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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 16, 2021 to January 30, 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted by the Council on 25th June 2018
Deviations:
yes
Remarks:
External fetal sex was compared with internal sex only in foetuses examined for soft tissue malformations, in foetuses examined for skeletal malformations it was not possible. This deviation did not affect the reliability of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-methylaniline
EC Number:
202-870-9
EC Name:
N-methylaniline
Cas Number:
100-61-8
Molecular formula:
C7H9N
IUPAC Name:
N-methylaniline
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar CRL
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Dědinská 893/29, 161 00 Prague 6, Czech Republic, RČH CZ 11760500
- Age at study initiation: 12 week in the dose-range finding experiment and 11 – 12 weeks in the main study
- Weight at study initiation:
- Bedding: sterilized clean shavings of soft wood or sterilized LIGNOCEL (raw material – spruce; producer: J.Rettenmaier & söhne, Germany).
- Diet (e.g. ad libitum): 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. Not specified if ad libitum
- Water (e.g. ad libitum): free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry.
- Acclimation period: 6 days in the dose-range finding experiment and 13 days in the main study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The application forms (the test item in olive oil) were prepared daily just before administration. The weighed amount of the test item was dissolved in portion of olive oil into beaker. Then replenished to the required volume. The application form was by magnetic stirrer (3.5 cm stirrer) at 500 rpm for 30 minutes and then during the administration. You do not reduce the speed, when you removing the application form. It can be taken in the whole volume and is stable for 120 minutes. The concentrations of all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.

VEHICLE
- Vehicle (if other than water): olive oil
- Concentration in vehicle: 10 mg/ 10 ml and 1000mg/10 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See section 8. Analytical methods
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 2 females
- Proof of pregnancy: Vaginal smears were carried out daily in the morning to monitor 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.
Duration of treatment / exposure:
From the 5th to the 19th day of pregnancy
Frequency of treatment:
Daily
Duration of test:
From June 16, 2021 to January 30, 2022
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
No. of animals per sex per dose:
around 24 pregnant females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses were selected based on results of a dose range finding test
- Time of day for (rat) dam blood sampling: Blood samples from the females which were found to be pregnant 20th day of gestation were assessed for serum levels of thyroid hormones

Examinations

Maternal examinations:
HEALTH CONDITIONS: Yes
- Time schedule: daily - during the acclimatization, mating and pregnancy. . In administration period this observation was performed before application and immediately after application.

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily - during the administration period. Females were observed in natural conditions in their cages after application, once a day at the similar time each day.

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy. The body weight of pregnant females was recorded on automatic balances with group mean computing module. Weight increments were computed as a mean per group (in grams).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy. Food consumption was determined at three-day intervals; it coincided with the terms of body weight recording.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus (incl. cervix) and thyroid gland.
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.
Ovaries and uterine content:
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 numbers of corpora lutea of both ovaries were recorded. Uteri of non-pregnant females were examined to confirm the non-pregnant status
Blood sampling:
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 - Triiodothyronine, T4 - Thyroxine, TSH – Thyroid Stimulating Hormone) by kits (manufacturer BioVendor, Brno, Czech Republic).
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 and genital papilla were observed.
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 alterations according to the internal SOP M/6 - Prenatal Developmental Toxicity. 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.
Note: Evaluation of pathological examination of foetuses was performed according to The harmonized nomenclature for developmental toxicology, based on the IFTS (The International Federation of Teratology Societies) terminology
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 results statistically significant on probability level 0.05 are indicated in the summary tables.

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 statistically 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 m
Indices:
Reproduction parameters
In utery, the 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): (corpora lutea - implantations)/corpora lutea x 100
Postimplantation loss – IUDL (Intra Uterine Death Late): resorptions/implantations × 100

Historical control data:
Historical control data available but not reported in the study report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs of diseases were recorded during the acclimatization, mating period and application period in the control and treated females of all dose levels.
No clinical changes indicating the dysfunction of organism were found in the control and treated groups during the whole study.
Mortality:
no mortality observed
Description (incidence):
No unscheduled death of females was recorded during the study.
Body weight and weight changes:
effects observed, treatment-related
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 of body weight was performed for all intervals from the 5th to 20th day of pregnancy.
The body weights of treated females at all dose levels were comparable to the control group during whole study. No statistically significant changes were observed.
The body weight increment of treated females was statistically significantly decreased at the dose level 40 mg/kg bw/day compared with the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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. The statistical evaluation was performed from the 5th to 20th day of pregnancy.
Mean food consumption was statistically significantly decreased at all treated groups in comparison with the control group from the 8th to the 20th day of pregnancy, on the 8th day of the pregnancy with dose dependence.
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).
The concentration of T4 was statistically insignificantly increased at the dose level 15 mg/kg bw/day. The serum level of TSH was decreased at the dose level 5 mg/kg bw/day and sligthly increased at the dose level 40 mg/kg bw/day. The concentration of T3 was comparable in treated groups with control.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Uterus
The uteri of all females were weighed, but only females which were found to be pregnant on the 20th day of gestation were used for calculation of the 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.
Absolute and relative weight of uterus in all treated groups were similar with the control females. Statistically significant differences in uterus biometry were not detected in females of any dose level.
Absolute weight of uterus was insignificantly increased at the dose level 5 mg/kg bw/day in comparison with control. The relative weights of uterus in all dose levels were comparable with control. Statistically significant differences in uterus biometry were not detected in females of any dose level.
Body Weight - Corrected
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. Mean values of corrected body weight of treated females were slightly reduced at the dose level 15 and 40 mg/kg bw/day, with dose dependence.

Thyroid gland
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 levels.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination was performed in all females (including non-pregnant females).
In all treated females at the dose level 40 mg/kg bw/day were recorded enlarged and/or dark spleens.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The histopathology of the thyroid glands was performed for all treated and control females. Histological examination of thyroid glands revealed mild atrophy, mild hypertrophy C cells, mild degeneration, hyperplasia C cells.
The histopathological examination of all spleens was performed, because macroscopical findings in spleens were recorded during pathological examination in all females at the highest dose level. Histological examination of spleens revealed congestion, extramedullary hematopoiesis, hemosiderosis and haemorrhage.

Maternal developmental toxicity

Description (incidence and severity):
For evaluation of IUDE and IUDL females with foetuses and implantations were used. The statistical evaluation was performed for the number of corpora lutea, implantations, resorptions and also for preimplantation and postimplantation losses.
The numbers of implantations and corpoa lutea were similar in all treated groups with control group. The number of resorptions was increased at the dose level 40 mg/kg bw/day compared to the control group.
Preimplantation losses (IUDE) were lower in treated groups in comparison with control group. Postimplantation losses (IUDL) were increased at the dose level 40 mg/kg bw/day compared to the control group. Statistically significant differences were not detected in these all parameters.
Description (incidence and severity):
Five dead foetuses fotetuses were found at the dose level 40 mg/kg bw/day.
Description (incidence and severity):
The number of females with foetuses on the 20th day of pregnancy was comparable at all treated groups with control group (20 – 23 – 24 – 20). The number of non-pregnant females was 4, 1, 0 and 3, respectively, for the four groups: 0, 5, 15 and 40 mg/kg bw/day.
One female, which became pregnant and then all implanted conceptuses in a uterus were totally resorbed (female without foetuses but with implantations), was observed only at the dose level 40 mg/kg bw/day (female No.193).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: No adverse effects at this dose level

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: spleen

Results (fetuses)

Description (incidence and severity):
The mean body weights of all foetuses, males and females were decreased at the dose level 40 mg/kg bw/day, in male foetuses with statistical significance. At the dose level 5 mg/kg bw/day, the body weights of all foetuses and female foetuses were statistically significantly increased compared to the control group.
Description (incidence and severity):
Five dead foetuses fotetuses were found at the dose level 40 mg/kg bw/day. The total number of live foetuses (males and females) in group were increased at the dose level 5 and 15 mg/kg bw/day. The total number of foetuses (males and females) per litter in all treated groups was similar to the control group.
Statistically significant differences of above-mentioned parameters were not detected in any dose level.
Description (incidence and severity):
Data reported for control, 5, 15 and 40 mg/kg bw dose group:
Total number of live foetuses 283, 361, 363 and 300
Number of live foetuses – males 129, 187, 180 and 147
Number of live foetuses – females 154, 174, 183 and 153
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 of males was slightly decreased with statistical significance at the dose level
40 mg/kg bw/day. Other AGD and corrected AGD of males and females were similar with the control group.
Description (incidence and severity):
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.
Five dead foetuses were found at the dose level 40 mg/kg bw/day. The dead foetuses could not be examined due to autolysis. One foetus without tail was found at the dose level 5 mg/kg bw/day. No other external changes were recorded.
Description (incidence and severity):
The proportions of affected litters (%) were expressed in numeric form in sequence: control – 5 – 15 – 40 mg/kg bw/day further in the text.
The statistical evaluation of skeletal findings was performed. The data from control group were compared with data from treated groups using the generalized linear mixed models with Poisson distribution. Statistically significant differences were recorded during the statistical evaluation of numbers of the litters/foetuses with skeletal findings. The findings that statistically significantly changed are indicated in the summary tables No. 23 and 25.

Examination of foetal cranium revealed mainly incomplete ossification of cranial bones. Incompletely ossified were mostly nasal bone, frontal bone, parietal bone, interparietal bone, supraoccipital bone, squamosal part of temporal bone, arcus zygomaticus, less frequently premaxilla, mandibula and basisphenoid. High incidence of incomplete ossification interparietal bone and supraoccipital bone were recorded at all dose levels as well as in control group. The incidence of litters with incomplete ossification of nasal bone, premaxilla and mandibula in treated groups were lower or comparable with control group. The proportions of litters with incomplete ossification of frontal bone at treated groups (70.00 % – 69.57 % – 87.50 % – 80.00 %) were similar or slightly increased in comparison with control group. The proportion of litters with incomplete ossification of parietal bone (80.00 % – 86.96 % – 79.17 % – 100.00 %) was increased at the dose level 40 mg/kg bw/day. The incidence litters with hole in the supraoccipital bone (65.00 % – 86.96 % – 62.50 % – 30.00 %) was increased at the dose level 5 mg/kg bw/day. The proportion of litters with incomplete ossification of arcus zygomaticus and squamosal part of temporal bone were lower in all treated groups in comparison with control group. The increased proportion of litters with incomplete ossification of basisphenoid (15.00 % – 8.70 % – 25.00 % – 30.00 %) was recorded at the dose levels 15 and 40 mg/kg bw/day.

During examination of the foetal skeletons incomplete ossification, bipartite ossification and unossified ossification sites of sternebra were recorded. The incidence of litters with incomplete ossification of ossification sites of sternebra was high in treated groups as well as in control group. The statistically significantly increased numbers of foetuses with incomplete ossification of ossification sites of sternebra were detected at the dose levels 15 and 40 mg/kg bw/day (83.01 % – 88.95 % – 95.34 % – 97.48 %), but in the case of conversion to litter no statistical significance was found.
The proportions of litters with bipartite ossification of ossification sites of sternebra were statistically insignificantly increased (5.00 % – 8.70 % – 12.50 % – 30.00 %) at the dose levels 15 and 40 mg/kg bw/day, with dose dependence. The incidence of litters with unossified ossification sites of sternebra in treated groups was lower or comparable with the control group (80.00 % – 47.83 % – 66.67 % – 80.00 %). The incidence of affected foetuses with this skeletal finding was decreased with statistical significance at the dose level 5 mg/kg bw/day (33.33 % – 10.00 % – 23.83 % – 41.51 %).

Examination of vertebrae revealed mainly incomplete, bipartite and dumbbell ossification of vertebrae thoracic centrum. The incomplete ossification of vertebrae thoracic centrum was statistically significantly decreased at the dose level 5 mg/kg/day (60.00 % – 17.39 % – 37.50 % – 55.00 %). The proportion of litters with bipartite ossification was sligthly increased at the dose levels 5 and 15 mg/kg bw/day (10.00 % – 21.74 % – 25.00 % – 10.00 %). The increased proportions of litters with asymmetric bipartite ossification of vertebrae thoracic centrum were recorded at the dose levels 5 and 15 mg/kg bw/day (5.00 % – 13.04 % – 25.00 % – 5.00 %). The increases proportions of litters with dumbell ossification of vertebrae thoracic centrum (70.00 % – 65.22 % – 95.83 % – 75.00 %) and with asymmetric dumbell ossification of vertebrae thoracic centrum (40.00 % – 43.48 % – 62.50 % – 50.00 %) were recorded at the dose level 15 mg/kg bw/day.

Anomaly of ribs – supernumerary ribs – lumbar ossification site and wavy ribs were recorded. The proportion of litters with supernumerary ribs – lumbar ossification site was increased at the dose level 5 mg/kg bw/day (60.00 % – 91.30 % – 62.50 % – 75.00 %). The incidence of litters with wavy ribs was lower or comparable with the control group.

The incomplete ossification of scapula was similar or lower at all dose levels in comparison with control group (25.00 % – 4.35 % – 25.00 % – 25.00 %).
Description (incidence and severity):
Detailed gross dissections of foetuses were performed. Location and morphology of organs and big vessels were reviewed during examination of internal alterations. No findings were found at all dose levels and in the control group.

Effect levels (fetuses)

open allclose all
Dose descriptor:
LOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
other: mean AGD
Remarks on result:
other: The adverse effects observed in foetuses at 40 mg/kg bw are considered as secondary to maternal toxicity
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
other: No adverse effects observed at this dose level

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL (No Observed Adverse Effect Level) for the test item N-Methylaniline for TOXICITY in pregnant females was established as 15 mg/kg bw/day.
This NOAEL value is based on no mortality of females, no changes in health condition status, no alteration to thyroid hormone measurements, no pathological findings in the dams and no
dose-related changes in reproduction parameters.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established as 15 mg/kg bw/day. This NOAEL is based on no altered growth and no significant treatment-related structural abnormalities in foetuses of pregnant rats. However, due to signs of toxicity observed at 40 mg/kg bw/day in pregnant females, the adverse effects observed on foetuses at the same dose level are considered as secondary to maternal toxicity.
Executive summary:

Maternal animals


The oral administration of the test item, N-Methylaniline, to pregnant females by gavage from the 5th to the 19th day of pregnancy at the dose levels 5, 15 and 40 mg/kg bw/day did not cause mortality of pregnant females.


The body weight of treated maternal animals increased equally with controls for the whole time of pregnancy and was adequate to species, sex and age of animals used in the study. The body weight increment was statistically significantly decreased at the dose level 40 mg/kg bw/day. The reduction of body weight increment correlated with the decrease of food consumption in treated females at the highest dose level. The changes in body weight increment and food consumption at the dose 40 mg/kg bw/day were considered to be adverse.


The corrected body weight of treated females was slightly decreased at the dose levels 15 and 40 mg/kg bw/day, with dose dependence and without statistical significance. Food consumption was statistically significantly decreased at all treated groups in comparison with the control group from the 8th to the 20th day of pregnancy, on the 8th day of the pregnancy with dose dependence. The reduced food consumption is not adverse effect at the dose levels 5 and 15 mg/kg bw/day, because the body weights and body weight increments in these dose levels were similar to the control group. Reduced food consumption without concomitant effect on body weight gain is likely a transient effect and probably not adverse (Ronald D. Hood: Developmental and Reproductive Toxicology: A Practical Approach, Third Edition).   


Clinical examination of treated maternal animals detected no clinical symptoms of toxicity related to treatment with the test item. The behavior, health condition and clinical status of treated maternal animals were similar compared to the control animals.


Pathological examination of pregnant and non-pregnant females revealed pathological finding probably related to the test item treatment at the dose level 40 mg/kg bw/day. In this dose, an enlarged and/or dark spleens were recorded in all females. Histological examination of spleens revealed congestion (mild to marked) in all 24 females at this dose level. Extramedullary hematopoiesis was found in 23 females and this finding is not a physiological condition. Drugs and chemicals that damage blood cells can cause intense extramedullary hematopoiesis.  Hemosiderosis of spleen was found in 23 females and ­this finding is associated with destruction of red blood cells of the spleen. The last finding was haemorrhage, which was found in 4 females at this dose level. The observed pathological changes are multiple, significant, consistent and related to the treatment of the test item.


Evaluation of uterus weights did not demonstrate a negative effect of the test item treatment. Absolute weight of uterus was insignificantly increased at the dose level 5 mg/kg bw/day in comparison with control. The relative weights of uterus in all dose levels were comparable with control.


The number of live foetuses, early and late intrauterine death were evaluated on the basis of examination of uterus content.


The reproductive parameters as numbers of implantations and corpora lutea were comparable between treated and control groups. Number of resorptions was slightly increased at the dose level 40 mg/kg bw/day, but without statistical significance and dose dependence. One female at the same dose level became pregnant and then all implanted conceptuses in uterus were totally resorbed. Postimplantation losses (IUDL) were increased at the dose level 40 mg/kg bw/day without statistical significance and dose dependence. These findings in reproductive parameters at the dose level 40 mg/kg bw/day (resorption, IUDL) were caused probably by the test item treatment.


Absolute and relative weight of thyroid gland and histological examination of thyroid glands did not reveal any changes associated with the application of the test item.


Examination of serum levels of thyroid hormone revealed increased serum level of T4 at the dose level 15 mg/kg bw/day, but without statistical significance and without dose dependence. The concentration of TSH was decreased at the dose level 5 mg/kg bw/day and slightly increased at the dose level 40 mg/kg bw/day, both without dose dependence and statistical significance. 


Histological examination of thyroid glands revealed mild atrophy, mild degeneration, mild hypertrophy C cells and/or hyperplasia C cells. The found changes are isolated, weakly expressed and do not deviate from the natural viability of the thyroid histological picture of clinically healthy animals. These findings were also found at the control group and are not related to the treatment of the test item.


 Foetuses


The influence of the test item on the development of the conceptus in uterus was assessed according to the results of weighting, careful necropsy and skeletal/soft tissue examination of foetuses.


Five dead foetuses were found at the highest dose level. The numbers of live foetuses (males and females) in treated groups were similar or increased in comparison with control group. The total number of live foetuses per litter in all treated groups was similar to the control group.


The mean value of foetal body weight was decreased at the dose level 40 mg/kg bw/day, with statistical significance in male foetuses and without dose dependence. This reduction of fetal body weight is related to the decrease of body weight increments of pregnant females at the same dose level. The statistically significantly increased foetal body weight was observed in all foetuses and female foetus at the dose level 5 mg/kg bw/day. These increases are without biological and toxicological significance.


The mean AGD of males was slightly decreased at the dose level 40 mg/kg bw/day with statistical significance. This reduction is related to the decreased body weight of male foetuses at the same dose level, because the corrected AGD was similar to the control group. Other mean AGD and corrected AGD in male and female foetuses at other dose levels were comparable to the control group.


Treatment with the test item was not associated with the occurence of external and visceral variations and malformations. Only at the dose level 40 mg/kg bw/day was found five dead foetuses. One foetus without tail was found at the dose level 5 mg/kg bw/day.


Examination of foetal skeletons indicated mainly delayed development of the skeleton at all dose levels as well as in the control group. According to the OECD 414 the litter as the unit for data analysis should be used for statistical evaluation. The statistical evaluation was performed for the number of foetuses with skeletal findings and also for number of litters with skeletal findings in this study. Statistically significant differences without toxicological significance were recorded during the statistical evaluation of the number of foetuses/litters with skeletal findings.


Incomplete ossification of foetal cranium was observed during the examination in all groups, including the control group. The delayed development in nasal bone, premaxilla, mandibula, frontal bone, parietal bone, interparietal bone, supraoccipital bone, arcus zygomaticus, squamosal part of temporal bone and basisphenoid were not related to the treatment because the occurrence of these findings in litters were high in all groups, including the control group.


The incidence of litters with incomplete ossification of nasal bone, premaxilla and mandibula in treated groups were lower or comparable with control group. The proportion of litters with incomplete ossification of frontal bone at treated groups (70.00 % – 69.57 % – 87.50 % – 80.00 %) was similar or slightly increased in comparison with control group. The proportion of litters with incomplete ossification of parietal bone (80.00 % – 86.96 % – 79.17 % – 100.00 %) was increased at the dose level 40 mg/kg bw/day.


High incidence of litters with affected foetuses with incomplete ossification of interparietal bone (95.00 % – 100.00 % – 95.83 % – 100.00 %) and supraoccipital bone (100.00 % – 100.00 % – 100.00 % – 100.00 %) were recorded at all dose levels as well as in control group. These findings were not related to the treatment, because the occurrence of these findings was comparable to the control group. The incidence of litters with hole in the supraoccipital bone (65.00 % – 86.96 % – 62.50 % – 30.00 %) was increased at the dose level 5 mg/kg bw/day but without statistical and toxicological significance and dose dependence. The proportion of litters with incomplete ossification of arcus zygomaticus and squamosal part of temporal bone were lower in all treated groups in comparison with control group. The increased proportion of litters with incomplete ossification of basisphenoid (15.00 % – 8.70 % – 25.00 % – 30.00 %) was recorded at the dose levels 15 and 40 mg/kg bw/day. The increased incidence of this variation was without statistical significance.


During examination of the foetal skeletons incomplete ossification, bipartite ossification and unossified ossification sites of sternebra were recorded. It is a normal variability in the schedule of ossification; ossification of sternum has not to be complete on the 20th day of gestation.


The incidence of litters with incomplete ossification sites of sternebra was high in treated groups as well as in control group. The statistically significantly increased numbers of foetuses with incomplete ossification of ossification sites of sternebra were detected at the dose levels 15 and 40 mg/kg bw/day (83.01 % – 88.95 % – 95.34 % – 97.48 %), but in the case of conversion to litter no statistical significance was found.


The proportions of litters with bipartite ossification of ossification sites of sternebra were statistically insignificantly increased at the dose levels 15 and 40 mg/kg bw/day (5.00 % – 8.70 % – 12.50 % – 30.00 %), with dose dependence. The incidence of litters with unossified ossification sites of sternebra in treated groups was lower or comparable with the control group (80.00 % – 47.83 % – 66.67 % – 80.00 %). Percentage occurrence this finding was on the border of statistical significance (pvalue = 0.057) at the dose level 5 mg/kg bw/day. This reduced incidence of unossified ossification sites of sternebra at the lowest dose level (33.33 % – 10.00 % – 23.83 % – 41.51 %) was without biological and toxicological significance.


Examination of vertebrae revealed incomplete, dumbbell and bipartite ossification of vertebrae thoracic centrum in all test groups including control group.


The proportion of litters with incomplete ossification of vertebrae thoracic centrum was statistically significantly decreased at the dose level 5 mg/kg bw/day (60.00 % – 17.39 % – 37.50 % – 55.00 %). This decrease was without biological and toxicological significance. The incidences of litters with bipartite ossification were slightly increased at the dose levels 5 and 15 mg/kg bw/day (10.00 % – 21.74 % – 25.00 % – 10.00 %). These increases were without dose dependence and statical significance. The increase proportions of litters with asymmetric bipartite ossification of vertebrae thoracic centrum (5.00 % – 13.04 % – 25.00 % – 5.00 %) were recorded at the dose levels 5 and 15 mg/kg bw/day. The increased proportions of litters with dumbell ossification of vertebrae thoracic centrum (70.00 % – 65.22 % – 95.83 % – 75.00 %) and with asymmetric dumbell ossification of vertebrae thoracic centrum (40.00 % – 43.48 % – 62.50 % – 50.00 %) were recorded at the dose level 15 mg/kg/day. These increases are without statistical significance and dose dependence.


Changes in the size, shape, or symmetry of sternebrae or vertebral centra are transient and have no implications for the health or survival of the offsprings (John M. DeSesso, Anthony R. Scialli: Bone development in laboratory mammals used in developmental toxicity studies).


Anomaly of ribs – wavy ribs and supernumerary ribs – lumbar ossification site was recorded.


The proportion of litters with supernumerary ribs – lumbar ossification site was increased at the dose level 5 mg/kg bw/day (60.00 % – 91.30 % – 62.50 % – 75.00 %), without dose dependence. This increase was statistically non-significant. The proportion of litters with wavy ribs was comparable or lower at all dose levels in comparison with control group (35.00 % – 13.04 % – 29.17 % – 45.00 %).


The incomplete ossification of scapula was comparable or lower at all dose level in comparison with control group (25.00 % – 4.35 % – 25.00 % – 25.00 %).