6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

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:

experimental study

Adequacy of study:

key study

Study period:

2012-09-03 to 2012-08-22

Reliability:

1 (reliable without restriction)

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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

Remarks:

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

Limit test:

no

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)

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

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

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

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

Duration of treatment / exposure:

Males: 28 days; Females: approx. 54 days

Frequency of treatment:

7 days/ week

Details on study schedule:

Arrival of the Test Item: 30 March 2012

Study Initiation Date: 03 September 2012

Experimental Starting Date: 12 September 2012

Experimental Completion Date: 04 November 2012

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

Completion Date of Delegated Phase(Formulation Analysis): 08 March 2013

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested

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
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 100, 300, 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 were evaluated in group 1 and 4. 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.

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):

piloerection, salivation, moving the bedding, nasal discharge (red) in most animals of MD or HD groups but with no 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):

in HD group, blue pigment in macrophages was seen in the testis and epididymis in the males and in endometrial macrophages of the uterus in the females (in one female also in the cervix)

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Day of sacrifice
All male animals were sacrificed on day 29. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating. Lactating females along with pups were sacrificed on respective post natal day 4.

Clinical observation and mortality
There were clinical signs recorded in control and treated group animals during the study period. The clinical signs piloerection, salivation, moving
the bedding, nasal discharge (red) were seen in most animals of MD or HD groups. All animals in HD group had blue discoloured skin and all animals of LD, MD and HD groups had blue discoloured faeces starting from day 8 of study. Clinical signs namely piloerection, salivation and nasal
discharge (red) were also seen in few isolated males and females of control or LD groups. The above clinical signs were likely to be due to treatment
with no adversity.

Body weight and body weight change
In males, there were no effect on body weight noted throughout the study period in treated groups when compared with controls. In females, there
was statistically significant decrease noted for body weight in female HD group (GD 14 and 20) and body weight change in female HD group (between GD 0-20). This decrease was due to slightly lower weight gain of 3/10 females in HD group. Hence, considering the decrease in limited number of
animals, the effect was unlikely to be considered as an adverse effect due to treatment.

Food consumption
In males, there were no effect on food consumption during the study period. However, in females there was slight decrease in food consumption during gestation and lactation days of female HD group without attaining the statistical significance. During gestation days the changes in food consumption did not show dose response pattern. Hence, this finding in the absence of statistical significance and/or absence of dose response pattern was not considered to have toxicological relevance.

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 70% MD group 90% and HD group 100%. The decrease in pregnancy rate in LD and MD groups
in the absence of dose response was considered to be incidental in nature.

Gross pathology
At necropsy, male and female rats showed Blue discoloration of a number of organs was observed in a dose-related manner in all dose groups, males and females. In LD group, organs discoloured were kidney, testis, epididymis, oviduct, uterus and cervix. In MD and HD groups, in addition, male
accessory reproductive organs, adrenal gland, skin and mammary gland, thymus, salivary glands, ovary, vagina, urinary bladder, rectum, bone and
lymph nodes were also concerned. 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.
At necropsy one female of MD group (animal 70) showed one elongated and dead pup in its uterus. This was an isolated incident and considered to
be incidental due to its finding only in an one isolated animal of MD 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:
Blue pigment in interstitial macrophages was seen at a mild degree in the testis in all males and at a minimal degree in the epididymis in a proportion of males of HD group. This change was considered to represent test item deposition in macrophages. In all females of this dose group, minimal or
mild amounts of blue pigment were noted in endometrial macrophages in the uterus (in one female also in the cervix). These changes were
considered to be caused by deposition of the coloured test item.

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.

At terminal sacrifice, one control female (No. 44), three females of LD group (Nos. 51, 56, 60) and one female of MD group (No. 66) did not show
any indication of recent pregnancy. Histomorphology of their reproductive organs indicated physiological sexual cycling.

Other organs:
In the kidney, minimal or mild amounts of blue pigment were observed in the corticotubular epithelium, in 5/10 males and 10/10 females of HD
group.

This change was considered to be caused by deposition of the coloured test item and was not further evaluated in the intermediate dose groups.

Minimal or mild degrees of cortical basophilic tubules were also seen in a low proportion of high dose animals, but were not considered a test
item-related effect, as they may be seen in untreated rats of this strain and age.

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 108.2%, 111.0% and 94.8% of the nominal concentration, respectively.

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 98.8 and 109.6% of the nominal value, and 97.5 and 91.9% for HD dose group. The coefficients of variation of the different sampling
locations (top, middle, bottom) were 10.6 and 1.0% in LD dose group, and 5.4 and 8.9% in HD dose group.

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 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 in the absence of
statistical significance and dose response pattern the changes were not considered to have toxicological relevance.

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 % pre implantation loss was higher in MD and HD groups and % post implantation loss was higher in MD group. But the mean corpora lutea, implantation sites and live pups on PND 0 being comparable between the treated groups and the corresponding controls, the changes in percent
pre and post implantation loss was not considered to have toxicological relevance.

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 (except for one female in MD group, Animal 70) resulted in normal births and therefore delivery index remained unaffected in all treated groups. At necropsy one female of MD group (animal 70) showed one elongated and dead pup in its uterus. At terminal sacrifice, one control female, three females of LD group and one female of MD group did not show any indication of recent pregnancy. There was no dose relationship, and this was therefore 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), animal 65 (MD group) and 80 (HD 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 dark or black spot, white spot noted on head or thoracic region or limbs in few isolated pups of control groups. These were considered to
be spontaneous and incidental in nature. Few pups of MD group and most pups of HD group were smeared blue colour of test item which could be
due licking by dam post treatment.

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 40529/A in sterile water to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed no major toxicological findings.

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40529/A, the no observed adverse effect level
(NOAEL) is considered to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Executive summary:

The aim of this study was to assess the possible effect ofFAT 40529/Aon male, female fertility and embryofetal developmentin Wistar rats.

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 with100, 300 and 1000 mg/kg body weight per dayofFAT 40529/Aat 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 were sacrificed on their respective day 26 after the evidence of mating.

Clinical Observation and Mortality

The clinical signs piloerection, salivation, moving the bedding, nasal discharge (red) were seen in most animals of MD or HD groups. All animals in HD group showed blue discoloured skin and all animals of LD, MD and HD groups showed blue discoloured faeces. Clinical signs namely piloerection, salivation and nasal discharge (red) were also seen in few isolated males and females of control or LD groups.Theaboveclinical signs 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 no effect on body weight and body weight change were noted during the study period. In females, there were statistically significant decrease in body weight (GD 14 and 20) and body weight change (between GD 0-20) noted in HD group. The finding was considered unlikely to be an adverse effect due to treatment.

Food Consumption

In males and females, there were no effect on food consumption during the study period.

 

Litter Weight data

There were no treatment related changes noted for group mean litter weight, total litter weight, male and female litter weight measured on PND 0 and PND 4 in treated groups when compared to corresponding control.

 

Precoital interval and duration of gestation

There was no treatment related effect observed on the duration of gestation andprecoital interval in the treated groupswhen 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 70% MD group 90% and HD group 100%.

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 pre implantation loss and percent post implantation loss in the treated groups when compared with corresponding controls.

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.

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

Blue discoloration of a number of organs was observed in a dose-related manner in all dose groups, males and females. In LD group, organs discoloured were kidney, testis, epididymis, oviduct, uterus and cervix. In MD and HD groups, a number of other organs were also concerned. These color changes were considered to be caused by the color of the test item itself.

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, in HD group, blue pigment in macrophages was seen in the testis and epididymis in the males and in endometrial macrophages of the uterus in the females (in one female also in the cervix). These changes were considered to be caused by deposition of the coloured test item. In the absence of other structural changes or functional impairment of these organs, they were considered non-adverse and were not further evaluated in the intermediate groups. No test item-related histological findings were noted in the other male and in the female reproductive organs.

One control female, three females of LD group and one female of MD group did not show any indication of recent pregnancy at terminal sacrifice. Histomorphology of their reproductive organs indicated physiological sexual cycling, and in the lack of dose relationship their non-gravid state was considered unrelated to the treatment.

In the kidney, minimal or mild amounts of blue pigment were observed in the corticotubular epithelium, in males and females of HD group, and was considered to be caused by deposition of the coloured test item. In the absence of other structural changes or functional impairment of the kidney, it was considered non-adverse and was not further evaluated in the intermediate dose groups.

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 108.2%, 111.0% and 94.8% of the nominal concentration, respectively.

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 98.8 and 109.6% of the nominal value, and 97.5 and 91.9% for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 10.6 and 1.0% in LD dose group, and 5.4 and 8.9% 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

The aim of this reproduction/developmental toxicity screening test, performed according to OECD 421 and GLP, was to assess the possible effect of FAT 40529/A on male and female fertility, and embryofetal development in Wistar rats. 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 100, 300 and 1000 mg/kg body weight per day of FAT 40529/A 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 108.2%, 111.0% and 94.8% of the nominal concentration, respectively. 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 98.8 and 109.6% of the nominal value, and 97.5 and 91.9% for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 10.6 and 1.0% in LD dose group, and 5.4 and 8.9% in HD dose group. 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 were sacrificed on their respective day 26 after the evidence of mating.

The clinical signs piloerection, salivation, moving the bedding, nasal discharge (red) were seen in most animals of MD or HD groups. All animals in HD group showed blue discoloured skin and all animals of LD, MD and HD groups showed blue discoloured faeces. Clinical signs namely piloerection, salivation and nasal discharge (red) were also seen in few isolated males and females of control or LD groups. The above clinical signs were likely to be due to treatment with no adversity. There were no mortalities observed in males or females during the study period. In males no effect on body weight and body weight change were noted during the study period. In females, there were statistically significant decrease in body weight (GD 14 and 20) noted in HD group. The finding was considered unlikely to be an adverse effect due to treatment. In males and females, there were no effect on food consumption during the study period. There were no treatment related changes noted for group mean litter weight, total litter weight, male and female litter weight measured on PND 0 and PND 4 in treated groups when compared to corresponding control. 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 70% MD group 90% and HD group 100%. There were no treatment related changes 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. 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. The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control. Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to the control. There were no treatment related gross external findings observed in pups from the treated groups on PND 0 and 4. Blue discoloration of a number of organs was observed in a dose-related manner in all dose groups, males and females. In LD group, organs discoloured were kidney, testis, epididymis, oviduct, uterus and cervix. In MD and HD groups, a number of other organs were also concerned. These color changes were considered to be caused by the color of the test item itself. 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. Histologically, in HD group, blue pigment in macrophages was seen in the testis and epididymis in the males and in endometrial macrophages of the uterus in the females (in one female also in the cervix). These changes were considered to be caused by deposition of the coloured test item. In the absence of other structural changes or functional impairment of these organs, they were considered non-adverse and were not further evaluated in the intermediate groups. No test item-related histological findings were noted in the other male and in the female reproductive organs. One control female, three females of LD group and one female of MD group did not show any indication of recent pregnancy at terminal sacrifice. Histomorphology of their reproductive organs indicated physiological sexual cycling, and in the lack of dose relationship their non-gravid state was considered unrelated to the treatment. In the kidney, minimal or mild amounts of blue pigment were observed in the corticotubular epithelium, in males and females of HD group, and was considered to be caused by deposition of the coloured test item. In the absence of other structural changes or functional impairment of the kidney, it was considered non-adverse and was not further evaluated in the intermediate dose groups.

In conclusion, the repeated dose administration of FAT 40529/A in sterile water to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed no major toxicological findings. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40529/A, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg body weight for reproduction and developmental toxicity.

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.