Registration Dossier

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:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The test substance, Gelbpigment E4GN was tested for prenatal developmental toxicity using the Method B.31 , Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in OJ. L142, 2008 and OECD Test Guideline No. 414 Prenatal Developmental Toxicity Study, Adopted by the Council on January 22nd 2001.Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with untreated males. The test substance was then administered to pregnant females by gavage - daily from the 5th to the 19th day of pregnancy. The study included five groups of females - 3 treated groups and 2 control groups (vehicle only). The dose levels for the study - 250, 500 and 1000 mg/kg/day - have been chosen based on the results of a dose-range finding experiment.The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during the study. On the 20th day of pregnancy the maternal animals were euthanized, the uterine contents were examined and the foetuses were evaluated for external, soft tissue and skeletal alterations.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test substance name:Gelbpigment E4GNOther names: Pigment Yellow 150EC-Number (from ECHA): 939-379-0Muiticomponent substance1st component:lUPAC name (English):Nickel, 5,5' -azobis-2,4,6(l H,3H,5H)-pyrimidinetrione complexesCAS name: Nickel, 5,5 ' -azobis-2,4,6( I H,3H,5H)-pyrimidinetrione complexesCAS No.: 68511-62-6 (main component)EECNo.: 270-944-8 (main component)Molecular formula: C8H4N606NiMolecular weight: 338.9 g/mol2nd component:CAS name: MelamineCAS No.:108-78-1Composition of test substance:56.1 % Nickel Complex Dye of the test item Gelbpigment E4GN (CAS 68511-62-6)2.1 % Nickel Free Dye of the test item Gelbpigment E4GN39.7% Melamine (CAS 108-78-1)1.7 % Water0.4 % Potassium chlorideAppearance: yellowish powder
Specific details on test material used for the study:
Test substance name: Gelbpigment E4GN Other names: Pigment Yellow 150EC-Number (from ECHA): 939-379-0Multicomponent substance1st component:IUPAC name (English):Nickel, 5,5´-azobis-2,4,6(1H,3H,5H)-pyrimidinetrione complexesCAS name: Nickel, 5,5´-azobis-2,4,6(1H,3H,5H)-pyrimidinetrione complexesCAS No.:68511-62-6 (main component) EEC No.:270-944-8 (main component)Molecular formula:C8H4N6O6NiMolecular weight:338.9 g/mol 2nd component:CAS name: MelamineCAS No.:108-78-1Composition of test substance56.1 % Nickel Complex Dye of the test item Gelbpigment E4GN (CAS 68511-62-6)2.1 % Nickel Free Dye of the test item Gelbpigment E4GN 39.7 % Melamine (CAS 108-78-1)1.7 % Water 0.4 % Potassium chloride Appearance:yellowish powder Storage:at room temperature in dark in the supplied container

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Species:Albino laboratory rat Selection of animal species: laboratory rat has been chosen because this species is recommended according to the test guideline and because our testing laboratory has long experience with this species Strain: Wistar CRL (SPF quality - guaranteed) Supplier: SPF breeding Charles River delivered by VELAZ s.r.o., Únětice, Czech Republic, RČH CZ 11760500Sex: sexually adult females, 12 weeks old and mean body weight 245.65 g (pilot experiment)/250.79 g (main study) at the beginning of the mating (+ males, 12 weeks old - only for mating)Acclimatization: 12 days (pilot experiment)/13 days (main study)Total number of animals:main study – approx. 20 pregnant females for each group (total number of animals before mating: 128 females and 32 males (3 females = sentinel animals for pathological, bacteriological and parasitological examination) pilot experiment – approx. 5 pregnant females for each dose level (total number of animals before mating: 28 females and 10 males) Housing conditions:All the study was conducted in the SPF (specified pathogens free) animal room of CETA in conditions according to SOP No. 12 – Control of SPF animal house conditions.Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 22+3°C, a relative humidity of 30-70% and 12-hour light/12-hour dark cycle. Animals were housed in plastic cages containing sterilised clean fibres of soft wood - sterilized LIGNOCEL (raw material - spruce; producer: J.Rettenmaier&Söhne, Germany). Before mating 2 rats of the same sex were housed in one cage, during the mating period one male and two females were housed in one cage. Pregnant females were placed individually (polycarbonate cages TECNIPLAST, type 1291H for rats).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: mixture of ethanol, Kolliphor HS 15 and tap water (10%, 40% and 50% - v/v/v)
Details on exposure:
The test substance was administered to the stomach by gavage as a suspension in vehicle (mixture of ethanol, Kolliphor HS15 and tap water - 10%, 40% and 50% - v/v/v). The test substance was mixed with Ethanol and melted Kolliphor (under stirring) and finally the tap water was added (under stirring). The application form was then mixed by magnetic stirrer and it was still mixed during administration. A description of precise method for preparation of application form is archived as raw data. The application form was prepared fresh daily.The test substance was administered to pregnant females in graduated dose levels from the implantation (the 5th day after fertilisation) to one day prior to the day of scheduled euthanasia (the 19th day after fertilisation).
Analytical verification of doses or concentrations:
no
Details on mating procedure:
After acclimatisation females were mated with males (1 male and 2 females). Mating of animals was performed for a restricted time period per day: approx. from 3 pm to approx. 7 am on the following day. Vaginal smears were carried out daily in the morning until all females needed for the study were inseminated. The presence of sperms was examined in the vaginal smears. Day 0 of pregnancy was the day on which sperms in vaginal smears were found out. Probably pregnant females were randomly selected to the experimental groups: 2S inseminated females were allocated to each group (to achieve about 20 pregnant females per group). The identity of parental males was recorded for each litter.
Duration of treatment / exposure:
The test substance was administered to pregnant females in graduated dose levels from the implantation (the 5th day after fertilisation) to one day prior to the day of scheduled euthanasia (the 19th day after fertilisation).
Frequency of treatment:
once daily
Duration of test:
20 days
No. of animals per sex per dose:
20 female rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with untreated males. The test substance was then administered to pregnant females by gavage - daily from the 5th to the 19th day of pregnancy. The study included five groups of females – 3 treated groups and 2 control groups (vehicle only). The dose levels for the main study – 250, 500 and 1000 mg/kg/day – have been chosen based on the results of a dose-range finding experiment.

Examinations

Maternal examinations:
The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during the study. On the 20th day of pregnancy the maternal animals were euthanized, the uterine contents were examined and the foetuses were evaluated for external, soft tissue and skeletal alterations
Ovaries and uterine content:
Pathological examination of females and uterine weights: On the 20th day of pregnancy the females were sacrificed. The macroscopic examination for any structural abnormalities andpathological changes was perfonned. The gravid uterus (incl. the cervix) was removed and weighed. In uterus number of viable foetuses, number of dead foetuses, number of implantation sites, number of early resorptions (implantation without recognisableembryo/foetus) and number of late resorptions (dead embryo or foetus with external degenerative changes) were recorded. Placental pathologic findings were recorded. Number of corpora lutea on ovaries was 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: weight of uterus x 100/ body weight.
Fetal examinations:
Sex and individual body weights of live foetuses were recorded. Each foetus (incl. dead foetuses) 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.One half of each litter (about one half of female foetuses and about one half of male foetuses) was examined for soft tissue alterations using detailed gross dissection (with a magnifying lens).Placing and morphology of the following organs were viewed: nasopharyngeal tract, thymus, thyroid gland, trachea, oesophagus, lungs, heart, great vessels, diaphragm, liver, stomach, spleen, pancreas, intestines, kidneys, adrenal glands, gonads, ureters, urinary bladder, eyes, brain, spinal cord. The necropsy was also done in dead foetuses.The second half of each litter was processed and microscopically examined for skeletal alterations: the foetuses were fixed in ethanol, macerated in potassium hydroxide solution, stained with Alizarin red for visualization of ossified skeletal structures and placed in glycerine-based solution. The skeletal examination was performed under a stereo microscope and included examination of bones of the scull, clavicle, scapula, vertebrae, sternum, ribs, pelvic girdle, forelimb skeleton and hindlimb skeleton.The evaluation of foetal pathological findings was performed according to The Harmonized Nomenclature for Developmental Toxicology, based on the IFTS (The International Federation of Teratology Societies) tenninology(available: [http://www.devtox.org/nomenclature/organ.phpj, last update: October 2012).
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used. The data from control group I 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 (inc. corrected body weight) and fetuses•food consumption (per interval)•body weight increment•uterine weights (absolute and relative weight )•preimplantation (IUDE) and postimplantation loss (IUDL)As the first step the test for normality (Shapiro-Wilk test) was performed. For pre and post –implantation losses the Arcsin transformation of data was used and t-test was done. For the data not normally distributed (relative weight of uterus, food consumption 14th day - control I, body weight of fetuses – males, females, both sex) the transformation of data was done (Box-Cox transformation). Due to still not normal distribution (relative weight of uterus, food consumption 14th day – control I, body weight of fetuses – males, females, both sex) non-parametric tests were performed (Kruskal-Wallis Test) for comparison of the medians. If data were normally distributed the variance check was used (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are 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.
Indices:
For each group fertility and gestation indexes were calculated.Fertility index:(number of pregnant females/number of sperm-positive females) x 100Gestation index:(number offemales with live foetuses/number of pregnant females) x 100In uterus number of viable foetuses and number ofresorptions (early and late resorptions incl. dead foetuses) were recorded. Number of corpora lutea on ovaries was noted. Preimplantation and postimplantation losses were calculated from number of implantations (= number of foetuses plus number of resorptions), corpora lutea and resorptions were also counted.Preimplantation loss - IUDE (Intrauterine Death Early):(corpora lutea - implantations/corpora lutea) x 100Postimplantation loss - IUDL (Intrauterine Death Late):(resorptions/implantations) x 100
Historical control data:
Historical control data for basic parameters and fetal alterations are available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No adverse changes of health condition and no clinical symptoms of intoxication were found in females at any dose level after administration of the test substance. In females of the dose levels 500 and 1000 mg/kg/day a finding related to the colour of the test substance (yellow colour of faeces) was observed.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths of females during the study at any dose level.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the control of body weight increments and food consumption in pregnant females at all dose levels: 250, 500 and 1000 mg/kg/day toxicologically significant treatment-related effects were not detected
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of treated mothers was relatively balanced with controls except the increase in the last measured interval at the dose levels 250 and 1000 mg/kg/day. It is probably related to a higher number of foetuses (litter size) in mothers of these dose levels. The difference was not dependent on dose level therefore it was not considered to be of toxicological significance.
Food efficiency:
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
Description (incidence and severity):
The behaviour, health condition and clinical status of treated maternal animals were similar compared to controls and no serious changes were found.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopical structure of organs of pregnant females and values of reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses), were unaffected by treatment with the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
In females of the dose level 250 mg/kg/day statistically significant decrease of postimplantation loss compared to Control I was noted.The number of corpora lutea and implantations was slightly higher in dosed groups compared to control groups. Preimplantation loss was incidentally increased in both control groups.The value of postimplantation loss was increased insignificantly in females of the dose level 1000 mg/kg/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of females achieving pregnancy were analogous in treated and control groups. Abortions/total resorptions did not occur in any group. The numbers of females with live foetuses (on the 20th day of pregnancy) were well-balanced in control and treated groups. No significant decrease of fertility and gestation indexes was recorded in females of any dose level.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The number of resorptions was negligible in females of any dose levels.
Dead fetuses:
no effects observed
Description (incidence and severity):
The numbers of females with live foetuses (on the 20th day of pregnancy) were well-balanced in control and treated groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The number of females with live foetuses was well-balanced in control and treated groups and the gestation rate was not affected by treatment.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The numbers of females with live foetuses (on the 20th day of pregnancy) were well-balanced in control and treated groups.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
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 decreased at the lowest and the middle dose levels. At the highest dose level the foetal body weight was similiar to control although the number of foetuses was higher.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): 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 decreased at the lowest and the middle dose levels. At the highest dose level the foetal body weight was similiar to control although the number of foetuses was higher.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of females with live foetuses was well-balanced in control and treated groups and the gestation rate was not affected by treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio was well balanced in treated and control groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
All females of the control and treated groups were inseminated. The numbers of females achieving pregnancy were analogous in treated and control groups. Abortions/total resorptions did not occur in any group. The numbers of females with live foetuses (on the 20th day of pregnancy) were well-balanced in control and treated groups. No significant decrease of fertility and gestation indexes was recorded in females of any dose level.A statistical evaluation was performed for the body weight of live foetuses. No statistically significant differences were detected at any dose level. The fetal body weight was non-significantly decreased at the dose levels 250 and 500 mg/kg/day. Vice versa at the dose level 1000 mg/kg/day the fetal body weight was well balanced with control.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
A statistical evaluation of external alterations was not performed due to their rare occurrence.External malformations were observed very sporadically without relation to the test substance treatment: Anasarca and microcephaly was diagnosed in one dead foetus of Control II. Discolored skin and short tail was detected in one live extremely small foetus of the dose level 500 mg/kg/day. The placenta of this foetus was small. Spontaneous external alterations were observed only sporadically in foetuses of control groups and at the dose levels 500 and 1000 mg/kg/day. Small or extremely small dead foetus was noted in one litter of Control I, in one litter of Control II and in one litter of the dose level 1000 mg/kg/day. The placenta of both control foetuses was reduced in size and in the foetus of Control II it was accompanied by discolored skin.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected only sporadically without relation to the test substance treatment: misshapen arcus zygomaticus in one foetus at the Control I, one foetus at the Control II and in one foetus at the dose level 1000 mg/kg/day; misshapen humerus in two foetuses (in 2 different litters) at the Control II and in 3 foetuses (in 3 different litters) at the dose level 250 mg/kg/day (cf. Table 22). The incidence of misshapen humerus at the dose level 250 mg/kg/day was statistically significant for the portion of affected foetuses per group if compared with control I, however incidence was comparable to findings in control II and not dose-related so that treatment relationship was not evident for this finding.Statistically significant increase of affected foetuses portion was detected for incomplete ossification of some cranial bones (cf. Table 18). Incomplete ossification of frontal bone, interparietal bone, nasal bone and arcus zygomaticus was noted at the dose levels 500 and 1000 mg/kg/day though generally without dose relation (higher incidence of incomplete ossification occurred in treated groups also compared qualitatively to Control II). The portion of foetuses with incomplete ossification of nasal bone was significantly increased also at the dose level 250 mg/kg/day but not dose-related and quite comparable to the incidence in control group II at the 250 mg/kg and 1000 mg/kg dose levels. The ossification of supraoccipital bone was significantly delayed at the dose level 1000 mg/kg/day, however without dose relation. The frequency of bipartite ossification of supraoccipital bone was significantly increased only at the dose level 500 mg/kg/day. The extent of ossification of other cranial bones was changed non-significantly or was analogous to control in foetuses of the dose levels 250, 500 and 1000 mg/kg/day. Incomplete ossification of sternum was recorded in control foetuses and in foetuses of the dose levels 250, 500 and 1000 mg/kg/day in similar frequency. The ossification of sacral vertebra was significantly delayed at the dose level 1000 mg/kg/day. The difference was evident also in qualitative comparison with the control group II. The ossification of other vertebra, ribs, clavicle, scapula, pelvis and limbs was not delayed in foetuses of the dose levels 250, 500 and 1000 mg/kg/day.The following structural skeletal variations were detected sporadically without treatment relation: misaligned sternebra, supernumerary ribs (statistically significantly increased at the dose levels 250 and 1000 mg/kg/day in comparison to control I, however lower incidence than in control II), wavy ribs (statistically significantly reduced at the dose levels 250 and 1000 mg/kg/day). The occurrence of hole and/or unossified area in basisphenoid was non-significantly increased in foetuses of the dose levels 250, 500 and 1000 mg/kg/day (not dose related) and incidence per litter was sporadic too.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A statistical evaluation of external alterations was not performed due to their rare occurrence.External malformations were observed very sporadically without relation to the test substance treatment: Anasarca and microcephaly was diagnosed in one dead foetus of Control II. Discolored skin and short tail was detected in one live extremely small foetus of the dose level 500 mg/kg/day. The placenta of this foetus was small. Spontaneous external alterations were observed only sporadically in foetuses of control groups and at the dose levels 500 and 1000 mg/kg/day. Small or extremely small dead foetus was noted in one litter of Control I, in one litter of Control II and in one litter of the dose level 1000 mg/kg/day. The placenta of both control foetuses was reduced in size and in the foetus of Control II it was accompanied by discolored skin.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A finding related to the yellow colour of the test substance (i.e. yellow colour of faeces) was observed in females of the middle and the highest dose level.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 1000 mg/kg/day was the highest applied dose

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Overview of the single group composition, female code numbers and the coding used for labelling of groups in the other tables:

       Groups of animals
 Treatment  Group code  Code numbers of females
 Control I  C - I  No. 101 - 125
 Control II  C - II  No. 126 - 150
 250 mg/kg/day  250  No. 151 - 175
 500 mg/kg/day  500  No. 176 - 200
 1000 mg/kg/day  1000  No. 200 - 225

Reproduction parameters:

Fertility parameters

All females of the control and treated groups were inseminated. The numbers of females achieving pregnancy were analogous in treated and control groups. Abortions/total resorptions/early deliveries and stillbirths did not occur in any group. The numbers of females with live foetuses (on the 20th day of pregnancy) were well-balanced in control and treated groups. No significant decrease of fertility and gestation indexes was recorded in females of any dose level.

                Fertility parameters (per group)

 

             Group code

 Parameter

 C - I  C - II  250  500  1000
 Females started (N)  25  25  25  25  25
 Sperm-positive females (N)  25  25  25  25  25
 Pregnant females (N)  21  23  21  22  20
 Non-pregnant females (N)  4  2  4  3  5
 Females with totally resorbed foetuses (N)  0  0  0  0  0
 Females with live foetuses on the day of necropsy (N)  21  23  21  22  20
 Fertility index (%) 84   92  84  88  80
Gestation index (%)  100  100  100 100   100

Intrauterine Death Early and Intrauterine Death Late

Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) were used for calculation of mean and standard deviation values. The statistical evaluation was performed for the number of corpora lutea, implantations, resorptions and also for preimplantation and postimplantation losses. In females of the dose level 250 mglkglday statistically significant decrease of postimplantation loss compared to Control I was noted. Statistically significant differences of other mentioned parameters were not detected in females of any dose levels. The number of corpora lutea and implantations was slightly higher in dosed groups compared to control groups. Preimplantation loss was incidentally increased in both control groups. The number of resorptions was negligible in females of any dose levels. The value of postimplantation loss was increased insignificantly in females of the dose level 1000 mg/kg/day.

                Parameter of reproduction (number per female, mean +/- SD
               Group code
 Parameter  C -I  C - II  250  500  1000
 Corpora lutea  16.10±2.83   15.78±1.41

  17.05±2.09

  16.14±2.12

  16.95±2.21

 Implantations

 14.57±3.52

 14.74±1.81

 16.14±2.29

15.27±1.93 

 16.15±2.32

 Preimplantation loss

 1.52 ± 2.14

 1.04 ± 1.19

0.90 ± 0.89 

 0.86 ± 1.17

0.80 ± 1.01 

 Resorptions (Postimplantation loss)

  0.81±0.93

  0.65±0.88

  0.33±0.80

  0.41±0.67

  0.90±1.02

               I U D E and I U D L (% per female, mean±SD)
               Group code
 Parameter  C -I  C - II  250  500  1000
 IUDE  9.72±13.46   6.64±7.62

  5.43±5.41

 5.10±6.27

 4.81±6.64

 IUDL

 5.53±6.32

 4.22±5.62

 2.01±4.66

2.58±4.20

6.20±8.64

Note: IUDE = Intrauterine Death Early % (= preimplantation loss) IUDL = Intrauterine Death Late % (= postimplantation loss). The value statistically significant on probability level 0.05 is shaded.

Number of foetuses

Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) 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 the number of dead foetuses per litter. Statistically significant differences of above mentioned parameters were not detected in females of any dose level. Dead foetuses were found out very sporadically in control groups and once at the dose level 1000 mg/kg/day. The total numbers of live foetuses and also the mean numbers of foetuses per litter at all dose levels were incidentally non-significantly increased compared to group Control I and as well higher than in control group II. The sex ratio was well balanced in treated and control groups.

                Total number of foetuses in groups
  Group code             
 Parameter  C - I  C - II  250  500  1000

 Total number of live foetuses

 289  324  332  327  305

 Total number of live male foetuses

 148  165  158  173  155
 Total number of live female foetuses  141  159  174  154  150
 Total number of dead foetuses  1  2  0  0  1

         Number of foetuses (per litter, mean±SD)
               Group code
 Parameter  C - I  C - II  250  500  1000

 Mean number of live foetuses

 13.76±3.45  14.09±1.68  15.81±2.25  14.86± 1.88  15.25±2.81

 Mean number of live male foetuses

 7.05±2.62  7.17±2.08  7.52±2.04  7.86±1.58  7.75±2.1 2
 Mean number of live female foetuses  6.71±2.1 0  6.91±1.86  8.29±2.49  7.00±2.23  7.50±1.85
 Number of dead foetuses  0.05±0.22  0.09±0.29  0.00  0.00  0.05±0.22

Body weight of females and body weight increment:

Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) were used for calculation of mean and standard deviation values. The statistical evaluation was performed for body weight on the 8th, 11th, 14th, 17th, 20th day of pregnancy and for corrected body weight increment. Statistically significant differences were not detected in females of any dose level. The body weights of treated females well balanced with the control females. The higher body weight observed in females of the dose level 250 mg/kg/day on the 20th day of pregnancy was considered unrelated to treatment of the test substance. Corrected body weight increment of treated females was analogous to the body weight increment of the control females.

Body weight in grams (mean ± SD)
               Group code
 Day of Pregnancy  C - I  C - II  250  500  1000

 1st day

 262.71 ±13.23  264.20± 12.50  263.86± l5.57  264.20±14.64  264.31 ±16.22

 5th day

 277.93± 13.S9  281.29± 12.84  280.94± 18.09  281.14±16.12  282.25± 16.64
 8th day  288.09±14.41  291.16± 14.83  292.04± 18.36  289.43± 18.33  292.87±18.79
 11th day  304.73±16.32  307.22±15.16  308.23±20.66  303.49±20.39  307.65±18.89
 14th day  322.82±15.43  327.20±18.14  328.53±24.80  321.11±22.28  326.73±20.21
 17th day  356.19±21.64  360.86±21.73  365.46±28.46  353.55±28.06  361.09±23.00
 20th day  407.24±30.87  414.55±27.26  425.23±38.55  407.28±34.97  416.97±29.14
 Total weight increment 20th day - 1st day  144.52±26.52  150.36±24.18  161.38±28.93  143.08±24.85  152.66±23.98
 Corrected1 weight  increment 20th day - 1st day  68.94±17.74  72.52±18.25  75.38±23.24  63.88±14.04 68.63±16.15 

1: minus uterine weight

Note: Statistically significant differences on probability level 0.05 were not detected.

Uterine weight

Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) were used for calculation of mean and standard deviation values. The statistical evaluation of absolute and relative weight of uterus was perfonned. Statistically significant differences of uterus weights were not detected in females of any dose level. Higher absolute and relative uterine weights were observed in females of all dosed groups and with a higher increase at levels of 250 and 1000 mg/kg/day. These increases were noted in relation to incidentally higher litter size and without clear dose response and thus did not relate to the test substance treatment.

 Uterine weights (mean ± SD)
               Group code
 Parameter  C - I  C - II  250  500  1000

 Absolute weight of uterus (g)

  

75.5829± 18.7295

  

77.8352± 13.0424

  

85.9914± 16.8290

  

79.1928±15.7589

  

84.0290± 14.4628

 Relative weight of uterus (%)

  

18.4022± 3.6946

  

18.7045± 2.3855

  

20.1754± 3.1586

  

19.3118± 2.6854

  

20.1085± 2.9416

Number of foetuses

Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) 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 the number of dead foetuses per litter. Statistically significant differences of above mentioned parameters were not detected in females of any dose level.

Dead foetuses were found out very sporadically in control groups and once at the dose level 1000 mg/kg/day. The total numbers of live foetuses and also the mean numbers of foetuses per litter at all dose levels were incidentally non-significantly increased compared to group Control I and as well higher than in control group II. The sex ratio was well balanced in treated and control groups.

Total number of foetuses in groups
               Group code
 Parameter  C - I  C - II  250  500  1000

Total number of live foetuses

  

289

  

324

  

332

  

327

  

305

 Total number of live male foetuses

  

148

  

165

  

158

  

173

  

155

 Total number of live female foetuses 141  159  174  154  150
 Total number of dead foetuses 1 2  0  0 1

 Mean number of live foetuses

 13.76±3.45

 14.09±1.68

15.81±2.25 

 14.86±1.88

15.25±2.81 

 Mean number of live male foetuses

 7.05±2.62

7.17±2.08 

 7.52±2.04

7.86±1.58 

 7.75±2.1 2

 Mean number of live female foetuses

 6.71±2.1 0

 6.91±1.86

8.29±2.49 

 7.00±2.23

7.50±1.85 

 Mean number of dead foetuses

 0.05±0.22

0.09±0.29 

 0.00

 0.00

0.05±0.22 

Note: Statistically significant differences on probability level 0.05 were not detected.

Examination of external alterations

The statistical evaluation of external alterations was not performed due to their rare occurrence.

External malformations were observed very sporadically without relation to the test substance treatment:

Anasarca and microcephaly was diagnosed in one dead foetus of Control II. Discolored skin and short tail was detected in one live extremely small foetus of the dose level 500 mg/kg/lday. The placenta of this foetus was small.

Spontaneous external alterations were observed only sporadically in foetuses of control groups and at the dose levels 500 and 1000 mg/kg/day. Small or extremely small dead foetus was noted in one litter of Control I, in one litter of Control II and in one litter of the dose level 1000 mg/kg/day. The placenta of both control foetuses was reduced in size and in the foetus of Control II it was accompanied by discolored skin.

Total number of foetuses in groups /Total number of foetuses (per litter, mean +/- SD)

 

             Group code

 Parameter

 C - I

 C - II

 250

 500

 1000

Total number of live foetuses

  

289

  

324

  

332

  

327

  

305

 Total number of live male foetuses

  

148

  

165

  

158

  

173

  

155

 Total number of live female foetuses

141

 159

 174

 154

 150

 Total number of dead foetuses

1

2

 0

 0

1

 Mean number of live foetuses

 13.76±3.45

 14.09±1.68

15.81±2.25 

 14.86±1.88

15.25±2.81 

 Mean number of live male foetuses

 7.05±2.62

7. 17±2.08 

 7.52±2.04

7.86±1.58 

 7.75±2.1 2

 Mean number of live female foetuses

 6.71±2.10

 6.91±1.86

8.29±2.49 

 7.00±2.23

7.50±1.85 

 Number of dead foetuses

 0.05±0.22

0.09±0.29 

 0.00

 0.00

0.05±0.22 

The statistical evaluation of external alterations was not performed due to their rare occurrence.

External malformations were observed very sporadically without relation to the test substance treatment: Anasarca and microcephaly was diagnosed in one dead foetus of Control II. Discolored skin and short tail was detected in one live extremely small foetus of the dose level 500 mglkglday. The placenta of this foetus was small. Spontaneous external alterations were observed only sporadically in foetuses of control groups and at the dose levels 500 and 1000 mg/kg/day. Small or extremely small dead foetus was noted in one litter of Control I, in one litter of Control II and in one litter of the dose level 1000 mg/kg/day. The placenta of both control foetuses was reduced in size and in the foetus of Control II it was accompanied by discolored

External alterations - number of affected foetuses

 

             Group code

 Alteration

 C - I

 C - II

 250

 500

 1000

Total number of examined foetuses

290

  326

 332

327

  305

 Total number of examined litters

 21

  23

  21

 22

 20

 Dead foetus: extremely small, small placenta (reduced)

1

 0

 0

0

 0

 Dead foetus: * small head (microcephaly)

a generalized subcutaneous oedema (anasarca)

0

1

 0

 0

0

 Dead foetus: extremely small, small placenta

(reduced), pale colour of skin (discolored skin)

0

1

0

 0

0

Dead foetus: small

 0

0

 0

0

 1

 Live foetus: extremely small, hyperaemia of

skin (discoloured skin), * short tail, small

placenta

0

 0

0

 1

0

 Number of examined foetuses in litter

(mean ± SD)

 13.81±3.50

14.17±1.61 

15.81±2.25

14.86±1.88

15.30±2.89 

 Total number of foetuses with alteration  1  2  0  1  1

 Number of foetuses with alteration in litter

(mean ± SD)

0.05±0.22

 0.09±0.29

 0.00±0.00

 0.05±0.21

  0.05±0.22

 Portion of foetuses witb alteration in litter

(%, mean ± SD)

 0.26±1.21

 

0.64±2.14

  0.00±0.00

  0.32±1.52

  0.25±1.12

Note: * short tail = malformation, microcephaly = malformation

Examination of soft tissue alterations

The statistical evaluation of soft tissue alterations was not performed due to their rare occurrence.

Malformations were not detected in foetuses of any dose level. Spontaneous soft tissue alterations were observed sporadically in foetuses of control groups and the dose level 1000 mg/kg/day. In two dead foetuses (one of the control group I, one of the dose level 1000 mg/kg/day) only autolysis of organs was found out. Anasarca and cardiomegaly accompanied by mild autolysis of digestive system organs were detected in one dead foetus of the control group II (same as already described above). In another dead foetus of the control group II discolored organs and mild autolysis of organs were noted.

Soft tissue alterations - number of foetuses

 

             Group code

 Alteration

 C - I.

 C - II.

 250

 500

 1000

Total number of examined foetuses

140

  155

 155

155

  145

 Total number of examined litters

 21

  23

  21

 22

 20

Dead foetus: autolysis of organs

1

 0

 0

0

 1

Dead foetus: generalized subcutaneous oedema

(anasarca), enlargement of heart

(cardiomegaly), mild autolysis of organs of

digestive system

0

1

 0

 0

0

 Dead foetus: pale colour of organs

(discolored), mild autolysis of organs

0

1

0

 0

0

Number of examined foetuses in litter

(mean± SD)

 

 6.67±1.68

 

 6.74±0.92

 

 7.38±1.07

 

 7.05±0.79

  

 7.25±1.41

 Total number of foetuses with alteration

1

 2

0

 0

1

 Number of foetuses with alteration in litter

(mean± SD)

 0.05±0.22

0.09±0.29 

0.00±0.00

0.00±0.00

0.05±0.22 

 Portion of foetuses with alteration in litter

(%, mean ± SD)

 

0.53±2.42

  

1.35±4.47

 

0.00±0.00

  

0.00±0.00

  

0.50±2.24

                   Skeletal alterations (number of affected foetuses per group)
                  Group Code
    Alteration  C- I.  C - II.  250  500  1000
    Number of examined foetuses  150  171  177  172  161
  Cranium  frontal bone - i.o.  24  25  38  49  45
   Cranium  interparietal bone - i.o.  75  84  104  111  108
   Cranium  maxilla - i.o.  0  0  3  4  0
   Cranium  premaxilla - i.o.  0  0  1  0  2
   Cranium  nasal bone - i.o.  8  16  24  31  23
   Cranium  palatinum - i.o.  10  15  22  21  25
   Cranium  parietal bone - i.o.  63  78  86  95  78
   Cranium  supraoccipital bone - i.o.  72  80  92  102  100
   Cranium  basisphenoid - u.a.  2  5  3  8  15
   Cranium  basisphenoid - hole  1  5  19  18  12
   Cranium  arcus zygomaticus -i.o.  24  34  41  47  46
   Cranium

 * arcus zygomaticus - misshapen

 1  1  0  0  1
   Cranium  arcus zygomaticus - small  0  0  2  0  0
   Cranium  supraoccipital bone - bipartite ossification  1  0  3 10   0
 Sternum  misaligned sternebra  1  3  6  5  5
  Sternum  sternebra - unossified  120  128  135  144  128
  Vertebrae  vertebrae lumbar - i.o  0  1  1  0  0
 Vertebrae  vertebrae sacral - i.o.  12  19  24  13  34
 Ribs  supernumerary  0  7  1  0  5
 Ribs  wavy  17  6  6  20  6
 Ribs  bent  0  0  0  0  0

    clavicula

 0  0  0

    scapula

 0  0  0  0  0

    pelvis

 0  0  0  0  0

    * limbs - humerus - misshapen

 0  2  3  0  0

Note: i.o. - incomplete ossification, u.a. - unossified area

* arcus zygomaticus - misshapen = malformation, humerus - misshapen = malformation

Skeletal alterations (% age of affected foetuses per group)
                  Group Code
    Alteration  C - I.  C - II.  250  500  1000
    Number of examined foetuses  150  171  177  172  161
  Cranium  frontal bone - i.o.  16.00 14.62  21.47 28.49 27.95
   Cranium  interparietal bone - i.o.  50.00  49.12 58.76  64.53 67.08
   Cranium  maxilla - i.o.  0.00  0.00  1.69  2.33  0.00
   Cranium  premaxilla - i.o.  0.00  0.00  0.56  0.00  1.24
   Cranium  nasal bone - i.o.  5.33  9.36 13.56  18.02  14.29
   Cranium  palatinum - i.o.  6.67  8.77  12.43  12.21 15.53
   Cranium  parietal bone - i.o.  42.00  45.61  48.59  55.23  48.45
   Cranium  supraoccipital bone - i.o.  48.00  46.78  51.98  59.30  62.11
   Cranium  basisphenoid - u.a. 1.33  2.92  1.69 4.65  9.32
   Cranium  basisphenoid - hole 0.67   2.92  10.73  10.47 7.45
   Cranium  arcus zygomaticus -i.o. 16.00 19.88  23.16  27.33  28.57
   Cranium

  arcus zygomaticus - misshapen

 0.67  0.58  0.00  0.00  0.62
   Cranium  arcus zygomaticus - small  0.00  0.00  1.13  0.00  0.00
   Cranium  supraoccipital bone - bipartite ossification 0.67  0.00  1.69 5.81   0.00
 Sternum  misaligned sternebra 0.67 1.75  3.39 2.91  3.11
  Sternum  sternebra - unossified  80.00  74.85  76.27 83.72  79.50
  Vertebrae  vertebrae lumbar - i.o  0.00  0.58  0.56  0.00  0.00
 Vertebrae  vertebrae sacral - i.o.  8.00  11.11  13.56  7.56  21.12
 Ribs  supernumerary  0.00  4.09  0.56  0.00  3.11
 Ribs  wavy  11.33  3.51  3.39  11.63  3.73
 Ribs  bent  0.00  0.00  0.00  0.00  0.00

    clavicula

 0.00 0.00   0.00 0.00  0.00

    scapula

 0.00    0.00   0.00   0.00    0.00

    pelvis

  0.00    0.00    0.00    0.00    0.00

    * limbs - humerus - misshapen

   0.00  1.17  1.69    0.00    0.00

Note: The value statistically significant on probability level 0.05 is shaded.

                   Skeletal alterations (number of litters with affected foetuses)
                  Group Code
    Alteration  C- I.  C - II.  250  500  1000
    Number of examined litters  21  23  21  22  20
  Cranium  frontal bone - i.o.  8  6  9  11  12
   Cranium  interparietal bone - i.o.  19 20  20  19  19
   Cranium  maxilla - i.o.  0  0  2  1  0
   Cranium  premaxilla - i.o.  0  0  1  0  1
   Cranium  nasal bone - i.o.  2  3  4 5  4
   Cranium  palatinum - i.o.  2 6 9 8  4
   Cranium  parietal bone - i.o.  16  18  18  18  17
   Cranium  supraoccipital bone - i.o.  16  22  19  19  17
   Cranium  basisphenoid - u.a.  1  4  3 5  6
   Cranium  basisphenoid - hole  1  5  12  12  9
   Cranium  arcus zygomaticus -i.o.  8  9  10  10  10
   Cranium

  arcus zygomaticus - misshapen

 1  1  0  0  1
   Cranium  arcus zygomaticus - small  0  0  2  0  0
   Cranium  supraoccipital bone - bipartite ossification  1  0  1  0
 Sternum  misaligned sternebra  1  2  3  4  4
  Sternum  ossification sites - decreased number  19  22  20  22  18
  Vertebrae  vertebrae lumbar - i.o  0  1  1  0  0
 Vertebrae  vertebrae sacral - i.o.  2  4  3 2  4
 Ribs  supernumerary  0  1  1  0  3
 Ribs  wavy  7  3  5  6  4
 Ribs  bent  0  0  0  0  0

    clavicula

 0  0  0

    scapula

 0  0  0  0  0

    pelvis

 0  0  0  0  0

    * limbs - humerus - misshapen

 0  2  3  0  0

Skeletal alterations (%age of litters with affected foetuses)
                  Group Code
    Alteration  C - I.  C - II.  250  500  1000
    Number of examined litters 21  23  21 22  20
  Cranium  frontal bone - i.o.  38.10 26.09  42.86 50.00 60.00
   Cranium  interparietal bone - i.o.  90.48  86.96 95.24  86.36 95.00
   Cranium  maxilla - i.o.  0.00  0.00  9.52  4.54  0.00
   Cranium  premaxilla - i.o.  0.00  0.00  4.76  0.00  5.00
   Cranium  nasal bone - i.o.  9.52  13.04 19.05  22.73  20.00
   Cranium  palatinum - i.o.  9.52  26.09  42.86  36.36 20.00
   Cranium  parietal bone - i.o.  76.19  78.26  85.71 81.82  85.00
   Cranium  supraoccipital bone - i.o.  76.19  95.65  90.48  86.36 85.00
   Cranium  basisphenoid - u.a. 4.76  17.39  14.29 22.73  30.00
   Cranium  basisphenoid - hole 4.76  21.74  57.14  54.54 45.00
   Cranium  arcus zygomaticus -i.o. 38.10 39.13  47.62 45.45  50.00
   Cranium

  arcus zygomaticus - misshapen

4.76 4.35  0.00  0.00  5.00
   Cranium  arcus zygomaticus - small  0.00  0.00  9.52  0.00  0.00
   Cranium  supraoccipital bone - bipartite ossification 4.76  0.00  4.76 18.18  0.00
 Sternum  misaligned sternebra 4.76 8.70 14.29 18.18  20.00
  Sternum  sternebra - unossified  90.48  95.65  95.24 100.00  90.00
  Vertebrae  vertebrae lumbar - i.o  0.00  4.35  4.76  0.00  0.00
 Vertebrae  vertebrae sacral - i.o. 9.52  17.39  14.29  9.09 20.00
 Ribs  supernumerary  0.00  4.35  4.76  0.00 15.00
 Ribs  wavy  33.33  13.04 23.81  22.27 20.00
 Ribs  bent  0.00  0.00  0.00  0.00  0.00

    clavicula

 0.00 0.00   0.00 0.00  0.00

    scapula

 0.00    0.00   0.00   0.00    0.00

    pelvis

  0.00    0.00    0.00    0.00    0.00

    limbs - humerus - misshapen

   0.00  8.70  14.29    0.00    0.00

Note: The value statistically significant on probability level 0.05 is shaded.

Summary of foetal malformations

External malformations and skeletal malfonnations were observed very sporadically without relation to the test substance treatment. Malformations of soft tissues were not detected in foetuses of any dose level.

Malformations - Summary

 

             Group code

 0 - I.

 0 - II.

 250

 500

 1000

Total number of examined foetuses -

external alterations (incl./excl. dead

foetuses)

290/289

  326/324

 332/332

327/327

  306/305

 Total number of examined foetuses - skeletal alterations

 150

  171

  177

 172

 161

Total number of examined litters

21

 23

21

22

 20

Microcephaly (small head), anasarca

(generalized subcutaneous oedema), cardiomegaly

0

1*

 0

 0

0

Short tail, discolored skin (hyperaemia of skin),

small placenta (reduced)

0

0

0

 1

0

Misshapen arcus zygomaticus

 1

 

 1

 

 0

 

 0

  

 1

Misshapen humerus

0

 2

3

 0

0

 Total number of foetuses/litters

with mal formation (incl. dead foetuses)

1/1

4/4

3/3

1/1

1/1 

Total number of foetuses/litters

with malformation (excl. dead foetuses)

 1/1

  3/3

 3/3

  1/1

  1/1

 Portion of foetuses with malformation

(excl. dead foetuses) - % in group

 0.35

 0.93

 0.90

 0.31

 0.33

 Portion of litters with malformation

(excl. dead foetuses) - % in group

 4.76

 13.04

 14.29

 4.55

 5.00

Note: * -dead foetus

Findings classified as malfonnat ions are in bold print.

Discussion:

The oral administration of the test substance, Gelbpigment E4GN to pregnant females by gavage from the 5th to the 19th day of pregnancy at the dose levels 1000, 500 and 250 mg/kg/day did not cause mortality of pregnant females.

The test substance had no negative effect on 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 mothers was relatively balanced with controls except the increase in the last measured interval at the dose levels 250 and 1000 mg/kg/day. It is probably related to a higher number of foetuses (litter size) in mothers of these dose levels. The difference was not dependent on dose level therefore it was not considered to be of toxicological significance. Evaluation of uterus weights did not demonstrate a negative effect of the test substance treatment too.

Clinical examinations of treated mothers detected no clinical symptoms of toxicity related to the test substance. The behaviour, health condition and clinical status of treated maternal animals were similar compared to controls and no serious changes were found. Only a finding related to the yellow colour of the test substance (i.e. yellow colour of faeces) was observed in females of the middle and the highest dose level. Also pathological examination of females revealed only non-pathological findings related to the colour of the test substance: test substance-coloured chymus in stomach and intestines. The organs of the digestive system were without pathological findings. Other macroscopic changes were observed sporadically without relation to the test substance treatment.

The number of females with live foetuses was well-balanced in control and treated groups and the gestation rate was not affected by treatment. The reproductive parameters – number of live and dead foetuses, intra uterine death early and late (i.e. pre- and postimplantation loss) were evaluated following the examination of uterus content. The number of corpora lutea and implantations was incidentally slightly higher in dosed groups compared to control groups. Preimplantation loss was incidentally slightly increased in both control groups. The postimplantation loss was not affected by the test substance treatment: administration of the test substance did not cause mortality of foetuses and the number of live foetuses in treated litters was even slightly higher than in control litters. On the contrary the postimplantation loss was significantly decreased at the lowest dose level. The sex ratio in treated litters was well balanced with control.

The influence of the test substance on the development of the conceptus in uterus was assessed according to the results of weighing, careful necropsy and skeletal examination of foetuses. 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 decreased at the lowest and the middle dose levels. At the highest dose level the foetal body weight was similiar to control although the number of foetuses was higher. The test substance treatment did not evoke occurrence of external and visceral variations and malformations. Skeletal malformations were detected very sporadically without relation to the test substance treatment. The incidence of misshapen humerus was significantly increased only at the lowest dose level compared to control I but the occurrence was comparable to control II. Test substance treatment relation was not evident for this finding.

Increased incidence of delayed ossification of some cranial bones (frontal bone, interparietal bone, supraoccipital bone, arcus zygomaticus) was observed at the middle and the highest dose level.

The incidence of delayed ossification of nasal bone was increased at all dose levels compared to control I but it was not dependent on dose level. Compared to control group I higher incidence of incomplete ossification was also reported in control group II, but no statistical comparison between control group I and control group II or control group II and dose groups was performed. The higher incidence correlated with higher litter size in treated groups and the finding was observed mainly in foetusses with low foetal body weight (less than 2.5 g). The group 1000 mg/kg/day showed a significant increase of incomplete ossification of sacral vertebra compared to Control I and II. But this delay occurred only in litters with low foetal body weight too.

The above mentioned significant differences of ossification (excl. changes observed mostly in “small” foetuses) were qualitatively compared with results of other simultaneous developmental studies: the slight delay of ossification of supraoccipital bone and arcus zygomaticus was still evident. Though these changes were not considered to be adverse due to their reversibility in postnatal life. The degree of ossification of sternum, other vertebra, ribs, clavicle, scapula, pelvis and limbs was not changed in foetuses of treated groups compared to control groups. Structural skeletal variations (misaligned sternebra, wavy ribs) were detected sporadically without treatment relation. At the dose level 250 and 1000 mg/kg the incidence of wavy ribs was significantly reduced compared to Control I. Supernumerary ribs were observed more frequently in foetuses of the lowest and the highest dose level compared to control I but comparable to the incidence in control II so that findings are considered to be spontaneous due to sporadic occurrence and comparability with controls. Incidence of findings of basisphenoid bone (unossified area/hole) was slightly increased in foetuses of treated groups, however without statistical significance and/or without dose relationship so that a substance related effects is not assumed.

Applicant's summary and conclusion

Conclusions:
The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 1000 mg/kg/day. All changes observed in females were considered to be of no toxicological significance.The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established at 1000 mg/kg/day. The ossification of cranial bones in foetuses of the dose levels 500 and 1000 mg/kg/day was slightly delayed. But due to absence of external and visceral malformations and other adverse developmental effects and due to high variability in litter size and decreased foetal weight in these foetuses the delay of ossification was not considered as adverse.
Executive summary:

Study performance

Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with untreated males. The test substance was then administered to pregnant females by gavage - daily from the 5th to the 19th day of pregnancy. The study included five groups of females - 3 treated groups and 2 control groups (vehicle only). The dose levels for the main study - 250, 500 and 1000 mg/kg/day - have been chosen based on the results of a dose-range finding experiment.

The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during the study. On the 20th day of pregnancy the maternal animals were euthanized, the uterine contents were examined and the foetuses were evaluated for external, soft tissue and skeletal alterations.

Results

Maternal animals and reproduction parameters:

There were no unscheduled deaths of females during the study at any dose level. No adverse changes of health condition and no clinical symptoms of intoxication were found in females at any dose level after administration of the test substance. In females of the dose levels 500 and 1000 mg/kg/day a finding related to the colour of the test substance (yellow colour of faeces) was observed. During the control of body weight increments and food consumption in pregnant females at all dose levels: 250, 500 and 1000 mg/kg/day toxicologically significant treatment-related effects were not detected. Evaluation of uterine weights (incl. absolute and relative weight of uterus) did not reveal toxicologically significant treatment-related effects. Macroscopical structure of organs of pregnant females and values of reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses), were unaffected by treatment with the test substance.

Foetuses:

Test substance-related foetal mortality was not evident at any dose level. The foetal body weight was minimally decreased at the dose levels 250 and 500 mg/kg/day (without statistical significance and in relation to incidentally higher litter size). Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.

Structural skeletal variations and skeletal malformations were detected only sporadically without relation to the test substance treatment. Statistically significant increase in incomplete (delayed) ossification was detected for some cranial bones at the dose levels 500 and 1000 mg/kg/day though generally without dose relation. The ossification of sacral vertebra was significantly delayed at the dose level 1000 mg/kg/day (but associated with low foetal body weight). The ossification of other vertebra, ribs. clavicle, scapula. pelvis and limbs was not delayed in foetuses of the dose levels 250, 500 and 1000 mg/kg/day.

Conclusion

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 1000 mg/kg/day. All changes observed in females were considered to be of no toxicological significance.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established at 1000 mg/kg/day. The ossification of cranial bones in foetuses of the dose levels 500 and 1000 mg/kg/day was slightly delayed. But due to absence of external and visceral malformations and other adverse developmental effects and due to high variability in litter size and decreased foetal weight in these foetuses the delay of ossification was not considered as adverse.