main component 1 (isomer 1): 2-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonapht-7-ylamino]-1,3,5-triazin-2-ylamino}-3-{6-fluoro-4-[3-(1,5-disulfonaphth-2-ylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 1 (isomer 2): 2-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-3-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 2: 2,3-bis-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 3: 2,3-bis-{6-fluoro-4-[3-(1,5-disulfonaphth-2-ylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 September 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:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-610-1

EC Name:

-

Molecular formula:

multi constituent

IUPAC Name:

tetracosasodium 2-[(1E)-2-[6-({4-[(1-{[4-({6-[(1E)-2-(2,5-disulfonatophenyl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propan-2-yl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]benzene-1,4-disulfonate 2-[(1E)-2-[6-({4-[(2-{[4-({6-[(1E)-2-(2,5-disulfonatophenyl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propyl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]naphthalene-1,5-disulfonate 3-[(1E)-2-[6-({4-[(1-{[4-({6-[(1E)-2-(4,8-disulfonatonaphthalen-2-yl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propan-2-yl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]naphthalene-1,5-disulfonate 5-[(1E)-2-[6-({4-[(2-{[4-({6-[(1E)-2-(2,5-disulfonatophenyl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propyl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]naphthalene-1,6-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Name: FAT 40557/B
Batch No.: BOP 01-12
Physical State: powder
Colour: Red Brown
Purity: Sum of coloured constituents: 86 %
Main constituents: 64.5 %
Storage Conditions: at room temperature
Expiry Date: 30.01.2012

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM:
Young healthy male and nulliparous non pregnant female rats [strain: Wistar rat Crl:WI(Han)] (Full-Barrier), were used in this study. The animals were derived from a controlled full barrier maintained breeding system (SPF) (Source: Charles River, 97633 Sulzfeld, Germany)
According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals were bred for experimental purposes. At the start of treatment the age of the animals was 10-11 weeks. The range of the body weight was:
Females: 179-220 g, (mean: 199.53g, ± 20 %= 39.91 g)
Males: 264-311 g, (mean: 285.05 g, ± 20 %= 57.01 g)

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 housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 190612) except during mating period when individual male and female rats were cohabited.
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female rats, during gestation period and up to post natal day 3 in female rats. The male rats were dosed until the minimum total dosing period of 28 days is completed.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.

For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.

Details on mating procedure:

Animals were mated in the ratio of 1:1 (Male to Female). The subsequent morning and the next morning then onwards the vaginal smear of females
were checked to confirm the pregnancy. The day of sperm positive vaginal smear was considered as gestation day (GD) 0. Cages were arranged in
such a way that possible effects due to cage placement are minimised.

Females showing no evidence of copulation up to 14 day mating period were sacrificed 26 days after the last day of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle
was 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).

The dose formulation analysis was performed at BSL Bioservice Scientific Laboratories GmbH.

Duration of treatment / exposure:

Males: 28 days; Females: approx. 54 days

Frequency of treatment:

7 days/ week

Details on study schedule:

Arrival of the Test Item: 12 July 2012

Study Initiation Date: 03 September 2012

Experimental Starting Date: 12 September 2012

Experimental Completion Date: 06 November 2012

Completion Date of Delegated Phase (Histopathology): 14 August 2013

Completion Date of Delegated Phase (Formulation Analysis): 09 September 2013

Study Completion Date: 16 September 2013

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

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). The study included three dose groups
(LD, MD and HD) and one control group (C).

Preparation of the animals:
The rats were assigned to the dose/control groups using a randomization procedure based on stratified body weight to ensure harmonized group mean body weights between the groups. Each animal was assigned a unique identification number and caged individually. The animals were acclimatised for at least five days before the first dose administration.

Dosage:
In consultation with the sponsor the doses 50, 200, 1000 were selected for the 3 dose groups (LD, MD and HD):
The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectionem (sterile water) the same volume as used for the treatment groups.


Administration of doses:
The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female rats, during gestation period and up to post natal day 3 in female rats. The male rats were dosed until the minimum total dosing period of 28 days is completed. The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.

Mating:
Animals were mated in the ratio of 1:1 (Male to Female). The subsequent morning and the next morning then onwards the vaginal smear of females were checked to confirm the pregnancy. The day of sperm positive vaginal smear was considered as gestation day (GD) 0. Cages were arranged in such a way that possible effects due to cage placement are minimised. Females showing no evidence of copulation up to 14 day mating period were sacrificed 26 days after the last day of the mating period.


Clinical observation:
General clinical observations were made twice a day except during weekend and holidays where observation was made daily once, approximately at the same time each day and considering the peak period of anticipated effects after dosing.

Body weight and food Consumption:
All animals were weighed at randomisation, male rats weighed weekly during the entire study period and at termination. Females were weighed weekly during pre mating period, on GD 0, 7, 14, 20 and on PND 0 (within 24 hours of parturition) and PND 4 along with pups. The food consumption was measured on corresponding day of body weight after the beginning of the dose administration. The food consumption was not measured during mating period in both male and female rats.

Litter observations:
The duration of gestation was recorded and was calculated from day 0 of 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. Litters were weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by marking with tattoo. In addition to the observations on parent animals, any abnormal behavior 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 and ovaries, uterus with oviduct and cervix of all female adult animals were weighed. Paired organs were weighed separately.

Histopathology:
All organs and tissues listed in Table 2 were evaluated in group 1 and 4 animals. Macroscopic changes other than discolorations due to the test item were also evaluated in the intermediate dose groups. For paired organs marked with (*), both sides were examined.

Tissues evaluated microscopically
Cervix
Seminal vesicle *
Coagulating gland *
Ovary *
Testis *
Uterus
Epididymis *
Vagina
Prostate gland
Macroscopic lesions

For testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions were examined microscopically. Processing and histopathological evaluation was performed at GLP-certified test site Propath UK Ltd Willow Court, Netherwood Road, GB - Hereford HR2 6JU and KALEIDIS –Consultancy in Histopathology, 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, staining and
professional evaluation was performed according to the corresponding SOPs of the test site.

Examinations

Parental animals: Observations and examinations:

Body weight, food consumption, clinical signs, pathology, organ weight (reproductive organs), histopathology (reproductive organs)

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not Examined

Litter observations:

The duration of gestation was recorded and was calculated from day 0 of 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. Litters were weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups
were identified by marking with tattoo. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

yes

Postmortem examinations (offspring):

not examined

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control- and test groups. Statistical analysis was performed with GraphPad Prism (version V) software and p<0.05 was considered as statistical significant.

Reproductive indices:

Copulation, fertility, delivery indices

Offspring viability indices:

yes

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were few clinical signs namely pilorection, red nasal discharge, salivation, alopecia recorded in few animals of MD group. The clinical signs piloerection, salivation, moving the bedding, nasal discharge (red), discoloured faeces were seen in most animals of HD group transiently. The clinical signs in MD and HD groups were likely to be due to treatment with no adversity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males and females, there were no treatment related changes noted in treated groups when compared with controls. The statistical analysis of data revealed significant increase in body weight change in male LD and MD groups during mating and post-mating days 7-14. In the absence of dose response pattern this change was not related to treatment. In females, statistically significant increase in body weight change was noted in MD and HD groups. The body weight change in female MD and HD groups appeared to be within normal range of variation and hence the changes were not related to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males and females, there were no treatment related changes noted in treated groups when compared with controls. However, there was slight increase in food consumption without dose response pattern in male and/ or female treated groups when compared with control. The statistical analysis of data revealed significant increase in female LD and MD groups during 2nd week of treatment (premating day 7-14) without dose response pattern.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Reproductive organs:
Orange pigment in interstitial macrophages was seen at a minimal or mild degree in the testis and epididymis in all males and at a minimal degree in the seminal vesicle, coagulating gland and/or prostate gland in the majority of males of HD group. In MD group, a minimal amount of orange pigment in interstitial macrophages was seen in the epididymis of one single male. In the majority of females of HD group, a minimal to moderate amount of orange pigment was noted in endometrial macrophages in the uterus, and in some females in macrophages in the ovary or cervix. These changes were considered to be caused by deposition of the coloured test item in macrophages, and in the absence of other structural changes or indication of functional impairment of the organs concerned, were considered non adverse.

No other test item-related histological findings were noted in the male and female reproductive organs.

Reproductive organs of most 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 of HD group.

One control female (No. 44), one female of LD group (No. 117), two females of MD group (Nos. 123 and 125) and one female of HD group (No. 133) did not show any indication of recent pregnancy at terminal sacrifice.
Histomorphology of their reproductive organs indicated physiological sexual cycling.

Other organs:
In the kidney, orange pigment was observed in the corticotubular epithelium, in a dose-related manner, in all three test item-treated groups in the males and of HD group in the females. In HD group, the change was mostly mild to moderate in degree. This change was considered to be caused by deposition of the coloured test item and to corroborate the macroscopically noted colour changes. As it was associated, in HD group, in a proportion of animals only, with minimal tubular degeneration and/or corticotubular dilation, it was considered adverse in this dose group.

Furthermore, in the male dose group of HD group, hyaline droplets in the corticotubular epithelium, considered to represent male rat-specific α2-microglobulin, were noted at an increased incidence, when compared to the usual finding in untreated rats of this strain and age.

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):

The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control.
All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups. One control female, one female of LD group, two females of MD group and one female of HD group did not show any indication of recent pregnancy at terminal sacrifice. As there was no dose relationship this was considered to be unrelated to treatment.

Details on results (P0)

Precoital interval and duration of gestation:
There was no treatment related effect observed on the duration of gestation and precoital interval in the treated groups when compared with controls.

All females in control and treated groups (except one female in LD group, Animal no. 117) showed evidence of copulation during 14 days mating period. Successful mating of females resulted in pregnancy rate as follows, Control 90 %, LD group 100 % MD group 80 % and HD group 90 %.

Dose formulation analysis:
Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 93.1 %, 126.9 % and 78.5 % of the nominal concentration, respectively. Stability of formulation samples was investigated in study week 1 for LD and HD dose groups. After 6 hours storage at room temperature recovery compared to starting value was 99.9 % and 109.0 %. Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. In study week 1 the mean recovery observed for LD dose group was 99.8 % of the nominal value, and 96.8 % for HD dose group. In study week 5 the mean recovery observed for LD dose group
was 76.2 of the nominal value, and 29.0 % for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were in both study weeks between 0.5 and 2.8 % for LD and HD dose group.

In week 5, the concentration verification of sample no. 10 of HD group revealed low recovery (29.4 %) and sample no. 11 of MD group revealed the higher recovery (209.4 %). This is assumed to be have caused due to an improper homogenization of formulation sample before sample collection. The raw data review did not indicate mistake in the test item and vehicle measurement and showed that the formulations were prepared correctly and the animals received the correct dose concentration during week 5. The formulation samples are prepared freshly every day before dose administration and the error is considered to be isolated one caused on one specific day of a week during formulation preparation (date 16 October 2012, week 5).

In support to above argument, the histopathological kidney findings were in a dose related manner in all male treated groups and in female HD group. The changes in HD group were mild to moderate in degree, but was considered to be adverse in this dose group. This indicates that the animals in MD and HD groups received the right amount dose concentration during the study period.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on minimal tubular degeneration and/or corticotubular dilation in the kidney of some animals of the high dose group.

2

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related changes were noted up to the highest dose tested.

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:

not examined

Histopathological findings:

not examined

Details on results (F1)

Litter weight data:
There was no treatment related changes noted on group mean litter weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4. However, there was slight decrease in female litter weight in treated groups measured on PND 0 and PND 4, but the statistical analysis revealed no significant change. This decrease in female litter weight was considered to be due to slightly lower number of female pups, which is compensated by slightly higher number of male pups and subsequently slightly higher male litter weight. Hence, the changes were not considered to have toxicological relevance.

Pre and post natal data:
There were no treatment related changes noted for group means of corpora lutea, implantation sites, live pups born on PND 0, percent preimplantation loss and percent post implantation loss in the treated groups when compared with corresponding controls. However, the mean values
of % pre implantation loss was slightly higher in MD and HD groups, but without dose response pattern. The statistical analysis of data revealed no significant changes between the treated and control group values.

Litter data:
There was no treatment related effect observed on the number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4. There were no statistically significant difference noted between the treated and control groups.

Reproductive indices:
The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control. All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups. One control female, one female of LD group, two females of MD group and one female of HD group did not show any indication of recent pregnancy at terminal sacrifice. As there was no dose relationship this was considered to be unrelated to treatment.

Pup survival data:
The survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to control. However, 1 pup each from animal 50 (C group) and animal 115 (LD group) were found dead between PND 1 and 2. These findings were considered to be incidental.

Pup external findings:
There were no treatment related gross external findings observed in pups of the treated groups on PND 0 and 4. However, there were few findings namely black spot, pale and dry skin in isolated pup of control or treated groups. These were considered to be spontaneous and incidental in nature.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related changes were noted up to the highest dose tested.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40557/B, the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 200 mg/kg body weight while the NOAEL for reproduction/ developmental toxicity is considered to be 1000 mg/kg body weight.

Executive summary:

A GLP-compliant reproduction / developmental toxicity screening test after oral administration in Wistar rats with FAT 40557/B was carried out according to OECD guideline 421. In this study, four groups comprised of 10 adult male and 10 non pregnant nulliparous female rats [Wistar Crl:WI(Han)] were dosed daily by oral gavage with 50, 200 and 1000 mg/kg body weight per day of FAT 40557/B at dose volume of 5 mL/kg body weight. The test item was formulated in sterile water with an administration volume of 5 mL/kg body weight. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups. The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both males and in females, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted weekly based on the most recent body weight measurement. Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period. After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards, vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. 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. Males and females were sacrificed on day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females and the female that was not copulated were sacrificed on their respective day 26 after the evidence of mating and/or from the last day of mating period.

 

Clinical Observation and Mortality:

There were clinical signs namely piloerection, salivation, moving the bedding, nasal discharge (red), discoloured faeces noted in mid dose (MD) and high dose (HD) groups. These findings were seen in most animals of HD group transiently. The clinical signs in MD and HD groups were likely to be due to treatment with no adversity. There were no mortalities observed in males or females during the study period.

 

Body Weight Development:

In males and females, there were no treatment related changes noted in treated groups when compared with controls.

 

Food Consumption:

In males and females, there were no treatment related changes noted intreated groups when compared with controls.

 

Litter Weight data:

There was no treatment related changes noted on group mean litter weight, total litter weight, male litter weight and female litter weight of treated and control groups measured on PND 0 and PND 4.

 

Precoital interval and duration of gestation:

There was no treatment related effect observed on the duration of gestation and precoital interval in the treated groups when compared with controls. All females in control and treated groups showed evidence of copulation during 14 days mating period. Successful mating of females resulted in pregnancy rate as follows, Control 90 %, LD group 100 % MD group 80 % and HD group 90 %.

 

Pre and post natal data:

There were no treatment related changes noted for group means of corpora lutea, implantation sites, live pups born on post natal development (PND) 0, percent pre implantation loss and percent post implantation loss in the treated groups when compared with corresponding controls. However, the mean values of % pre implantation loss was slightly higher in MD and HD groups, but without statistical significant difference and dose response pattern.

 

Litter data:

There was no treatment related effect observed on the number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4.

 

Reproductive indices:

The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control. All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups. One control female, one female of low dose (LD) group, two females of MD group and one female of HD group did not show any indication of recent pregnancy at terminal sacrifice. As there was no dose relationship this was considered to be unrelated to treatment.

 

Pup survival data:

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to the control.

 

Pup External findings:

There were no treatment related gross external findings observed in pups from the treated groups on PND 0 and 4.

 

Gross Pathology:

At terminal sacrifice, red discoloration of a number of reproductive and other organs was noted in all animals of HD group. Kidney was found red or light discoloured in a proportion of males of LD and MD groups. In each one male of these dose groups, in addition, reproductive organs were also red discoloured. Red discoloration was considered to be related to the colour of the test item.

 

Organ Weight:

In males and females, there were no treatment related changes observed in the absolute and relative organ weights of the treated groups when compared with the controls.

 

Histopathology:

Histologically, orange pigment in interstitial macrophages was seen at minimal or mild degree in the testis and epididymis and at a minimal degree in the accessory glands of males of HD group. In MD group, minimal orange pigment in interstitial macrophages was seen in the epididymis of one single male (evaluated because of macroscopic colour change). In females of HD group, a minimal to moderate amount of orange pigment was noted in endometrial macrophages in the uterus, and in some females in macrophages in the ovary or cervix. These changes were considered to be due to deposition of the coloured test item in macrophages. No other test item-related histological findings were noted in the male and female reproductive organs. One control female, one female of LD group, two females of MD group and one female of HD group did not show any indication of recent pregnancy at terminal sacrifice. Histomorphology of their reproductive organs indicated physiological sexual cycling, and their non-gravid state was considered unrelated to the treatment. In the kidney, orange pigment was observed in the corticotubular epithelium, in a dose-related manner, in all three test item-treated groups in the males and in female HD group. As this finding was associated, in HD group, in a proportion of males and females only, with minor tubular degeneration and/or corticotubular dilation, it was considered adverse in this dose group. Furthermore, in males of HD group, hyaline droplets in the corticotubular epithelium, considered to represent male rat-specificα2-microglobulin, were seen and not considered to be relevant for risk assessment in humans. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40557/B, the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 200 mg/kg body weight while the NOAEL for reproduction/ developmental toxicity is considered to be 1000 mg/kg body weight.