Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 06, 2017- April 8th , 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Preliminary Prenatal Developmental Toxicity Study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 19. Sep. 2016 to 12. Oct. 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

In order to select the dosages for a main study (Reproduction/Developmental Toxicity Screening Test), the test item is administrated orally at the dose levels of 50, 200 and 800 mg/kg/day and the effects on pregnancy and embryo-foetal development in the rat are investigated in a study similar to OECD Guideline 414.

GLP compliance:

no

Remarks:

This study was exempt from compliance with Good Laboratory Practice regulations of the OECD. However, it was carried out in a GLP compliant facility.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italy S. P. A., Calco, Lecco, Italy
- Age and weight at study initiation:
♀ 9 weeks old; 200 to 225 g
♂ at least 11 weeks old; at least 340 g
- Housing: limited access rodent facility
During the pre-pairing period and after mating: no more than 5 of one sex to a cage, in polisulphone cages measuring 59.5 × 38 × 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate VA, Italy). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 3 times a week.
During the mating period: 1 male and 1 female per cage; clear polycarbonate cages measuring 42.5 × 26.6 × 18.5 cm with stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
- Diet: ad libitum; commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese MI, Italy)
- Water: ad libitum; via water bottles
- Acclimation period: 25 days
- Identification: tattoo on the hind feet

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ±2 °C
- Humidity: 55 % ±15 %
- Air changes: 15 to 20 per hour
- Photoperiod: 12 hours artificial light each day

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was dissolved in the vehicle. The formulations were prepared daily (concentrations of 5, 20 and 80 mg/mL) and the concentrations were calculated and expressed in terms of test item as supplied. The formulations were prepared using a homogeniser and were stable up to 2 hours from preparation (Sponsor’s communication). The formulations were maintained under magnetic stirring until completion of dosing.

DOSING:
- 10 mL/kg bw daily from day 6 to day 19 post coitum.

Details on mating procedure:

- M/F ratio per cage: paired one to one in the home cage of the male
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made; presence of sperm in the vaginal smear or by the presence of a copulation plug; referred to as day 0 of gestation / day 0 post coitum
- After unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- After successful mating: each pregnant female was weighed and allocated to groups, no more than 3 per cage, by computerised stratified randomisation to give approximately equal initial group mean body weights

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

once a day

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

medium dose

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

6 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
- The dose levels of 50, 200 and 800mg/kg/day were selected by the Sponsor, based on the results of a previous study (subacute 28 day oral toxicity study in Wistar rats) in which the dose of 50 mg/kg/day was identified as NOEL and doses of 200 and 1000 mg/kg/day showed changes due to treatment mainly in female rats.

ALLOCATION TO GROUPS:
- On the day of allocation (Day 0 post coitum), all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 3 to a cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.

Positive control:

No

Parental animals: Observations and examinations:

MORTALITY:
- Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays, a similar procedure was followed except that the final check was carried out at approximately mid-day.
- Time schedule: daily

CLINICAL SIGNS:
- All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: on days 0, 3, 6, 9, 12, 15 and 20 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE: no

WATER CONSUMPTION AND COMPOUND INTAKE: no

Litter observations:

PARAMETERS EXAMINED
- The following parameters were examined in offspring: litter data (pre-, post- and total implantation loss), mean foetal weight and sex ratios
- Pre-implantation loss was calculated as a percentage from the formula:
Pre-implantation loss % = ((no. corpora lutea) - (no. implantations)) / (no. corpora lutea) × 100
- Post-implantation loss was calculated as a percentage from the formula:
Post-implantation loss % = ((no. implantations) - (no. live foetuses)) / (no. implantations) ×100
- Total implantation loss was calculated as a percentage from the formula:
Total implantation loss % = ((no. corpora lutea) - (no. live foetuses)) / (no. corpora lutea) ×100
- Sex ratios of the foetuses were calculated as the percentage of males per litter.
- All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Postmortem examinations (parental animals):

SACRIFICE
- All test animals were killed on Day 20 post coitum and necropsied. All animals in extremis and those that had completed the scheduled test period were killed with carbon dioxide.
- All test animals, including those found dead, were subjected to necropsy, supervised by a pathologist.

GROSS NECROPSY
- The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The ovaries and uteri were examined to determine: gravid uterine weight; number of corpora lutea; number of implantation sites; number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); number of intra-uterine deaths; gross evaluation of placentae.
- Intra-uterine deaths were classified as: early resorptions (only placental remnants visible); late resorptions (placental and foetal remnants visible).
- Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.

Postmortem examinations (offspring):

SACRIFICE
- Foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia at the termination of the study (day 20 post coitum)
- These animals were subjected to postmortem examinations: litter data, mean foetal weight and sex ratios

EXAMINATION OF THE FOETUSES:
- All live foetuses were examined externally.
- Foetuses with apparent malformations were retained in Bouin fixative.
- Structural deviations were classified as follows:
Malformations: major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies: minor abnormalities that are detected relatively frequently.
Variants: a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Controls: 1 test animal demonstrated moderate hair loss on the head (day 20 only) and neck (days 15 to 19)
50 mg/kg bw/day: 1 test animal demonstrated slight hair loss on the head (days 8 to 19)
200 mg/kg bw/day: 1 test animal demonstrated moderate scabs on the head and slight hair loss on the head (day 20 only)
800 mg/kg bw/day: 4 test animals demonstrated red staining on the tail (days 13 to 20 (2 animals) and day 20 only (2 animals))

Scabs and hairloss: The isolated cases of hairloss and/or scabs noted are commonly observed in animals of this species under the same experimental conditions and are not considered related to treatment.
Staining: no toxicological relevance was attributed to this finding, since it was most likely due to the red colour of the test item.

No signs of reactions to treatment were noted during the dosing period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animals died during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences in terminal body weight and absolute weight gain were observed between control and treated groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

PREGNANCY RATE:
One female which received 50 mg/kg bw/day and one female which received 800 mg/kg bw/day were not pregnant.
Number of females with live foetuses on gestation day 20:
Control: 6
50 mg/kg bw/day: 5
200 mg/kg bw/day: 6
800 mg/kg bw/day: 5

FOETUS ANOMALIES:
50 mg/kg bw/day: 1 test animal carried 1 foetus of small weight (carrying 17 foetuses in total). This was considered incidental.

LITTER DATA:
Litter data, mean foetal weight and sex ratios were not affected by treatment.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Based on:

test mat.

Sex:

female

Remarks on result:

other: The high dose level for the subsequent main study could be up to 800 mg/kg bw/day

Conclusions:

The high dose level for the subsequent Reproduction/Developmental Toxicity Screening Test could be up to 800 mg/kg/day.

Executive summary:

Screening for reproductive/developmental toxicity was evaluated in a Preliminary Prenatal Developmental Toxicity Study according to the OECD Guideline 414 (2001). 24 female, Sprague-Dawley rats were mated with males (day 0) then administered doses of 50, 200 and 800 mg/kg bw/day of substance (6 rats per dose level) by oral gavage, from 6 to 20 days post coitum. A control group of an additional 6 rats were administered solely the vehicle, softened water. All animals were sacrificed on day 20.

Body weight was recorded on days 0, 3, 6, 9, 12, 15 and 20; and mortality and clinical signs were recorded daily. All females were caesarean-sectioned on day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and foetus sex was recorded. All foetuses were examined for external abnormalities.

Mortality

No animals died during the study. One female in the low dose group and one female in the high dose group were not pregnant. The number of females with live foetuses on gestation Day 20 was 6 in each of the control and mid-dose groups and 5 in each of the low and high dose groups.

Clinical signs

No signs of toxicological significance were noted during the study and no signs of reactions to treatment were observed during the dosing period. Red staining of the tail was noted in 4 females of the high dose group from gestation Day 13 or on the day of necropsy. This change was likely due to the colour of the test item and was considered of no toxicological relevance.

Body weight and body weight gain

Body weight and body weight gain were comparable between the control and treated groups. Terminal body weight, uterus weight and absolute weight gain No differences in terminal body weight, gravid uterus weight and absolute weight gain were observed in treated groups, when compared to the control group.

Litter data and sex ratios

Litter data, mean foetal weight and sex ratios were not affected by treatment.

Macroscopic examination

The most relevant change observed in high dose females was red staining of the tail. This change was likely due to the colour of the test item. The remaining changes observed were incidental or characteristically seen in untreated Sprague Dawley rats of the same age.

External examination of foetuses

At the external examination, one small foetus was present in the low dose group. This isolated case was considered incidental and not related to treatment.

Based on the results observed, the substance does not induce reproductive or developmental toxicity in pregnant female rats or their foetuses at doses of up to and including 800 mg/kg bw/day orally.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

EC Number:

418-220-4

EC Name:

Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

Molecular formula:

C52H32CrN18Na3O20S4

IUPAC Name:

chromium(3+) trisodium bis(6-amino-2-{4-[2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]benzenesulfonamido}-5-[2-(3-nitro-2-oxido-5-sulfonatophenyl)diazen-1-yl]pyrimidin-4-olate)

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: yes, virgin
- Age: males, females: 7-9 weeks on arrival
- Weight at study initiation: males 221 - 227 g; females 186 - 208 g on arrival
- Housing: from arrival to pairing, animals were housed up to 5 of one sex to a cage, in polisulphone solid bottomed cages measuring 59.5×38×20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polycarbonate cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material, which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Suitable nesting material was provided
and changed as necessary.
- Diet: a commercially available laboratory rodent diet was offered ad libitum throughout the study, with the exception of the days in which animals were deprived of food for hormone determination.
- Water: Drinking water was supplied ad libitum to each cage via water bottles.
(There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking tap water or the diet)
- Acclimation period: 25 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
-Allocation to groups: 7 days prior to the start of treatment, all animals were weighed. Animals at the extremes of theweight distribution and some female animals showing irregular cycle were excluded to leave the required number of animals. The rats were allocated to the
groups by computerised stratified randomisation to give approximately equal initial group mean bodyweights. Individualswere uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage. The cages were identified by a label
recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were evenly distributed across the battery to minimise possible environmental effects and/or contamination. In addition, 5 females showing irregular cycle during the period between allocation and the start of treatment were replaced with surplus female animals selected from the same batch. Females were selected on the basis of pre-exposure oestrous cyclicity and animals that failed to exhibit regular cycles were not
included in the groups.
ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Humidity: 55 % ± 15 %
- Air changes: 15 to 20 air changes per hour
- Photoperiod: artificial light for 12 hours each day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened by reverse osmosis

Details on exposure:

FORMULATION PROCEDURE
The required amount of test item was dissolved/suspended in the vehicle. The formulations were prepared daily or weekly (concentrations of 5, 15 and 45 mg/ml). The formulations were prepared using a homogeniser and were maintained under magnetic stirring until completion of dosing. Concentrations were calculated.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Details on mating procedure:

Pairing was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in the range from 0.5 to 50 mg/ml. Linearity, accuracy and precision were within the limits stated in for suspensions (r > 0.98; accuracy 90-110 %; precision CV < 5 %). Stability was verified in the range from 0.5 to 50 mg/mL and was found to be 28 hours at room temperature and 8 days at +5 °C ±3 °C. The proposed formulation procedure for the test item was checked in the range from 0.5 to 50 mg/ml by chemical analysis (concentration and homogeneity) to confirm that the method was suitable. Final results for all levels were within the acceptability limits for concentration (85-115 %) and homogeneity (CV < 10 %). Samples of the formulations prepared on Day 1 and Week 5 were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115 % for concentration and CV < 10 % for homogeneity). The software used for this activity was the Empower® 2 Build No. 2154.

Duration of treatment / exposure:

-Males: for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, for a minimum of 35 days.
-Females: for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight.

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight.

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight.

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight.

No. of animals per sex per dose:

10 males and 10 females for each dose level and control

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected on basis of the information from a preliminary non-GLP study. In this study, the test item was administered during the gestation period, to 6 female rats per group at dose levels of 50, 200 and 800mg/kg/day for a total of 13 days. No clinical signs, changes in body weight, gravid uterus weight, litter data and macroscopic observation were noted in the treated females, when compared to controls. Furthermore, the dose selection was also based on the results of a previous study (subacute 28-d oral toxicity study in Wistar rats) in which the dose of 50 mg/kg/day was identified as NOEL and the doses of 200 and 1000 mg/kg/day showed slight to moderate changes due to treatment mainly in case of female rats.

Examinations

Parental animals: Observations and examinations:

MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem
examinations to be carried out during the working period of that day.
CLINICAL SIGNS
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval (approximately 1.5 - 2 hour post-dose) was selected
taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.
BODY WEIGHT
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1, 4, 7 and 13 post partum and on the day of necropsy.
FOOD CONSUMPTION
The weight of food consumed by each cage ofmales and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Days 7
and 13 post partum starting from Day 1 post partum.
PARTURITION CHECK AND DURATION OF GESTATION
A parturition check was performed from Day 20 to Day 25 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition is defined complete (Day 0 post partum).

Oestrous cyclicity (parental animals):

Females were evaluated for oestrous cyclicity 7 days before allocation to groups and during the allocation phase. Animals that exhibited irregular cycle were not included in the study. Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made for females assigned to groups. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken on the day of necropsy.

Litter observations:

PUPS IDENTIFICATION, WEIGHT AND OBSERVATION
As soon as possible, after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. Pups killed or dying during the lactation periodwereweighed before the despatch to necropsy. Observation was performed once daily for all litters. All pups (surviving to treatment and not selected for culling) were sacrificed with the dams on Day 14 post partum.
ADJUSTMENT OF LITTER SIZE (CULLING) AND PUPS SELECTION FOR BLOOD COLLECTION AT NECROPSY
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. Litters were not culled to less than 8 pups, except for 1 dam (no. 75). Retained female pups in each litter were not below 2 (except for dam no.75).
NIPPLE COUNT AT DAY 13 POST PARTUM
The number of nipples/areolae in male pups were counted in each pup on Day 13 post partum (no abnormalities were observed, therefore these data were not reported).
ANOGENITAL DISTANCE (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.
THYROID HORMONES DETERMINATION (T4 and TSH)
Samples were assayed to determine the serum levels of Total thyroxine (total T4) and thyroid stimulating hormone (TSH) by a multiplex assay, using Luminex Magpix system and the MILLIPLEX MAP Rat Thyroid Hormone Magnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K).
As a part of the necropsy procedure, samples of blood were taken from all males, females, culled pups, and pups at Day 14 post partum. In the first instance, the determination was restricted as detailed below:
1. All males from all groups
2. Samples collected in pups at Day 14 post partum
All remaining samples (from pups at Day 4 post partum and from parental females at Day 14 post partum) will be destroyed after finalisation of the report.

Postmortem examinations (parental animals):

Parental animals were killed by exsanguination under isofluorane anaesthesia. The males were killed after the mating of all females, 35 days of treatment period. The surviving females with live pups were killed on Day 14 post partum.
NECROPSY AND BLOOD COLLECTION
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding the animal found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
BLOOD COLLECTION
Blood samples for thyroid hormones determination were taken from the vena cava in dams and adult males and by intra-cardiac puncture from pups according to the following schedule:
– All dams at termination on Day 14 post partum
– All adult males, at termination
Approximately 0.8mL of blood samples were taken from all parental males and females. The blood collected was centrifuged and the serum obtained was stored at -80 °C until analysis.
ORGAN WEIGHTS
From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
TISSUES FIXED AND PRESERVED
Samples of the tissues were fixed and preserved in 10 % neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70 % ethyl alcohol).
HISTOPATHOLOGICAL EXAMINATION
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological
evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed in all animals in the control and high dose groups killed at the end of the study.

Postmortem examinations (offspring):

Pups that had completed the scheduled test period (Day 4 post partum orDay 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
NECROPSY AND BLOOD COLLECTION
All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonads inspection.
BLOOD COLLECTION
Blood samples were taken by intra-cardiac puncture from pups according to the following schedule:
– At least two pups (1 sample of 0.2mL/sex if possible) per litter on Day 4 post partum
– At least two pups (1 sample of 0.5mL/sex) per litter on Day 14 post partum.
ORGAN WEIGHTS
At Day 14 post partum, Thyroid was weighed from one male and one female pup selected for blood collection of hormones determination and preserved in 10 % neutral buffered formalin for possible histopathological examination. The thyroid weight was determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings were carried out by means of the nonparametric Kolmogorov-Smirnov test if n is more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No signs of toxicological relevance were observed during the study.
Slight to marked (with severity increasing with the dose level) red staining in the cage tray and slight to moderate red faeces were observed during the pre-mating, post mating/gestation and post partum phases in cages of mid- and high dose animals.
Slight to marked red staining in the cage tray and moderate red faeces were observed during the mating phase in some cages of the low dose group animals an in all cages of mid- and high dose animals.
The above observations were considered due to the excretion of metabolised test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female from the high dose group was found dead on Day 12 of the pre-mating phase of the study. No clinical signs were observed in this animal up death.
At post mortem macroscopic observations, the most relevant findings observed in this animal included:
– Dark red and/or red soft or granular contents of gastrointestinal tract (ie: stomach, jejunum, ileum, caecum and colon). Such staining was considered related to the colour of the test item.
– Dark red soft contents in the thoracic cavity, red foamy contents in trachea and oesophagus.
On the basis of these findings, the cause of death of this animal was attributed to a misdosing. All other gross pathological findings were considered incidental.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in body weight were observed in the treated animals, when compared to controls.
No changes were observed on terminal body weight of treated animals, when compared to the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both sexes during the study.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted. The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No significant differences were observed in oestrous cycle between treated groups and controls.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

SPERMATOGENIC CYCLE
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups. All females mated.
Males: the copulatory and fertility indices were 100% for all groups.
Females: the copulatory and fertility indices were 100% for all groups.
The number of pregnant females, with live pups on Day 14 post partum was: 10, 10, 10 and 9 in control, low (50 mg/kg/day), mid- (150 mg/kg/day) and high dose (450 mg/kg/day) groups, respectively

Details on results (P0)

LITTER DATA OF FEMALES AND SEX RATIO OF PUPS
No significant differences in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights were observed among the treated dams and the controls.
THYROID HORMONES IN ADULT MALES
No significant differences in T4 and TSH hormone levels were described between the control and the treated parental males.
IMPLANTATION, PRE-BIRTH LOSS DATA AND GESTATION LENGTH OF FEMALES
Gestation periods were similar between treated and control group females. All pregnant dams gave birth on Day 22 post coitum (mean value). Corpora lutea, implantation sites, total litter size and pre-natal loss (percentage) were similar in control and treated groups. A high pre-implantation loss (66.7%) was observed in a single dam of the high dose groups. Since no other significant differences were observed between treated groups and controls, this change was considered to be incidental.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Cold to touch, pale aspect, apparently no food intake (milk) and small appearance, scab(s) in thoracic region and hair loss were the main clinical signs noted in control and treated pups. In addition, found dead pups were observed both in control and treated groups, without dose relation.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were observed in the weight of thyroid in treated pups when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Autolysed abdominal and/or thoracic organs were generally observed in pups found dead at birth and in pups which died during the lactation period. No significant findings were seen in pups killed on Day 14 post partum.

Details on results (F1)

THYROID HORMONES IN MALE AND FEMALE PUPS PERFORMED ON DAY 14 POST PARTUM
No significant differences in T4 and TSH hormone levels were seen in treated pups, when compared to controls.
ANOGENITAL DISTANCE AND NIPPLE COUNT
No differences in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups.
No nipples were observed in male pups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated. No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment-related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio, thyroid hormones (T4 and TSH) did not show any changes of toxicological relevance. Necropsy findings in pups did not reveal any treatment-related effect. No other treatment related changes were observed at macroscopic and microscopic examination. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages and a regular layering in the germinal epithelium was described.

No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated. No relevant treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (50, 150 and 450 mg/kg body weight/day).

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for general toxicity and for fertility and reproduction parameters was considered to be 450 mg/kg body weight/day for males and females.

Executive summary:

In order to provide information on toxicity as well as any possible effects of the test item on male and female reproductive performance, a study was performed according to the OECD Guideline. 421 (2016).

The effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring were investigated, when administered daily by oral gavage to male (up to 36 days) and female (from 51 to 54 days) Sprague Dawley SD rats. The vehicle was water softened by reverse osmosis.

All doses (50, 150 and 450 mg/kg body weight/day) were administered at a constant volume of 10 ml/kg body weight. The treatment groups were composed by 10 males and 10 females.

The following investigations were performed on parental animals of all groups: mortality check, clinical signs, body weight, body weight gain, food consumption and mating performance, thyroid hormone determination only for parental males, litter data, macroscopic observations, organ weights and histopathological examination. Clinical signs, anogenital distance, external and internal examination of pups at necropsy were also recorded. In addition, thyroid hormone levels were determined in 1 pup/sex/group randomly selected at Day 14 post partum. Histopathological evaluation of reproductive organs/tissues was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation. The identification of the stages of the spermatogenic cycle was performed in all males of the control and high dose groups.

The following results were obtained:

Mortality: one female from the high dose group was found dead on Day 12 of the pre-mating phase of the study. The cause of death was attributed to a misdosing represented by dark red soft contents in the thoracic cavity and red foamy contents in trachea and oesophagus.

Fate of females: the number of pregnant females, with live pups on Day 14 post partumwas: 10, 10, 10 and 9 in control, low (50 mg/kg/day), mid- (150 mg/kg/day) and high dose (450 mg/kg/day) groups, respectively.

Clinical signs: no relevant clinical signs were observed during the study. Slight to marked red staining and/or red faeces in the cage tray of animals from all treatedgroups were observed during the study.

Body weight and body weight gain: throughout the study, no difference in body weights was recorded in animals of both sexes compared to the control group.

Food consumption: no effects on food consumption were observed in both sexes.

Thyroid hormone determination: in adult males no differences in hormone levels were described between the control and the treated parentalmales; in male and female pups performed on Day 14 post partum Hormone levels in treated pups were similar to controls.

Oestrous cycle, reproductive parameters, pairing combination and mating performance: oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.

Implantation, pre-birth loss data and gestation length of females: no significant differences were observed for implantation, pre-implantation loss, pre-birth/post implantation loss and gestation length between the treated groups and the controls. Allpregnant dams gave birth on Day 22 post coitum (mean value).

Litter data of females and sex ratio of pups: no significant differences in total litter size, live litter size, mean pup loss, sex ratio and meanpup weights were observed among the treated dams and the controls.

Clinical signs of pups: there were no compound-related effects. Pre-weaning clinical signs were comparable between treated and control groups.

Anogenital distance and nipple count: no differences in the anogenital distance (normalised value) were seen between the controland the treated groups both for male and female pups. No nipples were observed in any pup.

Necropsy findings in pups: necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

Pups thyroid weights: no relevant changes were observed in the pups thyroid weights.

Terminal body weight and organ weights: no relevant changes were observed on terminal body weight, absolute and relative organweights of treated animals, when compared to the controls.

Macroscopic observations: no remarkable differences were noted at post mortem examination in treated animals, when compared to the controls.

Microscopic observations: no treatment-related changes were observed in treated animals, when compared to thecontrols.

In conclusion, no relevant treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (50, 150 and 450 mg/kg body weight/day). No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated. On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for fertility and reproduction parameters was considered to be 450 mg/kg body weight/day for males and females.