Reaction mass of pentasodium 2-{[4-chloro-6-(ethyl{3-[(2-sulfonatoethyl)sulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate and tetrasodium 2-[(4-chloro-6-{ethyl[3-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

reproduction/ developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2013 to 10 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2013 to 10 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See overall remarks, attachment section

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name: FAT 40824/B TE
Batch No.: BOP 01-12 (ROE 805)
Physical State: solid at 20 °C
Storage Conditions: room temperature
Expiry Date: 20 April 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were 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: 276 – 318 g (mean: 301.13 g, ± 20 % = 240.9 – 361.35 g)
females: 175 - 214 g (mean: 196.35 g, ± 20 % = 157.08 – 235.62 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. 0801)
- 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 kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (240113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was weighed into a tarred plastic vial on a precision balance and the required volume of vehicle (sterile water) was added and subjected to homogenization using a homogenizer (ultra turrax). The homogeneity of the test item formulation was ensured by vortexing the formulation on vortex machine for minimum 1 minute. The test item formulation was prepared freshly on each administration day before the administration procedure. The vehicle was also used as control item.

The following doses were evaluated
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Mid Dose: 300 mg/kg body Weight
High Dose: 1000 mg/kg body Weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. This dose also happens to be the limit dose. 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 aqua ad injectabile using the dose volume 5 ml/ kg body weight.

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. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (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 the day of sample collection and were stored at -20 °C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 134527.

Duration of treatment / exposure:

The female animals were treated with the test item formulation or vehicle on 7 days per week basis for a period of 54 days, i.e. during 14 days of pre-mating and 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 was completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Arrival of the Test Item: 23 July 2013
Study Initiation Date: 24 September 2013
1st Amendment to Study Plan: 21 October 2013
2nd Amendment to Study Plan: 24 October 2013
3rd Amendment to Study Plan: 15 January 2014
Experimental Starting Date: 26 September 2013
Experimental Completion Date: 11 November 2013
Completion Date of Delegated Phase (Histopathology): 26 May 2014
Completion Date of Delegated Phase (Formulation Analysis): 24 February 2014

Dose / conc.:

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

Remarks:

Group 1 (Control)

Dose / conc.:

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

Remarks:

Group 2 (Low dose)

Dose / conc.:

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

Remarks:

Group 3 (Middle dose)

Dose / conc.:

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

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Positive control:

Not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Time Schedule: Daily
General clinical observations were made once a day approximately at the same time each day 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. 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:
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.

FOOD CONSUMPTION:
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.

Oestrous cyclicity (parental animals):

Not evaluated

Sperm parameters (parental animals):

Not Evaluated

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 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 tattoo mark on the paws. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded. 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 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 post-natal day 4 using anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24432, expiry date: 01/2015 and Serumwerk, lot no: 00512, expiry date: 07/2014). All surviving pups were sacrificed by decapitation on PND 4. Non-pregnant females were sacrificed after day 26 from the last day of mating period. All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were preserved in modified Davidson’s Solution for one day and afterwards transferred into 70 % Ethanol. The numbers of implantation sites and corpora lutea were recorded for each parental female at necropsy.

Organ Weights
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 together.
 
Histopathology
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in Control and HD animals.
All gross lesions macroscopically identified were 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 will be 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 GLP-certified 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). 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 tissue processing issued a detailed phase plan which was attached to the study plan per amendment. The principal histopathological investigator provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

Postmortem examinations (offspring):

Pups sacrificed on day 4 post partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p <0.05 was considered as statistically significant).

Clinical signs:

no effects observed

Description (incidence and severity):

However, there were clinical signs including nasal discharge (red coloured), slight to moderate salivation in male HD group animals. Incidences of nasal discharge and slight salivation were also in single isolated males of LD or MD groups. In female HD group animals, there were incidences of slight to severe pilorection, moderate salivation, moving the bedding, slight abnormal breathing. There were also lower incidences of alopecia, eschar, nasal discharge, slight to moderate salivation in isolated animals of control or LD or MD groups. The clinical signs observed in both male and female animals of control and/ or dose groups were transient and had no toxicological relevance. Alopecia in various parts of the body was seen in some animals of control and MD group and these were observed in few isolated animals. Thus, this finding was considered to be incidental.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment related changes in food consumption of male and female animals of the dose groups, when compared to control group during the treatment period. There were no statistically significant differences between the control and dose groups. There was a slightly higher (but not statistically significant) food intake observed in HD group males, when compared to controls. As values were within the normal range of variations, this effect was not assumed to be toxicologically relevant.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

However, microscopically, the treatment-related findings were recorded in the kidney that was examined as the gross lesions of the HD group. Slight to moderate hyaline droplets in the proximal tubules were recorded in all kidneys with macroscopic alteration, except for one male (no. 38: the tissues had not been preserved after necropsy). There were no further indicators of renal injury, and hence, this was considered not to be adverse under the condition of this study.
Unilateral yellow spot in the epididymis recorded in male no. 23 of MD group was correlated microscopically with spermatic granuloma, and was spontaneous lesion which is commonly seen in male rats. The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. There were not treatment-related effects on the completeness of stages or cell populations of the testes. Some histologic alterations were recorded at a minimum severity in a few animals from both of the control and high-dose males, but all of these were considered to be within the range of normal background lesions which are occasionally observed in males of this strain and age. Nothing special was observed in ovaries, uterus with cervix and vagina from female no. 71 which failed to produce a pregnancy.
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Reproductive Indices:
There were no treatment related effects observed for the reproductive indices (Copulation, fertility, delivery and viability indices) in dose groups when compared with the control group.
However, the copulation and fertility indices were slightly reduced in HD group, when compared to controls. The fertility index in HD group was 90% when compared to control, LD and MD groups wherein the fertility index was 100 %. Three animals in HD group (Animals 71, 77 and 78) did not show the evidence of mating through vaginal smear, but two females (Animals 77 and 78) were pregnant and littered normally. Female no. 71 did not show the evidence of mating and was non pregnant. Histopathological evaluation of this animal and its male partner did not show any evidence of toxicological relevance. The slight reduction in viability index in the HD group (98.99 %) was due to mortality of 1 female pup (pup no.11, Dam no. 77). This being noted in single isolated female animal, the finding was considered to be incidental.

Pre-coital Interval and Duration of Gestation:
There were no treatment related effects on the duration of pre-coital interval and the duration of gestation of dose group females when compared to control. There were no statistically significant changes noted.

Pre- and Post-Natal Data:
There were no changes of toxicological relevance noted for the pre and post-natal data including number of corpora lutea, number of implantation sites, number of live pups and percent pre- and post- implantation loss in dose groups when compared to the control. There were no statistically significant changes noted. One female (no. 60) of LD group did not deliver the pups and appeared non gravid. However, at necropsy the uterus showed the implantation sites. As these intrauterine deaths were in single female of LD group, the finding was not considered to be of toxicological relevance. However, there was higher (slight to marked) percent pre-implantation loss in dose groups when compared to control. But in the absence of statistical significance and dose related response, the finding was not considered to have any toxicological relevance.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the study findings

Critical effects observed:

no

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:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Litter Data:
There were no changes of toxicological relevance noted for the litter data including total number of pups born, number of still births, number of runts on PND 0 and number of live pups, number of male and female pups, sex ratio on PND 0 and PND 4 when compared to controls. There were no statistically significant changes noted for the litter data between the dose and the control groups. However, there was a slightly, but not statistically significantly lower number of live pups and number of female pups observed on PND 0 and PND 4 in LD and HD groups when compared to controls. As this was not dose related effect, these changes had no toxicological relevance.

Litter Weight Data:
There were no changes of toxicological relevance noted for litter weight data including pup mean weight, total litter weight and male litter and female litter weight in dose groups when compared to the control. There were no statistically significant changes noted. However, there was a slightly lower total litter weight and male litter weight in LD group and slightly lower female litter weight in LD and HD groups when compared to Control group on PND 0 and 4. But there was no dose related response and no statistically significant decrease noted. Thus, the changes were considered to be incidental.

Precoital interval and duration of gestation:
There were no treatment related effects on the duration of precoital interval and the duration of gestation of dose group females when compared to control. There were no statistically significant changes noted.

Pre and Postnatal Data:
There were no changes of toxicological relevance noted for the pre and post natal data including number of corpora lutea, number of implantation sites, number of live pups and percent pre- and post- implantation loss in dose groups when compared to the control. There were no statistically significant changes noted. One female (no. 60) of LD group did not deliver the pups and appeared non gravid. However at necropsy the uterus showed the implantation sites. As these intrauterine deaths were in single female of LD group, the finding was not considered to be of toxicological relevance. However, there was higher (slight to marked) percent pre implantation loss in dose groups when compared to control. But in the absence of statistical significance and dose related response, the finding was not considered to have any toxicological relevance.


Reproductive Indices:
There were no treatment related effects observed for the reproductive indices (Copulation, fertility, delivery and viability indices) in dose groups when compared with the control group.
However, the copulation and fertility indices were slightly reduced in HD group, when compared to controls. The fertility index in HD group was 90 % when compared to control, LD and MD groups wherein the fertility index was 100 %. Three animals in HD group (Animals 71, 77 and 78) did not show the evidence of mating through vaginal smear, but two females (Animals 77 and 78) were pregnant and littered normally. Female no. 71 did not show the evidence of mating and was non pregnant. Histopathological evaluation of this animal and its male partner did not show any evidence of toxicological relevance. The slight reduction in viability index in the HD group (98.99 %) was due to mortality of 1 female pup (pup no.11, Dam no. 77). This being noted in single isolated female animal, the finding was considered to be incidental.

Pup Survival Data
There was no treatment related changes observed on the survival of the pups from PND 0 to PND 4 in dose groups when compared to the control groups. One female pup (pup no. 11) from female no. 77 of HD group was missing on PND 2. This missing pup was considered to be cannibalised by the dam. This being single isolated pup of single isolated female animal, the finding was considered to be incidental.

Pup External Findings:
There were no gross external abnormalities of toxicological relevance observed in dose groups when compared to the control. However, there were few incidences including crust on head and snout (pup no.1, Dam no. 54) in LD group and small pup (pup no. 11, Dam no. 77) in HD group. These findings were observed in single pup of single isolated female of LD or HD groups. Thus, the findings were considered to be incidental in origin.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on overall study findings

Reproductive effects observed:

not specified

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 104.3 %, 98.1 % and 101.9 % 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 129.2 (for LD dose) and 98.8 % (for HD dose). 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 between 119.0 % and 105.8 % of the nominal value and between 109.6 % and 82.6 % of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle and bottom) were between 11.8% and 0.9% in LD dose group, between 12.3 % and 11.6 % in HD dose group.

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with FAT 40824/B in male and female Wistar rats, the NOAEL was considered to be 1000 mg/kg body weight/ day for reproduction toxicity and offspring developmental toxicity.

Executive summary:

A GLP-compliant study was carried out to assess the possible effects of FAT 40824/B on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. The study was carried out according to OECD guideline 421. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and 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 total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The doses evaluated were 0, 100, 300 and 1000 mg/kg body weight/ day. The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad iniectabilia 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 body weight. During the period of administration, the animals were observed each day for signs of toxicity. 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 males and females, 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 mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery 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 after day 26 from the last day of mating period. The numbers of implantation sites and corpora lutea were recorded for each parental female at necropsy. Pups sacrificed (by decapitation) on postnatal day 4 and those found dead, were carefully examined for gross external abnormalities. A histopathological evaluation of Testes, epididymis, ovaries, uterus with cervix, vagina, and accessory sex organs (prostate, seminal vesicle with coagulating gland) was performed on high dose and control animals. Gross lesion macroscopically identified was examined microscopically in all animals.

 

Summary Results

No mortality occurred in the control or any of the dose groups during the treatment period of this study. There were no clinical signs of toxicological relevance noted in dose groups when compared to the controls. However, there were clinical signs including nasal discharge (red coloured), slight to moderate salivation in male HD group animals and there were incidences of slight to severe piloerection, moderate salivation, moving the bedding and slight abnormal breathing in female HD groups. There were also few incidences of eschar, nasal discharge, slight to moderate salivation and abnormal breathing males and/ or females of LD and/ or MD groups. These clinical signs observed in both male and female animals were transient and had no toxicological relevance. There were no treatment related changes for body weight, body weight gain and food consumption of male and female animals of dose groups when compared to the control group during the study. There were no changes of toxicological relevance for litter data including total number of pups born, number of still births, number of runts on PND 0 and number of live pups, number of male and female pups, sex ratio, on PND 0 and PND 4. There were no changes of toxicological relevance for litter weight data including pup mean weight, total litter weight, male and female litter weight in dose groups when compared to the control. There were no treatment related effects observed on the duration of precoital interval and the duration of gestation of dose group females when compared to controls. There were no changes of toxicological relevance in number of corpora lutea, number of implantation sites, number of live pups and percent pre and post implantation loss in dose groups when compared to the control.

There were no treatment related effects observed for the reproductive indices (Copulation, fertility, delivery and viability indices) in dose groups when compared with the control group. The fertility index in HD group was 90% when compared to 100% fertility index of control, LD and MD groups.

There were no treatment related changes observed on the survival of the pups from PND 0 to PND 4 in dose groups when compared to the control groups. There were no gross external abnormalities of toxicological relevance observed in dose groups when compared to the control.

Gross pathological observation of females at necropsy revealed no treatment related findings. The kidneys of 9/10 males of the HD group were discolored dark or red, which correlated microscopically with hyaline droplets in the proximal tubules. This change was not considered to be an adverse effect of treatment, as there were no further indicators of renal injury. Histopathologically there was no evidence of toxicity in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. In addition, 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 remainder of findings was within the range of normal background lesions which may be recorded in animals of this strain and age. Formulation analysis was performed on samples of all dose groups collected at various intervals during the study. The concentration verification of samples of all dose groups was investigated in study weeks 1, 3, 5 and 7. The mean recoveries in LD, MD and HD groups were 104.3 %, 98.1 % and 101.9 % of the nominal concentration, respectively. The Stability of formulation samples of LD and HD dose groups was investigated in study week 1.the recoveries after 6 hours storage at room temperature compared to starting value was 129.2 (for LD dose) and 98.8 % (for HD dose). The Homogeneity of formulation samples of LD and HD dose groups was determined in study week 1 and 5. The mean recoveries observed for LD dose group was between 119.0 % and 105.8 % of the nominal value and between 109.6 % and 82.6 % of the nominal value for HD dose group. The coefficients of variation of the different sampling levels (top, middle and bottom) were between 11.8 % and 0.9 % in LD dose group, between 12.3 % and 11.6 % in HD dose group. On the basis of this reproduction/ developmental toxicity screening test with FAT 40824/B TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight/ day the following conclusions can be made: At a dose level of 1000 mg/kg body weight/ day slight to moderate hyaline droplets in the proximal tubules were recorded in all kidneys with macroscopic alteration. There were no indications of renal injury, and hence, this was considered not to be adverse under the condition of this study. There were no signs of reproduction and developmental toxicity observed in this study. Thus, the NOAEL in this study was considered to be 1000 mg/kg body weight/ day for adult animals (both males and females) including reproduction toxicity and offspring developmental toxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline: reproduction/ developmental toxicity screening test

Deviations:

yes

Remarks:

See overall remarks, attachment section

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Name: FAT 40824/B TE
Batch No.: BOP 01-12 (ROE 805)
Physical State: solid at 20 °C
Storage Conditions: room temperature
Expiry Date: 20 April 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Specific details on test material used for the study:

Name: FAT 40824/B TE
Batch No.: BOP 01-12 (ROE 805)
Physical State: solid at 20 °C
Storage Conditions: room temperature
Expiry Date: 20 April 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 were 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: 276 – 318 g (mean: 301.13 g, ±20 % = 240.9 – 361.35 g)
females: 175 - 214 g (mean: 196.35 g, ±20 % = 157.08 – 235.62 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: 10x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0801)
- 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 kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (240113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was weighed into a tarred plastic vial on a precision balance and the required volume of vehicle (sterile water) was added and subjected to homogenization using a homogenizer (ultra turrax). The homogeneity of the test item formulation was ensured by vortexing the formulation on vortex machine for minimum 1 minute. The test item formulation was prepared freshly on each administration day before the administration procedure. The vehicle was also used as control item.

The following doses were evaluated
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Mid Dose: 300 mg/kg body Weight
High Dose: 1000 mg/kg body Weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. This dose also happens to be the limit dose. 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 aqua ad injectabile using the dose volume 5 ml/ kg body weight.

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 in the last week of the study (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 the day of sample collection and were stored at -20 °C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 134527.

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. 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:

The female animals were treated with the test item formulation or vehicle on 7 days per week basis for a period of 54 days, i.e. during 14 days of pre-mating and 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 was completed.

Frequency of treatment:

7 days/week

Duration of test:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and 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 total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3 (Middle dose)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Time Schedule: Daily
General clinical observations were made once a day approximately at the same time each day 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. 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:
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.

FOOD CONSUMPTION:
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.

Fetal examinations:

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 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 tattoo mark on the paws. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p <0.05 was considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

However, there were clinical signs including nasal discharge (red coloured), slight to moderate salivation in male HD group animals. Incidences of nasal discharge and slight salivation were also in single isolated males of LD or MD groups. In female HD group animals, there were incidences of slight to severe pilorection, moderate salivation, moving the bedding, slight abnormal breathing. There were also lower incidences of alopecia, eschar, nasal discharge, slight to moderate salivation in isolated animals of control or LD or MD groups. The clinical signs observed in both male and female animals of control and/ or dose groups were transient and had no toxicological relevance. Alopecia in various parts of the body was seen in some animals of control and MD group and these were observed in few isolated animals. Thus, this finding was considered to be incidental.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in food consumption of male and female animals of the dose groups, when compared to control group during the treatment period. There were no statistically significant differences between the control and dose groups. There was a slightly higher (but not statistically significant) food intake observed in HD group males, when compared to controls. As values were within the normal range of variations, this effect was not assumed to be toxicologically relevant.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

The necropsy showed discoloured (dark or red) kidneys in 9 out of 10 male HD group animals. This correlated microscopically with hyaline droplets in the proximal tubules, which was not considered to be adverse under the condition of this study. Besides, there were no macroscopic abnormalities in this study. A unilateral yellow spot in the epididymis was recorded in male no. 23 of MD group. This correlated microscopically with spermatic granuloma and was spontaneous lesion which is commonly seen in male rats.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

However, microscopically, the treatment-related findings were recorded in the kidney that was examined as the gross lesions of the HD group. Slight to moderate hyaline droplets in the proximal tubules were recorded in all kidneys with macroscopic alteration, except for one male (no. 38: the tissues had not been preserved after necropsy). There were no further indicators of renal injury, and hence, this was considered not to be adverse under the condition of this study.
Unilateral yellow spot in the epididymis recorded in male no. 23 of MD group was correlated microscopically with spermatic granuloma, and was spontaneous lesion which is commonly seen in male rats. The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. There were not treatment-related effects on the completeness of stages or cell populations of the testes. Some histologic alterations were recorded at a minimum severity in a few animals from both of the control and high-dose males, but all of these were considered to be within the range of normal background lesions which are occasionally observed in males of this strain and age. Nothing special was observed in ovaries, uterus with cervix and vagina from female no. 71 which failed to produce a pregnancy.
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no changes of toxicological relevance noted for the pre and post natal data including number of corpora lutea, number of implantation sites, number of live pups and percent pre- and post- implantation loss in dose groups when compared to the control. There were no statistically significant changes noted.
One female (no. 60) of LD group did not deliver the pups and appeared non gravid. However at necropsy the uterus showed the implantation sites. As these intrauterine deaths were in single female of LD group, the finding was not considered to be of toxicological relevance.
However, there was higher (slight to marked) percent pre implantation loss in dose groups when compared to control. But in the absence of statistical significance and dose related response, the finding was not considered to have any toxicological relevance.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

One female pup (pup no. 11) from female no. 77 of HD group was missing on PND 2. This missing pup was considered to be cannibalised by the dam. This being single isolated pup of single isolated female animal, the finding was considered to be incidental.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: Overall study findings

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not specified

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

One female pup (pup no. 11) from female no. 77 of HD group was missing on PND 2. This missing pup was considered to be cannibalised by the dam. This being single isolated pup of single isolated female animal, the finding was considered to be incidental.

External malformations:

no effects observed

Description (incidence and severity):

However, there were few incidences including crust on head and snout (pup no.1, Dam no. 54) in LD group and small pup (pup no. 11, Dam no. 77) in HD group. These findings were observed in single pup of single isolated female of LD or HD groups. Thus, the findings were considered to be incidental in origin.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Details on embryotoxic / teratogenic effects:

There were no embryotoxic or teratogenic effects seen.

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: Overall study findings

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 104.3 %, 98.1 % and 101.9 % 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 129.2 (for LD dose) and 98.8 % (for HD dose). 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 between 119.0 % and 105.8 % of the nominal value and between 109.6 % and 82.6 % of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle and bottom) were between 11.8 % and 0.9 % in LD dose group, between 12.3 % and 11.6 % in HD dose group.

Applicant's summary and conclusion

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with FAT 40824/B in male and female Wistar rats, the NOAEL was considered to be 1000 mg/kg body weight/ day for developmental toxicity.

Executive summary:

A GLP-compliant study was carried out to assess the possible effects of FAT 40824/B on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. The study was carried out according to OECD guideline 421. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and 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 total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The doses evaluated were 0, 100, 300 and 1000 mg/kg body weight/ day. The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad iniectabilia 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 body weight. During the period of administration, the animals were observed each day for signs of toxicity. 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 males and females, 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 mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery 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 after day 26 from the last day of mating period. The numbers of implantation sites and corpora lutea were recorded for each parental female at necropsy. Pups sacrificed (by decapitation) on postnatal day 4 and those found dead, were carefully examined for gross external abnormalities. A histopathological evaluation of Testes, epididymis, ovaries, uterus with cervix, vagina, and accessory sex organs (prostate, seminal vesicle with coagulating gland) was performed on high dose and control animals. Gross lesion macroscopically identified was examined microscopically in all animals.

Summary Results

No mortality occurred in the control or any of the dose groups during the treatment period of this study. There were no clinical signs of toxicological relevance noted in dose groups when compared to the controls. However, there were clinical signs including nasal discharge (red coloured), slight to moderate salivation in male HD group animals and there were incidences of slight to severe piloerection, moderate salivation, moving the bedding and slight abnormal breathing in female HD groups. There were also few incidences of eschar, nasal discharge, slight to moderate salivation and abnormal breathing males and/ or females of LD and/ or MD groups. These clinical signs observed in both male and female animals were transient and had no toxicological relevance. There were no treatment-related changes for body weight, body weight gain and food consumption of male and female animals of dose groups when compared to the control group during the study. There were no changes of toxicological relevance for litter data including total number of pups born, number of still births, number of runts on PND 0 and number of live pups, number of male and female pups, sex ratio, on PND 0 and PND 4. There were no changes of toxicological relevance for litter weight data including pup mean weight, total litter weight, male and female litter weight in dose groups when compared to the control. There were no treatment related effects observed on the duration of precoital interval and the duration of gestation of dose group females when compared to controls. There were no changes of toxicological relevance in number of corpora lutea, number of implantation sites, number of live pups and percent pre and post implantation loss in dose groups when compared to the control.

There were no treatment related effects observed for the reproductive indices (Copulation, fertility, delivery and viability indices) in dose groups when compared with the control group. The fertility index in HD group was 90% when compared to 100 % fertility index of control, LD and MD groups.

There were no treatment related changes observed on the survival of the pups from PND 0 to PND 4 in dose groups when compared to the control groups. There were no gross external abnormalities of toxicological relevance observed in dose groups when compared to the control.

Gross pathological observation of females at necropsy revealed no treatment related findings. The kidneys of 9/10 males of the HD group were discolored dark or red, which correlated microscopically with hyaline droplets in the proximal tubules. This change was not considered to be an adverse effect of treatment, as there were no further indicators of renal injury. Histopathologically there was no evidence of toxicity in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. In addition, 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 remainder of findings was within the range of normal background lesions which may be recorded in animals of this strain and age. Formulation analysis was performed on samples of all dose groups collected at various intervals during the study. The concentration verification of samples of all dose groups was investigated in study weeks 1, 3, 5 and 7. The mean recoveries in LD, MD and HD groups were 104.3 %, 98.1 % and 101.9 % of the nominal concentration, respectively. The stability of formulation samples of LD and HD dose groups was investigated in study week 1.the recoveries after 6 hours storage at room temperature compared to starting value was 129.2 (for LD dose) and 98.8 % (for HD dose). The Homogeneity of formulation samples of LD and HD dose groups was determined in study week 1 and 5. The mean recoveries observed for LD dose group was between 119.0 % and 105.8 % of the nominal value and between 109.6% and 82.6% of the nominal value for HD dose group. The coefficients of variation of the different sampling levels (top, middle and bottom) were between 11.8 % and 0.9 % in LD dose group, between 12.3 % and 11.6 % in HD dose group. On the basis of this reproduction/ developmental toxicity screening test with FAT 40824/B in male and female Wistar rats, the NOAEL was considered to be 1000 mg/kg body weight/ day for developmental toxicity.