses. The interpretation of this finding is questionable, due to the high incidence of this finding also in the control foetuses. The dumbbell ossification of vertebrae thoracic centrum is included in the Transitional Findings (grey zone). These may be upgraded to malformation or downgraded to variations status, depending on severity and/or frequency of occurrence.

Less frequent were findings of bipartite ossification of vertebrae thoracic centrum. Test item treatment relation was not evident for these findings.

The changes such as wavy ribs and ribs-supernumerary site were detected also in all groups. The portions of litters with wavy ribs and ribs-supernumerary site were similar or lower at all dose levels compared to the control group. These findings did not relate with test item treatment.

The incidence of icomplete ossification of scapula was recorded only in litters at treated groups, without dose relationship and statistical significance. This variation is not considered to be adverse.

Visceral malformations:

no effects observed

Description (incidence and severity):

Detailed gross dissections of foetuses were performed. Placing and morphology of organs and big vessels were reviewed during examination of internal alterations. No findings were found at all dose levels and control group.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Results in tabular form:

	dose (mg/kg)				notes
	0	100	300	750
Number of dams with abortions	0	0	0	0
Number of dams with early deliveries	0	0	0	0
Number of dams with stillbirths	0	0	0	0
Number of dams with resorptions	0	0	0	1
Total number of live foetuses	329	367	300	316
Number of live foetuses – males	167	187	164	164
Number of live foetuses – females	162	180	136	152
Number of dead foetuses	0	0	0	1
Number of implantations per female	15.45 ± 3.81	15.46 ± 3.23	15.35 ± 3.48	16.40 ± 1.73	The numbers of implantations and corpora lutea in treated groups were comparable with the control group. The numbers of resorptions were similar or lower at all dose levels in comparison with the control group. Preimplantation losses (IUDE) and postimplantation losses (IUDL) in all treated groups were similar or decreased in comparison with control group. Statistically significant differences were not detected.
Number of resorptions per female	0.50 ± 0.60	0.17 ± 0.38	0.35 ± 0.49	0.60 ± 0.88
Number of corpora lutea per female	16.41 ± 2.46	16.13 ± 2.54	16.65 ± 2.58	16.85 ± 1.84
Pre-implantation loss, percent per female	7.31 ± 19.08	4.81 ± 10.93	8.00 ± 13.72	2.52 ± 4.65
Post-implanation loss,percent per female	7.41 ± 20.99	1.06 ± 2.52	2.37 ± 3.42	3.70 ± 5.33
Pre-implantation loss, number	not reported	not reported	not reported	not reported
Post-implanation loss, number	not reported	not reported	not reported	not reported
Body weight at 1st and 20th day of pregnancy, grams	284.7 ± 18.7 440.8 ± 34.3	291.2 ± 20.4 446.4 ± 29.6	287.3 ± 19.9 434.8 ± 31.1	289.3 ± 20.5 440.7 ± 36.0	Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) were used for calculation of mean body weights. The statistical evaluation was performed from the 5th to 20th day of pregnancy. The body weights of treated females at all dose levels were similar to the control group during whole study. No statistically significant changes were observed. The body weight increments at all dose levels were comparable with the body weight increment of the control females. The lowest body weight increment was recorded at the middle dose level.
Body weight increment, grams	156.1	155.1	147.5	151.5
Absolute weight and relative (%) weight of the thyroid	0.0313 ± 0.0027 0.0071 ± 0.0008(%)	0.0312 ± 0.0023 0.0070 ± 0.0006(%)	0.0309 ± 0.0024 0.0071 ± 0.0007 (%)	0.0311 ± 0.002 0.0071 ± 0.0008 (%)	The thyroid glands of all females were weighed (after fixation), but only females which were found to be pregnant 20th day of gestation were used for calculation of mean weight of thyroid gland. The absolute weight of thyroid gland was recorded and the relative weight of thyroid gland was computed. The statistical evaluation of absolute and relative weight of thyroid glands was performed. Absolute and relative weights of the thyroid glands were similar in the treated and control females. Statistically significant differences of thyroid weights were not detected in females of any dose level
Histopathology of the thyroid	no effects	not examined	not examined	no effects	The histopathology of the thyroid glands was performed for the control females and high dose in first instance. Histological examination of thyroid glands revealed no pathological changes. Because the treatment related changes were not recorded, histopathology of thyroid gland at doses 100 and 300 mg/kg/day was not performed.
Results of the thyroid hormone (T4, T3 and TSH)	no effects	no effects	no effects	no effects	No statistically significant differences were recorded in serum levels of thyroid hormones T3, T4 and TSH in females from treated groups against control females.
Mean number of live offspring, litter	15.67 ± 2.13	15.29 ± 3.20	15.00 ± 3.51	15.80 ± 1.96
Percent of live offspring, litter	not reported	not reported	not reported	not reported
Mean foetal/pup body weight by sexes combined	3.81 ± 0.84 (M+F)	3.87 ± 0.85 (M+F)	4.26 ± 0.96 (M+F)	3.48 ± 0.98 (M+F)
Mean foetal/pup body weight by sex	3.90 ± 0.86 (M) 3.71 ± 0.83(F)	3.99 ± 0.88 (M) 3.75 ± 0.82 (F)	4.36 ± 0.98 (M) 4.14 ± 0.99 (F)	3.57 ± 1.02 (M) 3.39 ± 0.94 (F)
AGD (M+F) and corrected AGD (M+F), mm	3.47 (M)/2.13(F) 2.21(M)/1.39(F)	3.45(M)/2.12(F) 2-19(M)/1.38(F)	3.63(M)/2.23(F) 2.23(M)/1.40(F)	3.34(M)/2.15(F) 2.20(M)/1.45(F)	The AGD and corrected AGD of male foetuses at all dose levels were comparable with control. The statistically significantly increased female AGD was recorded at the middle dose level, but the corrected AGDs of female foetuses at all dose levels were comparable with control.
Number (and percent) of foetuses and litters with malformation (including runts) and/or variations, external	0/0	0/0	0/0	1/0.29	Examination of symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla were performed. One dead foetus was found out in the highest dose level (0 – 0 – 0 – 1). No external changes were recorded.
Description and incidences of malformations and main variations	no effects	no effects	no effects	no effects
Macroscopic changes of soft tissues – individual foetuses
Total number of examined foetuses	155	171	140	148
Total number of examined litters	21	24	20	20
With pathological findings - total (number of affected foetuses)	0	0	0	0
Number of examined foetuses in litter	7.38	7.13	7.00	7.40
(mean ± SD)	±0.97	±1.62	±1.84	±0.99
Total number of foetuses with alteration	0	0	0	0
Number of foetuses with alteration in litter	0.00	0.00	0.00	0.00
(mean ± SD)	±0.00	±0.00	±0.00	±0.00
Portion of foetuses with alteration in litter	0.00	0.00	0.00	0.00
(% mean ± SD)	±0.00	±0.00	±0.00	±0.00
Skeletal findings, litter	no effects	no effects	no effects	no effects	No statistically significant differences were recorded during the statistical evaluation of skeletal findings. Only the incidence of incomplete ossification of ossification sites of sternebra in foetuses at the highest dose level was statistically significantly decreased compared to the incidence in control foetuses, but this finding has no toxicological significance. Examination of foetal skeleton indicated mainly delayed development of the skeleton at all dose levels and control group.

 

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 750 mg/kg/day. This NOAEL value is based on no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established as 750 mg/kg/day. This NOAEL is based on no altered growth in treated foetuses. The occurrence of the dumbbell ossification of vertebrae thoracic centrum in 100 % of litters at the dose level 750 mg/kg/day was not considered as adverse due to the high incidence in control litters and because the incidence of affected foetuses with this finding at treated groups was quite comparable with control group.

Executive summary:

The test item was tested for prenatal developmental toxicity following the OECD Test Guideline 414.

 

Study performance

Before the start of experiments with laboratory animals the stability and homogeneity of application form were determined at the test facility.

A dose-range finding experiment (DRFE) was performed to determine the dose levels for the main study.

Wistar rat females and males of SPF quality were used for testing. After acclimatization the females were mated with males in the main study.

The test item was then administered to pregnant females by gavage, daily from the 5^th to the 19^th day of pregnancy, at dose levels of 0, (vehicle), 100, 300 and 750 mg/kg/day.

 

The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during developmental toxicity study. On the day of necropsy, blood was collected from pregnant females to determine thyroid hormone levels (T3, T4, TSH). On the 20th day of pregnancy the maternal animals were euthanized, the uterine contents were examined and the foetuses were assessed for changes on soft tissues and skeleton. The foetuses were weighed and the anogenital distances were measured. The thyroid glands were removed from the females and histological examination was performed.

 

Results

Mothers

The test item had no negative effecton the growth of maternal animals. The body weight of treated maternal animals increased equally with controls for the whole time of pregnancy and individual variability of body weight increments was adequate to species, sex and age of animals used in the study. Also food consumption of treated females was balanced with control. 

Clinical examinations of treated mothers detected no clinical symptoms of toxicity related to the test item treatment. The behavior, health condition and clinical status were similar compared to the control and no serious changes were found.

Also pathological examination revealed no pathological finding related to the test item treatment. Evaluation of uterus weights did not demonstrate a negative effect of the test item treatment too.

The examinations of reproductive parameters revealed that the numbers of implantations, corpora lutea were comparable at dose levels with control group. The numbers of resorptions were similar or lower in treated groups in comparison with control group. Preimplantation losses (IUDE) and postimplantation losses (IUDL) in all treated groups were similar or decreased compared to the control group. Reproductive parameters were unaffected by treatment with the test item.

Examination of the thyroid gland– absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones,did not reveal any changes associated with the application of the test item.

Foetuses

One dead foetus was only found at the highest dose level. The average total number of live foetuses in litter was well balanced in all groups.

Based on statistical evaluation of mean values of foetal body weight no significant growth retardation was detected in treated groups. The foetal body weight was non-significantly slightly decreased at the highest dose level, although litter size was comparable in all groups. Males were heavier than females in all groups (include control). This weight imbalance is common.

The mean anogenital distance andcorrected AGD of male foetuses at the all dose levels were balanced with control.The mean anogenital distances of female foetuses was statistically significantly increased at the middle dose level compared to the control. This may be related to the fact that in the middle dose there were foetuses with a higher body weight than in other dose levels, becausethe mean corrected anogenital distance of female foetuses was comparable at all dose levels with control group.

The test item treatment did not evoke occurrence of external and visceral variations and malformations.

 No statistically significant differences were recorded during the statistical evaluation of skeletal findings. Only the incidence of incomplete ossification of ossification sites of sternebra in foetuses at the highest dose level was statistically significantly decreased compared to the incidence in control foetuses, but this finding has no toxicological significance.

Examination of foetal skeleton indicated mainly delayed development of the skeleton at all dose levels and control group.

Incomplete ossification of foetal cranium was found out during examination in all groups including control group. With delayed development were affected mostly parietal bone, interparietal bone, supraoccipital bone, arcus zygomaticus, squamous part of temporal bone, less frequently frontal bone, basioccipital bone, basisphenoid and nasal bone. This delayed development was not related probably to the treatment, because the occurrence of these findings are seen commonly also in high percentage in control foetuses. The highest portions of litters with incomplete ossification of parietal bone, interparietal bone, supraoccipital bone, frontal bone, nasal bone and basisphenoid were recorded at the highest dose level, this may be associated with the lower body weight of the foetuses at this dose level.

Other findings on cranial bones which were observed in all test groups including control group were hole in the supraoccipital bone. The frequency of holes in the supraoccipital bone was similar or lower at dose levels compared to the control group. Other changes of foetal cranium were found only sporadically. 

 Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebrawere recorded in foetuses of all treated groups as well as control. It is normal variability in the schedule of ossification; ossification of sternum have not to be complete on the 20^thday of pregnancy. These findings were not related to the treatment, because the occurence of these findings was comparable or lower to the control group.

Examination of vertebrae revealed high incidence of dumbbell ossification of vertebrae thoracic centrum in all test groups including control group. There was recorded increased dose-dependent incidence of litters with dumbbell ossification of vertebrae thoracic centrumin the treated groups compared to the control group (76.19 %–79.17 %–85.00 %–100.00 %) but the incidence of affected foetuses with this finding at treated groups was quite comparable with control group (33.33 % – 33.16 % – 31.25 % – 40.63 %), highlighting no dose-dependency of the effect. The statistical significance was not detected. This delay development is not related to the lower body weight of foetuses. The interpretation of this finding is questionable, due to the high incidence of this finding also in the control foetuses. The dumbell ossificationof vertebrae thoracic centrum is included in the Transitional Findings (grey zone). These may be upgraded to malformation or downgraded to variations status, depending on severity and/or frequency of occurrence.

Less frequent were findings of bipartite ossification of vertebrae thoracic centrum.Test item treatment relation was not evident for these findings.

The changes such as wavy ribs andribs-supernumerary sitewere detected also in all groups. The portions of litters with wavy ribs and ribs-supernumerary site were similar or lower at all dose levels compared to the control group. These findings did not relate with test item treatment.

 The incidence of incomplete ossification of scapula was recorded only in litters at treated groups, without dose relationship and statistical significance.This variation is not considered to be adverse.

 

The NOAEL for toxicity in PREGNANT FEMALES was established as 750 mg/kg/day. This NOAEL value is based on no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters up to this dose level.

 

The NOAEL for PRENATAL DEVELOPMENT was established as 750mg/kg/day. The occurrence of the dumbbell ossification of vertebrae thoracic centrum in all litters at the dose level 750 mg/kg/day was not considered as adverse due to the high incidence in control litters and because the incidence of affected foetuses with this finding at treated groups was quite comparable with control group.