[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 26 November 2012; Experiment start date - 04 December 2012; Experiment completion date - 27 January 2013; Study completion date - 17 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:

other: OECD Guideline 421 (Reproduction/ Development Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): FAT 40'348/B
- Lot/batch No.: PV Op.2/UO
- Expiration date of the lot/batch: July 1993
- Stability under test conditions: stable for at least 2 hours when diluted

Specific details on test material used for the study:

Name: FAT 40348/F TE
Batch No: BOP 01-12 (Lot: BS-1109708)
Physical State: solid powder
Colour: red-brown
Storage Conditions: at room temperature
Molecular weight: 982.96
Active components: sum of all coloured substance: 82.37 %
Main constituents: 46.60 %
Date of Analysis: 21 August 2012
Expiry Date: 06 February 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

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 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)

Administration / exposure

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.

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.

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.

Duration of treatment / exposure:

Males: 28 days; Females: approx. 54 days

Frequency of treatment:

7 days/ week

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 group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Mid dose group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High dose group

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 tattooing. 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, epididymites, 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 epididymites 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 epididymites 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, epididymites, 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.

Examinations

Maternal examinations:

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

Fetal examinations:

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.

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 significance.

Indices:

Copulation, fertility, delivery indices.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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 and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

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.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

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.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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.

Details on embryotoxic / teratogenic effects:

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. There were no affect on copulation in females of control and treated groups 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.

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.

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.

Effect levels (fetuses)

Dose descriptor:

NOAEL

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.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40348/F, 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 of FAT 40348/F on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. This study was conducted according to OECD test guideline 421 in a GLP-certified laboratory. 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 received aqua ad injection, 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 post-natal day 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 water and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 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. The administration volume was 5 mL/kg body weight.

 

Summary Results

There was no mortality caused due to treatment. However, one animal 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. Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40348/F, 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.