2-{4-[4-[4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazin-2-ylamino]phenylazo]phenylazo}naphthalene-4,6,8-trisulfonate, trisodium salt

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2012 to 14 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: 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:

442-230-8

EC Name:

-

Cas Number:

321679-52-1

Molecular formula:

Hill formula: C31 H25 F N9 Na3 O12 S4 CAS formula: C31 H28 F N9 O12 S4 · 3 Na

IUPAC Name:

trisodium 7-(2-{4-[2-(4-{[4-({2-[2-(ethenesulfonyl)ethoxy]ethyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}phenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-1,3,5-trisulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Name: FAT 40800/A TE
Batch No.: WP 2/01
CAS No.: 321679-52-1
Physical State: solid
Colour: Dark orange
Storage Conditions: at room temperature
Purity: 84.79 % sum of all coloured substances, main constituent: 62.18 %
Expiry Date: 12 July 2015

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 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. 0702)
- 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. The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same dose volume. 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 analysed on the day of sample collection and were stored at -20 °C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 124666.

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: 05 September 2012
Study Initiation Date: 23 October 2012
1st Amendment to Study Plan: 11 April 2013
Experimental Starting Date: 31 October 2012
Experimental Completion Date: 22 December 2012
Proposed Completion Date of Delegated Phase (Histopathology): December 2013
Completion Date of Delegated Phase (Formulation Analysis): 06 September 2013

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control (Group 1)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low dose (Group 2)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Middle dose (Group 3)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High dose (Group 4)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Administration of Doses:
The animals were treated with the test item formulation or vehicle daily for a 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 was completed. 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.

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
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 together. Organ weights of animals found dead were not taken. A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanized due to morbidity. 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. 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.
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.5.01 software or 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

Description (incidence and severity):

Males
No clinical signs or observations were observed in males during the study.
Females
At 400 mg/kg bw/day; an eschar on the right ear was noted in one female between day 2 and 15 of the gestation period. At 200 mg/kg bw/day, one female had nasal discharge immediately after administration with the test item at the end of the gestation period. At 100 mg/kg bw/day, alopecia was observed on abdomen or forepaws in one female during the first week of the gestation period and in another female within the same group on thorax and cervical area at the end of the gestation period and throughout the lactation period. Same female was noted to nasal reddish discharge on gestation day 15. Type and frequency of these clinical signs were not indicative of any test item-related effect.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males, statistically significantly lower mean body weight gain which occurred at 200 mg/kg bw/day between day 7 and 14 of the pre-mating period did not follow a dose dependent pattern, therefore considered to be of incidental nature.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

However, at 200 and 400 mg/kg bw/day, decrease in food consumption was observed during the lactation period without achieving the statistical significance. This effect was not considered to be adverse as it did not affect the body weight development.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Spermatic granuloma was seen in two control males and two males 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 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.

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

Description (incidence and severity):

All paired females achieved pregnancy and gave birth; therefore the copulation, fertility and delivery indexes were 100 % in all groups. The viability index was not affected by the treatment with the test item. In order of ascending dose levels, viability index was: 100.0 %, 98.32 %, 97.33 %, and 98.75 %.

Effect levels (P0)

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: Based on the overall study findings

Target system / organ toxicity (P0)

Critical effects observed:

no

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 of the litter data was observed such as the total number of pups born, number of male and females, sex ratio, live pups on PND 0. At 400 mg/kg bw/day, on PND 4, sex ratio was statistically significantly lower, as the mean number of males was slightly lower, this was considered to be of incidental nature. No still birth or runts were observed at birth.

Litter Weight Data
Statistical analysis of litter weight data revealed statistically significant decrease in mean litter weight on PND 0 in 400 mg/kg bw/day. However, no such effect was observed on PND 4. As difference was marginal and weights were recovered on PND 4, this statistically significant effect on mean litter weight on PND 0 was considered to be toxicologically irrelevant. The statistically significant decrease in male litter weight at 200 mg/kg bw/day and 400 mg/kg bw/day was considered to be incidental as no such significant effect was observed on total litter weight.
This effect could also be attributed to decrease in sex ratio in 200 mg/kg bw/day and 400 mg/kg bw/day groups.

Precoital Interval and Duration of Gestation
The precoital time and duration of the gestation were not affected by the treatment with the test item. In order of ascending dose levels, mean precoital time was: 1.89, 3.00, 2.11 and 1.11 days; the duration of the gestation was: 22.56, 21.78, 22.44 and 21.89 days.

Pre- and Post-Natal Data
Mean number of corpora lutea, number of implantation sites, and number of live pups born on PND 0 and on PND 4 were comparable between treatment groups and the control group. At 400 mg/kg bw/day, the incidence of pre-implantation losses was higher when compared with the control group, this was mainly due to one female which had the 70 % of pre-implantation loss. Excluding this female, incidence of pre-implantation losses was similar to the control, therefore not considered to be a test item-related effect. At 100 mg/kg bw/day and 200 mg/kg bw/day, no test item-related effects were observed in the incidence of pre-implantation loss. The incidence of post-implantation losses was not affected by the treatment with the test item.
 
Reproductive Indices
All paired females achieved pregnancy and gave birth; therefore the copulation, fertility and delivery indexes were 100 % in all groups. The viability index was not affected by the treatment with the test item. In order of ascending dose levels, viability index was: 100.0 %, 98.32 %, 97.33 %, and 98.75 %.

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. Between PND 0 and PND 4, the pups missing were: one pup from female no. 52, 57 at 100 mg/kg bw/day, one pup from female no. 70 at 200 mg/kg bw/day group, and one pup from female No. 79 in 400 mg/kg bw/day dose group. These missing pups from various treatment groups were presumed to be cannibalized by the female.

Pup External Findings
The external findings noted in few pups were: dry skin, dark area on shoulders or on foot region, or discolored dark snout. Type and incidence of findings did not give any indication of a test item-related effect. At necropsy, except for three pups which had a wound on the cervical region or on forepaw or injury on the dorsal area, no other findings were observed.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on overall study findings

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for parental generation and offspring was established at 400 mg/kg bw/day.

Executive summary:

A GLP-compliant, reproduction/ developmental toxicity screening test with FAT 40800/A was carried out according to OECD guideline 421. The aim of this study was to assess the possible effects of test substance on male and female fertility and embryofetal development after repeated dose administration in 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 received aqua ad injectionem, 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 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 respective post natal day 4. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on postnatal day 4 were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Any gross lesion macroscopically identified were examined microscopically in all animals.

The doses evaluated were 0, 100, 200 and 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 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.

 

Summary Results

No mortality occurred during the entire duration of the study. In males no clinical signs were observed. In females, the clinical signs noted during the course of the study were not considered to be of adverse nature.

No effects were observed in mean body weight and mean body weight gain in both males and females. The lower mean food consumption in females at 200 and 400 mg/kg bw/day during the lactation was not considered to be adverse as it did not affect the body weight.

Gross pathological observation of male and females at scheduled necropsy revealed no treatment-related findings. There was no statistically significant difference in the absolute and relative organ weights of the male and female treatment groups observed when compared with the control group.

In the male and female reproductive organs, no histopathological lesions were found in treated groups which were considered to be test item-related.

At 400 mg/kg bw/day, mean litter weight was statistically significantly lower on postnatal day 0. However, no such effect was observed on PND 4 and difference was marginal and weights were recovered on PND 4.

Reproduction indexes, mean precoital time, and duration of the gestation were not affected by the treatment with the test item. Mean number of corpora lutea, incidence of pre- and post-implantation losses, survival rate between PND 0 and PND 4 were also not affected. No test item-related external and macroscopic findings were observed in pups at all dose levels.

No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.

 

In conclusion, reproduction/ developmental toxicity screening test with FAT 40800/A TE in male and female Wistar rats with dose levels of 100, 200, and 400 mg/kg body weight, no adverse effects were observed in males and females during the whole study and no test item-related morphologic alterations in sexual organs were observed during the histopathologic evaluation.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for parental generation and offspring was established at 400 mg/kg bw/day.