Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 2012 to 16 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD guideline 421 (Reproduction developmental toxicity screening test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate
EC Number:
402-170-5
EC Name:
Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate
Cas Number:
106359-91-5
Molecular formula:
Hill formula: C26 H23 F N9 O13 S4 Na3
IUPAC Name:
trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate
Details on test material:
Name: FAT 40224/H TE
Batch No.: TZ 2382-BB-503C81
Physical State: powder
Colour: red
Density: 1.46 g/cm3 (20°C)
pH: 6.5 to 7.5 conc.(% w/w): 1%
Melting Point: > 200°C
Purity / Active Components: sum of all coloured substances: 85.4%, main constituents: 50.6%
Date of Analysis: 23.07.2012
Storage Conditions: at room temperature
Expiry Date: 28.01.2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ 2382-BB-503C81
- Expiration date of the lot/batch: 28 January 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 10-11 weeks old, females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups: males:
272 - 303 g (mean: 288.10 g ± 20 % = 230.48 – 345.72 g), females: 184 - 217 g (mean: 199.48 g ± 20 % = 159.58 – 239.37 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare [7] the animals were bred for experimental purposes.

Housing and Feeding Conditions
-Full barrier in an air-conditioned room
-Temperature: 22 ± 3 °C
-Relative humidity: 55 ± 10 %
-Artificial light, sequence being 12 hours light, 12 hours dark
-Air change: 10 x / hour
-Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0856)
-Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
-The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 030512)
-Certificates of food, water and bedding are filed at BSL BIOSERVICE
-Adequate acclimatization period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples).
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose formulations (4 samples).


Samples for the nominal concentration verification was taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) from all groups (16 samples).

Samples for homogeneity analysis was taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples).

Samples for stability analysis was taken in the first week of the study, 0 hours after the preparation (at room temperature), from high and low dose formulations (2 samples).

The dose formulation analysis was performed at BSL Bioservice Scientific Laboratories GmbH.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle for a period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.
Frequency of treatment:
7 days/ week
Duration of test:
54 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
Middle dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Number and sex of the animals
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Preparation of the animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Dosage
In consultation with the sponsor the following doses (Table 1) were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectionem using the same volume as used for the high dose group.

Administration of doses:
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Mating:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. Females with unsuccessful mating will be allowed to mate with other male of the same group. The cages were arranged in such a way that possible effects due to cage placement were minimised.

Clinical observation:
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination. Once before the first exposure, and at least once a week thereafter, detailed clinical observations will be made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Pathology:
Gross necropsy:
Males were sacrificed after the completion of mating period (total dosing of 28 days), pregnant females were sacrificed on respective post natal day 4 and non pregnant females sacrificed on their respective day 26 after the evidence of mating or completion of mating period by using Ketamine/Xylazine (2:1) at the dose volume of 1.4 ml/kg body weight. At the time of sacrifice the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. The pups were killed on post natal day 4 by decapitation. Dead pups and pups killed on day 4 post-partum were carefully examined for gross external abnormalities. The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin. Testes and epididymides were fixed in modified Davidson’s Solution for 24 hours and then transferred to 10 % neutral buffered formalin.

Organ weight:
The testes and epididymides of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed. Paired organs were weighed separately. Organ weights of animals found dead were not taken.

Histopathology
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in Control and HD animals. All organs showing gross lesions were examined in all groups. All non-pregnant female animals were examined histopathologically. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Examinations

Fetal examinations:
Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
male: none; female: slight piloerection (9/10 HD; 1/10 C ), moderate piloerection (4/10 HD ; 0/10 C ), slight salivation (4/10 HD ; 0/10 C ), and moving the bedding (5/10 HD ; 0/10 C ), indicating discomfort, assumed to be test-item related.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, a slight tendency towards a decreased body weight development was observed for HD animals during the whole treatment period.
In this regard, a significantly decreased body weight gain in HD group was found during treatment days 1-7.
In female animals, no test item related influence on body weight development during the pre-mating period was measured. A significantly decreased (p<0.05) body weight gain during pre-mating days 1-7 (C: +3.8 g; HD: -2.4 g) is considered to be not of toxicological relevance since it recovered during the second week of the pre-mating period. During gestation and lactation, no significant influence on body weight development of female animals was observed.
For both – male and female animals – a test item relation is possible. However, due to the mild characteristics as well as the partially reversibility an adverse toxicological relevance cannot be assumed.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, the relative total testes weight (to body weight) was significantly increased (p<0.05) in HD group (1.0325 %) when compared to C group (0.9484 %). In this regard, also the relative weight (to body weight) of the left testis was significantly (p<0.05) increased in HD group (0.5203 %) when compared to C group (0.4751 %).
Since no finding was present in histopathology, this weight increase is not considered to be test item related and of toxicological relevance.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histologically, in the males, yellow-brown pigment in interstitial macrophages was seen at a minimal or mild degree in the testis and at a minimal degree in the epididymis in all rats treated at 1000 mg/kg/day. This change confirmed the macroscopic observation of yellow discoloration in some of these organs and was considered to represent test item deposition in macrophages. As there was no indication of other structural changes or functional impairment of these organs, the pigmentation was considered non-adverse.
No test item-related histological findings were noted in the other male and in the female reproductive organs.

As a conclusion, only minor test item-related pathological findings were noted in this study and were considered to be directly caused by test item deposition in macrophages and to be non-adverse.
Other effects:
no effects observed
Description (incidence and severity):
Precoital interval and duration of gestation
No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group.
The values were comparable between the groups. All pregnancies resulted in normal births.
Successful mating resulted in 6 pregnancies in the control, 9 in low, and 10 in medium and high dose group animals. The reduced number of pregnancies in C and LD group is considered to be incidental.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly reduced number of implantation sites was counted for HD group (p>0.05). In particular, 9.3 (HD) and 11.67 (C) were counted/calculated. Due to a high variance and a missing significance, this is not assumed to be test item related.
Furthermore, an increased percentage of pre-implantation loss was detected in HD group: 23.26 % (HD) and 7.83 (C) were calculated. This was not statistical significant (combined with a high variance). Furthermore, when comparing the total number of corpora lutea and implantation sites these values were no significantly different among the groups. Hence, a test item relation is not assumed.
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Study findings

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No difference was found for mean litter weights at PND 0 and PND 4 of treatment groups when compared to C group. The total litter weight was slightly (p>0.05) reduced in HD group. Furthermore, the male litter weight was slightly (p>0.05) decreased, too, at PND0 and PND4 in HD group.
This belongs mainly to the reduced litter size and a reduced number of male animals which hence leads to a reduced total litter weight.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly decreased total of pubs born was observed for HI) animals. In particular, 9.1 (HD and 11.17 (C) were counted / calculated. This correlates with a slightly decreased of male pubs (3.6 (HD) in comparison to 5.83 (C)). However, this was not statistical significant (combined with a high variance) and the degree of decrease was rather mild.
One still birth was detected in LD group (animal No. 59) as well as MD group (animal No. 61). However. since no other still births were detected (especially in HD group) this is not assumed to be test item related but incidental.
In correlation with PND 0. the total number of living pubs was still slightly (p>0.05) decreased in HD group at PND 4: 9.1 (HD and 11.17 (C) were counted.
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No difference was found for mean litter weights at PND 0 and PND 4 of treatment groups when compared to C group. The total litter weight was slightly (p>0.05) reduced in HD group. Furthermore, the male litter weight was slightly (p>0.05) decreased, too, at PND0 and PND4 in HD group.
This belongs mainly to the reduced litter size and a reduced number of male animals which hence leads to a reduced total litter weight.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
A slightly decreased total of pubs born was observed for HI) animals. In particular, 9.1 (HD and 11.17 (C) were counted / calculated. This correlates with a slightly decreased of male pubs (3.6 (HD) in comparison to 5.83 (C)). However, this was not statistical significant (combined with a high variance) and the degree of decrease was rather mild.
One still birth was detected in LD group (animal No. 59) as well as MD group (animal No. 61). However. since no other still births were detected (especially in HD group) this is not assumed to be test item related but incidental.
In correlation with PND 0. the total number of living pubs was still slightly (p>0.05) decreased in HD group at PND 4: 9.1 (HD and 11.17 (C) were counted.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related gross external findings were observed in any of the treated groups. Few incidences of external findings were observed but none of them was considered to be test item related.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Study findings

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

A reduced fertility index (number of pregnant females/ number of copulated females X 100) was observed in the C (60 %) and medium dose group (90 %). MD, and HD groups exhibited 100 %. The copulation index ((No. of rats copulated / No. of pairs) X 100) as well as delivery index (No. of dams with live newborns / No.of pregnant dams) X 100) were 100 % for each group. The viablity index was 100 % for C, LD, MD, and HD animals. The reduced fertility index is assumed to be incidental.

Applicant's summary and conclusion

Conclusions:
Based on the reproduction/ developmental toxicity screening test after oral administration in Wistar rats with FAT 40224/H, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for developmental toxicity in males and females.
Executive summary:

The aim of this study was to assess the possible effects of FAT 40224/H on male and female fertility and embryofetal development after repeated dose administration in Wistar rats according to OECD guideline 421. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injection (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. Three doses used in the study were 100, 300 and 1000 mg/kg body weight. The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad injection (sterile water) and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28-30 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on postnatal day 4 and those found dead, were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.


 


Summary Results:


No mortality occurred in the control or any of the dose groups during the treatment period of this study. For female animals, more clinical symptoms were found in HD group when compare to C group. In particular, slight piloerection (9/10 HD animals; 1/10 C animals), moderate piloerection (4/10 HD animals; 0/10 C animals), slight salivation (4/10 HD animals; 0/10 C animals), and moving the bedding (5/10 HD animals; 0/10 C animals) were observed. In male animals, a slight tendency towards a decreased body weight development was observed for HD animals during the whole treatment period. In this regard, a significantly decreased body weight gain in HD group was found during treatment days 1-7. In female animals, no test item related influence on body weight development during the pre-mating period was measured. A significantly decreased (p<0.05) body weight gain during pre-mating days 1-7. In male animals, food consumption was not different in treatment groups when compared to C group. In female animals, a significantly increased (p<0.05) food consumption was recognized for HD group during premating days 7-14. A slightly decreased total number of pups born was observed for HD animals. In particular, 9.1 (HD) and 11.17 (C) were counted/calculated. In correlation with PND 0, the total number of living pups was still slightly (p>0.05) decreased in HD group at PND 4. No difference was found for mean litter weights at PND 0 and PND 4 of treatment groups when compared to C group. The total litter weight was slightly (p>0.05) reduced in HD group. Furthermore, the male litter weight was slightly (p>0.05) decreased, too, at PND0 and PND4 in HD group. No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group. Successful mating resulted in 6 pregnancies in the control, 9 in low, and 10 in medium and high dose group animals. A slightly reduced number of implantation sites was counted for HD group (p>0.05).


In this regard, an increased percentage of pre-implantation loss was detected in HD group: 23.26 % (HD) and 7.83 (C) were calculated. No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls. No treatment-related gross external findings were observed in any of the treated groups. Few specific gross pathological changes were recorded for the male and female animals. The most common were discoloured yellow testes (2/10 HD; 0/10 C) and discoloured yellow oviduct and cervix (2/10 HD; 0/10 C).


In male animals, the relative weight of testes (to body weight) was significantly increased (p<0.05) in HD group. In this regard, also the relative weight (to body weight) of the left testis was significantly (p<0.05) increased in HD group when compared to C group. Histologically, in the males, yellow-brown pigment in interstitial macrophages was seen at a minimal or mild degree in the testis and at a minimal degree in the epididymis in all rats treated at 1000 mg/kg/day.


No test item-related histological findings were noted in the other male and in the female reproductive organs. Reproductive organs of those control and high dose females having undergone pregnancy showed typical post-partum histomorphology. So, based on the reproduction/ developmental toxicity screening test after oral administration in Wistar rats with FAT 40224/H, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for developmental toxicity in males and females.