Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to OECD guideline 421 and in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 27 July 1995
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
Version / remarks:
EPA 712-C-00-367, July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Specific details on test material used for the study:
Identification: FAT 40045/Z TE
Common Name : NOVACRON TURQUOISE P-GR Reactive Blue 072
Batch No. AT-2486840

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: Crl: WI(Han) (Full Barrier)
Sex : male and female; the female animals were non-pregnant and nulliparous.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: males: 10-11 weeks old, females: 10-11 weeks old.
- Weight at study initiation: males: 266 - 305 g, (mean: 285.70 g, ± 20 % = 228.56 – 342.84 g); females: 185 - 214 g (mean: 200.90 g, ± 20 % = 160.72 – 241.08 g)
- Housing: The animals were kept in groups of 2 animals/ sex/ cage in IVC cages), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 1526) (except during the mating period when one female were paired with one male and during gestation/lactation period when females were housed individually)
- Diet : Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 131113)
- Water : Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period:Adequate acclimatisation period (at least 5 days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 55 ± 10 %
- Air changes: 10 x / hour
- Photoperiod: Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua ad injectionem
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item FAT 40045/Z TE was weighed into a tared plastic vial on a precision balance and suspended in aqua ad injectionem, respectively. Homogeneity of the test items in the vehicle was maintained by vortexing.
The test item formulations were prepared at least every ten days. Prepared test item formulations were stored at 2-8 °C, but warmed to room temperature before dose administration.
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. Females with unsuccessful mating will be allowed to mate with other male of the same group. 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 was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle was analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from C, Csup, LD, MD, HDsup and HD group (week 1 only) (in total 21 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom in study week 1 (LD group, HD group, HDsup group) and 5 (LD group and HDsup group) (in total 15 samples).
Samples for stability analysis were taken, 0 hours after the preparation, 6 hours after the preparation (at room temperature) and another sample 10 days after the preparation (stored at 2-8 °C), in the first week of the study from formulations of HD, HDsup and LD groups (9 samples).
Each sample was retained twice (sample A, sample B). The dose formulation analysis was performed by BSL BIOSERVICE Scientific Laboratories GmbH, in accordance with GLP. The exact procedure was described in a phase plan which was amended to the study plan.
All formulation samples were stored at -15 to -35 °C. The A samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140342. The B samples were retained at BSL until the analysis has been performed, and discarded after completion of the final study report.
Duration of treatment / exposure:
The test item was administered daily in graduated doses to 3 groups of test animals (low dose: LD, medium dose: MD and high dose: HD), one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.
Frequency of treatment:
daily
Details on study schedule:
- Arrival of the Test Item: 07 January 2014
- Study Initiation Date: 01 April 2014
1st Amendment to Study Plan: 10 April 2014
2nd Amendment to Study Plan: 20 May 2014
3rd Amendment to Study Plan: 18 July 2014
- Delivery of Animals: 27 March 2014 and 03 April 2014
- Acclimatisation Period: 27 March 2014 - 04 April 2014; 03 April 2014 - 11 April 2014
- Experimental Starting Date: 02 April 2014
- Treatment Period: 04 April 2014 – 04 June 2014
- Experimental Completion Date: 04 June 2014
- Proposed Completion Date of Delegated Phase (Histopathology): 25 September 2014
- Proposed Completion Date of Delegated Phase (Formulation Analysis): 25 June 2014
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control/Control sup
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low Dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium Dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose/High Dose sup
No. of animals per sex per dose:
120 animals (60 males and 60 females) will be included in the study (10 male and 10 female animals per group).
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
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 AND COMPOUND INTAKE (if feeding study):
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.
Litter observations:
Litter Observations
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
Pathology
All surviving male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on postnatal day 4 along with pups using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24664, expiry date: 06/2015, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015, lot no: 01213, expiry date: 10/2015) was used. Males of HD group were euthanized on study day 7 without measurement of body weight, food consumption, organ weights and without clinical or macroscopic observations. Organs or tissue of these animals for possible histopathological evaluation were not preserved. 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.

Histopathology:
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanized due to morbidity. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and examined in Csup and HDsup animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate,
seminal vesicle with coagulating gland) were also examined in the mating partners of the non -pregnant female LD and MD animals. These examinations were extended to animals of all other dosage groups for treatment-related changes that are observed in the HDsup group. For adequate evaluation of these examinations in other dosage groups C group was used for comparison. Only organs and tissues of the other dosage groups showing changes in the HDsup group were examined. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. 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.
Histological processing of tissues to microscope slides was performed at the GLPcertified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01
December 2012) [9]. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites. The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights was performed for each gender by comparing values of all groups with each other (C, Csup, LD, MD, HDsup) using a Tukey-Test (used in conjunction with ANOVA). These statistics were performed with GraphPad Prism V.6.01 software (p <0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Discoloured blue faeces were noted in both males and females of the LD and the MD group from the 4th day of treatment and in both males and females of the HDsup group from the 3rd day of treatment until the end of the study, respectively. Discolouration of the skin and the faeces was considered to be based on the blue colour of FAT 40045/Z TE. On gestation day 26, the health status of female no. 41 of the C group was moderately reduced (wasp waist, slightly reduced spontaneous activity, dark urin and pale eyes (probably anaemic). Due to parturition difficulties this female did not litter before the final planned sacrifice on gestation day 26. At necropsy, a uterine torsion was noted as likely reason for a pregnancy complication. One dead foetus was found in the uterus. Uterine torsion is a rare phenomen, but can occur incidentally. Moderate or slight salivation was noted transitorily in all males of the HDsup group, three females of the HDsup group and one female of the LD group. Moving the bedding was observed occasionally in all males of the HDsup group and 3 females of the HDsup group. The symptoms of salivation and moving the bedding were only noted transitorily presumably as a local effect of the test item and thus, they are not considered to be toxicologically relevant. Low incidences of slight clinical signs like alopecia on various body parts, nasal discharge, a crust at the cheek or slight piloerection were noted in isolated males and/or females of the dose groups and/or the control groups. As these findings were mostly transient and seen irrespective of the groups in isolated animals, they are considered to be incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female No. 70 from MD group was found dead on Mating day 2. The cause of its death was considered to be most likely related to an aspiration of the test item after regurgitation and is seen as an effect of bolus administration of the dosing formulation via gavage. One animal of the MD group (female no. 70) was found dead on mating day 2. The cause of its death was considered to be most likely related to to an aspiration of the test item after regurgitation. All other animals survived the scheduled treatment period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In both males and females, the mean body weight increased with the progress of the study during premating, mating/postmating and gestation period in the control groups, the LD, the MD and the HDsup group. Values were within the normal range of variation of historical control data. There were no test item-related effects of toxicological relevance noted on body weight and body weight gain in both males and females. Generally, body weight of both the male and female Csup and HDsup group was slightly lower compared to the C, LD and MD group, respectively (up to 6 % in males and up to 8 % in females).This finding can be attributed to the fact that these supplementary animals were delivered in an extra batch. Throughout the premating, mating and post-mating periods of male animals there were no statistically significant differences in body weights between male animals of LD or MD and C groups and between male animals of HDsup and the Csup group. Moreover, no statistically significant difference in body weight was observed in female animals between LD or MD and C groups and between HDsup and the Csup group during premating, gestation and lactation. However, there was a statistically significantly lower body weight gain in week 2 of premating period in LD males (14.7 g) when compared to the C group (23.2 g). During this time no significant difference was observed between MD and C group and between HDsup and Csup groups. Thus this transiently lower body weight gain in male animals of the LD group is not assumed to be related to toxic characteristics of the the test item. Moreover, no decrease in body weight gain was observed in female animals treated with FAT 40045/Z TE during this time period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no biologically significant effects on food consumption during the treatment period in both males and females of the dose groups, when compared to the respective controls. In correlation to the body weight and body weight change, the food consumption of the control groups, the LD, MD and HDsup group increased with progress of the study for males during premating period and for females during premating and first two weeks of gestation. In the last week of gestation food consumption for females remained roughly constant in Csup and HDsup group or decreased slightly In C, LD and MD group, but all values were within the normal range of variation for animals of this strain and age. A slightly – but statistically significantly higher mean food consumption in male animals of the HDsup group (342.4 g), when compared to Csup group (310.4 g), during the second week of premating is not assumed to be toxicologically relevant. During the first week of the premating period, a tendency towards higher mean food consumption was seen in the female animals of LD (215 g) and MD group (208 g) when compared to the Control group (186.4 g). As there was no dose dependency and values were within the range of historical control data, statistically significant differences are not assumed to be biologically relevant.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Under the conditions of this study, the test item FAT 40045/Z TE produced no histological evidence of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix, and vagina. There were also no reveal effects noted on the completeness of stages or cell populations. There was no indication for maturation arrest, reabsorption of sperm or any other degenerative type. The cause of the unsuccessful pregnancy and/or infertility of female no. 101 and 102 of the Csup group and their male pairing partners no. 81 and 82, respectively, could not be established from the reproductive organs examined from these animals. Organs and tissues with the macroscopic finding of blue/green or greenish discolouration were not subjected to microscopic examination, as this finding was due to the blue colour of the test item used in this study. All other gross lesions recorded were considered to be within the range of normal background alterations.
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
Description (incidence and severity):
There were no treatment-related effects of FAT 40045/Z TE on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control groups. A slightly reduced copulation index (number of copulated females / number of pairs x 100) was observed in the Csup, LD and MD group (90 % compared to 100 % in C group and HDsup group) and therefore is considered to be incidental. The fertility index (number of pregnant females/ number of copulated females x 100) was also slightly reduced in the Csup group (88.9 %) compared to 100 % in all other groups. The cause of the unsuccessful copulation and/or pregnancy and/or infertility of females no. 101 and 102 of the Csup group as well as their male pairing partners (no. 81 and 82), respectively, could not be established from the reproductive organs examined histopathologically from these animals. A slightly reduced delivery index in the C group (90%) compared to 100% in the Csup group and the dose groups was based on pregnant female no. 41 of the C group which did not litter before the end of the study due to parturition difficulties. At necropsy one dead foetus was found in a contorted uterus. The remaining pregnant females of the C group littered normally. The viability index (no. of live offspring at day 4 / no. of live offspring at birth X 100) was marginally lower in the Csup group (98.96%) and the LD group (87.3%) when compared to 100% in each the C and MD and HDsup group. This was attributed to the death of one complete litter from a dam of the LD group. But without dose-dependency, this finding is considered to be incidental.

Details on results (P0)

Litter Data
There were no effects of toxicological relevance of FAT 40045/Z TE on litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes noted for these litter data. A slightly lower mean total number of pups were noted in the HDsup group compared to the Csup group. As this finding was not statistically significant and was within the range of historical control data, it was considered to be incidental.

Litter Weight Data
There were no statistically or biologically significant effects of FAT 40045/Z TE on the litter weight data including pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4. Values were within the normal range of variation for animals of this strain.

Precoital Interval and Duration of Gestation
There were no statistically or biologically significant effects of FAT 40045/Z TE on the duration of precoital interval and the duration of gestation in the dose groups, when compared to the control groups. Values were within the normal range of variation of historical control data.

Pre- and Post-Natal Data
There were no statistically or biologically significant effects of FAT 40045/Z TE on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of post-implantation loss in the dose groups when compared to the control groups. The HDsup group was noted to have a moderately, statistically significantly higher percentage of pre-implantation loss (30.53%) compared to the Csup group (10.25 %). As values were within the range of historical control data and did not follow a dose-dependent pattern (LD group: 5.08%, MD group: 5.59 %), this effect was not considered to be related to the treatment with the test item.

Pup Survival Data
There were no statistically or biologically significant effects of FAT 40045/Z TE on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group. A higher mortality of pups between PND 1 and PND 4 was observed in the LD group (12.7 %) and a marginally higher mortality of pups was observed in the Csup group (1.04 %) when compared to 0.0 % in the C, MD and HDsup group. As this finding did not achieve statistical significance and did not show a dose response pattern, it is considered incidental and not related to the test item. Regarding female no. 103 of the Csup group one pup was missing on PND 1. In female no. 55 none of the 14 pups was found on PND 2. The loss of pups was attributed to cannibalism by the dam, respectively. One pup of female no. 52 also of the LD group was found dead on PND 2.

Pup External Findings
No gross external abnormalities of toxicological relevance were observed in any of the groups. A dark snout observed in one single pup of the C group, one single pup of the Csup group and two pups of the LD group and is considered to be incidental. There were few incidental further external findings such as a wound in two pups of the C group and one pup of the MD group as well as a swollen eye in one single pup of the LD group and a cold pup with pale skin in the MD group. These findings are considered to be spontaneous and not related to the test item.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects were seen

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
There were no statistically or biologically significant effects of FAT 40045/Z TE on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group. A higher mortality of pups between PND 1 and PND 4 was observed in the LD group (12.7 %) and a marginally higher mortality of pups was observed in the Csup group (1.04 %) when compared to 0.0 % in the C, MD and HDsup group. As this finding did not achieve statistical significance and did not show a dose response pattern, it is considered incidental and not related to the test item. Regarding female no. 103 of the Csup group one pup was missing on PND 1. In female no. 55 none of the 14 pups was found on PND 2. The loss of pups was attributed to cannibalism by the dam, respectively. One pup of female no. 52 also of the LD group was found dead on PND 2.

Pup External Findings:
No gross external abnormalities of toxicological relevance were observed in any of the groups. A dark snout observed in one single pup of the C group, one single pup of the Csup group and two pups of the LD group and is considered to be incidental. There were few incidental further external findings such as a wound in two pups of the C group and one pup of the MD group as well as a swollen eye in one single pup of the LD group and a cold pup with pale skin in the MD group. These findings are considered to be spontaneous and not related to the test item.

GROSS PATHOLOGY (OFFSPRING):
There were no macroscopic findings of toxicological relevance in any of the groups. However, at necropsy, a treatment-related macroscopic finding consisted of green, greenish or blue discolouration of several organs in males and females from the LD, MD and HDsup group was found. Since this finding was due to the blue colour of the test item used in this study, the green, greenish or blue discoloured organs (except for some organs from the animal found dead) were not subjected to microscopic examination. All other gross lesions recorded were considered to be within the range of normal background alterations, which may be seen in rats of this strain, age. Female no. 41 of the C group did not litter before the end of the study (gestation day 26) presumably because of parturition difficulties. At necropsy, one dead foetus was found in a contorted uterus which was assumed to be the reason for the pregnancy
complication. Furthermore, a node (20x10mm) was seen in the uterus. The left ovary, oviduct and cervix could not be found. The spleen was enlarged and the liver discoloured yellowish. The macroscopic findings and the resulting deteriorated health condition of female no. 41 are presumably based on a spontaneous uterine torsion which is considered incidental.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects on developmental parameters

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Dose Formulation Analysis:

Concentration of FAT 40045/Z TE was determined in study weeks 1, 3, 5 and in the last week for all dose groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HDsup) groups were 99.7 %, 98.4 % and 96.9 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations did not differ from nominal concentration by more than 15 %. Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HDsup dose groups. After 6 hours storage at room temperature recovery compared to starting value was 98.9% and 100.7% for LD and HDsup dose, respectively. All samples were stable, as concentration after storage did not differ from start value by more than 15 %. After 10 days storage at 2-8 °C recovery compared to starting value was 101.5 % and 99.8% for LD and HDsup dose, respectively. All samples were stable, as concentration after storage did not differ from start value by more than 15 %. Homogeneity of formulation samples was determined in study weeks 1 and 5 for the LD and HDsup dose groups. The mean recovery observed for the LD dose group was 96.3 % and 96.9 % of the nominal value and 93.7 % and 98.6 % of the nominal value for HDsup dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 1.6% and 3.6% in LD dose group and 2.8 % and 0.6 % in HDsup dose group. All samples were homogenous, as COV was below or equal 15 %.

Applicant's summary and conclusion

Conclusions:
The NOAEL of FAT 40045/Z TE in this study is considered to be 1000 mg/kg bw/day.
Executive summary:

The aim of this study was to assess the possible effects of FAT 40045/Z TE on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals (low dose: LD, medium dose: MD and high dose: HD), one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of one additional control group were handled identically as the dose groups but received aqua ad injectionem, the vehicle used in this study. The control group was shared with BSL Study No. 140329. The 4 groups comprised 10 male and 10 female Wistar rats. On day 7 HD animals were excluded from the study due to wrong dosing and a supplementary group HDsup was introduced (10 males and 10 females). As these animals were taken from a different batch, additionally a supplementary control group Csup was introduced (10 males and 10 females; also shared with BSL Study No. 140329). During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 4. Non-pregnant females were sacrificed on day 26. 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. A full histopathological evaluation of the tissues was performed on Csup and HDsup animals and in non-pregnant female animals of the LD and MD animals. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the HDsup group. Any gross lesion macroscopically identified will be examined microscopically in all animals.

The following doses were evaluated: Control/Control sup: 0 mg/kg/d Control sup: 0 mg/kg/d Low Dose: 100 mg/kg/d Medium Dose: 300 mg/kg/d High Dose/High Dose sup: 1000 mg/kg body weight The test item formulation was prepared at least every ten days. Prepared test item formulations will be stored at 2-8°C, but warmed to room temperature before dose 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 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg bw.

Female No. 70 from MD group was found dead on Mating day 2. The cause of its death was considered to be most likely related to an aspiration of the test item after regurgitation and is seen as an effect of bolus administration of the dosing formulation via gavage. All other animals survived the scheduled study period. There were no clinical signs of toxicological relevance in the dose groups, when compared to the control groups. The skin of all male and female animals of the HDsup group was observed to be discoloured blue from the 7th day of treatment until the end of the study. Blue coloured faeces were noted in both males and females of the LD and the MD group from the 4th day of treatment and in both males and females of the HDsup group from the 3rd day of treatment until the end of the study, respectively. Discolouration of the skin and the faeces was considered to be based on the the blue colour of FAT 40045/Z TE . There were no test item-related effects of toxicological relevance noted on body weight and body weight gain in both males and females. There were no biologically significant effects on food consumption during the treatment period in both males and females of the dose groups, when compared to the respective controls. There were no effects of toxicological relevance on litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4. There were no statistically or biologically significant effects on the litter weight data including pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4. There were no statistically or biologically significant effects on the duration of precoital interval and the duration of gestation in the dose groups, when compared to the control groups. There were no statistically or biologically significant effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of pre- and post-implantation loss in the dose groups, when compared to the control groups.

There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control groups. There were no statistically or biologically significant effects on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group. No gross external abnormalities of toxicological relevance were observed in any of the groups. There were no macroscopic findings of toxicological relevance in any of the groups. However, at necropsy, a treatment-related macroscopic finding consisted of green, greenish or blue discolouration of several organs in males and females from the LD, MD and HDsup group. Since this finding was due to the blue colour of the test item used in this study, the green, greenish, blue discoloured organs (except for some organs from the animal found dead) were not subjected to microscopic examination. The test item produced no histological evidence of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix, and vagina. As a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. The cause of the unsuccessful pregnancy and/or infertility of female no. 101 and 102 of the Csup group and their male pairing partners no. 81 and 82, respectively, could not be established from the reproductive organs examined histopathologically from these animals. Concentration of FAT 40045/Z TE in dose formulations was determined in study weeks 1, 3, 5 and in the last week for all dose groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HDsup) groups were 99.7%, 98.4% and 96.9% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations did not differ from nominal concentration by more than 15%. Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HDsup dose groups. After 6 hours storage at room temperature recovery compared to starting value was 98.9% and 100.7% for LD and HDsup dose, respectively. All samples were stable, as concentration after storage did not differ from start value by more than 15%. After 10 days storage at 2-8°C recovery compared to starting value was 101.5% and 99.8% for LD and HDsup dose, respectively. All samples were stable, as concentration after storage did not differ from start value by more than 15%. Homogeneity of formulation samples was determined in study weeks 1 and 5 for the LD and HDsup dose groups. The mean recovery observed for the LD dose group was 96.3% and 96.9% of the nominal value and 93.7% and 98.6% of the nominal value for HDsup dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 1.6% and 3.6% in LD dose group and 2.8% and 0.6% in HDsup dose group. All samples were homogenous, as COV was below or equal 15%

On the basis of this reproduction/ developmental toxicity screening test with FAT 40045/Z TE in male and female Wistar rats with dose levels of 100, 300 and 1000 mg/kg bw/day the following conclusions can be made: No adverse effects of FAT 40045/Z TE were found at dose levels of 100, 300 and 1000 mg/kg bw/day. Thus, the NOAEL of FAT 40045/Z TE in this study is considered to be 1000 mg/kg bw/day.