2-{[(2-{3-[(4-{Ethyl[3-(vinylsulfonyl)phenyl]amino}-6-fluoro-heteromonocycle-2-yl)amino]-2- (hydroxy-O)phenyl}hydrazono-N2)(phenyl)methyl]diazenyl-N1}benzoato(6-)-O]cuprate(4-) sodium salt, polysulfo

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April 2012 - 25 January 2013

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 - 302 g, (mean: 288.48 g, ± 20% = 230.78 – 346.17 g), females: 167 - 210 g,
(mean: 186.53 g, ± 20% = 149.22 – 223.83 g).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Adequate acclimatisation period (at least 5 days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3°C
- Humidity (%): 55 +- 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: From: 09 May 2012 To: 22 June 2012

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

aqua ad injectionem

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 20 mg/ml, 60 mg/ml, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required): Manufacturer: Diprom; Batch No.: 10952-1
- Purity: 100 %

The test item was administered daily during 14 days pre-mating and 14 days mating period in both male and female animals,
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 has been completed.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL/kg body weight.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 weeks
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged (how):individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for the nominal concentration verification were taken in study week 5, study week 6, and study week 7 of freshly prepared control, low,
medium, and high dose groups (12 samples in total).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 5 and 6
(12 samples in total)
All concentration samples were stored frozen (approximately -20°C) until the analysis was performed.
The determination of test item concentration in the dosing formulations was performed by the analytics department of BSL BIOSERVICE Scientific
Laboratories GmbH under the BSL study no. 120827 in accordance with GLP.

Duration of treatment / exposure:

Males are dosed for a minimum of four weeks and up to day before scheduled kill (this includes a minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating).
Females are dosed throughout the study. This includes two weeks prior to mating (two estrous cycles), till conception, during gestation and up to
four days after parturition.

Frequency of treatment:

Once per Day. 7 Days per week.

Details on study schedule:

- F1 parental animals not mated

Doses / concentrations

Remarks:

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

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). The study included one control (C)
and three dose groups (LD, MD, HD).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
- Rationale for animal assignment (if not random): random

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily except weekend and public holidays where it was performed once daily


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: In the male animals the body weight was taken weekly during the entire study period.
In the female animals the body weight was taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0
(within 24 hours of parturition) and post-natal day 4 along with pups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration.
Food consumption was not measured during the mating period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
[testis weight, epididymis weight, weight of prostate with seminal vesicles and coagulating gland]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external abnormalities]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30
- Maternal animals: All surviving animals on post-natal day 4 along with pups

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The testes, epididymides, and prostate, seminal vesicles with coagulating glands as a whole of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately. Organ weights of animals found dead were not taken.

A histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period.

Postmortem examinations (offspring):

SACRIFICE
All F1 offspring on PND4

GROSS NECROPSY
- Gross necropsy consisted of [external evaluation.]

HISTOPATHOLOGY / ORGAN WEIGTHS
No

Statistics:

A statistical assessment of the results of the body weight, food consumption and absolute and relative organ weights was performed for each
gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed
with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

fertility index, copulation index, delivery index, viability index

Offspring viability indices:

No

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A slightly higher incidence of clinical symptoms in the HD group could be mentioned

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the testis, a minimal or mild amount of greyish pigment in interstitial macrophages was observed in 8/10 males treated at 1000 mg/kg/day.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Mortality
No mortalities were recorded at any dose levels.

Clinical Observations
A few clinical symptoms were noticed during the application period of male and female animals and their incidence was slightly higher in
treatment groups.
For the male animals no clinical symptom was found in C and LD group. In MD group, one individual male animal exhibited salivation.
In HD group, 1 animal exhibited salivation, 2 animals exhibited a swollen cheek, one animal exhibited a swollen chin, and 2 animals exhibited
bluish skin.
One individual female animal had eschar at the right corner of the mouth. In the LD group eschar at the right shoulder as well as salivation
were observed at one respective individual animal. In the MD group salivation, moving the bedding, and red nasal discharge could be exhibited
at one respective animal. An increase in clinical symptoms was found in the HD group: the most prominent were salivation (4/10 female animals),
and piloerection (2/10 female animals). Other clinical findings in female HD group were moving the bedding, red nasal discharge, alopecia, and a
little wound.
The slightly higher incidence of clinical symptoms in the HD group is assumed to be test item related. However, a toxicological relevance within
this study cannot be clearly mentioned.

Body Weight Development
In both males and females, the mean body weight increased with the progress of the study in the C, LD, MD, and HD groups.
Throughout the treatment period, body weights were within the normal range of variation for this strain.

Food Consumption
In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in the C, LD, MD, and HD groups.
No considerable effect of FAT 40853/A TE on food consumption was found in any of the other groups of male animals and in female animals.

Precoital Interval and Duration of Gestation
No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared the treatment groups with the control group. The values were comparable between the groups. All pregnancies resulted in normal births.

Reproductive Indices
A reduced fertility index (number of pregnant females/ number of copulated females X 100) was observed in the LD group (80 %) as compared
to control (90 %), MD (90 %) and high dose group (90 %). This is not assumed to be test item related.
No difference was found in treatment groups for copulation index, delivery index, as well as viability index when compared to C group.

Gross findings
Few specific gross pathological changes were recorded for the male and female animals in C and treatment groups. Discoloured red mesenteric
lymph nodes were observed in 1/10 male C, 5/10 male LD, and 3/10 male MD animals. Yellow spots (up to 1 cm) on the right epididymides were
observed in 2/10 male LD animals. 1/10 female HD animals exhibited a fluid distension in the uterus. These findings were not considered to be
test item related but occurred spontaneously.
Due to the colour of the test item, several tissues and organs were discoloured blue in treatment groups. The following blue discolourations
could be found for male animals: kidneys, ileum, testes, peyer´s patches, stomach, skin, axillary lymph nodes, epididymides, caecum, colon, rectum, urinary bladder, eyes, mandibular lymph nodes, parotid/sublingual salivary glands, mesenteric lymph nodes, seminal vesicles, thymus, and prostate. These discolourations were most prominent in HD animals, followed by MD and LD animals. It is not assumed to be of toxicological relevance.

Organ Weight
In males and females, there was no statistically significant difference in the absolute and relative organ weights of the treatment groups when
compared with the control group. No biological relevant differences could be mentioned. The only slight difference was found for the weights
of the ovaries in the HD group, which were slightly higher than the weights of the ovaries in the C group. However, due to the high standard
deviation and due to the slight difference, this is not assumed to be of toxicological relevance.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fertility, no effects on fertility were observed in male and female rats exposed to 100, 300 and 1000 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

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

Details on results (F1)

Litter Data
The total number of pups born, the sex ratio and the number of viable pups on postnatal day 0 or 4 were comparable among the groups.

Litter Weight Data
No treatment-related effect on litter weight was observed despite a significant increase in litter weight in the MD. However, as no dose-relatinship was present, the effect is considered as incidental.

Uterine content
A slight but not statistically significant increase in the number of corpora lutea was observed in mid- and high-dose females whereas the number of implantations sites and post-implantation loss were unremarkable compared to the control group. A significant increase in the percentage of pre-implantation loss in the high-dose group of 29.85% compared with 8.75% in the control group was determined. However, it has to be considered that the number of corpora lutea was increased about 19% in high-dose females without affecting the number of implantation sites or the number of post-implantation loss. Further, no effects were observed on the number of born pups and their viability. Thus, the biological significance of the observed pre-implantation loss is questionable.


Pup External Findings
No treatment-related gross external findings were observed in any of the treated groups neither on day 0 nor on day 4. Few incidences of external findings were observed in the LD (dark snout), the MD (dark snout) and the HD (hematoma on top of head, dark snout) which were considered to be spontaneous and not related to the test item.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity, no substance-related effects were observed on development, growth and viabilty of fetuses, no treatment-related increase in malformations

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The male wistar rats after being treated with FAT 40853/A TE in aqua ad injectionem for the period of 28 days at the dosage of 100, 300 and 1000
mg/ kg body weight revealed no clear signs of toxicity in the treatment groups when compared to the corresponding controls. Hence, the no
observed adverse effect level (NOAEL) for sytemic toxicity to male adult animals is assumed to be 1000 mg/kg body weight.
The female rats were dose administered for approximately 54 days at the same dosage. There was no treatment related changes observed for any of the parameters when compared to the control. Hence, the no observed adverse effect level (NOAEL) for maternal and systemic toxicity is assumed to be 1000 mg/kg body weight.
No effects were observed on the examined litter data including number of pups born, sex ratio, litter weight, number of viable pups and gross external findings. Therefore, a NOAEL > 1000 mg/kg bw/day is assumed for developmental toxicity.

Executive summary:

The aim of this study was to assess the possible effect of the test substance on male and female fertility and embryofetal development in Wistar rats. In this study, four groups comprising 10 adult male and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with100, 300 and 1000 mg/kg body weight per day at a dose volume of 5 mL/kg body weight. The test item was formulated in aqua ad injectionem (sterile water). Control animals were handled identically as treated groups and received aqua ad injectionem (sterile water) in a similar volume as the treated groups. The test item formulations were prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation up to post natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted based on the most recent body weight measurement. Animals were examined daily for clinical signs and mortality. Body weight and food consumption was measured weekly except the mating period. After 14 days of treatment, animals were paired (1:1) for a maximum of 14 days. The subsequent morning, vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. Males and females were sacrificed on day 29 and post natal day 4, respectively and subjected to necropsy. Non pregnant females were sacrificed on their respective gestation day 26 after the evidence of mating or completion of mating period. A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. The examinations of these organs were extended to animals of the medium and low dose groups if treatment-related changes were observed in high dose groups.

 

Summary Results

No mortalities were recorded at any dose levels.

A slightly higher incidence of clinical symptoms in the HD group was determined including salivation, swollen cheek or chin, pale bluish skin, moving of the bedding, nasal discharge, piloerection, alopecia at forelimbs and abdomen. Throughout the treatment period, body weights were within the normal range of variation for this strain and no difference between C and treatment group was discovered. No considerable effect on food consumption was found in any of the groups of male and female animals. No treatment-related effect on the litter data was observed such as the total number of pups born, number of male and females, sex ratio, and live pups on PND 0 and PND 4. Litter weights were comparable among the groups on PND 0 and PND 4. Precoital intervals and duration of gestation did not reveal differences in the test and control groups. The group mean numbers of corpora lutea, number of implantation sites, number of live pups born on PND 0, and percentage of post-implantation loss remained unaffected. A significant increase in the percentage of pre-implantation loss was determined in the high-dose group. However, as the number of corpora lutea was increased in high-dose females without affecting the number of implantation sites, the number of post-implantation loss or the number of born pups and their viability, the biological significance of the observed increase in pre-implantation loss is questionable. Despite the LD group, fertility indices of treated animals were comparable to controls. Copulation index, delivery index and viability index were comparable among the groups. No treatment-related gross external findings were observed in any of the treated groups. Due to the colour of the test item, several tissues and organs were discoloured blue in treatment groups. These discolourations were most prominent in HD animals, followed by MD and LD animals.

In males and females, there was no statistically significant difference in the absolute and relative organ weights of the treatment groups when compared with the control group. In the testes, a minimal or mild amount of greyish pigment in interstitial macrophages was observed in 8/10 males treated at 1000 mg/kg/day. No test item-related histological findings were noted in the other male and in the female reproductive organs.