Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
Based on ECHA decision number: TPE-D-2114453637-41-01/F
Type of information:
experimental study
Remarks:
A pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: OECD TG 414) in a first species (rat or rabbit), oral route using the registered substance was conducted based on ECHA decision number: TPE-D-2114453637-41-01/F.
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Pregnant rats were treated with the test item starting from the 6th and lasting until the 20th day of pregnancy (the 'critical' phase of organogenesis and the fetal development). Their development was observed during the gestation period (day of mating is day 0 of pregnancy). On gestation day 21, the dams were laparotomised and examined for corpora lutea, implantation sites and resorptions in the uterus and for the condition of the fetuses.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
black powder
Details on test material:
Nigrosin WLF content: 85.4%
No correction factor was used.
Specific details on test material used for the study:
The dose levels refer to the supplied test item, no correction factor was used.

Test animals

Species:
rat
Strain:
other: CD/Crl:CD(SD)
Details on test animals or test system and environmental conditions:
Animals
For this study mature female CD® rats, a strain bred by Charles River Laboratories Research Models and Services Germany GmbH, were used. The animals were held for at least 5 days for adaptation.
Health checks were performed on the day of delivery and at the beginning of matings. Each animal was given a thorough examination. Animals were judged to be healthy on the basis of general observations (physical examination). No replacements due to illness had to be carried out.
Species Rat
Strain / Stock CD® / Crl:CD(SD)
Breeder Charles River Laboratories,
Adaptation period 5 days
Age
(on day 0 of gestation) 8 - 9 weeks
Body weight
(on day 0 of gestation) 188.7 - 271.4 g
Selection of species The rat is a commonly used rodent species for such embryotoxicity studies.
Animal identification Each rat received a continuous number. Additionally the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception and dates of administration.
Number of animals 100 female mated animals
Treated animals:
Groups 1 - 4: 25 females per group
Evaluated dams and litters:
Groups 1 - 4: 20 dams per group

Diet, housing and drinking water
Diet
Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered daily ad libitum. Food residue was removed and weighed.
Certificates of analysis of the composition and for contaminants were provided by the manufacturer and were included in the raw data.
The phytoestrogen content of the diet used was below 350 µg genistein equivalents/g diet.

Housing
The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm at a room temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55% ± 10% (maximum range). Deviations from the maximum range caused for example during cleaning procedures were dealt with in SOPs.
The rooms were alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) was used as bedding material in these cages. The cages were cleaned and changed once a week.
The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.

Drinking water
Tap water was offered in 500 mL drinking water bottles (MAKROLON) ad libitum.
Samples of drinking water were taken periodically by the Wasserwerk Wankendorf (24601 Wankendorf, Germany), and periodic analyses were performed.



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Administration
Route of administration Oral, via gavage
Frequency of administration Once daily
Treatment period Day 6 to 20 of gestation
Vehicle Tap water
Administration volume 10 mL/kg b.w./day
Selection of route of administration According to OECD guideline 414
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item preparation
The test item formulations were freshly prepared on each day of dosing.
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg bw once daily from the 6th to the 20th day of gestation. The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the homogeneity and concentration of the test item mixture were monitored.
The male rats for mating remained untreated.
Details on mating procedure:
Sexually mature ('proven') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until 25 mated dams were available for each group.
The non-pregnant rats were excluded from the analysis of the results and replaced by other pregnant animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
Duration of study
- 5 adaptation days
- 1 mating day
- 15 administration days from gestation days 6 to 20
- laparotomy on gestation day 21
Frequency of treatment:
Daily.
Duration of test:
15 administration days from gestation days 6 to 20.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Number of pregnant rats to be evaluated: 20 f
Dose / conc.:
250 mg/kg bw/day
Remarks:
Low dose
Number of pregnant rats to be evaluated: 20 f
Dose / conc.:
500 mg/kg bw/day
Remarks:
Intermediate dose
Number of pregnant rats to be evaluated: 20 f
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
Number of pregnant rats to be evaluated: 20 f
No. of animals per sex per dose:
25 female animals per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
Group size / Dose levels
Four (4) groups of pregnant rats were established, each obtained from matings which were carried out on a daily basis. Vaginal smears were taken each morning. When positive, the animals were assigned to the test groups by mating day using a randomisation scheme based on the body weight of the animals.
Day 0 of pregnancy was the day on which sperm was found in the vaginal smear.

Justification for dose selection
The dose levels were selected based on available toxicological data derived from a dose-range-finding study.
In this dose-range-finding for a prenatal developmental toxicity study, the test item was administered orally to female rats at dose levels of 250, 500 or 1000 mg/kg b.w./day from the 6th to 20th day of pregnancy.
No prematurely deceased dams were noted during the study. No changes in behaviour or external appearance were noted. The dark discoloured faeces of the animals of the dose groups were considered to be due to the black colour of the administered test item. No test item-related changes were noted for the body weight, the body weight gain or the food consumption.
Under the present test conditions, no toxicologically relevant observation was seen in any dose group including 1000 mg/kg b.w./day for the dams.

Examinations

Maternal examinations:
Clinical signs
Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness, or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Special attention was paid to ascertain if there are any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity etc.

Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion or premature delivery occurred in the study.

Body weight
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 - 21). Furthermore, the carcass weight and the net weight gain from day 6 was given.

Carcass weight = Terminal body weight-gravid uterine weight

Net weight change from day 6 = Carcass weight-body weight on day 6

These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

Food and drinking water consumption
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of gestation day 6 to 21.
The relative food consumption (g/kg b.w./day) was calculated using the following formula:

Daily food consumption [g/kg b.w./day] = (Total food intake [g])/(Body weight [kg] )

Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

Thyroid hormone (T3, T4, TSH) determination
In order to obtain approximately 2 x 150 µL serum for each endocrine endpoint (T3, T4, TSH), a sufficient volume of blood was taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight following a randomisation scheme. Blood samples were taken always at the same time of day (approximately from 7:00 a.m. to 10:00 a.m.).
Sampling schedule for thyroid hormone.
Sampling time Sampled animals Number of samples/endpoint
Before sacrifice All evaluated dams 80

The samples were divided into aliquots, if possible, and stored at 20°C ± 10% at LPT until analysis using ELISA.
The T3, T4 and TSH ELISA (commercial kits: IBL International GmbH , instrument: Tecan Sunrise ) was conducted at LPT.

Necropsy
Examination of the dams
On the 21st day of gestation the rats were laparotomised under CO2 narcosis. The ovaries, thyroids and the uteri were removed.
Organs weighed.: Thyroid (2) (including parathyroids), Gravid uterus including cervix.

In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of sacrifice. The thyroids and any organs with macroscopic findings of all dams were fixed in 7% buffered formalin for histopathological examination.
Organs preserved: Thyroid (2) (including parathyroids), Gross lesions.






Ovaries and uterine content:
Evaluation / Parameters
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam, % per litter
- distribution in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions < 2 mm, number and % per litter
- late resorptions > 2 mm, number and % per litter
- % litters with resorption per group

Weight of placentae
- individual data per fetus
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group

Fetuses
- total number of litters
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive + dead) per sex and dam
- male/female ratio (alive + dead) per group
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- mean % per sex and group

Runts
- number per dam
- number per group
Fetal examinations:
Examination of the fetuses
The fetuses were removed and the following examinations were carried out:
(a) Macroscopic inspection (gross evaluation) of the placentae, e.g. for focal indurations.
(b) Number of fetuses (dead and alive) and number of placentae.
(c) Determination of sex and viability of fetuses (spontaneous movement, spontaneous breathing).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus are determined.
(f) Weight of fetuses and placentae were determined (fetuses are considered as runts if their weight was less than 70% of the mean litter weight).
(g) The ano-genital distance (AGD) of all live fetuses was determined by using a scale.
(h) Fetuses were inspected externally for damages, especially for malformations.
(i) The fetuses were sacrificed in an ether atmosphere.
(j) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 50 per cent of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and the peritoneal cavity (without damage to ribs and sternum) were opened and the location, size, and condition of the internal organs are determined. The skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
2) The remaining 50 per cent of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.
(k) External fetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue malformations).
(l) Indication of incomplete testicular descent/cryptorchidism was noted in male fetuses.

The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.

Dead fetuses
- number per group
- mean per dam

Malformed fetuses
- type of malformation
- individual data per fetus
- number and incidence (%) per group and litter
Total malformation rate [%] = (Malformed fetuses per group)/(Fetuses per group)*100

Fetuses with variations
- type of variation
- individual data per fetus
- number and incidence (%) per group9 and litter
Total variation rate [%] = (Fetuses per group with variations)/(Fetuses per group)*100

Fetuses with retardations
- type of retardation
- individual data per fetus
- number and incidence (%) per group9 and litter
Total retardation rate [%]=(Fetuses per group with retardations)/(Fetuses per group)*100


Statistics:
Parametrical data
The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.0.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Significantly different data are indicated in the summary tables of the result sections of the report.

Non-parametrical data
The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi2 test with the StatXact 4.0.1 software:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Indices:
Indices of pre-implantation loss and post-implantation loss:
Calculation of group indices
Pre implantation loss [%]=(Corpora lutea (per group)-implantations (per group))/(Corpora lutea (per group))*100

Post implantation loss [%]=(Implantations (per group)-living fetuses (per group))/(Implantation (per group))*100

Calculation of mean indices per litter
Pre implantation loss [%]=(Sum of pre-implantation losses per litter in a group [%])/(Number of litters in a group)*100
Post implantation loss [%]=(Sum of post-implantation losses per litter in a group [%])/(Number of litters in a group)*100

# Dams with total loss of implantation sites are considered with a post-implantation loss of 100% in the mean value.
Historical control data:
No data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour or the external appearance were noted for the control dams or the dams treated orally with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day. None of the animals died prematurely.
However, for all dose groups, dark discoloured faeces were noted from GD 7 until study termination on GD 21. The dark discoloration of the faeces was considered to be due to the black colour of the administered test item and therefore, was considered to be not of toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related differences in body weight were noted between the dams of the control group and the dose groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences in food consumption were noted between the female animals of the control group and the female animals of the treatment groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day by oral administration) during the gestation period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weights
No test item-related differences were noted between the control group and the female animals dosed with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day.

Gravid uterus weight and carcass weight
No test item-related changes in the gravid uterus weight and the carcass weight (terminal body weight minus gravid uterine weight) in comparison to the control group were noted for the dams of the treatment groups (oral treatment with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day).

Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were noted for the female animals of the treatment groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day p.o.).
Necropsy of female no. 8 (control group) revealed a bilateral dilation of renal pelvis (left side: 5 mm, right side: 8 mm). This change is considered to be a spontaneous finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology (thyroid only)
Histopathologic examination of the thyroids revealed no test item-related changes.
The ultimobranchial remnants and the microfollicles observed in the thyroids are considered to be coincidential findings within the normal range of variations.

Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone concentration
In the dose groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day), no test item-related differences were noted for the concentrations of T3, T4 and TSH.
However, at 1000 mg Nigrosin WLF/kg b.w./day, a marginal but statistically significant increase was noted for the concentration of the thyroid hormone T3 (12.8% above the value of the control group, statistically significant at p ≤ 0.05). However, this small increase is considered to be spontaneous. There was no correlation between the histopathologic observation of an increased incidence of squamous cell cysts and an increase in the T3 levels. Furthermore, no differences were noted for the thyroid weights.
The severely individual varying serum concentrations of TSH were considered to be spontaneous and not test item-related.
It is known that histopathological examination of the thyroids is usually more sensitive than hormone levels (Beekhuijzen et al., 2016). The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weight was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormone values.”

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Parameter Group 1 Group 2 Group 3 Group 4
Pre-implantation loss per group 1.0 2.5 2.5 1.6
[%] mean per dam 0.9 2.1 2.4 2.0
Post-implantation loss per group 2.3 1.9 2.9 4.0
[%] mean per dam 2.3 1.8 3.3 4.3
Description (incidence and severity):
Parameter Group 1 Group 2 Group 3 Group 4
Total resorptions
total 7 6 9 12
mean per dam 0.4 0.3 0.5 0.6
Early or late resorptions:
no effects observed
Description (incidence and severity):
Parameter Group 1 Group 2 Group 3 Group 4
Early resorptions
total 5 4 5 8
mean per dam 0.3 0.2 0.3 0.4
Late resorptions
total 2 2 4 4
mean per dam 0.1 0.1 0.2 0.2
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group or in the treatment groups (oral treatment with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day).
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Findings
Examination of the dams
Mortality No premature deaths were noted.
Clinical signs No changes in behaviour or external appearance were noted for the treatment groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day).
Dark discoloured faeces were noted for all animals in the dose groups. However, this was considered to be due to the black colour of the test item and not to be of toxicological relevance.
Body weight and
body weight gain No test item-related influence was noted.
Food and drinking water
consumption No test item-related influence was noted.
The visual appraisal of the drinking water consumption revealed no differences between the control and the test item-treated animals.
Necropsy findings No test item-related changes were noted during the macroscopic inspection of the dams at necropsy.
Uterus and carcass
weights No test item-related influence was noted for the uterus and carcass weights.
Thyroid hormone
concentrations No test item-related changes were noted for the concentrations of the thyroid hormones T3, T4 and TSH.
Necropsy findings No test item-related changes were noted during the macroscopic inspection of the dams at necropsy.
Histopathology Microscopical evaluation of the thyroids revealed increased numbers of squamous cell cysts in the thyroids of the animals treated with the test item Nigrosin WLF compared to the control group.
Thyroid weighs No test item-related differences for the left and right thyroid weights were noted.
Reproduction data No test item-related influence was noted on the reproductive parameters (number of implantation sites, resorptions and fetuses).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
No test item-related differences for the fetal weights were noted in the dose groups (oral administration of the dams with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day) in comparison to the control group.
No test item-related difference in the occurrence of runts was noted between the control group and the dose groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences between the ratio of male and female fetuses were noted between the control group and the test item group (oral treatment with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day). The range is within the biological variability and the differences were without any toxicological relevance.
Group 1: 0.84
Group 2: 0.84
Group 3: 1.00
Group 4: 1.30

Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No test item-related macroscopically visible malformations or variations were noted for the fetuses of the test item group (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day) during the external fetal inspection at laparotomy.
One fetus of a high dose dam revealed multiple malformations of the head in the form of prosboscis (approx. 4 mm) accompanied by absent eyes, brachygnathia (small lower jaw), cleft palate and lip. These alterations are considered to be of spontaneous nature due to the single incidence of one affected fetus.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day) during skeletal examinations of the fetuses according to DAWSON.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test item-related malformations were noted for the fetuses of the control group and the fetuses of the test item groups (250, 500 or 1000 mg Nigrosin WLF/kg b.w./day) during the soft tissue examination according to WILSON.
Other effects:
no effects observed
Description (incidence and severity):
ABSTRACT and ASSESSMENT of fetal alterations
No dead fetuses and no test item-related increases in the incidence of runts were noted at any tested dose level.
No test item-related malformations or variations were noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON for the test item groups (oral treatment of the dams with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day). Furthermore, the skeletal examination according to DAWSON revealed no test item-related retardations (delay in ossification).
Details on embryotoxic / teratogenic effects:
No test item-related influence on the reproductive parameters (number of im-plantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) was noted for the dams of the test item group (oral treatment with 250, 500 or 1000 mg Nigrosin WLF/kg b.w./day). Nigrosin WLF did not show any teratogenic potential.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remarks'

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Examination of the fetuses

Mortality                     No dead fetuses were noted.

Weight of fetuses

and placentae              No test item-related differences were noted.

Fetal development       No test item-related differences between the control group and the dose groups were noted for the sex distribution, number of runts, ano-genital

distance and the testicular development.

Fetal alterations

Malformations           No test item-related malformation was noted during the external examination at laparotomy, the gross inspection of the internal organs, the skeletal

examination according to DAWSON and the soft tissue examination according to WILSON.

Variations                  The external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft

tissue examination according to WILSON revealed no test item-related variations.

Retardations              No test item-related retardations (delays in ossification) were noted during the skeletal examination according to DAWSON.

Maternal Body Weights - Summary

Sex: Female   

Day(s) Relative to Mating (Litter: A)

 

 

 

 0

1

2

 3

 4

 5

 6

 Gr.1:

control

Mean

SD

N

230.40

17.87

20

238.16

15.12

20

247.71

15.60

20

253.29

17.65

20

257.52

17.99

20 

264.23 

18.48

20

268.32 

18.28

20

 Gr.2:

250 mg/kg

Mean

SD

N

% Diff

233.08

15.65

20

1.2

239.66

15.14

20

0.6

247.22

17.46

20

-0.2

254.56

16.04

20

0.5

258.52

16.61

20

0.4

263.75 

15.75

20

-0.2

267.94 

16.85

20

-0.1

Gr.3:

500 mg/kg

Mean

SD

N

 % Diff 

232.33

15.64

20

0.8

240.77

15.97

20

1.1

248.94

15.84

20

0.5

254.12

15.59

20

0.3

259.26 

16.53

20

0.7

264.03

16.51

20

-0.1

268.35 

17.00

20

0.0

  

Gr.4:

1000 mg/kg

Mean

SD

N

 % Diff 

232.49

19.36

20

0.9

239.21

19.47

20

0.4

246.66

19.14

20

-0.4

252.58

19.14

20

-0.3

256.61 

16.93

20

-0.4

262.56

19.67

20

-0.6

266.75 

17.84

20

-0.6

Sex: Female   

Day(s) Relative to Mating (Litter: A)

 

 

 

 7

8

9

10

 11

 12

 13

 Gr.1:

control

Mean

SD

N

274.06

17.77

20

276.84

20.01

20

282.57

20.46

20

290.22

19.97

20

297.26

19.95

20 

301.91 

19.18

20

306.40 

19.19

20

 Gr.2:

250 mg/kg

Mean

SD

N

% Diff

272.94

16.38

20

-0.4

276.29

16.85

20

-0.2

281.29

15.07

20

-0.5

288.29

17.02

20

-0.7

294.87

18.35

20

-0.8

297.98

17.93

20

-1.3

302.27

17.79

20

-1.3

Gr.3:

500 mg/kg

Mean

SD

N

 % Diff 

271.59

15.12

20

-0.9

276.56

14.78

20

-0.1

282.52

14.25

20

0.0

290.08

15.23

20

0.0

295.24

15.95

20

-0.7

301.11

15.59

20

-0.3

305.07 

16.95

20

-0.4

  

Gr.4:

1000 mg/kg

Mean

SD

N

 % Diff 

270.52

18.93

20

-1.3

274.88

18.74

20

-0.7

280.81

18.75

20

-0.6

286.79

20.10

20

-1.2

291.68 

19.79

20

-1.9

295.44

19.94

20

-2.1

302.88 

20.83

20

-1.1

Sex: Female   

Day(s) Relative to Mating (Litter: A)

 

 

 

 

 14[a]

15[a1]

16[a1]

17[a1]

 18[a1]

 19[a]

 20[a]

 21

 Gr.1:

control

Mean

SD

N

310.11

23.63

20

316.86

26.65

20

326.44

31.69

20

338.65

35.63

20

355.74

30.38

20 

372.82

29.44

20

385.11

30.06

20

400.78

29.76

20

 Gr.2:

250 mg/kg

Mean

SD

N

% Diff

307.25

18.16

20

-0.9

316.02

18.47

20

-0.3

326.25

17.71

20

-0.1

341.20

17.34

20

0.8

354.82

18.62

20

-0.3

370.56

19.73

20

-0.6

383.03

22.93

20

-0.5

399.87

24.79

20

-0.2

Gr.3:

500 mg/kg

Mean

SD

N

 % Diff 

310.29

16.56

20

0.1

318.46

16.78

20

0.5

327.47

17.99

20

0.3

341.10

18.40

20

0.7

354.60

20.22

20

-0.3

369.87

21.84

20

-0.8

385.05 

22.47

20

0.0

399.27

24.84

20

-0.4

  

Gr.4:

1000 mg/kg

Mean

SD

N

 % Diff 

306.86

20.30

20

-1.0

315.48

20.58

20

-0.4

325.26

21.83

20

-0.4

338.17

21.94

20

-0.1

353.01 

19.64

20

-0.8

369.20

23.68

20

-1.0

382.80 

23.61

20

-0.6

398.61 

26.54

20

-0.5

[a] - Anova & Dunnett

[a1] - Anova & Dunnett(Rank)

Maternal Body Weight Gain - Summary ( Day(s) Relative to Mating (Litter: A))

 Sex: Female   

Period

(days)

(g)

Period

(days)

(g)

Period

(days)

(%)

 Period

(days)

(%)

 

 

 6 - 21

0 - 21 

6 - 21

 0 - 21

 Gr.1:

control

Mean

SD

N

132.46

18.81

20

170.38

21.90

20

49.44

6.80

20

74.27

10.27

20

 Gr.2:

250 mg/kg

Mean

SD

N

 % Diff

131.94

18.37

20

-0.4

166.80

21.65

20

-2.1

49.43

7.51

20

-

 71.97

11.28

20

-

Gr.3:

500 mg/kg

Mean

SD

N

 % Diff 

130.92

19.51

20

-1.2

166.94

19.49

20

-2.0

48.99

7.95

20

-

72.16

9.56

20

-

  

Gr.4:

1000 mg/kg

Mean

SD

N

 % Diff 

131.87

12.30

20

-0.4

166.12

12.54

20

-2.5

49.49

4.02

20

-

71.78

6.63

20

-

Gravid Uterine Weight, Carcass Weight and Net Body Weight Gain (Day(s): 21 Relative to Mating (Litter: A)

 Sex: Female   

Gravid

Uterus

(g)

[a]

Carcass

(g)

[a1]

Net Gain

from GD 6

(g)

[a] 

Gr.1:

control

Mean

SD

N

105.69

10.28

20

295.09

23.84

20

26.77

13.22

20

Gr.2:

250 mg/kg

Mean

SD

N

 % Diff 

107.99 

11.17

20

2.2

291.88

20.43

20

-1.1

23.95 

14.47

20

-

  

Gr.3:

500 mg/kg

Mean

SD

N

 % Diff 

104.63 

15.26

20

-1.0

294.64 

14.90

20

-0.2

26.30 

14.77

20

-

  

Gr.4:

1000 mg/kg

Mean

SD

N

 % Diff 

101.67 

16.48

20

-3.8

296.95 

28.09

20

0.6

30.20 

18.44

20

-

[a] - Anova & Dunnett(Rank)

[a1] - Anova & Dunnett

Thyroid Hormone Determination - Summary ( Day(s): GD21 Relative to Mating (Litter: A))

Sex: Female   

T4

concentrat.

(nmol/L)

[a]

T3

concentrat.

(ng/mL)

[a1]

TSH

concentrat.

(ng/mL)

[a1] 

Gr.1:

control

Mean

SD

N

19.5817

2.7097

20

1.5244

0.1685

20

1.0122

1.3841

20

 Gr.2:

250 mg/kg

Mean

SD

N

 % Diff 

19.1099 

4.1030

20

-2.4

1.5346

0.1565

20

0.7

0.7510

1.0412

20

-25.8

  

Gr.3:

500 mg/kg

Mean

SD

N

 % Diff 

18.3644 

3.8698

20

-6.2

1.6408

0.1542

20

7.6

1.5899

1.1455

20

57.1

  

Gr.4:

1000 mg/kg

Mean

SD

N

 % Diff 

18.9417 

4.7734

20

-3.3

1.7281**

0.3256

20

13.4

1.6777

1.2046

20

65.8

[a] - Anova & Dunnett(Log)

[a1] - Anova & Dunnett: ** = p ≤ 0.01

Maternal Macroscopic Findings at Necropsy, Placentae-Inspection - Summary -

 Removal Reason(s): ALL

Female

 
 

Gr.1:

control

Gr.2:

250

mg/kg

Gr.3:

500

mg/kg

Gr.4:

1000

mg/kg

 Number of Animals:

20 

 20

 20

 20

 KIDNEY, LEFT

Submitted

Normal

pelvis; Dilation

20

19

20

20

20

20

20

20

KIDNEY, RIGHT

Submitted

Normal

pelvis; Dilation

20

19

20

20

20

20

20

20

Chi-Squared & Chi-Squared - Pearson

The non-pregnant animal no. 81 of the high dose group was not considered.

Maternal Thyroid Weights - Summary

Sex: Female   

T

Thyroid/Par.

(left)

(g)

GD21

Gr.1:

control

Mean

SD

N

0.0103

0.0027

20

 Gr.2:

250 mg/kg

Mean

SD

N

 % Diff 

0.0099

0.0025

20

-4.4

  

Gr.3:

500 mg/kg

Mean

SD

N

 % Diff 

0.0098

0.0030

20

-4.9

  

Gr.4:

1000 mg/kg

Mean

SD

N

 % Diff 

0.0103

0.0024

20

0.0

Reproduction Data - Summary - Values per Group

Sex: Female Day(s)

Relative to Mating (Litter: A)

 

Gr.1:

control

 

Gr.2:

250 mg/kg

 

Gr.3:

500 mg/kg

 

Gr.4:

1000 mg/kg

 

 Evaluated dams with:

- viable fetuses

- only dead fetuses

- resorptions

- resorptions (%)

- complete resorption (NVF)

 

20

20

0

6

30.0

20

20

0

5

25.0

20

20

0

7

35.0

20

20

0

11

55.0

 Corpora Lutea per group

 

 310

 321

 315

 304

 Implantation sites per group

 

 307

 313

 307

 299

 No. of resorptions per group

 

 7

 6

 9

 12

 No. of early resorpt. per gr.

 

 5

 4

 5

 8

 No. of late resorpt. per gr.

 

 2

 2

 4

 4

 Evaluated dams with 1 Resorpt.

 Yes

 5

 4

 5

 10

 Evaluated dams with 2 Resorpt.

 Yes

 1

 1

 2

 1

 Evaluated dams with >2 Resorpt

 Yes

 0

 0

 0

 0

 Fetuses (alive + dead) / group

 

 300

 307

 298

 287

 M. fetuses (alive+dead) /group

 

 137

 140

 149

 162

 F. fetuses (alive+dead) /group

 

 163

 167

 149

 125

 M/F-Ratio (alive+dead) /group

 

 0.84

 0.84

 1.00

 1.30

 Fetuses (dead) per group

 

 0

 0

 0

 0

 Fetuses (alive) per group

 

 300

 307

 298

 287

 Pre-implant. Loss (%) /group

 

 1.0

 2.5

 2.5

 1.6

 Post-implant. Loss (%) /group

 

 2.3

 1.9

 2.9

 4.0

 Runts per Group

 

 1

 1

 0

 3

Non pregnant females (NP) are not considered in the calculation.

Results:

Pre-implantation loss: no statistically significant differences,

Post-implantation loss: no statistically significant differences.

Reproduction Data - Summary - Mean Values per Dam ( Day(s): 21 Relative to Mating (Litter: A))

 Sex: Female   

Gr.1:

control

Gr.2:

250 mg/kg

Gr.3:

500 mg/kg

Gr.4:

1000 mg/kg

 Considered

 20

 20

 20

 20

 Viab Fet Yes

 

 20

 20

 20

 20

 Viab Fet No

 

 0

 0

 0

 0

Corpora [a]

Lutea

Mean

SD

Sum

15.5 

2.0

310

16.1 

2.2

321

15.8

2.4

315

15.2

2.2

304

Implantation [a1]

sites

Mean

SD

Sum

15.4

1.9

307

15.7

1.9

313

15.4

2.2

307

15.0

2.6

299

Total Number [a1]

Resorptions

Mean

SD

Sum

0.4

0.6

7

0.3

0.6

0.5

0.7

0.6

0.6

12

Early [a1] 

Resorptions           

Mean

SD

Sum 

0.3

0.6

5

0.2

0.4

0.3

0.4

 0.4

0.5

8

Late [a1] 

Resorptions      

Mean

SD

Sum

0.1

0.3

2

0.1

0.3

 0.2

0.4

4

 0.2

0.4

4

Pre-implant. [a1]

loss (%)

Mean

SD

0.91

2.22

2.13

5.79

2.37

5.34

1.98

7.51

Post-implant. [a1]

loss (%)

Mean

SD

2.26 

3.72

 1.83

3.38

 3.27

5.71

 4.31

4.41

Fetuses [a1]

alive + dead

(Both)

Mean

SD

Sum 

15.0 

1.9

300

15.4 

1.8

307

 14.9

2.4

298

 14.4

2.8

287

Dead Fetuses [a1]

Mean

SD

Sum

0.0

0.0

0.0 n

0.0

0 n

0.0 n

0.0

0 n 

0.0 n

0.0

0 n 

Fetuses [a]

alive

(Both)

Mean

SD

Sum 

15.0 

1.9

300

 15.4

1.8

307

14.9 

2.4

298

14.4 

2.8

287

 Live fetuses

% of total

Mean

SD

100.00 

0.00

20

 100.00

0.00

20

100.00

0.00

20 

100.00

0.00

20 

[a] - Anova & Dunnett

[a1] - Anova & Dunnett(Rank): n - Inappropriate for statistics

Non pregnant females (NP) are not considered in the calculation.

Fetal and Placental Weights - Mean Values per Group ( Day(s): 21 Relative to Mating (Litter: A))

Sex: Female   

Gr.1:

control

Gr.2:

250 mg/kg

Gr.3:

500 mg/kg

Gr.4:

1000 mg/kg

Mean Fetal [a]

Weight (M)

(g)

Mean

SD

N

%Diff

5.28

0.32

20

5.34

0.27

20

1.1

5.28

0.28

20

-0.1

5.36

0.34

20

1.3

Mean Fetal [a]

Weight (F)

(g)

Mean

SD

N

%Diff

5.05

0.31

20

5.08

0.19

20

0.7

5.08

0.30

20

0.7

5.08

0.40

20

0.6

Mean Fetal [a]

Weight (both)

(g)

Mean

SD

N

%Diff

5.17

0.29

20

5.20

0.21

20

0.6

5.18

0.29

20

0.3

5.24

0.33

20

1.5

Mean Plac. [a1] 

Weight (M)

(g)

    

 Mean

SD

N

%Diff

0.505

0.054

20

0.507

0.059

20

0.5

0.499

0.107

20

-1.1

0.508

0.056

20

0.6

Mean Plac. [a1] 

Weight (F)

(g)

Mean

SD

N
%Diff

0.486

0.057

20

0.487

0.049

20

0.2

0.493

0.130

20

1.4

0.479

0.062

20

-1.5

Mean Plac. [a1] 

Wt (Both)

(g)

Mean

SD

N

%Diff

0.496

0.054

20

0.497

0.052

20

0.2

0.498

0.123

20

0.4

0.494

0.055

20

-0.3

[a] - Anova & Dunnett

[a1] - Anova & Dunnett(Rank)

Ano-genital Distance - Mean Values per Group ( Day(s): GD21 Relative to Mating (Litter: A))

Sex: Female   

Gr.1:

control

Gr.2:

250 mg/kg

Gr.3:

500 mg/kg

Gr.4:

1000 mg/kg

AnoGen [a]

Male

(Ocular units)

Mean

SD

N

%Diff

27.6

4.6

20

27.3

2.9

20

-2.0

26.5

1.8

20

-4.7

27.2

2.7

20

-2.2

AnoGen [a]

Female

(Ocular units)

Mean

SD

N

%Diff

14.5

2.5

20

14.2

1.5

20

-2.4

14.1

1.1

20

-2.7

14.0

1.8

20

-3.7

AnoGen [a]

Male

(Ocu. u./g bw)

Mean

SD

N

%Diff

16.00

2.61

20

15.61

1.46

20

-2.4

15.24

0.96

20

-4.7

15.55

1.29

20

-2.8

AnoGen [a]

Female

(Ocu. u./g bw)

    

 Mean

SD

N

%Diff

8.47

1.42

20

8.24

0.82

20

-2.7

8.23

0.72

20

-2.9

8.14

0.98

20

-3.8

[a] - Anova & Dunnett(Rank)

[a1] - Anova & Dunnett

Testicular Development of the Male Fetuses - Summary

 Exam Type: Testes Examination   

Gr.1:

control

Gr.2:

250 mg/kg

Gr.3:

500 mg/kg

Gr.4:

1000 mg/kg

Number of Litters Examined:

Number of Male Fetuses Examined (User defined): 

20

137 

20

140 

20

149 

20

162 

Position of testes

-, Normal

Fetuses N(%)

Litters N(%)

137(100.0)

20(100.0)

140(100.0) 

20(100.0)

149(100.0)

20(100.0)

162(100.0) 

20(100.0)

Cryptorchidism

-, Both testes present

Fetuses N(%)

Litters N(%

137(100.0)

20(100.0

140(100.0) 

20(100.0)

149(100.0)

20(100.0)

162(100.0) 

20(100.0)

[Fetuses N] - Chi-Squared & Chi-Squared - Pearson

Summary of All Classified and Unclass. Fetal External Observations - by Classification-Type -

Exam Type: All

Gr.1:

control

Gr.2:

250 mg/kg

Gr.3:

500 mg/kg

Gr.4:

1000 mg/kg

Dams with Viable Fetuses:

Total Number of Fetuses in Group:

Number of Fetuses Examined:

20

300

300

20

307

307 

20

298

298

20

287

287

All classifications

Number of Fetuses [f]

0

0

0

0

 Group % of Fetuses    0.0  0.0 0.0   0.3
 Number of Litters [f] Yes   0  0  0  1
   %  0.0  0.0 0.0   5.0
 Litter % of Fetuses  Mean  0.00  0.00 0.00   0.38
   Max  0.0  0.0  0.0  7.7
   Min  0.0  0.0  0.0  0.0
 Malformation          

Number of Fetuses [f]

 

 0

 0

 0

 1

  Group % of Fetuses

 

 0.0

 0.0

 0.0

 0.3

  Number of Litters [f]

 Yes

 0

 0

 0

 1

 

 %

 0

 0

 0

 5.0

  Litter % of Fetuses

 Mean

 0.00

 0.00

 0.00

 0.38

   Max 0.0  0.0  0.0  7.7
   Min 0.0  0.0  0.0  0.0

[f] - Chi-Squared & Chi-Squared - Pearson

Summary of All Classified and Unclass. Fetal External Observations - by Observation-Type -

Exam Type: External

Gr.1:

control

Gr.2:

250 mg/kg

Gr.3:

500 mg/kg

Gr.4:

1000 mg/kg

Dams with Viable Fetuses:

Total Number of Fetuses in Group:

Number of Fetuses Examined:

20

300

300

20

307

307 

20

298

298

20

287

287

Head/Neck

Head, Multiple Malformations -

Malformation

Fetuses N(%)

Litters N(%)

0(0.0)

0(0.0)

 0(0.0)

0(0.0)

 0(0.0)

0(0.0)

1(0.3)

1(5.0)

[Fetuses N] - Chi-Squared & Chi-Squared - Pearson

[Litters N] - Chi-Squared & Chi-Squared - Pearson

Summary Of All Classified And Unclassified Fetal Internal Observations - No. Fetuses/Group Day(s): 21 Relative to Mating (Litter: A)

Sex: Female 

No. Fetuses

(alive+dead)

Internal

Examination

With Intern

Malformation

 With Intern

Variation

 Gr.1:

control

 300

 150

 0

 0

 Gr.2:

250 mg/kg

 307

 154

 0

 0

 Gr.3:

500 mg/kg

 298

 149

 0

 0

 Gr.4:

1000 mg/kg

 287

 143

 0

 0

SUMMARY OF ALL CLASSIFIED FETAL SKELETAL OBSERVATIONS

 

 

 TEST GROUP 1

Control

TEST GROUP 2

250 mg/kg

TEST GROUP 3

500 mg/kg 

 TEST GROUP 2

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

20 

Fetuses Evaluated 

 N

 150

 154

 149

 144

 Live

 N

 150

 154

 149

 144

 Dead

 N

 0

 0

 0

 0

 TOTAL MALFORMATIONS

 

 

 

 

 

 Fetal Incidence

 N

 0

 0

 0

 0

 

 %

 0.0

 0.0

 0.0

 0.0

 Litter Incidence

 N

 0

 0

 0

 0

 

 %

 0.0

 0.0

 0.0

 0.0

 Affected Fetuses/Litter

MEAN% 

 0.0

 0.0

 0.0

 0.0

 

 S.D.

 0.0

 0.0

 0.0

 0.0

 TOTAL VARIATIONS

 

 

 

 

 

 Fetal Incidence ##

 N

 0

 0

 4*

 3

 

 %

 0.0

 0.0

 2.7

 2.1

 Litter Incidence

 N

 0

 0

 3

 3

 

 %

 0.0

 0.0

 15.0

 15.0

 Affected Fetuses/Litter

  MEAN% 

 0.0

 0.0

 2.4

 2.2

 

  S.D.

 0.0

 0.0

 6.4

 5.4

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test

## FETUSES AFFECTED BY SEVERAL CHANGES WILL BE COUNTED AS ONE FETAL INCIDENCE

SUMMARY OF ALL CLASSIFIED FETAL SKELETAL OBSERVATIONS

TOTAL RETARDATIONS

 

 

 

 

 

 Fetal Incidence ##

 134

131

 127

 121

 

 %

 89.3

 85.1

 85.2

 84.0

 Litter Incidence

 N

 20

 20

 20

 20

 

 %

 100.0

100.0 

 100.0

 100.0

 Affected Fetuses/Litter

MEAN% 

 88.1

 84.4

 86.2

 83.1

 

 S.D.

 16.8

 22.0

 14.7

 25.6

──────────────────────────────────────────────────────────────────────────────────────────────────────────────

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test)

## FETUSES AFFECTED BY SEVERAL CHANGES WILL BE COUNTED AS ONE FETAL INCIDENCE

SUMMARY OF FETAL SKELETAL MALFORMATIONS

 

 TEST GROUP 1

Control

TEST GROUP 3 

250 mg/kg

TEST GROUP 3 

500 mg/kg

TEST GROUP 4 

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

 20

 Fetuses Evaluated

 N

150

154

149

144

 Live

 N

150 

154

149

144 

 Dead

 N

 0

 0

 0

 TOTAL FETAL SKELETAL MALFORMATIONS

 

 

 

 

 

 Fetal Incidence

 0

 0

 0

 0

 

0.0

0.0

0.0

0.0

 Litter Incidence

 

 %

0.0 

0.0 

0.0 

0.0 

SUMMARY OF FETAL SKELETAL VARIATIONS

 

 

 TEST GROUP 1

Control

TEST GROUP 3 

250 mg/kg

TEST GROUP 3 

500 mg/kg

TEST GROUP 4 

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

 20

 Fetuses Evaluated

 N

150

154

149

144

 Live

 N

150 

154

149

144 

 Dead

 N

 0

 0

 0

 RIB(S) WAVY

 

 

 

 

 

 Fetal Incidence

 0

 0

 2

 0

 

0.0

0.0

1.3

0.0

 Litter Incidence

 

 %

0.0 

0.0 

5.0 

0.0 

 STERNEBRA(E) BIPARTITE

 

 

 

 

 

 Fetal Incidence

0

0

2

1

 

 %

0.0

0.0 

1.3 

0.7 

 Litter Incidence

0

0

 

 %

0.0

0.0

10.0 

5.0 

 STERNEBRA(E) MISALIGNED (SEVERITY: SLIGHT)

 

 

 

 

 

 Fetal Incidence

0

0

0

 

%

0.0 

0.0 

0.0 

1.4 

 Litter Incidence

 

0.0 

0.0 

0.0 

10.0 

 TOTAL FETAL SKELETAL VARIATIONS

 

 

 

 

 

 Fetal Incidence 

N

 4*

 

0.0 

0.0 

2.7 

2.1 

 Litter Incidence

N

 

0.0 

0.0 

15.0 

15.0 

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test)

## FETUSES AFFECTED BY SEVERAL CHANGES WILL BE COUNTED AS ONE FETAL INCIDENCE

SUMMARY OF FETAL SKELETAL RETARDATIONS

 

 

 TEST GROUP 1

Control

TEST GROUP 3 

250 mg/kg

TEST GROUP 3 

500 mg/kg

TEST GROUP 4 

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

 20

 Fetuses Evaluated

 N

150

154

149

144

 Live

 N

150 

154

149

144 

 Dead

 N

 0

 0

 0

 ABSENCE OF OSSIFICATION IN METACARPALIA 2 TO 5

 

 

 

 

 

 Fetal Incidence

1

6*

 2

1

 

0.7

3.9

1.3

0.7

 Litter Incidence

6* 

 

 %

5.0 

30.0 

10.0 

5.0 

 ABSENCE OF OSSIFICATION IN METATARSALIA 2 TO 5

 

 

 

 

 

 Fetal Incidence

0

0

1

0

 

 %

0.0

0.0 

0.7 

0.0 

 Litter Incidence

0

0

0

 

 %

0.0

0.0

5.0 

0.0 

 CAUDAL VERTEBRAL BODIES, ALL BODIES UNOSSIFIED

 

 

 

 

 

 Fetal Incidence

0

2

0

 

%

0.0 

1.3 

0.0 

0.7 

 Litter Incidence

 

0.0 

10.0 

0.0 

5.0 

 CAUDAL VERTEBRAL BODIES, ONLY ONE BODY OSSIFIED

 

 

 

 

 

 Fetal Incidence 

N

0

 

0.0 

0.0 

0.0 

1.4 

 Litter Incidence

N

 

0.0 

0.0 

0.0 

10.0

HYOID UNOSSIFIED

 

 

 

 

 

Fetal Incidence 

 N

 98

 78**

95 

87 

 

 %

65.3 

50.6 

63.8 

60.4 

Litter Incidence

20 

18 

19 

19 

 

100.0 

90.0

95.0 

95.0 

 OS PUBIS INCOMPLETELY OSSIFIED

 

 

 

 

Fetal Incidence 

N

 2

 2

 0

 1

 

%

 1.3

1.3 

 0.0

0.7 

Litter Incidence 

N

 2

 1

 0

10.0 

5.0 

0.0 

5.0 

 SACRAL VERTEBRAL ARCH/ARCHES INCOMPLETELY OSSIFIED

 

 

 

 

 Fetal Incidence 

 1

 0

 0

 0

 0.7

 0.0

 0.0

 0.0

Litter Incidence  

 1

 0

 0

 

 5.0

 0.0

 0.0

 0.0

 SACRAL VERTEBRAL BODY/BODIES UNOSSIFIED

 

 

 

 

 

 Fetal Incidence 

 N

 0

 1

 0

 0

 

 %

0.0 

 0.6

 0.0

0.0 

 Litter Incidence 

 N

 0

 0

 

 0.0

5.0 

0.0 

0.0 

 SKULL INCOMPLETE OSSIFICATION

 

 

 

 

 

  Fetal Incidence

 N

 6

13 

 7

 

 %

4.0 

2.6 

8.7 

4.9 

  Litter Incidence

 N

 6

 2

 

 %

 30.0

10.0 

30.0 

30.0 

 STERNEBRA(E) INCOMPLETELY OSSIFIED

 

 

 

 

 

 Fetal Incidence

 N

 6

 7

 5

 10

 

 %

4.0 

4.5 

 3.4

 6.9

 Litter Incidence

 N

 4

 5

 4

 6

 

20.0 

25.0 

 20.0

30.0 

 STERNEBRA(E) REDUCED IN SIZE

 

 

 

 

 

 Fetal Incidence

 N

92 

109 

 89

89 

 

 %

 61.3

70.8 

59.7 

61.8 

 Litter Incidence 

 N

 19

20 

20

20 

 

 %

 95.0

100.0

100.0 

100.0 

 STERNEBRA(E) UNOSSIFIED

 

 

 

 

 

Fetal Incidence

 N

 10

11 

10 

 

 %

 6.7

7.1 

6.7 

 5.6

Litter Incidence 

 N

 6

 7

 5

 

 30.0

 35.0

30.0 

25.0 

 THORACIC VERTEBRAL BODY/BODIES BIPARTITE

 

 

 

 

 

 Fetal Incidence

 8

 5

 8

 3

 

 5.3

 3.2

 5.4

 2.1

 Litter Incidence 

 6

 3

 6

 

 30.0

15.0 

 30.0

15.0 

 THORACIC VERTEBRAL BODY/BODIES DUMBBELL-SHAPED

 

 

 

 

 

 Fetal Incidence

 N

 12

 16

13 

10 

 

 %

8.0 

10.4 

 8.7

6.9 

 Litter Incidence 

 7

 8

 7

 5

 

 35.0

 40.0

35.0 

25.0 

 TOTAL FETAL SKELETAL RETARDATIONS

 

 

 

 

 

  Fetal Incidence

 134

 131

127 

 121

 

 89.3

85.1 

 85.2

84.0 

  Litter Incidence  

 20

 20

20 

 20

 

 100.0

100.0 

100.0 

100.0 

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test)

## FETUSES AFFECTED BY SEVERAL CHANGES WILL BE COUNTED AS ONE FETAL INCIDENCE

SUMMARY OF ALL CLASSIFIED FETAL SOFT TISSUE OBSERVATIONS

 

 

 TEST GROUP 1

Control

TEST GROUP 3 

250 mg/kg

TEST GROUP 3 

500 mg/kg

TEST GROUP 4 

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

 20

 Fetuses Evaluated

 N

150

154

149

143

 Live

 N

150 

154

149

143 

 Dead

 N

 0

 0

 0

TOTAL MALFORMATIONS

 

 

 

 

 

 Fetal Incidence

0

0

 0

1

 

0.0

0.0

0.0

0.7

 Litter Incidence

 

 %

0.0 

0.0 

0.0 

5.0 

 Affected Fetuses/Litter

MEAN% 

0.0

0.0 

0.0 

0.7 

S.D.

0.0

0.0

0.0

3.2

 TOTAL VARIATIONS

 

 Fetal Incidence

18

12

14 

10

 

%

12.0

7.8

9.4

7.0 

 Litter Incidence

 12

 8

13

60.0

40.0

65.0

35.0 

 Affected Fetuses/Litter

MEAN% 

11.5 

8.1

9.2 

6.4

 

 S.D.

11.1 

11.5 

7.5 

10.2 

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test)

## FETUSES AFFECTED BY SEVERAL CHANGES WILL BE COUNTED AS ONE FETAL INCIDENCE

SUMMARY OF FETAL SOFT TISSUE MALFORMATIONS

 

 

 TEST GROUP 1

Control

TEST GROUP 3 

250 mg/kg

TEST GROUP 3 

500 mg/kg

TEST GROUP 4 

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

 20

 Fetuses Evaluated

 N

150

154

149

143

 Live

 N

150 

154

149

143 

 Dead

 N

 0

 0

 0

 MULTIPLE MALFORMATIONS

 

 

 

 

 

 Fetal Incidence

0

0

0

1

 

0.0

0.0

0.0

0.7

 Litter Incidence

 

 %

0.0 

0.0 

0.0 

5.0 

 TOTAL FETAL SOFT TISSUE MALFORMATIONS

 

 

 

 

 

 Fetal Incidence

0

0

0

1

 

 %

0.0

0.0 

0.0

0.7 

 Litter Incidence

0

0

1

 

 %

0.0

0.0

0.0 

5.0 

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test)

SUMMARY OF FETAL SOFT TISSUE VARIATIONS

 

 

 TEST GROUP 1

Control

TEST GROUP 3 

250 mg/kg

TEST GROUP 3 

500 mg/kg

TEST GROUP 4 

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

 20

 Fetuses Evaluated

 N

150

154

149

143

 Live

 N

150 

154

149

143 

 Dead

 N

 0

 0

 0

 DILATATION OF CEREBRAL VENTRICLE

 

 

 

 

 

 Fetal Incidence

4

4

5

4

 

2.7

2.6

3.4

2.8

 Litter Incidence

3

 

 %

15.0 

20.0 

25.0 

20.0 

 DILATATION OF RENAL PELVIS

 

 

 

 

 

 Fetal Incidence

10

5

7

5

 

 %

6.7

3.3 

4.7

3.5 

 Litter Incidence

9

5

4

 

 %

45.0

25.0

30.0 

20.0 

 KIDNEY(S): MALPOSITIONED

 

 

 

 

 

 Fetal Incidence

1

2

1

 

%

0.7 

1.3 

0.7

0.0 

 Litter Incidence

 

5.0 

10.0 

5.0 

0.0 

LIVER: HAEMORRHAGIC FOCUS/FOCI

 

 

 

 

 

 Fetal Incidence 

N

1

1

 

2.0 

0.7 

0.7 

0.7 

 Litter Incidence

N

 

15.0 

5.0 

5.0 

5.0

TOTAL FETAL SOFT TISSUE VARIATIONS

 

 

 

 

 

Fetal Incidence 

 N

 18

 12

14 

10 

 

 %

12.0 

7.8 

9.4

7.0

Litter Incidence

12 

13 

7

 

60.0 

40.0

65.0 

35.0 

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test)

## FETUSES AFFECTED BY SEVERAL CHANGES WILL BE COUNTED AS ONE FETAL INCIDENCE

SUMMARY OF FETAL SOFT TISSUE UNCLASSIFIED OBSERVATIONS

 

 

 TEST GROUP 1

Control

TEST GROUP 3 

250 mg/kg

TEST GROUP 3 

500 mg/kg

TEST GROUP 4 

1000 mg/kg

 Litters Evaluated

 N

 20

 20

 20

 20

 Fetuses Evaluated

 N

150

154

149

143

 Live

 N

150 

154

149

143 

 Dead

 N

 0

 0

 0

HEART: SITUS INVERSUS

 

 

 

 

 

 Fetal Incidence

0

0

 0

1

 

0.0

0.0

0.0

0.7

 Litter Incidence

 

 

0.0 

0.0 

0.0 

5.0 

 THORACIC CAVITY: FILLED WITH BLOOD

 

 

 

 Fetal Incidence

N

0

1

3

1

 

 %

0.0

0.7

2.0

0.7

  Litter Incidence

0

1

1

 

%

0.0

5.0

15.0

5.0 

TOTAL FETAL SOFT TISSUE UNCLASSIFIED OBSERVATIONS

 

 Fetal Incidence

 N

0

1

3

1

%

0.0

0.7

2.0

0.7

 Litter Incidence

N

0

1

 

%

0.0

5.0

15.0

5.0

SIGNIFICANTLY DIFFERENT FROM CONTROL: * = P≤0.05 ** = P≤0.01 (Fisher or Chi-square test)

Applicant's summary and conclusion

Conclusions:
Fetuses
The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item. No dead fetuses and no test item-related malformations, variations or retardations were noted.
Under the conditions of the study, Nigrosin WLF did not show any teratogenic potential.
The no-observed-adverse effect level (NOAEL) for the fetal organism was above 1000 mg Nigrosin WLF/kg b.w./day
Executive summary:

In this prenatal developmental toxicity study, the test item Nigrosin WLF was administered orally to female rats at dose levels of 250, 500 or 1000 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Dams

No test item-related premature deaths of the dams were observed in any group.

The daily cage-side observations revealed no changes in behaviour and the external appearance. The dark discoloured faeces of the animals of the dose groups were considered to be due to the black colour of the administered test item. No test item-related influence was noted on body weight, the food and drinking water consumption. The macroscopic inspection at necropsy did not reveal any morphological changes which are considered to be related to the administration of the test item.

The histopathological examination of the thyroids also revealed no test item-related changes.

The NOAEL for maternal toxicity was 1000 mg Nigrosin WLF/kg b.w./day; the highest dose tested

Fetuses

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item. No dead fetuses and no test item-related malformations, variations or retardations were noted.

Under the conditions of the study, Nigrosin WLF did not show any teratogenic potential.

The no-observed-adverse effect level (NOAEL) for the fetal organism was above 1000 mg Nigrosin WLF/kg b.w./day.