Sodium 5-({4-chloro-6-[ethyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-(1-sulfonato-2-naphthyl)diazenyl]naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012-11-26 to 2013-09-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female;
the female animals will be 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: 253 - 296 g
(mean: 279.77 g, ± 20% = 223.82 – 335.73 g)
females: 173 - 209 g
(mean: 194.44 g, ± 20% = 155.55 – 233.33 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. 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 housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 160812) 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)

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 on weekly basis.

Details on mating procedure:

Mating was performed in 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.
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 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).
All formulation samples were analysed on same day of sample collection or were stored at -20°C until the analysis was performed.
All samples were analysed 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: 28 September 2012
Study Initiation Date: 26 November 2012
1st Amendment to Study Plan: 27 June 2013
Experimental Starting Date: 04 December 2012
Experimental Completion Date: 27 January 2013
Completion Date of Delegated Phase (Histopathology): 14 August 2013
Completion Date of Delegated Phase (Formulation Analysis): 09 September 2013

Remarks:

Doses / Concentrations:
50, 250, 1000
Basis:
nominal conc.

No. of animals per sex per dose:

10 animals/sex/dose

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
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. 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 doses 50, 250, 1000 were selected for the 3 dose groups (LD, MD and HD):
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of minimum 28 days for males and maximum of
54 days for females, 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.
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 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 on weekly basis.

Mating
Mating was performed in 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.
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. 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 and food Consumption
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 was measured 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 tatooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.



Pathology
Gross necropsy
All male animals were sacrificed after the completion of the mating period (minimum dosing period: 28 days) on study day 29 or 31, while female
animals were sacrificed on post-natal day 4 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 and lot no: 00512, expiry date: 07/2014) was used. All surviving pups were killed on post natal day 4 by decapitation.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
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.
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 fixed in modified Davidson’s Solution for 24 hours and
then transferred in 10 % neutral buffered formalin.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.


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.


Histopathology
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 C and HD animals. Macroscopic changes were evaluated in all study 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.

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 behavior 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 significants.

Reproductive indices:

Copulation, fertility, delivery indices

Offspring viability indices:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

test item related but without adversity

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:

effects observed, treatment-related

Description (incidence and severity):

minor degrees of red-brown pigment in interstitial macrophages

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Day of sacrifice
Except one animal in MD group (animals 70) all other animals in treated and control groups survived the treatment period. The animal (no. 70) had
consumed the gavaging cannula due to which the general health condition of the animal was deteriorated. The animal was euthanized on Gestation Day 15.


Clinical observation and mortality
There were clinical signs namely reddish nasal discharge, hematoma (ear), piloerection, alopecia, salivation, abnormal breathing, respiratory sound and moving the bedding seen transiently in most animals of treated groups when compared to controls. As the clinical signs were seen transiently,
the findings were not considered to be an adverse effect.

Body weight and body weight change
In males and females, there were no effects on body weight and body weight change throughout the study period in treated groups when compared with controls. The statistical analysis of data revealed no significant changes between the treated and control groups. However, there was decrease
in body weight change in male HD group on 1st week of premating without attaining the statistical significance. This was due to one single male
animal (Animal No. 39) which did not show weight gain during the first week, but during the later days gained the weight that was within the range
of variations.

Food consumption
In males and female, there was no effect on food consumption during the study period. The statistical analysis of data revealed no significant changes between the treated and control groups.

Precoital interval and duration of gestation
There were 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 90% and HD group 100%.
One control female (No. 48) and one female of MD group (No. 67) did not show any indication of recent pregnancy at terminal sacrifice.
There was no dose relationship, and this was therefore considered to be unrelated to treatment.





Gross pathology
Red or reddish discoloration of a number of organs was observed in all animals of HD group. Organs concerned were one or several of the following: male and female reproductive organs, kidney, mesenteric lymph node, axillary lymph node, mandibular lymph node, skin, adrenal gland, thymus and gastrointestinal tract. In MD group, kidney, mesenteric lymph node and/or testis/epididymis were found to be red or reddish discoloured in a
proportion of animals, predominantly in the males. These color changes were considered to be caused by the color of the test item itself.
Other macroscopic organ findings were very few and not considered to be test item-related, including proliferating tissue at the kidney of one male
of LD group.


Organ weight
In males and females, the organ weight data (absolute and relative to terminal body weight) showed no changes considered to be of toxicological
relevance. The statistical analysis of data indicated no significant changes in treated groups when compared to the corresponding control group.

Histopathology
Reproductive organs:
Red-brown pigment in interstitial macrophages was seen at a mostly minimal degree in the testis in all males and at a minimal degree in the
epididymis in a proportion of males of HD group. Minimal red-brown pigment was also noted in the testis of one single male of MD group.
In the prostate gland, minimal red-brown pigment in interstitial macrophages was seen in one single male of HD group. These changes were
considered to represent test item deposition in macrophages.

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

Reproductive organs of most control and all 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 and one female of MD group did not show any indication of recent pregnancy at terminal sacrifice. Histomorphology of their
reproductive organs did not indicate any underlying pathological process, and as there was no dose relationship, this observation was considered
to be unrelated to treatment.

Other organs:
According to the study plan, other organs were only examined in case of macroscopic observations made at necropsy.

In the kidney, minimal or mild amounts of red pigment were observed in the corticotubular epithelium, in 10/10 males and 8/10 females of HD group. It was also seen at a minimal degree in each one male and one female of MD group. This change was considered to be caused by deposition of the
coloured test item. In addition, in the groups of MD or HD groups, minimal to moderate numbers of corticoepithelial hyaline droplets were noted in
the males, and minimal to moderate corticotubular degeneration/regeneration was seen, predominantly in the females.

Nephroblastoma, confirming a macroscopic tissue proliferation at the kidney, was observed in one male of LD group. This tumor type is known to
occur sporadically in young rats of this strain and was therefore considered an incidental finding.

In the mesenteric lymph node, minimal or mild sinus histiocytosis, partially red pigmented, was seen in 9/10 males of HD group. Pigmented
histiocytes were not found in the mesenteric lymph node of two males of MD group and which had also presented gross color change. Likewise,
minimal sinus histiocytosis, partially red pigmented, was observed in the macroscopically discolored mandibular lymph node of two males of
HD group. Intestine was evaluated in one male of the HD group which had shown color change of the gastrointestinal tract at necropsy. In this animal, minimal numbers of red-pigmented histiocytes were seen in the small intestinal villi of jejunum and ileum. Lymph node and intestinal changes were considered to represent test item deposition in macrophages and not to be adverse, as no accumulations or degeneration were seen.

No test item-related histopathological lesions were noted in the other miscellaneous organs evaluated in this study.




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 97.3%, 100.1% and 98.6% 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 100.7% and 100.7%.

Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group was 99.4 and 94.1% of the nominal value and 97.3 and 103.8% for HD dose group.
The coefficients of variation of the different sampling locations (top, middle, bottom) were 0.7 and 6.2 % in LD dose group, and 4.1 and 1.0% in HD dose group.

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 250 and 1000 mg/kg body weight, minimal to moderate numbers of corticoepithelial hyaline droplets were noted in the males, and minimal to moderate corticotubular degeneration/regeneration was seen, predominantly in the females.

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 findings of toxicological relevance were observed up to the highest dose tested.

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

Litter weight data
There were 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. There were no statistically significant difference noted between the treated and control groups.

Pre and post natal data
There were no statistically significant difference noted for group means of corpora lutea, implantation sites, live pups born on 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 percent pre and post implantation loss was higher in HD group. Taking into consideration of higher mean values of
corpora Lutea, Implantation sites and live pups on PND 0 in HD group compared to other corresponding treated and control groups and also
considering the absence of statistical significance and/ or dose relationship for the percent implantation losses, the findings were not related to
adverse effect due to treatment.

Litter data
There were 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 except one isolated female in HD (animal 77) groups resulted in normal births. As there were no overt clinical signs noted in this
animal during the treatment period or there were any indications of structural impairment of reproductive organs recorded histopathologically.
Hence, taking this into account the changes in delivery index in HD group was not related to adverse effect due 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, 2 pups
(pup no. 1, 3) from animal 49 (C group) and 1 pup (pup no. 8) of animal 66 (MD group) were missing between PND 1 and 3. The missing pup is
assumed to be cannibalized by dam. These findings were in isolated animals and hence 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 dark head/ snout, injury (on neck/ snout), dark and swollen hindlimb, discoloured/ dry skin, red spot on head in few isolated pups of control or treated groups. These were considered to be spontaneous and incidental in nature.

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 findings of toxicological relevance were observed up to the highest dose tested.

Reproductive effects observed:

not specified

Conclusions:

In conclusion, the repeated dose administration of FAT 40348/F TE in sterile water to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 50, 250 and 1000 mg/kg body weight showed histopatholgical kidney changes. At 250 and 1000 mg/kg body weight, minimal to moderate numbers of corticoepithelial hyaline droplets were noted in the males, and minimal to moderate corticotubular degeneration/regeneration was seen, predominantly in the females. These findings were considered to be related to test item administration and to be adverse. No treatment related changes were noted for reproduction/ developmental parameters. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40348/F TE, the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 50 mg/kg body weight and the NOAEL for reproduction/ developmental toxicity is considered to be 1000 mg/kg body weight.

Executive summary:

The aim of this study was to assess the possible effects ofFAT 40348/F TEon 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 minimum treatment period of 28 days for males and a maximum treatment period of 54 days for females, 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. Animals of control group were handled identically as the dose groups but receivedaqua 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.

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 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 days 29 and 31 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 confirmed mating.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities.

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.

Testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in C and HD animals. Macroscopic changes were evaluated in all study animals.

 

The following doses were evaluated:

Control:                          0        mg/kg body weight

Low Dose:                     50       mg/kg body weight

Medium Dose:               250     mg/kg body weight

High Dose:                     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 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 a minimum dosing period of 28 days (half males of each group were treated 28 days and rest half males treated for 30 days). Dose volumes were adjusted individually based on weekly body weight measurements.Theadministration volume was 5 mL/kg body weight.

Summary Results

There was nomortality caused due to treatment. However, one animal (No. 70) was euthanized as the animal had consumed the gavaging cannula and as a result the general health condition of the animal was deteriorated.

There were clinical signs namely reddish nasal discharge, hematoma (ear), piloerection, alopecia, salivation, abnormal breathing, respiratory sound and moving the bedding seen transiently in most animals of treated groups when compared to controls. As the clinical signs were seen transiently, the findings were not considered to be an adverse effect.

In males and females, there were no effects on body weight, body weight change and food consumption noted throughout the study period in treated groups when compared with controls.

There were 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 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.

There were 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 90% and HD group 100%.

One control female (No. 48) and one female of MD group (No. 67) did not show any indication of recent pregnancy at terminal sacrifice. There was no dose relationship, and this was therefore considered to be unrelated to treatment.

There were no treatment related changes noted for group means of corpora lutea, implantation sites and live pups born on PND 0 in the treated groups when compared with corresponding controls. The mean values of percent pre and post implantation loss was higher in HD group, but these findings were not related to adverse effect due to treatment.

The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control.

All pregnancies except one isolated female in HD (animal 77) groups resulted in normal births. Considering the absence of overt clinical signs during the treatment period and also absence of any structural impairment of reproductive organs the above finding was not related to an adverse effect due to treatment.

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

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

 

Red or reddish discoloration of a number of organs was observed in all animals of HD group. Organs concerned were one or several of the following: male and female reproductive organs, kidney, mesenteric lymph node, axillary lymph node, mandibular lymph node, skin, adrenal gland, thymus and gastrointestinal tract. In MD group, kidney, mesenteric lymph node and/or testis/epididymis were found to be red or reddish discoloured in a proportion of animals, predominantly in the males. These color changes were considered to be caused by the color of the test item itself.

Other macroscopic organ findings were very few and not considered to be test item-related, including proliferating tissue at the kidney of one male of LD group.

In males and females, the organ weight data (absolute and relative to terminal body weight) showed no changes considered to be of toxicological relevance.

 

Histologically, in HD group, minor degrees of red-brown pigment in interstitial macrophages were seen in the epididymis and/or testis of all and in the prostate gland of one single male rat. In MD group, red-brown pigment was noted in the testis of one single male. These changes were considered to represent test item deposition in macrophages. No test item-related histological findings were noted in the other male and in the female reproductive organs. One control female and one female of MD group did not show any indication of recent pregnancy at necropsy. As there was no dose relationship, this was considered to be unrelated to treatment. In the kidney, minor amounts of red pigment were observed in the corticotubular epithelium, in the majority of animals of HD group, and in one male and one female of MD group. This change was considered to be caused by deposition of the coloured test item. In addition, in MD and HD groups, minimal to moderate numbers of corticoepithelial hyaline droplets were noted in the males, and minimal to moderate corticotubular degeneration/regeneration was seen, predominantly in the females. These findings were considered to be related to test item administration and to be adverse. Furthermore, in HD group only, minor sinus histiocytosis, partially red pigmented, was seen in the mesenteric lymph node and mandibular lymph node, and minimal numbers of red-pigmented histiocytes were noted in small intestinal villi of one single male. Lymph node and intestinal changes were considered to represent test item deposition in macrophages.

No test item-related histopathological lesions were noted in the other miscellaneous organs evaluated in this study.

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 97.3%, 100.1% and 98.6% 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 100.7% and 100.7%.

Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group was 99.4 and 94.1% of the nominal value and 97.3 and 103.8% for HD dose group.

The coefficients of variation of the different sampling locations (top, middle, bottom) were 0.7 and 6.2% in LD dose group, and 4.1 and 1.0% in HD dose group.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline study, klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No studies on the reproduction/developmental toxicity of FAT40817 were available. However, Article 13 of REACH states that, in case no appropriate animal studies are available for assessment, information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. One reproduction/developmental toxicity screening test is available for the structural analogue FAT 40348.

The aim of this reproduction/developmental toxicity screening test, performed according to OECD 421 and GLP, was to assess the possible effects of FAT 40348/F TE on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. In this study, four groups comprised of 10 adult male and 10 female rats were dosed daily by oral gavage with 0 (C), 50 (LD), 250 (MD) and 1000 (HD) mg/kg body weight per day of the test substance at dose volume of 5 mL/kg body weight. The test item was formulated in sterile water. 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 97.3%, 100.1% and 98.6% 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 100.7% and 100.7%. Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group was 99.4 and 94.1% of the nominal value and 97.3 and 103.8% for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 0.7 and 6.2% in LD dose group, and 4.1 and 1.0% in HD dose group. The test item formulation was prepared freshly on each day of administration and was 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 a minimum dosing period of 28 days (half males of each group were treated 28 days and rest half males treated for 30 days). Dose volumes were adjusted individually based on weekly body weight measurements.

During the period of administration, the animals were observed each day for signs of toxicity. 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 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 days 29 and 31 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 confirmed mating. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities. 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. Testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in C and HD animals. Macroscopic changes were evaluated in all study animals.

There was no mortality caused due to treatment. However, one animal (No. 70) was euthanized as the animal had consumed the gavaging cannula and as a result the general health condition of the animal was deteriorated. There were clinical signs namely reddish nasal discharge, hematoma (ear), piloerection, alopecia, salivation, abnormal breathing, respiratory sound and moving the bedding seen transiently in most animals of treated groups when compared to controls. As the clinical signs were seen transiently, the findings were not considered to be an adverse effect. In males and females, there were no effects on body weight, body weight change and food consumption noted throughout the study period in treated groups when compared with controls. There were 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 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. There were 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 90% and HD group 100%. One control female (No. 48) and one female of MD group (No. 67) did not show any indication of recent pregnancy at terminal sacrifice. There was no dose relationship, and this was therefore considered to be unrelated to treatment. There were no treatment related changes noted for group means of corpora lutea, implantation sites and live pups born on PND 0 in the treated groups when compared with corresponding controls. The mean values of percent pre and post implantation loss was higher in HD group, but these findings were not related to adverse effect due to treatment. The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control. All pregnancies except one isolated female in HD (animal 77) groups resulted in normal births. Considering the absence of overt clinical signs during the treatment period and also absence of any structural impairment of reproductive organs the above finding was not related to an adverse effect due to treatment. The survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to control. There were no treatment related gross external findings observed in pups of the treated groups on PND 0 and 4. Red or reddish discoloration of a number of organs was observed in all animals of HD group. Organs concerned were one or several of the following: male and female reproductive organs, kidney, mesenteric lymph node, axillary lymph node, mandibular lymph node, skin, adrenal gland, thymus and gastrointestinal tract. In MD group, kidney, mesenteric lymph node and/or testis/epididymis were found to be red or reddish discoloured in a proportion of animals, predominantly in the males. These color changes were considered to be caused by the color of the test item itself. Other macroscopic organ findings were very few and not considered to be test item-related, including proliferating tissue at the kidney of one male of LD group. In males and females, the organ weight data (absolute and relative to terminal body weight) showed no changes considered to be of toxicological relevance. Histologically, in HD group, minor degrees of red-brown pigment in interstitial macrophages were seen in the epididymis and/or testis of all and in the prostate gland of one single male rat. In MD group, red-brown pigment was noted in the testis of one single male. These changes were considered to represent test item deposition in macrophages. No test item-related histological findings were noted in the other male and in the female reproductive organs. One control female and one female of MD group did not show any indication of recent pregnancy at necropsy. As there was no dose relationship, this was considered to be unrelated to treatment. In the kidney, minor amounts of red pigment were observed in the corticotubular epithelium, in the majority of animals of HD group, and in one male and one female of MD group. This change was considered to be caused by deposition of the coloured test item. In addition, in MD and HD groups, minimal to moderate numbers of corticoepithelial hyaline droplets were noted in the males, and minimal to moderate corticotubular degeneration/regeneration was seen, predominantly in the females. These findings were considered to be related to test item administration and to be adverse. Furthermore, in HD group only, minor sinus histiocytosis, partially red pigmented, was seen in the mesenteric lymph node and mandibular lymph node, and minimal numbers of red-pigmented histiocytes were noted in small intestinal villi of one single male. Lymph node and intestinal changes were considered to represent test item deposition in macrophages. No test item-related histopathological lesions were noted in the other miscellaneous organs evaluated in this study.

In conclusion, the repeated dose administration of FAT 40348/F TE in sterile water to the male (28 and 30 days) and female (maximum 54 days) Wistar rats at dosages of 50, 250 and 1000 mg/kg body weight showed histopatholgical kidney changes. At 250 and 1000 mg/kg body weight, minimal to moderate numbers of corticoepithelial hyaline droplets were noted in the males, and minimal to moderate corticotubular degeneration/regeneration was seen, predominantly in the females. These findings were considered to be related to test item administration and to be adverse. No treatment related changes were noted for reproduction/ developmental parameters. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40348/F TE, the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 50 mg/kg body weight and the NOAEL for reproduction/ developmental toxicity is considered to be 1000 mg/kg body weight.

Short description of key information:
Under the conditions of the reproduction/ developmental toxicity screening test (OECD 421, GLP), the NOAEL for fertility and developmental toxicity was determined to be 1000 mg/kg bw.

Justification for selection of Effect on fertility via oral route:
Only study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the absence of adverse effect on reproductive organs or tissues in the reproduction/developmental toxicity screening test, classification is not necessary for toxicity to fertility in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Based on the absence of developmental effects in the reproduction/developmental toxicity screening test classification is not necessary for developmental toxicity in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.