Trisodium 5-[[5-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulphonatophenyl]azo]-1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-3-methanesulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-23 to 2014-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-740-3

EC Name:

-

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
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: 234 - 293 g (mean: 272.64 g, ± 20% = 218.11 – 327.17 g)
females: 174 - 213 g (mean: 193.90 g, ± 20% = 155.12 – 232.68 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 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. 1039)
- 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. 190612)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was weighed into a tarred plastic vial on a suitable precision balance and the vehicle (sterile water) was added to give the appropriate final concentration of the test item.
The vehicle has been selected as suggested by the sponsor and on the basis of the test item’s characteristics. The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation was recorded for all dosing formulations. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every
dose administration. The vehicle was also used as control item.

The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 200 mg/kg body weight
High Dose: 400 mg/kg body weight

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. 28-day oral toxicity study in rodents where the NOEL was established at 200 mg/kg, The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Details on mating procedure:

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 copulation. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.

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 in the last week of the study (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).
All formulation samples were stored at -20°C until the analysis were performed. All samples were analysed at BSL BIOSERVICE Scientific Laboratories GmbH.
All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH on particular day of sampling. Formulation analysis were performed in accordance with GLP and the procedures followed for the determination of test item concentration in the dosing formulations and control formulation was described in a separate study phase plan (BSL phase study No. 122442).

Duration of treatment / exposure:

The female animals were treated with the test item formulation or vehicle on 7 days per week basis for approximately 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 was completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Arrival of the Test Item: 27 September 2012
Study Initiation Date: 23 October 2012
1st Amendment to Study Plan: 16 April 2013
Experimental Starting Date: 31 October 2012
Experimental Completion Date: 12 December 2012
Proposed Completion Date of
Delegated Phase (Histopathology): December 2013
Completion Date of Delegated Phase
(Formulation Analysis): 06 September 2013

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 200, 400 mg/kg bw/day

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
This includes the control group animals shared with BSL study No.124658.

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Time schedule: daily
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.

BODY WEIGHT:
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study.
During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

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.

Postmortem examinations (parental animals):

SACRIFICE
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 along with pups using an anaesthesia (ketamine/xylazin, 2:1, medistar Arzneimittel, lot no: 00312, expiry date: 06/2014 and Serumwerk, lot no: 00312, expiry date: 05/2014) was used.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.
The number of corpora lutea and implantation sites was recorded for any females sacrificed 24 to 26 days after the end of the pairing period with no evidence of mating and for any females sacrificed on day 25 post-coitum due to non-delivery.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with 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, except for testes and epididymides which were preserved in modified Davidson’s Solution
and then transferred in 10 % neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in Control and HD animals and in non pregnant female animals of the LD and MD animals. These examinations were extended to animals of all other dosage groups for treatment-related changes that are observed in the HD group.
Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
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.

Postmortem examinations (offspring):

Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test.
These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Mortality
No mortality occurred during the whole duration of the study.

Clinical Observations
No test item-related clinical signs or observations were observed in males and females. The clinical signs noted were: nasal discharge after
administration, salivation, alopecia, lower incisor shorter or regurgitation after administration with the test item. All these clinical signs occurred
occasionally in single animals, thus not considered to be adverse effects of the treatment with the test item.

Body Weight Development
Mean body weight development and gain were not affected by the treatment with the test item throughout the study either in males or in females.
 
Food Consumption
Mean food consumption was not affected by the treatment with the test item at all dose levels in both males and females.

Pathology
During the macroscopic examination, yellow spots on epididymidis were observed in male in the control group, in three males at LD,
in one male at MD and HD group. These findings were considered to be of incidental nature.
No macroscopic findings were noted in the females.


Organ Weight
Mean absolute and relative weight of testes, epididymidis, prostate, ovaries and uterus were not affected by the treatment with the test item.
At MD, the higher statistically significant relative weight of right ovary was considered to be incidental as it did not follow a dose-dependent pattern. .

Histopathology
No test item-related histological findings were noted in the male and female reproductive organs.
Spermatic granuloma was seen in two control males and one male treated at 400 mg/kg/day and was considered incidental,
as spermatic granuloma may be observed spontaneously in untreated male rats of this strain and age.
Reproductive organs of all control and 9/10 high dose females showed typical post-partum histomorphology. The number of large
ovarian corpora lutea was not essentially different between control animals and animals treated at 400 mg/kg/day.
One female treated at 400 mg/kg/day (No. 134) did not show any indication of recent pregnancy at terminal sacrifice.
Histomorphology of its reproductive organs indicated physiological sexual cycling.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No findings of toxicological relevance were observed up to the highest dose tested.

2

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No findings of toxicological relevance were observed up to the highest dose tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Litter Data
No treatment-related effect on the litter data such as the total number of pups on PND 0 and 4 was observed. No runts or still birth were
observed in all groups.
The number of females was higher in all test item treated groups, consequently the sex ratio was statistically lower in all groups on PND0 and 4.
As there was no dose-dependency, this was considered to be incidental effect.


Litter Weight Data
No test item related effects were observed at any dose levels in mean litter weight on postnatal day 0 and 4.

Precoital Interval and Duration of Gestation
No treatment-related effect was observed in the pre-coital interval or in the duration of gestation when compared with the control group.
With the exception of female no. 134 at HD which was not pregnant, all the other females achieved a pregnancy.

 
Pre- and Post-Natal Data
Mean number of corpora lutea and of implantation sites were comparable among the all groups.
At HD, incidence of pre-implantation loss was higher when compared with the control, this was mainly due to female no. 140 which had
higher pre-implantation loss. This was an isolated occurrence and not considered to be related to the treatment with the test item.
Incidence of post implantation loss was also not affected by the treatment with the test item. In all test item-treated groups, the values were
lower than the control group.


Reproductive Indices
Copulation and delivery indexes were 100% in all dose groups. Fertility index was 100% in control, LD and MD groups and 90% at
HD as female no. 134 was not pregnant.
The viability index was 100% in the control, and LD groups, 99.0% at MD and 99.07% at HD. This slightly lower viability index was slightly
lower due to one pup loss in litter no. 127 and 133, respectively. This was considered to be incidental and not related to the
treatment with the test item.



Pup Survival Data
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.
One pup each went missing from MD (no. 127) and HD (no. 133) group between PND 1-4 and presumed to be cannibalized by the dam.

Pup External Findings
Findings such as dry skin, dark spot on navel were observed on few occasions in one litter in the control group and in two litters at LD and MD.
Frequency and type of these findings did not give any indication of a test item-related effect.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Development

Generation:

F1

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No findings of toxicological relevance were observed up to the highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with FAT 40277/D TE in male and female Wistar rats with dose levels of 100, 200, and 400 mg/kg bw/day the following conclusions can be made:
No effects of FAT 40277/D TE were observed at any dose level in male and female parental animals and in the offspring, therefore the NOAEL was established at 400 mg/kg bw/day for both generations.

Executive summary:

The aim of this study was to assess the possible effects of FAT 40277/D TE on male and female fertility and embryofetal development after repeated dose administrationin Wistar rats. 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 maximum 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 receive daqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the study, 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 from the day of sperm positive vaginal smear. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on postnatalday 4 were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed on high dose and control animals.

The following doses were evaluated:

Control:                   0        mg/kg body weight/day

Low Dose:                   100     mg/kg body weight/day

Medium Dose:             200     mg/kg body weight/day

High Dose:                  400     mg/kg body weight/day

The test item formulation was prepared freshly on each day of administration. The test item was dissolved inaqua ad injectionem(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. Theadministration volume was 5 mL/kg body weight.

Summary Results

All the animals survived until the scheduled necropsy and no test item-related clinical signs were observed.

No effect on body weight, body weight gain and food consumption was observed throughout the study.

No treatment-related effect on litter data and litter weight data was observed on PND 0 and 4.

Precoital interval, duration of gestation, pre and post natal data, pup survival data and pup external findings remained unaffected due to treatment with test item.

Macroscopic evaluation did not reveal any test item-related findings. Absolute and relative weight of organs was not affected. No test item-related morphologic findings were noted during the histopathologic evaluation.

The assessment of reproduction indexes, survival rate and litter data did not give any indication of a test item-related effect. Incidences of pre- and post-implantation losses were also not affected.

External and gross necropsy of pups revealed no treatment-related findings.

Conclusion

On the basis of this reproduction/ developmental toxicity screening test withFAT 40277/D TEin male and female Wistar rats with dose levels of 100, 200, and 400 mg/kg bw/day the following conclusions can be made:

No effects of FAT 40277/D TE were observed at any dose level in male and female parental animals and in the offspring, therefore the NOAEL was established at 400 mg/kg bw/day for both generations.